Eva Honsova

Rationale: Severe alcohol-associated hepatitis (SAH) is the most critical, acute, inflammatory phenotype within the alcohol-associated liver disease (ALD) spectrum, characterized by high 30- and 90-day mortality. Since several decades, corticosteroids (CS) are the only approved pharmacotherapy offering highly limited survival benefits. Contextually, there is an evident demand for 3PM innovation in the area meeting patients’ needs and improving individual outcomes. Fecal microbiota transplantation (FMT) has emerged as one of the new potential therapeutic options. In this study, we aimed to address the crucial 3PM domains in order to assess (i) the impact of FMT on mortality in SAH patients beyond CS, (ii) to identify factors associated with the outcome to be improved (iii) the prediction of futility, (iv) prevention of suboptimal individual outcomes linked to increased mortality, and (v) personalized allocation of therapy. Methods: We conducted a prospective study (NCT04758806) in adult patients with SAH who were non-responders (NR) to or non-eligible (NE) for CS between January 2018 and August 2022. The intervention consisted of five 100 ml of FMT, prepared from 30 g stool from an unrelated healthy donor and frozen at − 80 °C, administered daily to the upper gastrointestinal (GI) tract. We evaluated the impact of FMT on 30- and 90-day mortality which we compared to the control group selected by the propensity score matching and treated by the standard of care; the control group was derived from the RH7 registry of patients hospitalized at the liver unit (NCT04767945). We have also scrutinized the FMT outcome against established and potential prognostic factors for SAH — such as the model for end-stage liver disease (MELD), Maddrey Discriminant Function (MDF), acute-on-chronic liver failure (ACLF), Liver Frailty Index (LFI), hepatic venous-portal pressure gradient (HVPG) and Alcoholic Hepatitis Histologic Score (AHHS) — to see if the 3PM method assigns them a new dimension in predicting response to therapy, prevention of suboptimal individual outcomes, and personalized patient management. Results: We enrolled 44 patients with SAH (NR or NE) on an intention-to-treat basis; we analyzed 33 patients per protocol for associated factors (after an additional 11 being excluded for receiving less than 5 doses of FMT), and 31 patients by propensity score matching for corresponding individual outcomes, respectively. The mean age was 49.6 years, 11 patients (33.3%) were females. The median MELD score was 29, and ACLF of any degree had 27 patients (81.8%). FMT improved 30-day mortality (p = 0.0204) and non-significantly improved 90-day mortality (p = 0.4386). Univariate analysis identified MELD ≥ 30, MDF ≥ 90, and ACLF grade > 1 as significant predictors of 30-day mortality, (p = 0.031; p = 0.014; p = 0.034). Survival was not associated with baseline LFI, HVPG, or AHHS. Conclusions and recommendations in the framework of 3PM: In the most difficult-to-treat sub-cohort of patients with SAH (i.e., NR/NE), FMT improved 30-day mortality. Factors associated with benefit included MELD ≤ 30, MDF ≤ 90, and ACLF < 2. These results support the potential of gut microbiome as a therapeutic target in the context of 3PM research and vice versa — to use 3PM methodology as the expedient unifying template for microbiome research. The results allow for immediate impact on the innovative concepts of (i) personalized phenotyping and stratification of the disease for the clinical research and practice, (ii) multilevel predictive diagnosis related to personalized/precise treatment allocation including evidence-based (ii) prevention of futile and sub-optimally effective therapy, as well as (iii) targeted prevention of poor individual outcomes in patients with SAH. Moreover, our results add to the existing evidence with the potential to generate new research along the SAH’s pathogenetic pathways such as diverse individual susceptibility to alcohol toxicity, host-specific mitochondrial function and systemic inflammation, and the role of gut dysbiosis thereof.

