Molecular diagnostics of hepatobiliary and pancreatic neoplasias.

Neoplasias of the hepatopancreatobiliary tract are growing in numbers, have the poorest prognosis of all major cancer entities, and thus represent a rising clinical problem. Their molecular diagnostic has dramatically improved, contributing to tumor subtyping, definition of malignancy, and uncovering cases with hereditary predisposition. Most of all, predictive molecular testing allows to identify cases amenable to treatment with the rising number of approved targeted drugs, immune-oncological treatment, and clinical trials. In this review, the current state of molecular testing and its contribution to clinical decision-making are outlined.

[Microvascular changes in COVID-19]. / Mikrovaskuläre Veränderungen bei COVID-19.

BACKGROUND: Clinically, coronavirus disease 2019 (COVID-19) is associated with a wide range of symptoms, which can range from mild complaints of an upper respiratory infection to life-threatening hypoxic respiratory insufficiency and multiorgan failure. OBJECTIVE: The initially identified pulmonary damage patterns, such as diffuse alveolar damage in acute lung failure, are accompanied by new findings that draw a more complex scenario. These include microvascular involvement and a wide range of associated pathologies of multiple organ systems. A back-scaling of microstructural vascular changes is possible via targeted correlation of pathological autopsy results with radiological imaging. MATERIAL AND METHODS: Radiological and pathological correlation as well as microradiological imaging to investigate microvascular involvement in fatal COVID-19. RESULTS: The cases of two COVID-19 patients are presented. Patient 1 showed a relative hypoperfusion in lung regions that did not have typical COVID-19 infiltrates; the targeted post-mortem correlation also showed subtle signs of microvascular damage even in these lung sections. Patient 2 showed both radiologically and pathologically advanced typical COVID-19 destruction of lung structures and the case illustrates the damage patterns of the blood-air barrier. The perfusion deficit of the intestinal wall shown in computed tomography of patient 2 could not ultimately clearly be microscopically attributed to intestinal microvascular damage. CONCLUSION: In addition to microvascular thrombosis, our results indicate a functional pulmonary vasodysregulation as part of the pathophysiology during the vascular phase of COVID-19. The clinical relevance of autopsies and the integration of radiological imaging findings into histopathological injury patterns must be emphasized for a better understanding of COVID-19.

Notch inhibition counteracts Paneth cell death in absence of caspase-8.

Opposing activities of Notch and Wnt signaling regulate mucosal barrier homeostasis and differentiation of intestinal epithelial cells. Specifically, Wnt activity is essential for differentiation of secretory cells including Wnt3-producing Paneth cells, whereas Notch signaling strongly promotes generation of absorptive cells. Loss of caspase-8 in intestinal epithelium (casp8∆int) is associated with fulminant epithelial necroptosis, severe Paneth cell death, secondary intestinal inflammation, and an increase in Notch activity. Here, we found that pharmacological Notch inhibition with dibenzazepine (DBZ) is able to essentially rescue the loss of Paneth cells, deescalate the inflammatory phenotype, and reduce intestinal permeability in casp8∆int mice. The secretory cell metaplasia in DBZ-treated casp8∆int animals is proliferative, indicating for Notch activities partially insensitive to gamma-secretase inhibition in a casp8∆int background. Our data suggest that casp8 acts in the intestinal Notch network.

Serum levels of chemokines CCL4 and CCL5 in cirrhotic patients indicate the presence of hepatocellular carcinoma.

BACKGROUND: Most hepatocellular carcinomas (HCCs) are diagnosed at an advanced stage. The prognostic value of serum tumour markers alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) is limited. The aim of our study is to evaluate the diagnostic value of serum growth factors, apoptotic and inflammatory mediators of cirrhotic patients with and without HCC. METHODS: Serum samples were collected from cirrhotic potential liver transplant patients (LTx) with (n=61) and without HCC (n=78) as well as from healthy controls (HCs; n=39). Serum concentrations of CRP, neopterin and IL-6 as markers of inflammation and thrombopoietin (TPO), GCSF, FGF basic and VEGF, HMGB1, CK-18 (M65) and CK18 fragment (M30) and a panel of proinflammatory chemokines (CCL2, CCL3, CCL4, CCL5, CXCL5 and IL-8) were measured. Chi square, Fisher exact, Mann-Whitney U-tests, ROC curve analysis and forward stepwise logistic regression analyses were applied. RESULTS: Patients with HCC had higher serum TPO and chemokines (P<0.001 for TPO, CCL4, CCL5 and CXCL5) and lower CCL2 (P=0.008) levels than cirrhotic patients without HCC. Multivariate forward stepwise regression analysis for significant parameters showed that among the studied parameters CCL4 and CCL5 (P=0.001) are diagnostic markers of HCC. Serum levels of TPO and chemokines were lower, whereas M30 was significantly higher in cirrhotic patients than in HCs. CONCLUSIONS: High serum levels of inflammatory chemokines such as CCL4 and CCL5 in the serum of cirrhotic patients indicate the presence of HCC.

