Citations

PURPOSE Cancer of unknown primary (CUP) is a syndrome comprising metastatic cancers without a clinically identified primary site. Although patients with CUP have an unfavorable prognosis, treatment with site-specific therapies guided by clinical features, standard pathology, and molecular assays can improve overall survival. The 92-gene assay (CancerTYPE ID) is a gene expression–based classifier that helps identify the tissue of origin for metastatic cancers with unknown or uncertain diagnoses. This study reports the frequency of selected molecular aberrations of oncogenes, including KRAS, IDH1/2, BRCA1/2, and BRAF, in patients with CUP in the MOSAIC database to highlight potential treatment options. METHODS MOSAIC is a database of patients with CUP submitted for CancerTYPE ID testing and NeoTYPE biomarker testing. Tumor biopsy samples were analyzed by CancerTYPE ID for tumor type identification and further tested for molecular aberrations of oncogenes, including KRAS, IDH1/2, BRCA1/2, and BRAF. RESULTS CancerTYPE ID identified a specific tumor type in 92.5% (2,929 of 3,168) of CUP cases in the MOSAIC database. The most commonly identified histological type was adenocarcinoma (75.4%), with pancreaticobiliary being the most common molecularly diagnosed cancer (24.9%). Aberrations in KRAS, IDH1/2, BRCA, and BRAF genes were identified in 18.8% (n = 597) of biopsies. A cancer-specific US Food and Drug Administration (FDA)–approved or investigational targeted therapy was potentially available for 24.6% (n = 147) of these patients. CONCLUSION This retrospective analysis supports incorporating CancerTYPE ID into the evaluation for patients with CUP to help determine the tissue of origin and identify actionable genetic alterations. This approach may allow more patients with CUP to benefit from site-specific FDA-approved targeted therapies or enrollment into clinical trials.

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C•G-to-T•A conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA are predicted to remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences ( i.e. , the 5′-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5′-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5′ bases. These modified DdCBEs are herein referred to as αDdCBEs. Notably, 5′-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by αDdCBEs, which exhibited significant improvements in activity and specificity, regardless of the most 5′ bases of their TALE binding sites. Furthermore, we showed that αDdCBEs are compatible with the enhanced DddA tox variants DddA6 and DddA11, and we validated TALE shifting with αDdCBEs as an effective approach to optimize base editing outcomes. Overall, αDdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19–23 at the 5′ end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

ABSTRACT Wound infections, exacerbated by the prevalence of antibiotic-resistant bacterial pathogens, necessitate innovative antimicrobial approaches. Polymicrobial infections, often involving Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), present challenges due to biofilm formation and antibiotic resistance. Hypochlorous acid (HOCl), a potent antimicrobial agent, holds promise as an alternative therapy. An electrochemical bandage (e-bandage) that generates HOCl in situ via precise polarization controlled by a miniaturized potentiostat was evaluated for the treatment of murine wound biofilm infections containing both P. aeruginosa with “difficult-to-treat” resistance and MRSA. Previously, HOCl-producing e-bandage was shown to reduce murine wound biofilms containing P. aeruginosa alone. Here, in 5-mm excisional skin wounds containing 48-h biofilms comprising MRSA and P. aeruginosa combined, polarized e-bandage treatment reduced MRSA by 1.1 log 10 CFU/g ( P = 0.026) vs non-polarized e-bandage treatment (no HOCl production), and 1.4 log 10 CFU/g (0.0015) vs Tegaderm only controls; P. aeruginosa was similarly reduced by 1.6 log 10 CFU/g ( P = 0.0032) and 1.6 log 10 CFU/g ( P = 0.0015), respectively. For wounds infected with MRSA alone, polarized e-bandage treatment reduced bacterial load by 1.1 log 10 CFU/g ( P = 0.0048) and 1.3 log 10 CFU/g ( P = 0.0048) compared with non-polarized e-bandage and Tegaderm only, respectively. The e-bandage treatment did not negatively impact wound healing or cause tissue toxicity. The addition of systemic antibiotics did not enhance the antimicrobial efficacy of e-bandages. This study provides additional evidence for the HOCl-producing e-bandage as a novel antimicrobial strategy for managing wound infections, including in the context of antibiotic resistance and polymicrobial infections. IMPORTANCE New approaches are needed to combat the rise of antimicrobial-resistant infections. The HOCl-producing electrochemical bandage (e-bandage) leverages in situ generation of HOCl, a natural biocide, for broad-spectrum killing of wound pathogens. Unlike traditional therapies that may exhibit limited activity against biofilms and antimicrobial-resistant organisms, the e-bandage offers a potent, standalone solution that does not contribute to further resistance or require adjunctive antibiotic therapy. Here, we show the ability of the e-bandage to address polymicrobial infection by antimicrobial resistant clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa , two commonly isolated, co-infecting wound pathogens. Effectiveness of the HOCl-producing e-bandage in reducing pathogen load while minimizing tissue toxicity and avoiding the need for systemic antibiotics underscores its potential as a tool in managing complex wound infections.

