Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells.

A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.

Resilience and Patient-Reported Outcomes in Patients Undergoing Orthopedic Hand Surgery.

BACKGROUND: Previous studies have examined the impact of resiliency on postoperative outcomes in other orthopedic domains, but none to date have done so for hand surgery. METHODS: We performed a retrospective analysis of prospectively collected data of patients undergoing hand surgery at a single institution. We included patients with complete preoperative outcomes scores and 6-month follow-up. All patients completed the Brief Resilience Scale (BRS), Disabilities of the Arm, Shoulder, and Hand (DASH) Score, Quick Disabilities of the Arm, Shoulder, and Hand (QuickDASH), Veterans RAND 12-Item Health Survey (VR-12), and Numeric Rating Scale (NRS) for pain. Patients were stratified into high-resiliency (HR) and low-resiliency (LR) groups based on the preoperative BRS score, and outcomes between groups were compared. RESULTS: We identified 91 patients who underwent hand procedures and completed full preoperative and postoperative outcomes measures. There were no observed preoperative differences between the groups in all outcomes scores except the VR-12 Mental Component Score. Postoperatively, the HR group had superior DASH, QuickDASH, and VR-12 (mental and physical component) scores than the LR group. Postoperative pain, as measured by the NRS, was significantly lower in the HR group despite there being no preoperative difference. A larger percentage of patients in the HR group met the minimal clinically important difference in all outcomes except for the VR-12 Mental Component Scores. CONCLUSIONS: Patients with high preoperative resilience appear to have significantly better clinical outcomes following hand surgery with superior DASH, QuickDASH, and VR-12 scores at 6-month follow-up. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic study/Level IV evidence.

Pharmacogenetic actionability and medication prescribing in people with cystic fibrosis.

Although major advancements have been made in the therapeutics for people with cystic fibrosis (PwCF), many still require the use of multiple medications to manage acute exacerbations of disease and maintain health. Iterative trial and error processes of pharmacotherapeutic management can be optimized by assessing and incorporating pharmacogenetics. For 82 PwCF, we reviewed 2 years of medication use and response history and interrogated metabolizer status for common pharmacogenes, revealing 3336 medication exposure events (MEEs) to 286 unique medications. As expected, the more frequent MEEs were those commonly used to treat cystic fibrosis (CF), such as antibiotics and respiratory medications. Antibiotics also comprised 56.7% of the undesirable drug responses. The impact of gene variants on drug responses was assessed using Pharmacogenomics Knowledgebase (PharmGKB) and Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. Thirty-three (11.5%) medications have strong evidence of genetic influence on response as evidenced by gene-based dosing guidelines. 110 (38.5%) unique medications had at least one association with a very important pharmacogene, whereas 143 (50%) were associated with at least one clinical or variant annotation. Over 97% of participants had at least one actionable genotype. Eleven (13.4%) patients with an actionable genotype, taking the impacted medication, had an undesirable drug response described in the CPIC guidelines that could potentially have been mitigated with a priori knowledge of the genotype. PwCF take many medications for disease management, with frequent dose changes to elicit a desired clinical effect. As CF care evolves, implementing pharmacogenetics testing can improve efficiency and safety of prescribing practices using precision selection and dosing at medication initiation.

Outcomes of Proton Beam Therapy Compared With Intensity-Modulated Radiation Therapy for Uterine Cancer.

Purpose: To compare Patient-Reported Outcomes version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE) in patients with endometrial cancer receiving adjuvant pelvic radiation therapy with proton beam therapy (PT) versus intensity-modulated radiation therapy (IMRT). Materials and Methods: Patients with uterine cancer treated with curative intent who received either adjuvant PT or IMRT between 2014 and 2020 were identified. Patients were enrolled into a prospective registry using a gynecologic-specific subset of PRO-CTCAE designed to assess symptom impact on daily living. Questions included gastrointestinal (GI) symptoms of diarrhea, flatulence, bowel incontinence, and constipation in addition to other pertinent gynecologic, urinary, and other general symptoms. Symptom-based questions were on a 0- to 4-point scale, with grade 3+ symptoms occurring frequently or almost always. Patient-reported toxicity was analyzed at baseline, end of treatment (EOT), and at 3, 6, 9, and 12 months after treatment. Unequal variance t tests were used to determine if treatment type was a significant factor in baseline-adjusted PRO-CTCAE. Results: Sixty-seven patients met inclusion criteria. Twenty-two received PT and 45 patients received IMRT. Brachytherapy boost was delivered in 73% of patients. Median external beam dose was 45 Gy for both PT and IMRT (range, 45-58.8 Gy). When comparing PRO-CTCAE, PT was associated with less diarrhea at EOT (P = .01) and at 12 months (P = .24) than IMRT. Loss of bowel control at 12 months was more common in patients receiving IMRT (P = .15). Any patient reporting grade 3+ GI toxicity was noted more frequently with IMRT (31% versus 9%, P = .09). Discussion: Adjuvant PT is a promising treatment for patients with uterine cancer and may reduce patient-reported GI toxicity as compared with IMRT.