[EEF1A2 inhibits the p53 function in hepatocellular carcinoma via PI3K/AKT/mTOR-dependent stabilization of MDM4]. / EEF1A2 inhibiert über eine PI3K/AKT/mTOR-abhängige Stabilisierung von MDM4 die p53-Funktion im Leberzellkarzinom.

Upregulation of mouse double minute 4 (MDM4) is a frequent event in human hepatocellular carcinoma (HCC) but the underlying molecular mechanisms are poorly characterized. In this study a potential role of the phosphoinositide-3-kinase/v-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/AKT/mTOR) cascade was investigated in the regulation of MDM4 in HCC. Inhibition of the PI3K-AKT and/or mTOR pathways lowered MDM4 protein levels in HCC cells. Mechanistic protection from proteasomal degradation resulted from de-ubiquitination by ubiquitin-specific protease 2a and AKT-mediated phosphorylation of MDM4, thus increasing MDM4 protein levels. These findings were corroborated in a chimeric AKT mouse model. Upregulation of PI3K/AKT/mTOR signaling may result from overexpression of the eukaryotic elongation factor 1A2 (EEF1A2). Finally, a strong association between the expression of EEF1A2, phosphorylated AKT and MDM4 was observed in human HCC samples. Strong activation of the EEF1A2/PI3K/AKT/mTOR/MDM4 signaling pathway was observed in HCC patients with short survival suggesting that targeting this axis might be a promising approach in a subset of HCC patients.

[Genome-wide molecular screening for the identification of new targets in human hepatocellular carcinoma]. / Identifikation neuer Zielstrukturen beim humanen hepatozellulären Karzinom mittels genomweiter molekularer Screeninganalysen.

Molecular hepatocarcinogenesis represents a step-wise process which in most cases is associated with a well-defined chronic liver disease. By meta-analysis of classical comparative genomic hybridization (CGH) data an oncogenetic progression model could be generated (1q gain→ 8q gain → 4q loss → 16q loss → 13q loss). Array-based CGH allows the identification of etiology-dependent and independent genomic alterations. The Mouse Double Minute homologue 4 (MDM4) was shown to act as an oncogene of 1q32.1 gains in human hepatocellular carcinoma (HCC). Integration of genomic and epigenomic data facilitated the identification of tumor suppressor gene candidates in human HCC. For instance, Polo-like kinase 3 (PLK3) is frequently inactivated via promoter hypermethylation in combination with a loss of the second allele at 1p34.1. Both MDM4 overexpression and methylation-dependent inactivation of PLK3 represent potential targets for future therapeutic approaches.

Super-micro-bland particle embolization combined with RF-ablation: angiographic, macroscopic and microscopic features in porcine kidneys.

PURPOSE: To describe angiographic, macroscopic and microscopic features of super-micro-bland particle embolization in combination with RF-ablation in kidneys. Thereby, a special focus was given on the impact of the sequence of the different procedural steps. MATERIALS AND METHODS: In ten pigs, super-micro-bland particle embolization combined with RF-ablation was carried out. Super-micro-bland embolization was performed with spherical particles of very small size and tight calibration (40 ± 10 µm). In the left kidneys, RF-ablations were performed before embolization (I). In the right kidneys, RF-ablations were performed after embolization (II). The animals were killed three hours after the procedures. Angiographic (e.g. vessel architecture), macroscopic (e.g. long and short axes of the RF-ablations) and microscopic (e.g. particle distribution) study goals were defined. RESULTS: Angiography detected almost no vessels in the center of the RF-ablations in I. In II, angiography could not define the RF-ablations. Macroscopy detected significantly larger long and short axes of the RF-ablations in II compared to I (52.2 ± 3.2 mm vs. 45.3 ± 6.9 mm [P<0.05] and 25.1 ± 3.5mm vs. 20.0 ± 1.9 mm [P<0.01], respectively). Microscopy detected irregular particle distribution at the rim of the RF-ablations in I. In II, microscopy detected homogeneous particle distribution at the rim of the RF-ablations. Microscopy detected no particles in the center of the RF-ablations in I and II. CONCLUSION: The sequence of the different procedural steps of super-micro-bland particle embolization combined with RF-ablation impacts angiographic, macroscopic and microscopic features in kidneys in the acute setting.

