Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents.

Background: Treatment stratification and response assessment in pediatric sarcomas has relied on imaging studies and surgical/histopathological evidence of vital tumor cells. Such studies and evidence collection processes often involve radiation and/or general anesthesia in children. Cell-free circulating tumor DNA (ctDNA) detection in blood plasma is one available method of so-called liquid biopsies that has been shown to correlate qualitatively and quantitatively with the existence of vital tumor cells in the body. Our clinical observational study focused on the utility and feasibility of ctDNA detection in pediatric Ewing sarcoma (EWS) as a marker of minimal residual disease (MRD). Patients and methods: We performed whole genome sequencing (WGS) to identify the exact breakpoints in tumors known to carry the EWS-FLI1 fusion gene. Patient-specific fusion breakpoints were tracked in peripheral blood plasma using digital droplet PCR (ddPCR) before, during, and after therapy in six children and young adults with EWS. Presence and levels of fusion breakpoints were correlated with clinical disease courses. Results: We show that the detection of ctDNA in the peripheral blood of EWS patients (i) is feasible in the clinical routine and (ii) allows for the longitudinal real-time monitoring of MRD activity in children and young adults. Although changing ctDNA levels correlated well with clinical outcome within patients, between patients, a high variability was observed (inter-individually). Conclusion: ctDNA detection by ddPCR is a highly sensitive, specific, feasible, and highly accurate method that can be applied in EWS for follow-up assessments as an additional surrogate parameter for clinical MRD monitoring and, potentially, also for treatment stratification in the near future.

Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing-A Prospective Feasibility Study.

We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.

Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections.

Molecular techniques including the sequencing of fungal-specific DNA targets are increasingly used in the diagnosis of suspected invasive fungal infections. In contrast to established biomarkers like galactomannan or 1-3-ß-d-glucan, the clinical impact of these methods remains unknown. We retrospectively investigated the impact of ITS1-sequencing on antifungal treatment strategies in 71 patients (81 samples) with suspected invasive fungal infections. ITS-sequencing either confirmed already ongoing antifungal therapy (19/71 patients, 27%), led to a change in antifungal therapy (11/71, 15%) or supported the decision to withhold antifungal treatment (34/71, 48%) (in seven of 71 patients, ITS-sequencing results were obtained postmortem). ITS-sequencing results led to a change in antifungal therapy in a relevant proportion of patients, while it confirmed therapeutic strategies in the majority. Therefore, ITS-sequencing was a useful adjunct to other fungal diagnostic measures in our cohort.

Pleuropulmonary blastoma type I might arise in congenital pulmonary airway malformation type 4 by acquiring a Dicer 1 mutation.

Congenital pulmonary airway malformation (CPAM) occurs most commonly in infants. It is divided into 5 types. The most common types 1 and 2 are cystic, type 0 presents as bronchial buds without alveolar tissue, most likely corresponding to alveolar dysgenesis, while type 3 is composed of branching bronchioles and appears as a solid lesion. A defect in the epithelial-mesenchymal crosstalk might be the underlying mechanism for all. Type 4 is a peripheral cystic lesion with a thin cyst wall covered by pneumocytes. CPAM 4 has been mixed up with pleuropulmonary blastoma (PPB) type I and some authors question its existence. We investigated five cases of CPAM type 4 for the presence or absence of rhabdomyoblasts, and for markers associated with CPAM development. In addition, all cases were evaluated for mutations within the Dicer gene and for mutations of the RAS family of oncogenes. All five cases showed smooth muscle actin and desmin-positive cells; however, only one case showed a few cells positive for MyoD. The same case showed a mutation of Dicer 1. All cases were negative for mutations of the RAS family of genes. Fibroblast growth factor 10 was similarly expressed in all cases, and thus cannot be used to differentiate CPAM4 from PPB-I. Low expression of the proliferation marker Ki67 was seen in our CPAM 4 cases and the probable PPB-I case. YingYang-1 protein seems to play an active role in the development of PPB-I. CPAM 4 can be separated from PPB-I based on the presence of rhabdomyoblasts and mutations in Dicer 1 gene. These cells might not be numerous; therefore, all available tissue has to be evaluated. As CPAM 4 morphologically looks very similar to PPB-I, it might be speculated, that there exists a potential for progression from CPAM 4 to PPB-I, by acquiring somatic mutations in Dicer 1.

