Antimitochondrial Rather than Antinuclear Antibodies Correlate with Severe Drug-Induced Liver Injury.

INTRODUCTION: A proportion of patients with drug-induced liver injury (DILI) present with autoantibodies, which has led to the current concept of autoimmune-like DILI. However, no standardized definition exists and the clinical relevance has not been studied in detail yet. METHODS: 143 patients with DILI enrolled in a prospective study were analyzed. DILI diagnosis was based on the monocyte-derived hepatocyte-like cell test and supported by Roussel Uclaf Causality Assessment Method (RUCAM) and expert adjudication. Testing for antinuclear antibodies (ANA) and antimitochondrial antibodies (AMA) was performed using immunofluorescence. ANA titers ≥1:100 were considered positive and ≥1:400 clinically relevant; AMA positivity was considered at titers ≥1:100. RESULTS: 67% exhibited ANA ≥1:100 and 29% ANA ≥1:400; 10% were AMA positive. There was no significant correlation between the ANA titers and the causative drug, while AMA positive patients had taken nonsteroidal anti-inflammatory drugs more frequently. No difference was seen regarding clinical characteristics or laboratory parameters in patients with ANA ≥1:400, while patients with positive AMA presented with higher aminotransferases, bilirubin, and international normalized ratio. Significantly higher proportions of patients with ANA ≥1:400 or AMA positivity exhibited elevated immunoglobulin G levels. AMA positivity but not elevated ANA titers correlated with a higher proportion of Hy's law positivity. CONCLUSION: A closer look in a causality proven DILI cohort provided no evidence that presence of ANA titers is specific for DILI by a certain medication. AMA rather than ANA positivity was related to a more pronounced liver injury.

Development and Validation of a Test to Identify Drugs That Cause Idiosyncratic Drug-Induced Liver Injury.

BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (iDILI) is one of the most challenging diagnoses in hepatology. It is frequently impossible to identify the agent that has caused iDILI in patients who take multiple medicines. We developed an in vitro method to identify drugs that cause liver injury in patients, based on drug toxicity to monocyte-derived hepatocyte-like (MH) cells from patient blood samples. We then collected data on patients who were re-exposed to drugs found to be toxic in the MH test to validate test performance. METHODS: We performed a prospective study of patients referred to the University Hospital in Munich, Germany, with acute liver injury believed to be caused by medications (300 patients were enrolled in the study and we present data from 40 patients with iDILI and re-exposure to implicated drugs). We collected data from patients on medical history, laboratory test and imaging results, findings from biopsy analyses, and medications taken. Blood samples were collected from all patients and MH cells were isolated and cultured for 10 days. MH cells were then incubated with drugs to which each patient had been exposed, and toxicity was measured based on release of lactate dehydrogenase. Agents found to be toxic to MH cells were considered as candidates for the cause of liver injury. Patients were followed up for up to 6 months after liver injury and data on drug re-exposures and subsequent liver damage within the following 3 to 24 months were associated with findings from MH tests. RESULTS: Our test identified 10 drugs that were toxic to MH cells from 13 patients (amoxicillin/clavulanate to cells from 2 patients; diclofenac to cells from 2 patients; methylprednisolone to cells from 2 patients; and atorvastatin, metamizole, pembrolizumab, piperacillin/tazobactam, moxifloxacin, duloxetine, or sertraline each to cells from 1 patient). Thirteen patients had a recurrence of liver injury after inadvertent re-exposure to a single drug, and the MH test correctly identified 12 of the 13 drugs that caused these liver re-injury events. All 86 drugs that were not toxic to MH cells in our assay were safely resumed by patients and were not associated with liver re-injury in 27 patients. Therefore, the MH test identifies drugs that cause liver injury with 92.3% sensitivity and 100% specificity (1 false-negative and 12 true-positive results). CONCLUSIONS: We developed a test to identify drugs that cause liver injury in patients based on their toxicity to MH cells isolated from patients with DILI. We validated results from the assay and found it to identify drugs that cause DILI with 92.3% sensitivity and 100% specificity. The MH cell test could be a tool to identify causes of iDILI, even in patients taking multiple medications. ClinicalTrials.gov no: NCT 02353455.

Epigenetic silencing of the LDOC1 tumor suppressor gene in ovarian cancer cells.

PURPOSE: Due to very unspecific symptoms ovarian cancer often is diagnosed only at a late stage of the disease. Thus, morbidity and mortality of the patients are high. Even the established tumor marker CA12-5 shows only low specificity, rising the need for alternative biomarkers capable of detecting early stages of ovarian cancer. We analyzed the expression of the tumor suppressor candidate gene LDOC1 (leucine zipper downregulated in cancer 1) as a potential early biomarker in ovarian cancer cell lines. METHODS: A total of seven ovarian cancer cell lines were analyzed by RT-PCR (reverse transcriptase polymerase chain reaction) and real-time PCR for expression of LDOC1. Verification of promoter methylation was performed using methylation-specific primers on bisulfite-modified genomic DNA. RESULTS: Three out of seven ovarian cancer cell lines showed a complete loss of LDOC1 gene expression. LDOC1 silencing was caused neither by gene deletion nor gene rearrangements, but by methylation and subsequent inactivation of the concerned promoter as proofed by methylation specific primers. Similarly, promoter methylation could be inhibited by adding AdC (5-aza-2'-deoxycytidine), an inhibitor of DNA methyltransferases. As a result, a reactivation of the LDOC1 gene was seen. CONCLUSIONS: The tumor suppressor gene LDOC1 in ovarian cancer cell lines is downregulated by promoter methylation and thus may serve as an early biomarker. Further investigation will show if detection of methylated LDOC1 in peripheral blood has both adequate sensitivity and specificity for a timely non-invasive detection of ovarian cancer.