A Prospective Study of Mucosal Sparing Radiation Therapy in Resected Oropharyngeal Cancer Patients.

PURPOSE: Our objective was to report the prospective results of mucosal sparing radiation therapy in human papillomavirus-related oropharyngeal squamous cell carcinoma. METHODS AND MATERIALS: From March 2016 through May 2019, patients were enrolled in this institutional review board-approved prospective cohort study at a multisite institution. Inclusion criteria included p16+ American Joint Committee on Cancer seventh edition pathologic T1 or T2, N1 to N3, and M0 oropharyngeal cancers. Proton therapy (PT) was delivered to at-risk nodal regions, excluding the primary mucosal site. Secondary to insurance denial for PT, intensity modulated radiation therapy (IMRT) was allowed. European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module and Patient-Reported Outcomes Measurement Information System surveys (quality of life [QOL]) and modified barium swallowing impairment profiles (MBSImP) were obtained at baseline before radiation therapy, then 3 and 12 months after radiation therapy. Kaplan-Meier estimates were calculated for time-to-event clinical outcomes, and repeated measures mixed models were used to explore changes in QOL over time. A comparison of QOL and swallowing outcomes with standard-of-care treatment was analyzed. RESULTS: There were 61 evaluable patients with a median follow-up of 38 months (range, 10-64); 44 (72%) were treated with PT and 17 (28%) were treated with IMRT. The 2-year local control, locoregional control, distant metastasis-free survival, and overall survival were 98%, 97%, 98%, and 100%, respectively. There were 6 grade ≥3 events related to treatment. Two IMRT patients required percutaneous endoscopic gastrostomy tube placement during treatment secondary to significant nausea due to dysgeusia. Patients noted significant QOL improvement over time in the pain, swallowing, speech, social eating, social contact, mouth opening, and use of pain medication domains (all P < .02). The MBSImP overall severity score as well as oral and pharyngeal impairment scores showed stability with no significant change over time. For the 44 patients treated with PT, the mean D95 to the primary target was 10.7 Gy (standard deviation = 12.5 Gy). CONCLUSIONS: Mucosal sparing radiation is well tolerated in select resected human papillomavirus-related oropharyngeal squamous cell carcinoma with a low risk of recurrence at the mucosal primary site, a low rate of percutaneous endoscopic gastrostomy tube placement, and few radiation-related grade ≥3 adverse events.

Development and implementation of a Dependable, Simple, and Cost-effective (DSC), open-source running wheel in High Drinking in the Dark and Heterogeneous Stock/Northport mice.

Maintaining healthy and consistent levels of physical activity (PA) is a clinically proven and low-cost means of reducing the onset of several chronic diseases and may provide an excellent strategy for managing mental health and related outcomes. Wheel-running (WR) is a well-characterized rodent model of voluntary PA; however, its use in biomedical research is limited by economical and methodical constraints. Here, we showcase the DSC (Dependable, Simple, Cost-effective), open-source running wheel by characterizing 24-h running patterns in two genetically unique mouse lines: inbred High Drinking in the Dark line 1 [iHDID-1; selectively bred to drink alcohol to intoxication (and then inbred to maintain phenotype)] and Heterogeneous Stock/Northport (HS/Npt; the genetically heterogeneous founders of iHDID mice). Running distance (km/day), duration (active minutes/day) and speed (km/hour) at 13-days (acute WR; Experiment 1) and 28-days (chronic WR; Experiment 2) were comparable to other mouse strains, suggesting the DSC-wheel reliably captures murine WR behavior. Analysis of 24-h running distance supports previous findings, wherein iHDID-1 mice tend to run less than HS/Npt mice in the early hours of the dark phase and more than HS/Npt in the late hours of dark phase/early light phase. Moreover, circadian actograms were generated to highlight the broad application of our wheel design across disciplines. Overall, the present findings demonstrate the ability of the DSC-wheel to function as a high-throughput and precise tool to comprehensively measure WR behaviors in mice.

