Publications by authors named "Kirsten Utpatel"

39 Publications

[Quality assurance in dMMR and MSI diagnostics].

Pathologe 2021 Apr 9. Epub 2021 Apr 9.

Qualitätssicherungs-Initiative Pathologie QuIP GmbH, Berlin, Deutschland.

Deficient mismatch repair (dMMR) and microsatellite instability (MSI) have therapeutic relevance not only for colorectal carcinomas but also for carcinomas of other entities (endometrium, biliary tract, pancreas). In order to guarantee the knowledge and good technical quality necessary for adequate implementation of the corresponding analyses in pathology institutes, the Pathology Quality Assurance Initiative ("Die Qualitätssicherung-Initiative Pathologie") has been offering proficiency tests (PT) for years. It has been shown for the dMMR PT that various antibody clones from different manufacturers provide comparable results in immunohistological examinations, except for slight variations. The difficulty lies in the staining protocol (intensity of staining) and the interpretation of the staining results. The molecular pathological MSI PT has shown a positive trend at a high-quality level over the last three years. Success rates increased from 89 (2018) to 97% (2019/2020). The choice of assay, whether commercial or in-house tests with the designated cutoffs for this purpose, has not been shown to have a significant impact on the PTs in the selected EQA samples.
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http://dx.doi.org/10.1007/s00292-021-00930-zDOI Listing
April 2021

Identification and In-Depth Analysis of the Novel FGFR2-NDC80 Fusion in a Cholangiocarcinoma Patient: Implication for Therapy.

Curr Oncol 2021 Mar 8;28(2):1161-1169. Epub 2021 Mar 8.

Institute of Pathology, University of Regensburg, 93053 Regensburg, Germany.

Fibroblast growth factor receptor 2 (FGFR2) fusions have emerged as a new therapeutic target for cholangiocarcinoma in clinical practice following the United States Food and Drug Administration (FDA) approval of Pemigatinib in May 2020. FGFR2 fusions can result in a ligand-independent constitutive activation of FGFR2 signaling with a downstream activation of multiple pathways, including the mitogen-activated protein (MAPK) cascade. Until today, only a limited number of fusion partners have been reported, of which the most prevalent is BicC Family RNA Binding Protein (BICC1), representing one-third of all detected FGFR2 fusions. Nonetheless, in the majority of cases rare or yet unreported fusion partners are discovered in next-generation sequencing panels, which confronts clinicians with a challenging decision: Should a therapy be based on these variants or should the course of treatment follow the (limited) standard regime? Here, we present the case of a metastasized intrahepatic cholangiocarcinoma harboring a novel FGFR2-NDC80 fusion, which was discussed in our molecular tumor board. The protein NDC80 kinetochore complex component (NDC80) is an integral part of the outer kinetochore, which is involved in microtubule binding and spindle assembly. For additional therapeutic guidance, an immunohistochemical analysis of the predicted fusion and downstream effector proteins was performed and compared to cholangiocarcinoma samples of a tissue microarray. The FGFR2-NDC80 fusion resulted in strong activation of the FGFR2 signaling pathway. These supporting results led to a treatment recommendation of Pemigatinib. Unfortunately, the patient passed away before the commencement of therapy.
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http://dx.doi.org/10.3390/curroncol28020112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025813PMC
March 2021

Reply to "Does multiple intrahepatic cholangiocarcinoma worsen prognosis as "M1" stage?"; multiple primaries vs liver metastases.

Hepatology 2021 Feb 7. Epub 2021 Feb 7.

Medical Oncology / Institute of Cancer Sciences, The Christie NHS Foundation Trust / University of Manchester, Manchester, United Kingdom.

We thank Zhang and colleagues for their Letter to the Editor (1) regarding our work (2). Our manuscript ("Liver Metastases of Intrahepatic Cholangiocarcinoma: Implications for an Updated Staging System"(2)), suggested changes to the American Joint Committee on Cancer (AJCC) staging classification for intrahepatic cholangiocarcinoma (iCCA), by classifying "liver metastases" as stage IV rather than stage II/III in the absence/presence of lymph node metastases, respectively, as per AJCC v.8 (3).
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http://dx.doi.org/10.1002/hep.31740DOI Listing
February 2021

Viszerale Leishmaniose nach Therapie mit Immuncheckpoint-Inhibitoren.

J Dtsch Dermatol Ges 2021 Jan;19(1):119-121

Klinik und Poliklinik für Dermatologie, Universitätsklinikum Regensburg.

