Prospective comparison of two methods for assessing sarcopenia and interobserver agreement on retrospective CT images.

PURPOSE OF THE STUDY: To compare the relationships between two CT derived sarcopenia assessment methods, and compare their relationship with inter-rater and intrarater validations and colorectal surgical outcomes. STUDY DESIGN: 157 CT scans were identified across Leeds Teaching Hospitals National Health Service Trust for patients undergoing colorectal cancer surgery. 107 had body mass index data available, required to determine sarcopenia status. This work explores the relationship between sarcopenia, as measured by both total cross sectional-area (TCSA) and psoas-area (PA) and surgical outcomes. All images were assessed for inter-rater and intrarater variability for both TCSA and PA methods of sarcopenia identification. The raters included a radiologist, an anatomist and two medical students. RESULTS: Prevalence of sarcopenia was different when measured by PA (12.2%-22.4%) in comparison to TCSA (60.8%-70.1%). Strong correlation exists between muscle areas in both TCSA and PA measures, however, there were significant differences between methods after the application of method-specific cut-offs. There was substantial agreement for both intrarater and inter-rater comparisons for both TCSA and PA sarcopenia measures. Outcome data were available for 99/107 patients. Both TCSA and PA have poor association with adverse outcomes following colorectal surgery. CONCLUSIONS: CT-determined sarcopenia can be identified by junior clinicians, those with anatomical understanding and radiologists. Our study identified sarcopenia to have a poor association with adverse surgical outcomes in a colorectal population. Published methods of identifying sarcopenia are not translatable to all clinical populations. Currently available cut-offs require refinement for potential confounding factors, to provide more valuable clinical information.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade.

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Prevalence of frailty and reliability of established frailty instruments in adult elective colorectal surgical patients: a prospective cohort study.

PURPOSE: Large population studies now demonstrate that frailty is prevalent in all adult age groups. Limited data exist on the association between frailty and surgical outcome in younger patients. The aim of the study was to explore the agreement between frailty identification tools and collect pilot data on their predictive value for frailty-associated outcomes in an adult surgical population. STUDY DESIGN: Prospective cohort study. RESULTS: Frailty scores were recorded in 200 patients (91 men), mean (range) age 57 (18-92) years. The prevalence of prefrailty was 52%-67% and that of frailty 2%-32% depending on the instrument used. Agreement between the instruments was poor, kappa 0.08-0.17 in pairwise comparisons. Outcome data were available on 160 patients. Only the frailty phenotype was significantly associated with adverse outcomes, RR 6.1 (1.5-24.5) for postoperative complications. The three frailty scoring instruments studies had good sensitivity (Clinical Frailty Scale (CFS)-90%, Accumulation Deficit (AD)-96%, Frailty Phenotype (FP)-97%) but poor specificity (CFS-12%, AD-13%, FP-18%) for the prediction of postoperative complications. All three instruments were poorly predictive of adverse outcomes with likelihood ratios of CFS-1.02, AD-1.09 and FP-1.17. CONCLUSIONS: This study showed a significant prevalence of prefrailty and frailty in adult colorectal surgical patients of all ages. There was poor agreement between three established frailty scoring instruments. Our data do not support the use of current frailty scoring instruments in all adult colorectal surgical patients. However, the significant prevalence of prefrailty and frailty across all age groups of adult surgical patient justifies further research to refine frailty scoring in surgical patients.

The contribution of homology arms to nuclease-assisted genome engineering.

Designer nucleases like CRISPR/Cas9 enable fluent site-directed damage or small mutations in many genomes. Strategies for their use to achieve more complex tasks like regional exchanges for gene humanization or the establishment of conditional alleles are still emerging. To optimize Cas9-assisted targeting, we measured the relationship between targeting frequency and homology length in targeting constructs using a hypoxanthine-guanine phosphoribosyl-transferase assay in mouse embryonic stem cells. Targeting frequency with supercoiled plasmids improved steeply up to 2 kb total homology and continued to increase with even longer homology arms, thereby implying that Cas9-assisted targeting efficiencies can be improved using homology arms of 1 kb or greater. To humanize the Kmt2d gene, we built a hybrid mouse/human targeting construct in a bacterial artificial chromosome by recombineering. To simplify the possible outcomes, we employed a single Cas9 cleavage strategy and best achieved the intended 42 kb regional exchange with a targeting construct including a very long homology arm to recombine ∼42 kb away from the cleavage site. We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site.