Background and objectives: The histopathologic classification for ANCA-associated GN distinguishes four classes on the basis of patterns of injury. In the original validation study, these classes were ordered by severity of kidney function loss as follows: focal, crescentic, mixed, and sclerotic. Subsequent validation studies disagreed on outcomes in the crescentic and mixed classes. This study, driven by the original investigators, provides several analyses in order to determine the current position of the histopathologic classification of ANCA-associated GN. Design, setting, participants, & measurements: A validation study was performed with newly collected data from 145 patients from ten centers worldwide, including an analysis of interobserver agreement on the histopathologic evaluation of the kidney biopsies. This study also included a meta-analysis on previous validation studies and a validation of the recently proposed ANCA kidney risk score. Results: The validation study showed that kidney failure at 10-year follow-up was significantly different between the histopathologic classes (PP=0.98). In the meta-analysis, no significant difference in kidney failure was also observed when crescentic class was compared with mixed class (relative risk, 1.15; 95% confidence interval, 0.94 to 1.41). When we applied the ANCA kidney risk score to our cohort, kidney survival at 3 years was 100%, 96%, and 77% in the low-, medium-, and high-risk groups, respectively (P<0.001). These survival percentages are higher compared with the percentages in the original study. Conclusions: The crescentic and mixed classes seem to have a similar prognosis, also after adjusting for differences in patient populations, treatment, and interobserver agreement. However, at this stage, we are not inclined to merge the crescentic and mixed classes because the reported confidence intervals do not exclude important differences in prognosis and because an important histopathologic distinction would be lost.

Mutations in INF2 are frequently responsible for focal segmental glomerulosclerosis (FSGS), which is a common cause of end stage renal disease (ESRD); additionally, they are also connected with Charcot-Marie-Tooth neuropathy. INF2 encodes for inverted formin 2. This protein participates in regulation of the dynamics of the actin cytoskeleton, involving not only the polymerisation, but also the depolymerisation of filaments. The present study is the first mutational analysis of INF2 done in the Czech Republic. Mutational analysis of INF2 was performed on 109 patients (mean age at onset 41.44 ± 18.91 years) with FSGS or minimal change disease (MCD); and also in 6 patients without renal biopsy who had already developed chronic kidney disease (CKD)/ESRD at the time of diagnosis. We used high resolution melting method (HRM), with subsequent Sanger sequencing, in suspect samples from HRM analysis. The HRM method is an effective method for the screening of large cohorts of patients. Two pathogenic mutations (p.Arg214His and p.Arg218Gln) were detected in INF2. The first (p.Arg214His) was identified in the FSGS patient with a positive family history. The second mutation (p.Arg218Gln) was found in two brothers with ESRD of unknown etiology. The most frequent sequence change was the substitution p.P35P, the incidence of which corresponded with the frequencies available in the ExAC Browser and gnomAD Browser databases. This analysis also detected different exonic and intronic changes that probably did not influence the phenotype of the included patients. The INF2 mutational screening is useful in familial FSGS cases as well as in patients with an unknown cause for their ESRD, but with a positive family history. INF2 seems to be not only the cause of FSGS, but also of ESRD of unknown etiology. Our study has confirmed that the HRM analysis is a very useful method for the identification of single nucleotide substitutions.

11051 Background: Resistance against anticancer drugs significantly limits the clinical use and efficacy. One of the most important mechanisms of the multidrug resistance is low accumulation of the drug in cancer cells caused by an increased efflux (mediated mainly by ABCs) or by a decreased uptake (mediated by some members of SLCs). The aim of the present study was to investigate prognostic importance of 13 anticancer drug-relevant SLCs and all 49 human ABCs in PDAC patients in association with clinical and pathological characteristics and the tumor KRAS mutation status. Methods: Tumors and adjacent non-neoplastic pancreatic tissues were obtained from 50 patients with histologically verified PDAC. The transcript profile of ABCs and SLCs was assessed using quantitative real-time PCR. KRAS mutations in exon 2 were assessed by high-resolution melting analysis and sequencing. Associations of transcript levels with clinical parameters were assessed by non-parametric Mann-Whitney tests. The Kaplan-Meier method with the log-rank test and Cox regression were used for analysis of overall survival (OS). Results: Most transporters investigated were deregulated in PDAC and 14 ABCs or SLCs were associated with clinical characteristics. Up-regulation of 7 drug resistance-associated ABC efflux transporters and down-regulation of 5 drug uptake-relevant SLC transporters was observed in tumors vs. non-neoplastic tissues. Moreover, expression levels of SLC28A1 and SLC22A1 were associated with OS of all patients (P=0.001). High transcript levels of SLC29A3 and SLC22A3 significantly predicted longer OS in chemotherapy-treated patients (P=0.004 and P=0.038, respectively). No association of ABC and SLC expression with KRAS mutation status was observed. Conclusions: Up-regulation of numerous drug resistance-associated ABCs and down-regulation of SLCs implicated in drug uptake may explain the generally very poor response of PDAC to cytotoxic agents. This study also identified some ABCs and SLCs as potential prognostic and predictive factors in PDAC. Supported by the Czech Science Foundation grant P301/12/1734 and CZ.1.05/2.1.00/03.0076 European Regional Development Fund.