Renal artery embolization combined with radiofrequency ablation in a porcine kidney model: effect of small and narrowly calibrated microparticles as embolization material on coagulation diameter, volume, and shape.

The purpose of this study was to evaluate the effect of renal artery embolization with small and narrowly calibrated microparticles on the coagulation diameter, volume, and shape of radiofrequency ablations (RFAs) in porcine kidneys. Forty-eight RFAs were performed in 24 kidneys of 12 pigs. In 6 animals, bilateral renal artery embolization was performed with small and narrowly calibrated microparticles. Upper and lower kidney poles were ablated with identical system parameters. Applying three-dimensional segmentation software, RFAs were segmented on registered 2 mm-thin macroscopic slices. Length, depth, width, volume_segmented, and volume_calculated were determined to describe the size of the RFAs. To evaluate the shape of the RFAs, depth-to-width ratio (perfect symmetry-to-lesion length was indicated by a ratio of 1), sphericity ratio (perfect sphere was indicated by a sphericity ratio of 1), eccentricity (perfect sphere was indicated by an eccentricity of 0), and circularity (perfect circle was indicated by a circularity of 1) were determined. Embolized compared with nonembolized RFAs showed significantly greater depth (23.4 ± 3.6 vs. 17.2 ± 1.8 mm; p < 0.001) and width (20.1 ± 2.9 vs. 12.6 ± 3.7 mm; p < 0.001); significantly larger volume_segmented (8.6 ± 3.2 vs. 3.0 ± 0.7 ml; p < 0.001) and volume_calculated (8.4 ± 3.0 ml vs. 3.3 ± 1.1 ml; p < 0.001); significantly lower depth-to-width (1.17 ± 0.10 vs. 1.48 ± 0.44; p < 0.05), sphericity (1.55 ± 0.44 vs. 1.96 ± 0.43; p < 0.01), and eccentricity (0.84 ± 0.61 vs. 1.73 ± 0.91; p < 0.01) ratios; and significantly greater circularity (0.62 ± 0.14 vs. 0.45 ± 0.16; p < 0.01). Renal artery embolization with small and narrowly calibrated microparticles affected the coagulation diameter, volume, and shape of RFAs in porcine kidneys. Embolized RFAs were significantly larger and more spherical compared with nonembolized RFAs.

Expression of an ASCL2 related stem cell signature and IGF2 in colorectal cancer liver metastases with 11p15.5 gain.

BACKGROUND AND AIMS: Liver metastases are the leading cause of death in colorectal cancer. To gain better insight into the biology of metastasis and possibly identify new therapeutic targets we systematically investigated liver-metastasis-specific molecular aberrations. METHODS: Primary colorectal cancer (pCRC) and matched liver metastases (LMs) from the same patients were analysed by microarray-based comparative genomic hybridisation in 21 pairs and gene expression profiling in 18 pairs. Publicly available databases were used to confirm findings in independent datasets. RESULTS: Chromosome aberration patterns and expression profiles of pCRC and matched LMs were strikingly similar. Unsupervised cluster analysis of genomic data showed that 20/21 pairs were more similar to each other than to any other analysed tumour. A median of only 11 aberrations per patient was found to be different between pCRC and LM, and expression of only 16 genes was overall changed upon metastasis. One region on chromosome band 11p15.5 showed a characteristic gain in LMs in 6/21 patients. This gain could be confirmed in an independent dataset of LMs (n=50). Localised within this region, the growth factor IGF2 (p=0.003) and the intestinal stem cell specific transcription factor ASCL2 (p=0.029) were found to be over-expressed in affected LM. Several ASCL2 target genes were upregulated in this subgroup of LM, including the intestinal stem cell marker OLFM4 (p=0.013). The correlation between ASCL2 expression and four known direct transcriptional targets (LGR5, EPHB3, ETS2 and SOX9) could be confirmed in an independent expression dataset (n=50). CONCLUSIONS: With unprecedented resolution a striking conservation of genomic alterations was demonstrated in liver metastases, suggesting that metastasis typically occurs after the pCRC has fully matured. In addition, we characterised a subset of liver metastases with an ASCL2-related stem-cell signature likely to affect metastatic behaviour of tumour cells.