Atypical goblet cell hyperplasia occurs in CPAM 1, 2, and 3, and is a probable precursor lesion for childhood adenocarcinoma.

Congenital pulmonary airway malformation (CPAM) is a developmental disorder. Types 1-2-3 are the more common ones. Atypical goblet cell hyperplasia (AGCH) in CPAM might be a precursor lesion for pulmonary adenocarcinomas. In nine out of 33 CPAM cases, types 1-3 showed foci of goblet cell proliferations. As these cells completely replace normal epithelium, we prefer to name these proliferations AGCH. In 5 cases, adenocarcinomas were seen (AC). All cases were analyzed for proteins possibly being associated with CPAM development: fibroblast growth factor 10 (FGF10) and receptor 2 (FGFR2), forkhead box A1 (FOXA1) and A2 (FOXA2), MUC protein 5AC (MUC5AC), human epidermal growth factor receptor 2 (erbB2, HER2/neu), hepatocyte nuclear factor 4α (HNF4α), SOX2, and Ying Yang protein 1 (YY1). By next generation sequencing, AGCH and adenocarcinomas were evaluated for driver mutations. Expression for FGF10, FGFR2, FOXA1, and FOXA2 was seen in CPAM epithelium and stroma, but not differently in AGCH and AC. SOX2 was positive in CPAM epithelium and AGCH, however weakly in AC. YY1 and MUC5AC showed more intense staining in AGCH and AC than in CPAM epithelium. HER2 was intensely expressed in AC and less intensely in AGCH, but not in CPAM epithelium. KRAS mutation in exon 2 was detected in all AGCH and AC, but was absent in CPAM epithelia. AGCH can arise in CPAM types 1-3. Oncogenic KRAS mutation seems to be the oncogenic driver already in AGCH, proving its role as a precursor lesion for adenocarcinoma. It might upregulate HER2 at the protein level. YY1 seems to be involved in carcinogenesis.

HPV-negative penile squamous cell carcinoma: disruptive mutations in the TP53 gene are common.

The majority of penile squamous cell carcinomas is caused by transforming human papilloma virus (HPV) infection. The etiology of HPV-negative cancers is unclear, but TP53 mutations have been implicated. Archival tissues of 108 invasive squamous cell carcinoma from a single pathology institution in a low-incidence area were analyzed for HPV-DNA and p16ink4a overexpression and for TP53 mutations by ion torrent next-generation sequencing. Library preparation failed in 32/108 squamous cell carcinomas. Institutional review board approval was obtained. Thirty of 76 squamous cell carcinomas (43%; average 63 years) were HPV-negative with 8/33 squamous cell carcinomas being TP53 wild-type (24%; average 63 years). Twenty-five of 33 squamous cell carcinomas (76%; average 65 years) showed 32 different somatic TP53 mutations (23 missense mutations in exons 5-8, 6 nonsense, 1 frameshift and 2 splice-site mutations). Several hotspot mutations were detected multiple times (R175H, R248, R282, and R273). Eighteen of 19 squamous cell carcinomas with TP53 expression in immunohistochemistry had TP53 mutations. Fifty percent of TP53-negative squamous cell carcinomas showed mostly truncating loss-of-function TP53 mutations. Patients without mutations had longer survival (5 years: 86% vs 61%; 10 years: 60% vs 22%), but valid clinically relevant conclusions cannot be drawn due to different tumor stages and heterogeneous treatment of the cases presented in this study. Somatic TP53 mutations are a common feature in HPV-negative penile squamous cell carcinomas and offer an explanation for HPV-independent penile carcinogenesis. About half of HPV-negative penile cancers are driven by oncogenic activation of TP53, while a quarter is induced by loss of TP53 tumor suppressor function. Detection of TP53 mutations should be carried out by sequencing, as immunohistochemical TP53 staining could not identify all squamous cell carcinomas with TP53 mutations.