Loss of LDOC1 expression by promoter methylation in cervical cancer cells.

Cervical cancer lacks reliable prognostic factors for both progression and chemotherapeutic responsiveness. The expression of the LDOC1 tumor suppressor candidate was therefore investigated. In four of six cervical cancer cell lines tested, expression of LDOC1 was silenced. Downregulation of LDOC1 could also be shown in biopsies of cervical cancer specimens. PCR-based promoter methylation analysis revealed a significant association between promoter methylation and the loss of LDOC1 expression, which could be reverted by DNA methyltransferase inhibitors. This indicates that silencing of LDOC1 is a frequent event in cervical cancer and may be of interest as a molecular marker in cervical cancer.

SARS-CoV-2 antibody immunoassays in serial samples reveal earlier seroconversion in acutely ill COVID-19 patients developing ARDS.

OBJECTIVES: During the COVID-19 pandemic, SARS-CoV-2 antibody testing has been suggested for (1) screening populations for disease prevalence, (2) diagnostics, and (3) guiding therapeutic applications. Here, we conducted a detailed clinical evaluation of four Anti-SARS-CoV-2 immunoassays in samples from acutely ill COVID-19 patients and in two negative cohorts. METHODS: 443 serum specimens from serial sampling of 29 COVID-19 patients were used to determine clinical sensitivities. Patients were stratified for the presence of acute respiratory distress syndrome (ARDS). Individual serum specimens from a pre-COVID-19 cohort of 238 healthy subjects and from a PCR-negative clinical cohort of 257 patients were used to determine clinical specificities. All samples were measured side-by-side with the Anti-SARS-CoV-2-ELISA (IgG), Anti-SARS-CoV-2-ELISA (IgA) and Anti-SARS-CoV-2-NCP-ELISA (IgG) (Euroimmun AG, Lübeck, Germany) and the Elecsys Anti-SARS-CoV-2 ECLIA (Roche Diagnostics International, Rotkreuz, Switzerland). RESULTS: Median seroconversion occurred earlier in ARDS patients (8-9 days) than in non-ARDS patients (11-17 days), except for EUR N-IgG. Rates of positivity and mean signal ratios in the ARDS group were significantly higher than in the non-ARDS group. Sensitivities between the four tested immunoassays were equivalent. In the set of negative samples, the specificity of the Anti-SARS-CoV-2-ELISA (IgA) was lower (93.9%) compared to all other assays (≥98.8%) and the specificity of Anti-SARS-CoV-2-NCP-ELISA (IgG) was lower (98.8%) than that of Elecsys Anti-SARS-CoV-2 (100%). CONCLUSIONS: Serial sampling in COVID-19 patients revealed earlier seroconversion and higher signal ratios of SARS-CoV-2 antibodies as a potential risk marker for the development of ARDS, suggesting a utility for antibody testing in acutely diseased patients.

Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies.

Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)-based proteomics now allows highly specific and quantitative readout of the plasma proteome. Here, we employ Plasma Proteome Profiling to define quality marker panels to assess plasma samples and the likelihood that suggested biomarkers are instead artifacts related to sample handling and processing. We acquire deep reference proteomes of erythrocytes, platelets, plasma, and whole blood of 20 individuals (> 6,000 proteins), and compare serum and plasma proteomes. Based on spike-in experiments, we determine sample quality-associated proteins, many of which have been reported as biomarker candidates as revealed by a comprehensive literature survey. We provide sample preparation guidelines and an online resource ( www.plasmaproteomeprofiling.org) to assess overall sample-related bias in clinical studies and to prevent costly miss-assignment of biomarker candidates.

A second-derivate fitting algorithm for the quantification of free hemoglobin in human plasma.

BACKGROUND: Assessment of hemolysis in vivo is becoming increasingly relevant in critical care. Current methods (Harboe, 1959) for quantifying the free hemoglobin (fHb) content produce unsatisfactory results in case of hyperbilirubinemia, a frequent condition in patients at risk for intravascular hemolysis. METHODS: A novel evaluation method based on second-derivative fitting to quantify fHb content was developed. The method uses spectrophotometric data from 350 to 650 nm recorded with standard instruments as input. To evaluate the power of the new method, plasma of patients and non-icteric plasma of healthy volunteers were spiked with fHb concentrations up to 2000 mg/L and compared to methods described in the literature by Harboe, Noe and Fairbanks. All measurements were done in compliance with the bioanalytical method validation protocol from the European Medicines Agency. RESULTS: Both the second-derivative fitting algorithm as well as the methods of Harboe, Noe and Fairbanks quantified fHb accurately in non-icteric samples, with inaccuracy and imprecision below 10%. For icteric specimen, false high results were obtained with the established formulas for fHb concentrations below 700 mg/L. In contrast, no interference was found with the second-derivate fitting method for bilirubin concentrations up to 465 µmol/L. The lower limits of quantifications for the second-derivative fitting algorithm were specified in agreement with the EMA guideline with 25 mg/L fHb for both non-icteric and icteric specimens. CONCLUSIONS: A user-friendly, computer-based algorithm is reported that allows the accurate quantification of fHb concentrations in the presence of high bilirubin concentrations. The new method allows for uniform sample preparation with only a single dilution step and can be readily implemented in any laboratory on standard spectrophotometers using the provided supplementary Microsoft Excel macro.