Key Points Peripheral blood biomarkers may have value in monitoring kidney transplant recipients after treatment of acute rejection. The donor-derived cellfree DNA may be more sensitive to identifying antibody-mediated rejection and gene expression profile may be more sensitive to identifying acute cellular rejection. Background Persistent rejection is an increasingly recognized barrier to long-term kidney allograft survival. A noninvasive method to help identify patients with persistent rejection in need of biopsy would be valuable. Methods This was a post hoc analysis of a multicenter observational study. Patients who had a biopsy-proven acute rejection, had another biopsy within 9 months (270 days), and had a biopsy-paired biomarker sample were included. Results A total of 64 index rejections in 58 patients with repeat biopsies were identified with a median time to repeat biopsy of 100 days. Persistent rejection was present in 61%; 69% of follow-up biopsies were performed in clinically stable patients. Peripheral blood gene expression profile (GEP) demonstrated a sensitivity of 59%, specificity of 76%, positive predictive value (PPV) of 79%, and negative predictive value of 54%. Donor-derived cellfree DNA (dd-cfDNA) demonstrated a sensitivity of 62%, specificity of 86%, PPV of 88%, and negative predictive value of 56%. For repeat biopsies within 90 days of rejection in clinically stable patients (63% of repeat biopsies), both GEP and dd-cfDNA had specificities and PPVs of 100%. GEP was more likely to be positive in T-cell–mediated rejection while dd-cfDNA was more likely to be positive in antibody-mediated rejection. Conclusions Both GEP and dd-cfDNA may have utility in identifying clinically stable patients with persistent rejection in need of biopsy; however, they identify different types of rejections. Clinical Trial registration number: NCT01531257.

Abstract Background and Aims Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. Methods KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. Results KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. Conclusions This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

Abstract Background: Circulating levels of THC and CBD may be altered when administered with medications that strongly or moderately inhibit or induce CYP450 enzymes responsible for metabolizing both substances. THC and CBD levels can also theoretically be altered in individuals who carry germline genetic variants or atypical phenotypes of CYP450 enzymes. In individuals where drug and gene interactions occur together, there could be a heightened impact on the circulating levels of THC and CBD. Materials and Methods: We conducted a retrospective chart review using a database of participants who previously completed pharmacogenomic testing and reported oral cannabis use in a medical encounter. Two pharmacogenomic-trained pharmacists reviewed medications and atypical variants in each participant to characterize the predicted effect of cannabis-drug, cannabis-gene, and cannabis-drug-gene interactions on circulating levels of THC and CBD. Results: Oral cannabis use was confirmed in 71 participants, with an average age of 68.5 years and primarily White women. Regarding THC metabolism, drug-only interactions were noted in 1 participant; both drug and gene interactions were noted in 4 participants; single- or combination-gene-only interactions were noted in 38 participants; and 28 participants had no interactions. Concerning CBD metabolism, drug-only interactions were noted in 4 participants; both drug and gene interactions were noted in 4 participants; single- or combination-gene-only interactions were noted in 36 participants; and 27 participants had no interactions. Conclusion: This study is the first, to our knowledge, to conceptualize the potential impact of drugs and atypical phenotypes on altering circulating levels of THC and CBD in a clinical sample. The absence of published clinical outcomes in this field renders it challenging to estimate clinical significance of these findings. Until such data become available, leveraging the theoretical insights into drug interactions and atypical cannabis metabolism described in our study may serve as a tool to guide clinical care.

Abstract Objective To use an artificial intelligence (AI)‐powered large language model (LLM) to improve readability of patient handouts. Study Design Review of online material modified by AI. Setting Academic center. Methods Five handout materials obtained from the American Rhinologic Society (ARS) and the American Academy of Facial Plastic and Reconstructive Surgery websites were assessed using validated readability metrics. The handouts were inputted into OpenAI's ChatGPT‐4 after prompting: “Rewrite the following at a 6th‐grade reading level.” The understandability and actionability of both native and LLM‐revised versions were evaluated using the Patient Education Materials Assessment Tool (PEMAT). Results were compared using Wilcoxon rank‐sum tests. Results The mean readability scores of the standard (ARS, American Academy of Facial Plastic and Reconstructive Surgery) materials corresponded to “difficult,” with reading categories ranging between high school and university grade levels. Conversely, the LLM‐revised handouts had an average seventh‐grade reading level. LLM‐revised handouts had better readability in nearly all metrics tested: Flesch‐Kincaid Reading Ease (70.8 vs 43.9; P < .05), Gunning Fog Score (10.2 vs 14.42; P < .05), Simple Measure of Gobbledygook (9.9 vs 13.1; P < .05), Coleman‐Liau (8.8 vs 12.6; P < .05), and Automated Readability Index (8.2 vs 10.7; P = .06). PEMAT scores were significantly higher in the LLM‐revised handouts for understandability (91 vs 74%; P < .05) with similar actionability (42 vs 34%; P = .15) when compared to the standard materials. Conclusion Patient‐facing handouts can be augmented by ChatGPT with simple prompting to tailor information with improved readability. This study demonstrates the utility of LLMs to aid in rewriting patient handouts and may serve as a tool to help optimize education materials. Level of Evidence Level VI.