Alterations of canalicular ATP-binding cassette transporter expression in drug-induced liver injury.

BACKGROUND: Drug-induced liver injury (DILI) is a major clinical problem and its pathogenesis is poorly understood. The association of DILI with polymorphisms in hepatobiliary transport systems suggests a role for transport proteins in the pathogenesis of DILI. AIM: To investigate expression and tissue distribution of hepatobiliary transport systems in DILI. METHODS: Expression of the canalicular bile salt export pump BSEP (ABCC11), phospholipid flippase MDR3 (ABCB4) and bilirubin export pump MRP2 (ABCC2) was assessed immunohistochemically in liver biopsies from 23 patients with DILI. RESULTS: Of 12 patients with cholestatic DILI (mostly due to antibiotics), 8 displayed a marked reduction of MRP2, MDR3 and BSEP expression. Transporter staining was normal in 4 patients with cholestatic DILI. In 11 patients with necroinflammatory hepatocellular injury (most frequently caused by NSAIDs), transporter staining was normal in areas where hepatocyte morphology was preserved. Due to hepatocyte necrosis and the reduction of the hepatocyte number, overall transporter expression was reduced without a reduction in transporter expression at the single hepatocyte level. CONCLUSIONS: Canalicular ABC transporter expression is profoundly disturbed in most cases of cholestatic DILI. Drug-induced hepatitis does not lead to repression of transporter expression but to hepatocyte drop-outs with a numerical loss of bile canaliculi.

Sensitivity and specificity of in situ proximity ligation for protein interaction analysis in a model of steatohepatitis with Mallory-Denk bodies.

The in situ proximity ligation assay (isPLA) is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs). These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). We investigated the colocalization of all three key MDB components, namely keratin 8 (K8), keratin 18 (K18), and p62 (sequestosome 1) by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8-/- and Krt18-/- mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol.

Bile acids trigger cholemic nephropathy in common bile-duct-ligated mice.

UNLABELLED: Tubular epithelial injury represents an underestimated but important cause of renal dysfunction in patients with cholestasis and advanced liver disease, but the underlying mechanisms are unclear. To address the hypothesis that accumulation and excessive alternative urinary elimination of potentially toxic bile acids (BAs) may contribute to kidney injury in cholestasis, we established a mouse model for detailed in vivo time course as well as treatment studies. Three-day common bile duct ligation (CBDL) induced renal tubular epithelial injury predominantly at the level of aquaporin 2-positive collecting ducts with tubular epithelial and basement membrane defects. This was followed by progressive interstitial nephritis and tubulointerstitial renal fibrosis in 3-, 6-, and 8-week CBDL mice. Farnesoid X receptor knockout mice (with a hydrophilic BA pool) were completely protected from CBDL-induced renal fibrosis. Prefeeding of hydrophilic norursodeoxycholic acid inhibited renal tubular epithelial injury in CBDL mice. In addition, we provide evidence for renal tubular injury in cholestatic patients with cholemic nephropathy. CONCLUSION: We characterized a novel in vivo model for cholemic nephropathy, which offers new perspectives to study the complex pathophysiology of this condition. Our findings suggest that urinary-excreted toxic BAs represent a pivotal trigger for renal tubular epithelial injury leading to cholemic nephropathy in CBDL mice.

Genetic background effects of keratin 8 and 18 in a DDC-induced hepatotoxicity and Mallory-Denk body formation mouse model.

