NIS2+™, an optimisation of the blood-based biomarker NIS4® technology for the detection of at-risk NASH: A prospective derivation and validation study.

BACKGROUND & AIMS: NIS4® is a blood-based non-invasive test designed to effectively rule in/rule out at-risk non-alcoholic steatohepatitis (NASH), defined as non-alcoholic fatty liver disease activity score ≥4 and significant fibrosis (stage ≥2), among patients with metabolic risk factors. Robustness of non-invasive test scores across characteristics of interest including age, type 2 diabetes mellitus, and sex, and optimised analytical aspects are critical for large-scale implementation in clinical practice. We developed and validated NIS2+™, an optimisation of NIS4®, specifically designed to improve score robustness. METHODS: A well-balanced training cohort (n = 198) included patients from the GOLDEN-505 trial. The validation (n = 684) and test (n = 2,035) cohorts included patients from the RESOLVE-IT trial. Well-matched subgroups were created to avoid potential confounding effects during modelling and analysis of score robustness. Models were trained using logistic regressions for at-risk NASH detection and compared using Bayesian information criteria. Performance of NIS2+™ was compared with that of NIS4®, Fibrosis-4, and alanine aminotransferase using area under the receiver operating characteristic curve, and robustness was analysed through score distribution. RESULTS: Using the training cohort to compare all combinations of NIS4® biomarkers, NIS2 (miR-34a-5p, YKL-40) was identified as the best combination of parameters. To correct for the sex effect on miR-34a-5p (validation cohort), sex and sex ∗ miR-34a-5p parameters were added, creating NIS2+™. In the test cohort, NIS2+™ exhibited a statistically higher area under the receiver operating characteristic curve (0.813) vs. NIS4® (0.792; p = 0.0002), Fibrosis-4 (0.653; p <0.0001), and alanine aminotransferase (0.699; p <0.0001). NIS2+™ scores were not affected by age, sex, BMI, or type 2 diabetes mellitus status, providing robust clinical performances irrespective of patient characteristics. CONCLUSION: NIS2+™ constitutes a robust optimisation of NIS4® technology for the detection of at-risk NASH. IMPACT AND IMPLICATIONS: The development of non-invasive tests for accurate, large-scale detection of patients with at-risk non-alcoholic steatohepatitis (NASH; defined as NASH with non-alcoholic fatty liver disease activity score ≥4 and fibrosis stage ≥2) - who are at higher risk for disease progression and for developing liver-related life-threatening outcomes - is critical for identifying this patient population in the clinical setting and improving the screening process of NASH clinical trials. We report the development and validation of NIS2+™, a diagnostic test designed as an optimisation of NIS4® technology, a blood-based panel currently used to detect at-risk NASH in patients with metabolic risk factors. NIS2+™ showed improved performance for the detection of at-risk NASH compared with NIS4® and other non-invasive liver tests that was not impacted by patients' characteristics of interest, such as age, sex, type 2 diabetes mellitus, BMI, dyslipidaemia, and hypertension. This makes NIS2+™ a robust and reliable tool for the diagnosis of at-risk NASH among patients with metabolic risk factors, and an effective candidate for large-scale implementation in clinical practice and clinical trials.

Fine-scale geographical structure of genetic diversity in inland wild beet populations.

Introgression arising from crop-to-wild gene flow provides novel sources of genetic variation in plant species complexes. Hybridization within the Beta vulgaris species complex is of immediate concern; crop lineages (B. vulgaris ssp. vulgaris) hybridize easily with their wild relatives (B. vulgaris ssp. maritima) thereby threatening wild beet gene diversity with genetic swamping. Hybridization 'hotspots' occur in European seed production areas because inland ruderal wild beets occur and reproduce in sympatry with cultivated beets. We studied gene flow occurring between seed-producing cultivars and ruderal wild B. vulgaris in southwestern France to determine whether feral beets, arising from unharvested cultivated seed, represent an opportunity for crop-to-wild gene flow. We surveyed 42 inland ruderal beet populations located near seed production fields for nucleo-cytoplasmic variation and used a cytoplasmic marker diagnostic of cultivated lines. Occurrence of cultivated-type cytoplasm within ruderal populations clearly reflected events of crop seed escape. However, we found no genetic signatures of nuclear cultivated gene introgression, which suggests past introgression of cultivated cytoplasm into a wild nuclear background through seed escape rather than recent direct pollen flow. Overall, patterns of genetic structure suggested that inland ruderal wild beet populations act as a metapopulation, with founding events involving a few sib groups, followed by low rates of seed or pollen gene flow after populations are established. Altogether, our results indicate that a long-lived seed bank plays a key role in maintaining cultivated-type cytoplasm in the wild and highlight the need for careful management of seed production areas where wild and cultivated relatives co-occur.