[Highly differentiated liver tumors: recent developments and their diagnostic application]. / Hochdifferenzierte Lebertumoren: Neue Entwicklungen und ihre diagnostische Relevanz.

Biopsy diagnosis of early and highly differentiated liver tumors is one of the most challenging tasks in histopathology. During recent years its parameters have changed fundamentally due to shifting clinical algorithms. Modern histopathology has met this challenge by defining new, prognostically relevant subtypes of early hepatocellular carcinoma and by elaborating morphological algorithms and novel immunohistological markers for the differential diagnosis of highly differentiated hepatocellular tumors. In addition, a new, predictive molecular pathological and histological classification of liver cell adenoma has been developed. By means of the consequent application of these new diagnostic tools, together with the so-called 'matrix diagnosis', a reliable diagnosis is achieved in the vast majority of these difficult cases.

[Recent developments in biopsy diagnosis of early and undefined liver tumors]. / Neue Entwicklungen in der Biopsiediagnostik früher und unklarer Lebertumoren.

Biopsy diagnosis of early and highly differentiated liver tumors is difficult and complex. Modern pathology has met this challenge by several different means; elaborate morphological algorithms and novel immunohistological markers support the differential diagnosis of highly differentiated HCC and a new, predictive molecular pathological and histological classification of liver cell adenoma was developed. By these new diagnostic tools together with the so-called 'matrix diagnosis' a reliable diagnostic classification is now feasible in the vast majority of these difficult cases.

[Quality and quantity in hepatopathology. Diagnostic and clinically relevant grading for non-tumourous liver diseases]. / Mass und Zahl in der Hepatopathologie: Diagnostisch und klinisch relevante Graduierungen bei nichttumorösen Lebererkrankungen.

This article describes the grading and staging systems used in the clinical context for non-neoplastic liver diseases (chronic and autoimmune hepatitis, fatty liver and steatohepatitis, medicinal toxic liver damage, iron storage disease and gall duct diseases). Fibrotic parenchymal alterations can also be assessed as well as livers planned for transplantation, with respect to possible rejection reactions. The basis for the histopathological diagnostic procedure is the liver biopsy. The consistent and correct use of the histological scores is obligatory in the diagnostic assessment of non-neoplastic liver diseases. Different scores are available for the various liver diseases. These are qualitative and quantitative scores based on empiricism and the practical relevance has been effectively proven. Grading describes the inflammatory activity and staging the extent of fibrosis or structural disorders up to liver cirrhosis. In many instances staging is the histopathological criteria for the prognosis assessment and is, therefore, decisive for therapy indications and therapy initiation.

Detection of the ASPSCR1-TFE3 gene fusion in paraffin-embedded alveolar soft part sarcomas.

AIMS: Alveolar soft part sarcoma (ASPS) is a rare soft tissue tumour with unique morphology and a recurrent, non-reciprocal translocation der(17)t(X;17)(p11.2;q25) leading to the fusion of ASPSCR1 (also known as ASPL) to the transcription factor TFE3. Although diagnosis is straightforward in classical cases, tumours with atypical morphological features may be difficult to classify solely on the basis of conventional histopathology. The aim of this study was to analyse the chromosomal breakpoints in paraffin-embedded tissue. METHODS AND RESULTS: Three male and two female ASPS patients including one case with uncommon histology were investigated by fluorescence in situ hybridization with split- and fusion-probes. The presence of the resulting ASPSCR1-TFE3 fusion transcripts was assessed by reverse transcriptase-polymerase chain reaction. Hybridization results showed a t(X;17)(p11.2;q25) in all tumours with a duplication of the telomeric part of chromosome Xp. In addition to wild-type TFE3, ASPSCR1-TFE3 fusion transcripts (three type 1 and two type 2 transcripts) were detected in all cases. CONCLUSIONS: Molecular confirmation of ASPSCR1-TFE3 gene fusion is applicable to routinely processed archival and diagnostic tumour samples and aids in the differential diagnosis of ASPS.