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http://dx.doi.org/10.1111/ddg.14217_gDOI Listing
January 2021

Cabozantinib-based combination therapy for the treatment of hepatocellular carcinoma.

Gut 2020 Nov 3. Epub 2020 Nov 3.

Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California, USA

Objective: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer with limited treatment options. Cabozantinib, an orally bioavailable multikinase inhibitor is now approved by Food and Drug Administration (FDA) for HCC patients. We evaluated the therapeutic efficacy of cabozantinib, either alone or in combination, in vitro and in vivo.

Design: Human HCC cell lines and HCC mouse models were used to assess the therapeutic efficacy and targeted molecular pathways of cabozantinib, either alone or in combination with the pan-mTOR inhibitor MLN0128 or the checkpoint inhibitor anti-PD-L1 antibody.

Results: Cabozantinib treatment led to stable disease in c-Met/β-catenin and Akt/c-Met mouse HCC while possessing limited efficacy on Akt/Ras and c-Myc liver tumours. Importantly, cabozantinib effectively inhibited c-MET and ERK activity, leading to decreased PKM2 and increased p21 expression in HCC cells and in c-Met/β-catenin and Akt/c-Met HCC. However, cabozantinib was ineffective in inhibiting the Akt/mTOR cascade. Intriguingly, a strong inhibition of angiogenesis by cabozantinib occurred regardless of the oncogenic drivers. However, cabozantinib had limited impact on other tumour microenvironment parameters, including tumour infiltrating T cells, and did not induce programmed death-ligand 1 (PD-L1) expression. Combining cabozantinib with MLN0128 led to tumour regression in c-Met/β-catenin mice. In contrast, combined treatment with cabozantinib and the checkpoint inhibitor anti-PD-L1 antibody did not provide any additional therapeutic benefit in the four mouse HCC models tested.

Conclusion: c-MET/ERK/p21/PKM2 cascade and VEGFR2-induced angiogenesis are the primary targets of cabozantinib in HCC treatment. Combination therapies with cabozantinib and mTOR inhibitors may be effective against human HCC.
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http://dx.doi.org/10.1136/gutjnl-2020-320716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8089119PMC
November 2020

Liver metastases of intrahepatic cholangiocarcinoma: implications for a potential new staging system.

Hepatology 2020 Oct 18. Epub 2020 Oct 18.

Medical Oncology / Institute of Cancer Sciences, The Christie NHS Foundation Trust / University of Manchester, Manchester, United Kingdom.

Background: Intrahepatic cholangiocarcinoma (iCCA) with liver metastases (LM) are perceived to have a poor prognosis but the American Joint Committee on Cancer (AJCC) classifies them as early stage in the absence of lymph nodes or extrahepatic spread.

Methods: Patients with iCCA from the European Network for the Study of Cholangiocarcinoma (ENS-CCA) and Surveillance, Epidemiology, and End Results (SEER) registries with survival/staging (AJCCv.7) data were eligible. Modified staging was used (mAJCCv.7): Group-A: stages-I-III (excluding T2bN0); Group-B: stage-IVa (excluding T2bN1M0); Group-C: LM (T2bN0/1) and Group-D: stage-IVb (extra-hepatic metastases). Survival analysis (Kaplan Meier and Cox Regression) was performed in an ENS-CCA training cohort (TC) and findings internally [ENS-CCA (iVC)] and externally [SEER] validated. The aim was to assess if LM (Group-C) had a shorter survival compared to other early stages (Group-A). A modified version of AJCCv.8 (mAJCCv.8) is proposed.

Results: Total of 574 and 4,171 patients from the ENS-CCA and SEER registries were included. Following the new classification, 19.86% and 17.31% of patients from the ENS-CCA and SEER registries were reclassified into the Group-C, respectively. In the ENS-CCA TC, multivariable Cox Regression was adjusted for obesity (p-value 0.026) and performance status (p-value <0.001); patients in Group-C (HR 2.53 (95%CI 1.18-5.42); p-value 0.017) had a higher risk of death (vs Group-A). Findings were validated in the ENS-CCA iVC (HR 2.93 (95%CI 2.04-4.19); p-value <0.001) and in the SEER registry (HR of 1.88 (95%CI 1.68-2.09); p-value <0.001).

Conclusions: iCCA LM have a worse outcome than other early stages. As AJCCv.8 does not take this into consideration, a modification of AJCC v.8 (mAJCCv.8) including "liver metastases: multiple liver lesions, with or without vascular invasion" as a novel "M1a stage" is suggested.
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http://dx.doi.org/10.1002/hep.31598DOI Listing
October 2020

Visceral leishmaniasis after treatment with immune checkpoint inhibitors.