Proton therapy for isolated local regional recurrence of breast cancer after mastectomy alone.

Purpose/Objectives: To assess adverse events (AEs) and disease-specific outcomes after proton therapy for isolated local-regional recurrence (LRR) of breast cancer after mastectomy without prior radiotherapy (RT). Materials/Methods: Patients were identified from a multi-institutional prospective registry and included if diagnosed with invasive breast cancer, initially underwent mastectomy without adjuvant RT, experienced an LRR, and subsequently underwent salvage treatment, including proton therapy. Follow-up and cancer outcomes were measured from the date of RT completion. Results: Nineteen patients were included. Seventeen patients were treated with proton therapy to the chest wall and comprehensive regional lymphatics (17/19, 90%). Maximum grade AE was grade 2 in 13 (69%) patients and grade 3 in 4 (21%) patients. All patients with grade 3 AE received > 60 GyE (p=0.04, Spearman correlation coefficient=0.5). At the last follow-up, 90% of patients were alive with no LRR or distant recurrence. Conclusions: For breast cancer patients with isolated LRR after initial mastectomy without adjuvant RT, proton therapy is well-tolerated in the salvage setting with excellent loco-regional control. All grade 3 AEs occurred in patients receiving > 60 GyE.

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate.

The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of -5.7 to 3.5% (ELX), -3.2 to 6.0% (TEZ), and -3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Brain gene expression differences related to ethanol preference in the collaborative cross founder strains.

The collaborative cross (CC) founder strains include five classical inbred laboratory strains [129S1/SvlmJ (S129), A/J (AJ), C57BL/6J (B6), NOD/ShiLtJ (NOD), and NZO/HILtJ (NZO)] and three wild-derived strains [CAST/EiJ (CAST), PWK/PhJ (PWK), and WSB/EiJ (WSB)]. These strains encompass 89% of the genetic diversity available in Mus musculus and ∼10-20 times more genetic diversity than found in Homo sapiens. For more than 60 years the B6 strain has been widely used as a genetic model for high ethanol preference and consumption. However, another of the CC founder strains, PWK, has been identified as a high ethanol preference/high consumption strain. The current study determined how the transcriptomes of the B6 and PWK strains differed from the 6 low preference CC strains across 3 nodes of the brain addiction circuit. RNA-Seq data were collected from the central nucleus of the amygdala (CeA), the nucleus accumbens core (NAcc) and the prelimbic cortex (PrL). Differential expression (DE) analysis was performed in each of these brain regions for all 28 possible pairwise comparisons of the CC founder strains. Unique genes for each strain were identified by selecting for genes that differed significantly [false discovery rate (FDR) < 0.05] from all other strains in the same direction. B6 was identified as the most distinct classical inbred laboratory strain, having the highest number of total differently expressed genes (DEGs) and DEGs with high log fold change, and unique genes compared to other CC strains. Less than 50 unique DEGs were identified in common between B6 and PWK within all three brain regions, indicating the strains potentially represent two distinct genetic signatures for risk for high ethanol-preference. 338 DEGs were found to be commonly different between B6, PWK and the average expression of the remaining CC strains within all three regions. The commonly different up-expressed genes were significantly enriched (FDR < 0.001) among genes associated with neuroimmune function. These data compliment findings showing that neuroimmune signaling is key to understanding alcohol use disorder (AUD) and support use of these 8 strains and the highly heterogeneous mouse populations derived from them to identify alcohol-related brain mechanisms and treatment targets.

Patient-reported outcomes for patients with breast cancer undergoing radiotherapy: A single-center registry experience.