Keratin 8 (K8) and keratin 18 (K18) form the major hepatocyte cytoskeleton. We investigated the impact of genetic loss of either K8 or K18 on liver homeostasis under toxic stress with the hypothesis that K8 and K18 exert different functions. krt8⁻/⁻ and krt18⁻/⁻ mice crossed into the same 129-ola genetic background were treated by acute and chronic administration of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). In acutely DDC-intoxicated mice, macrovesicular steatosis was more pronounced in krt8⁻/⁻ and krt18⁻/⁻ compared with wild-type (wt) animals. Mallory-Denk bodies (MDBs) appeared in krt18⁻/⁻ mice already at an early stage of intoxication in contrast to krt8⁻/⁻ mice that did not display MDB formation when fed with DDC. Keratin-deficient mice displayed significantly lower numbers of apoptotic hepatocytes than wt animals. krt8⁻/⁻, krt18⁻/⁻ and control mice displayed comparable cell proliferation rates. Chronically DDC-intoxicated krt18⁻/⁻ and wt mice showed a similarly increased degree of steatohepatitis with hepatocyte ballooning and MDB formation. In krt8⁻/⁻ mice, steatosis was less, ballooning, and MDBs were absent. krt18⁻/⁻ mice developed MDBs whereas krt8⁻/⁻ mice on the same genetic background did not, highlighting the significance of different structural properties of keratins. They are independent of the genetic background as an intrinsic factor. By contrast, toxicity effects may depend on the genetic background. krt8⁻/⁻ and krt18⁻/⁻ mice on the same genetic background show similar sensitivity to DDC intoxication and almost resemble wt animals regarding survival, degree of porphyria, liver-to-body weight ratio, serum bilirubin and liver enzyme levels. This stands in contrast to previous work where krt8⁻/⁻ and krt18⁻/⁻ mice on different genetic backgrounds were investigated.

Alterations in lipid metabolism mediate inflammation, fibrosis, and proliferation in a mouse model of chronic cholestatic liver injury.

BACKGROUND & AIMS: The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice. METHODS: We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer. RESULTS: Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice. CONCLUSIONS: Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.

Cross ß-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions.

BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steatohepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross ß-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross ß-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, α1-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross ß-sheet conformation than K18, whereas p62 never had cross ß-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly α-helical to cross ß-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8⁻/⁻ mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.

The role of osteopontin and tumor necrosis factor alpha receptor-1 in xenobiotic-induced cholangitis and biliary fibrosis in mice.

Proinflammatory and profibrotic cytokines such as osteopontin (OPN) and tumor necrosis factor-alpha receptor-1 (TNFR(1)) may be critically involved in the pathogenesis of cholangiopathies and biliary fibrosis. We therefore aimed to determine the role of genetic loss of either OPN or TNFR(1) in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice as a model of xenobiotic-induced sclerosing cholangitis with biliary-type liver fibrosis using respective knock-out mice. OPN and TNFR(1) knock-out mice were fed a 0.1% DDC-supplemented diet for 4 weeks and compared with corresponding wild-type (WT) controls. Liver morphology (H&E staining), serum markers of liver injury and cholestasis (ALT, AP, bilirubin), markers of inflammation in liver (CD11b and F4/80 immunostaining, mRNA expression of iNOS, MCP-1, IL-1beta, INF-gamma, TNF-alpha and OPN), degree of ductular reaction (immunohistochemistry with morphometric analysis and western blotting for cholangiocyte-specific marker keratin 19) and degree of liver fibrosis (Sirius-red staining, hepatic hydroxyproline content for quantification) were compared between groups. DDC feeding in OPN and TNFR(1) knock-out mice and respective WT controls resulted in comparable extent of liver injury, inflammatory response, ductular reaction and liver fibrosis. Our data indicate that genetic loss of neither OPN nor TNFR(1) significantly effects on the pathogenesis of DDC-induced sclerosing cholangitis, ductular reaction and resulting biliary fibrosis.