J Dtsch Dermatol Ges 2021 Jan 11;19(1):119-121. Epub 2020 Aug 11.

Department of Dermatology, University Hospital Regensburg, Germany.

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http://dx.doi.org/10.1111/ddg.14217DOI Listing
January 2021

Staging more important than grading? Evaluation of malignancy grading, depth of invasion, and resection margins in oral squamous cell carcinoma.

Clin Oral Investig 2021 Mar 29;25(3):1169-1182. Epub 2020 Jun 29.

Department of Cranio-Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany.

Objectives: The present study evaluated the predictive value of staging and grading parameters concerning the presence of lymph-node metastases, overall survival (OS), and relapse-free survival (RFS) of patients with oral squamous cell carcinoma (OSCC).

Materials And Methods: HE-stains of 135 surgically treated (R0) primary OSCCs were analyzed using a both microscopic and software-based approach. Depth of invasion (DOI) and resection margins (RM) were measured, and each case was graded according to the malignancy grading system as described by Anneroth et al. and Bryne et al. on two different sites of the tumor (surface and invasion front; TS and IF).

Results: Parameters that could be identified as significant predictors of OS and RFS were UICC cancer stage (p = 0.009 and p = 0.012); pT-stage as defined in the 7th edition (p = 0.029 and 0.015) and, after restaging using DOI, 8th edition (p = 0.023 and p = 0.005) of the TNM classification of malignant tumors; the presence of lymphonodular metastases (LM) (p = 0.004 and p = 0.011); degree of keratinization (p = 0.029 and p = 0.042); and pattern of growth (p = 0.029 and p = 0.024) at the TS after applying a binary scale for both parameters. Also, when directly comparing the most extreme subgroups (scores 1 and 4) of lymphoplasmacytic infiltration at the IF, there was a significant difference in OS (p = 0.046) and RFS (p = 0.005). Invasion of blood vessels (p = 0.013) and perineural invasion (p = 0.023) were significantly associated with a lower OS. Age lower than 60 years (univariate p = 0.029, multivariate p = 0.031), infiltration of lymphatic vessels (p = 0.003), infiltration of nerves (p = 0.010), pT-stage (8th edition) (p = 0.014), degree of keratinization at the IF (p = 0.033), and nuclear polymorphism at the IF (p = 0.043) after conversion to a binary scale were found to be significant prognostic parameters regarding the presence of LM. DOI evolved as a significant predictor for OS (p = 0.006), RFS (p = 0.003), and LM (p = 0.032) in metric and grouped analysis.

Conclusions: The current evaluation revealed depth of invasion as strongest histologic predictor of metastatic tumor growth, overall survival, and relapse-free survival in OSCC, confirming the current adaption of the T-classification. Other distinct histologic grading parameters investigated during this study can give valuable indications of a tumor's potential aggressiveness, but the exact site, mode, and procedure need further exploration.

Clinical Relevance: Integrating measurement of DOI also into the pretherapeutic staging process could aid in treatment planning.
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http://dx.doi.org/10.1007/s00784-020-03421-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878266PMC
March 2021

Correlation of Ductoscopic and Histopathological Findings and Their Relevance as Predictors for Malignancy: A German Multicenter Study.

Anticancer Res 2020 Apr;40(4):2185-2190

Department of Obstetrics and Gynecology, University Hospital Tuebingen, Tuebingen, Germany.

Background/aim: The study aimed at investigating the correlation between ductoscopic and histopathological findings and clarify whether the former allow for accurate prediction of malignancy.

Patients And Methods: The prospective national multi-center study covered a sample of 224 patients with pathologic nipple discharge. A total of 214 patients underwent ductoscopy with subsequent extirpation of the mammary duct. The ductoscopic findings were categorized according to shape, number, color and surface structure of lesions and vascularity and compared to the histological results and analyses.

Results: Ductoscopy revealed lesions in 134 of 214 patients (62.2%). The criteria "multiple versus solitary lesion" differed significantly between malignant and benign lesions. All other criteria were not statistically significant. Malignant tumors were more frequently presented as multiple lesions, benign lesions or masses as solitary lesions (80% vs. 24.8%; p=0.018).