[The morphologic variants of KSHV/HHV 8-associated lymphoproliferations]. / Die Vielfalt der KSHV/HHV 8-assoziierten Lymphoproliferationen.

KSHV/HHV 8 infection is associated with primary effusion lymphoma, multicentric Castleman disease (MCD) and MCD-associated plasmablastic lymphoma. We report the case of an HIV-infected male with Kaposi sarcoma, MCD in the lymph node and development of a KSHV/HHV 8-associated plasmablastic lymphoma in the liver. Immunohistochemistry revealed an HHV 8 infection of plasmablasts showing cytoplasmic IgM/lambda expression. To the best of our knowledge, liver infiltration by a MCD-associated HHV 8 positive plasmablastic lymphoma has not been documented previously. The differential diagnosis is discussed.

[Malignant lymphomas of the liver: new diagnostic algorithms]. / Maligne Lymphome in der Leber. Neue diagnostische Algorithmen.

Primary hepatic lymphomas represent rare neoplasms, which are partly observed in association with chronic viral hepatitis, immunosuppression and autoimmune diseases. In contrast, secondary hepatic lymphomas are much more frequent and represent disseminated disease. Lymphomas involving the liver include, with decreasing frequency, diffuse large B-cell lymphoma, small lymphocytic lymphoma, Hodgkin's lymphoma, peripheral T-cell lymphoma, follicular lymphoma and extranodal marginal zone B-cell lymphoma. Many B-cell lymphomas in the liver reveal a characteristic infiltration pattern allowing a rapid and cost-effective diagnosis based on focused immunohistochemical analyses. In contrast, most T-cell lymphomas show a more diverse morphology, which is sometimes difficult to differentiate from a reactive condition. Therefore, additional molecular analyses are frequently necessary. The differential diagnosis includes hepatitis and inflammatory bile duct diseases, undifferentiated carcinoma, inflammatory myofibroblastic tumor as well as histiocytic and dendritic cell neoplasms.

Dysregulation of growth factor signaling in human hepatocellular carcinoma.

Dysregulation of pleiotropic growth factors, receptors and their downstream signaling pathway components represent a central protumorigenic principle in human hepatocarcinogenesis. Especially the Insulin-like Growth Factor/IGF-1 receptor (IGF/IGF-1R), Hepatocyte Growth Factor (HGF/MET), Wingless (Wnt/beta-catenin/FZD), Transforming Growth Factor alpha/Epidermal Growth Factor receptor (TGFalpha/EGFR) and Transforming Growth Factor beta (TGFbeta/TbetaR) pathways contribute to proliferation, antiapoptosis and invasive behavior of tumor cells. This review focuses on the relevant alterations in these pathways identified in human human hepatocellular carcinomas (HCCs). Resultant functional effects are modulated by multiple cross-talks between the different signaling pathways and additional tumor-relevant factors, such as cyclooxygenase-2 and p53. Several specific strategies are currently under development such as receptor kinase inhibitors, neutralizing antibodies and antagonistic proteins, which may improve the systemic treatment of human HCCs.

[Pathogenesis and pathomorphology of chronic viral hepatitis]. / Chronische Virushepatitis--pathogenetische und pathomorphologische Aspekte.

Chronic viral hepatitis represents the most common liver disease worldwide. It can be induced by HBV (eventually as HDV-coinfection) and HCV. From the pathologist's point of view chronic hepatitis represents portal accentuated inflammation of the liver associated with a variable degree of interface hepatitis and acinar damage. Although much research has been done to unravel the mechanisms which cause chronic viral hepatitis, many questions are unanswered. Up to now, liver biopsy is the gold standard for diagnosis of chronic viral hepatitis. On one hand it shows the grade of inflammation and the stage of disease, on the other hand it can highlight additional liver diseases, which might have an adverse influence. Therefore, liver biopsy allows the best prediction of disease progression. In a recent consensus statement, the scoring system of Desmet was recommended for grading and staging of chronic hepatitis.