Background: We present Patient-Reported Outcomes Version of the Common Terminology Criteria for Adverse Events (PRO-CTCAE) for patients undergoing adjuvant radiotherapy for breast cancer with curative intent. We describe the frequency and severity of PRO-CTCAE and analyze them with respect to dose fractionation. Methods: Patients were included in this study if they were treated with curative intent for breast cancer and enrolled on a prospective registry. Patients must have completed at least one baseline and one post-radiation survey that addressed PRO-CTCAE. For univariate and multivariate analysis, categorical variables were analyzed by Fisher's exact test and continuous variables by Wilcoxon rank sum test. PRO-CTCAE items graded ≥2 and ≥3 were analyzed between patients who received hypofractionation (HF) versus standard conventional fractionation (CF) therapy by the Chi-square test. Results: Three hundred thirty-one patients met inclusion criteria. Pathologic tumor stage was T1-T2 in 309 (94%) patients. Eighty-seven (29%) patients were node positive. Two hundred forty-seven patients (75%) experienced any PRO-CTCAE grade ≥2, and 92 (28%) patients experienced any PRO-CTCAE grade ≥3. CF was found to be associated with an increased risk of grade ≥3 skin toxicity, swallowing, and nausea (all p < 0.01). HF (OR 0.48, p < 0.01) was significant in the multivariate model for decreased risk of any occurrence of PRO-CTCAE ≥3. Conclusions: Our study reports one of the first clinical experiences utilizing multiple PRO-CTCAE items for patients with breast cancer undergoing radiation therapy with curative intent. Compared with CF, HF was associated with a significant decrease in any PRO-CTCAE ≥3 after multivariate analysis.

Plasma and cellular ivacaftor concentrations in patients with cystic fibrosis.

Access to cystic fibrosis transmembrane conductance regulator (CFTR) modulators has been gradually increasing for people with cystic fibrosis, the first of which was ivacaftor, a CFTR potentiator that is part of all clinically available modulator treatments. In this study, we hypothesized that the steady-state concentrations in blood and tissue are highly variable in patients taking ivacaftor in a real-world context, which may have an impact on the treatment approach. We collected nasal epithelial cells to estimate target site concentrations and blood samples to estimate pharmacokinetic parameters at a steady state. We found that patients on ivacaftor monotherapy have variable concentrations well above the maximal effective concentration and may maintain concentrations necessary for the clinical benefit even if dosing is reduced. We also are the first to provide detailed target site concentration data over time, which shows that tissue concentrations do not fluctuate significantly and do not correlate with plasma concentrations. These findings show that some patients may have higher-than-expected concentrations and may benefit from tailored dosing to balance clinical response with side effects or adherence needs.

Early Experience Using Proton Beam Therapy for Extremity Soft Tissue Sarcoma: A Multicenter Study.

Purpose: Proton beam therapy (PBT) may provide an advantage when planning well-selected patients with extremity soft tissue sarcoma (eSTS), specifically for large, anatomically challenging cases. We analyzed our early experience with PBT on toxicity and outcomes. Materials and Methods: A retrospective study was performed for eSTS treated between June 2016 and October 2020 with pencil beam scanning PBT at 2 institutions. Diagnostic, treatment, and toxicity characteristics were gathered from baseline to last follow-up or death. Wound complications were defined as secondary operations for wound repair (debridement, drainage, skin graft, and muscle flap) or nonoperative management requiring hospitalization. Statistical analysis was performed with R software. Results: Twenty consecutive patients with a median age 51.5 years (range, 19-78 years) were included. Median follow-up was 13.7 months (range, 1.7-48.1 months). Tumor presentation was primary (n = 17) or recurrent after prior combined modality therapy (n = 3). Tumor location was either lower extremity (n = 16) or upper extremity (n = 4). Radiation was delivered preoperatively in most patients (n = 18). Median pretreatment tumor size was 7.9 cm (range, 1.3 -30.0 cm). The 1-year locoregional control was 100%. Four patients (20%) had developed metastatic disease by end of follow-up. Maximum toxicity for acute dermatitis was grade 2 in 8 patients (40%) and grade 3 in 3 patients (15%). After preoperative radiation and surgical resection, acute wound complications occurred in 6 patients (35%). Tumor size was larger in patients with acute wound complications compared with those without (medians 16 cm, range [12-30.0 cm] vs 6.3 cm, [1.3-14.4 cm], P = .003). Conclusion: PBT for well selected eSTS cases demonstrated excellent local control and similar acute wound complication rate comparable to historic controls. Long-term follow-up and further dosimetric analyses will provide further insight into potential advantages of PBT in this patient population.