Conclusion: The ductoscopic criterion "solitary vs. multiple lesion" appears to have a low diagnostic prediction of malignancy or benignity.
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http://dx.doi.org/10.21873/anticanres.14179DOI Listing
April 2020

Full-Core Biopsy Systems Take Larger Liver Tissue Samples with Lower Fragmentation Rates Than Conventional Side-Notch Systems: A Randomized Trial.

Cancer Manag Res 2020 13;12:1121-1128. Epub 2020 Feb 13.

Department of Diagnostic and Interventional Radiology, Clinic Ernst von Bergmann, Potsdam, Germany.

Background: The aim of this study was to compare the histopathological quality and physical features of the specimen of a full-core end-cut biopsy system with that of the standard side-notch system for liver biopsies.

Methods: A full-core end-cut 16G biopsy device and a standard side-notch 16G needle were used to take biopsies of unclear liver lesions. Patients were randomized in two groups of 16 patients each. The primary endpoint of this prospective study was the core length measured using a dedicated microscope imaging software. Secondary endpoints were the quality of the specimen rated by an independent pathologist unaware of the device (scale from 1 to 5; with 1 as best and 5 as worst), the core diameter (determined by the microscopic imaging software) and presence of fragmentation (evaluated by the pathologist).

Results: For the full-core (FC) and side-notch (SN) groups, the mean core length was similar with 13,599 μm and 11,570 μm (p=0.131), respectively. The quality of the specimen was significantly better in the FC-group with an average rating of 1.68 vs 2.50 (p=0.009). The fragmentation rate in the FC-group was statistically significantly lower at 2/27 (7%) than in the SN-group at 13/33 (39%) (p=0.021). The diameter in the FC-group was 1042 μm vs 930 μm in SN-group (p=0.018).
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http://dx.doi.org/10.2147/CMAR.S209824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7025653PMC
February 2020

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells.

Int J Mol Sci 2020 Feb 13;21(4). Epub 2020 Feb 13.

Clinic for Plastic, Aesthetic and Hand Surgery, Otto von Guericke University Magdeburg, 39120 Magdeburg, Germany.

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µ), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the , , , , and gene expression in MCS, whereas , , , , , , , , and mRNAs were not significantly changed. and were elevated in AD, and , , , , , , and mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µ, the PC-3 cells showed significant downregulations of mRNA in AD and MCS, and , , and in AD and in MCS. In addition, we measured significant upregulations in , , , , , , , and mRNAs in AD and MCS, and increases in , , and in MCS as well as , , , , , and mRNAs in AD. and were not altered by s-µ. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.
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http://dx.doi.org/10.3390/ijms21041263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072928PMC
February 2020

mTORC2 Signaling Is Necessary for Timely Liver Regeneration after Partial Hepatectomy.

Am J Pathol 2020 04 5;190(4):817-829. Epub 2020 Feb 5.

Department of Bioengineering and Therapeutic Sciences, Liver Center, University of California, San Francisco, California. Electronic address:

Liver regeneration is a fundamental biological process required for sustaining body homeostasis and restoring liver function after injury. Emerging evidence demonstrates that cytokines, growth factors, and multiple signaling pathways contribute to liver regeneration. Mammalian target of rapamycin complex 2 (mTORC2) regulates cell metabolism, proliferation and survival. The major substrates for mTORC2 are the AGC family members of kinases, including AKT, SGK, and PKC-α. We investigated the functional roles of mTORC2 during liver regeneration. Partial hepatectomy (PHx) was performed in liver-specific Rictor (the pivotal unit of mTORC2 complex) knockout (Rictor) and wild-type (Rictor) mice. Rictor-deficient mice were found to be more intolerant to PHx and displayed higher mortality after PHx. Mechanistically, loss of Rictor resulted in decreased Akt phosphorylation, leading to a delay in hepatocyte proliferation and lipid droplets formation along liver regeneration. Overall, these results indicate an essential role of the mTORC2 signaling pathway during liver regeneration.
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http://dx.doi.org/10.1016/j.ajpath.2019.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180798PMC
April 2020

Oncogene-dependent function of BRG1 in hepatocarcinogenesis.