Hypocalcemia as a predictor of mortality and transfusion. A scoping review of hypocalcemia in trauma and hemostatic resuscitation.

BACKGROUND: Calcium plays an essential role in physiologic processes, including trauma's "Lethal Diamond." Thus, inadequate serum calcium in trauma patients exacerbates the effects of hemorrhagic shock secondary to traumatic injury and subsequently poorer outcomes compared to those with adequate calcium levels. Evidence to date supports the consideration of calcium derangements when assessing the risk of mortality and the need for blood product transfusion in trauma patients. This review aims to further elucidate the predictive strength of this association for future treatment guidelines and clinical trials. METHODS: Publications were collected on the relationship between i-Ca and the outcomes of traumatic injuries from PubMed, Web of Science, and CINAHL. Manuscripts were reviewed to select for English language studies. Hypocalcemia was defined as i-Ca <1.2 mmol/L. RESULTS: Using PRISMA guidelines, we reviewed 300 studies, 7 of which met our inclusion criteria. Five papers showed an association between hypocalcemia and mortality. CONCLUSIONS: In adult trauma patients, there has been an association seen between hypocalcemia, mortality, and the need for increased blood product transfusions. It is possible we are now seeing an association between low calcium levels prior to blood product administration and an increased risk for mortality and need for transfusion. Hypocalcemia may serve as a biomarker to show these needs. Therefore, hypocalcemia could potentially be used as an independent predictor for multiple transfusions such that ionized calcium measurements could be used predictively, allowing faster administration of blood products.

MOZ and Menin-MLL Complexes Are Complementary Regulators of Chromatin Association and Transcriptional Output in Gastrointestinal Stromal Tumor.

Gastrointestinal stromal tumor (GIST) is commonly characterized by activating mutations in the receptor tyrosine kinase KIT. Tyrosine kinase inhibitors are the only approved therapy for GIST, and complementary treatment strategies are urgently needed. As GIST lacks oncogene amplification and relies upon an established network of transcription factors, we hypothesized that unique chromatin-modifying enzymes are essential in orchestrating the GIST epigenome. We identified through genome-scale CRISPR screening that MOZ and Menin-MLL chromatin regulatory complexes are cooperative and unique dependencies in GIST. These complexes were enriched at GIST-relevant genes and regulated their transcription. Inhibition of MOZ and Menin-MLL complexes decreased GIST cell proliferation by disrupting interactions with transcriptional/chromatin regulators, such as DOT1L. MOZ and Menin inhibition caused significant reductions in tumor burden in vivo, with superior effects observed with combined Menin and KIT inhibition. These results define unique chromatin regulatory dependencies in GIST and identify potential therapeutic strategies for clinical application. SIGNIFICANCE: Although many malignancies rely on oncogene amplification, GIST instead depends upon epigenetic regulation of KIT and other essential genes. Utilizing genome-scale CRISPR dependency screens, we identified complementary chromatin-modifying complexes essential to GIST and characterize the consequences of their disruption, elucidating a novel therapeutic approach to this disease. This article is highlighted in the In This Issue feature, p. 1599.

Management of Severe Crush Injuries in Austere Environments: A Special Operations Perspective.

Crush injuries present a challenging case for medical providers and require knowledge and skill to manage the subsequent damage to multiple organ systems. In an austere environment, in which resources are limited and evacuation time is extensive, a medic must be prepared to identify trends and predict outcomes based on the mechanism of injury and patient presentation. These injuries occur in a variety of environments from motor vehicle accidents (at home or abroad) to natural disasters and building collapses. Crush injury can lead to compartment syndrome, traumatic rhabdomyolysis, arrythmias, and metabolic acidosis, especially for patients with extended treatment and extrication times. While crush syndrome occurs due to the systemic effects of the injury, the onset can be as early as 1 hour postinjury. With a comprehensive understanding of the pathophysiology, diagnosis, management, and tactical considerations, a prehospital provider can optimize patient outcomes and be prepared with the tools they have on hand for the progression of crush injury into crush syndrome.