Cell Death Dis 2020 02 4;11(2):91. Epub 2020 Feb 4.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Genomic studies have revealed that HCC is a heterogeneous disease with multiple subtypes. BRG1, encoded by the SMARCA4 gene, is a key component of SWI/SNF chromatin-remodeling complexes. Based on TCGA studies, somatic mutations of SMARCA4 occur in ~3% of human HCC samples. Additional studies suggest that BRG1 is overexpressed in human HCC specimens and may promote HCC growth and invasion. However, the precise functional roles of BRG1 in HCC remain poorly delineated. Here, we analyzed BRG1 in human HCC samples as well as in mouse models. We found that BRG1 is overexpressed in most of human HCC samples, especially in those associated with poorer prognosis. BRG1 expression levels positively correlate with cell cycle and negatively with metabolic pathways in the Cancer Genome Atlas (TCGA) human HCC data set. In a murine HCC model induced by c-MYC overexpression, ablation of the Brg1 gene completely repressed HCC formation. In striking contrast, however, we discovered that concomitant deletion of Brg1 and overexpression of c-Met or mutant NRas (NRAS) triggered HCC formation in mice. Altogether, the present data indicate that BRG1 possesses both oncogenic and tumor-suppressing roles depending on the oncogenic stimuli during hepatocarcinogenesis.
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http://dx.doi.org/10.1038/s41419-020-2289-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000409PMC
February 2020

Zoonotic spillover infections with Borna disease virus 1 leading to fatal human encephalitis, 1999-2019: an epidemiological investigation.

Lancet Infect Dis 2020 04 7;20(4):467-477. Epub 2020 Jan 7.

Department of Neuropathology, Technical University of Munich, Munich, Germany.

Background: In 2018-19, Borna disease virus 1 (BoDV-1), the causative agent of Borna disease in horses, sheep, and other domestic mammals, was reported in five human patients with severe to fatal encephalitis in Germany. However, information on case frequencies, clinical courses, and detailed epidemiological analyses are still lacking. We report the occurrence of BoDV-1-associated encephalitis in cases submitted to the Institute of Clinical Microbiology and Hygiene, Regensburg University Hospital, Regensburg, Germany, and provide a detailed description of newly identified cases of BoDV-1-induced encephalitis.

Methods: All brain tissues from 56 encephalitis cases from Bavaria, Germany, of putative viral origin (1999-2019), which had been submitted for virological testing upon request of the attending clinician and stored for stepwise diagnostic procedure, were systematically screened for BoDV-1 RNA. Two additional BoDV-1-positive cases were contributed by other diagnostic centres. Positive results were confirmed by deep sequencing, antigen detection, and determination of BoDV-1-reactive antibodies in serum and cerebrospinal fluid. Clinical and epidemiological data from infected patients were collected and analysed.

Findings: BoDV-1 RNA and bornavirus-reactive antibodies were detected in eight newly analysed encephalitis cases and the first human BoDV-1 isolate was obtained from an unequivocally confirmed human BoDV-1 infection from the endemic area. Six of the eight BoDV-1-positive patients had no record of immunosuppression before the onset of fatal disease, whereas two were immunocompromised after solid organ transplantation. Typical initial symptoms were headache, fever, and confusion, followed by various neurological signs, deep coma, and severe brainstem involvement. Seven of nine patients with fatal encephalitis of unclear cause were BoDV-1 positive within one diagnostic centre. BoDV-1 sequence information and epidemiological analyses indicated independent spillover transmissions most likely from the local wild animal reservoir.

Interpretation: BoDV-1 infection has to be considered as a potentially lethal zoonosis in endemic regions with reported spillover infections in horses and sheep. BoDV-1 infection can result in fatal encephalitis in immunocompromised and apparently healthy people. Consequently, all severe encephalitis cases of unclear cause should be tested for bornaviruses especially in endemic regions.

Funding: German Federal Ministry of Education and Research.
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http://dx.doi.org/10.1016/S1473-3099(19)30546-8DOI Listing
April 2020

Inhibition of MELK Protooncogene as an Innovative Treatment for Intrahepatic Cholangiocarcinoma.

Medicina (Kaunas) 2019 Dec 18;56(1). Epub 2019 Dec 18.

Institut für Pathologie, Universitätsklinikum Regensburg, 93053 Regensburg, Germany.

: Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. : We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against were investigated in iCCA cell lines. : Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients' shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when was silenced by specific siRNA in the same cells. At the molecular level, siRNA against triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. : Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167 (or other, more specific MELK inhibitors) with DNA-damaging agents might be a potentially effective therapy for human iCCA.
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http://dx.doi.org/10.3390/medicina56010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7023300PMC
December 2019

LEF1 supports metastatic brain colonization by regulating glutathione metabolism and increasing ROS resistance in breast cancer.

Int J Cancer 2020 06 11;146(11):3170-3183. Epub 2019 Nov 11.

Institute of Pathology, University of Regensburg, Regensburg, Germany.

More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.
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http://dx.doi.org/10.1002/ijc.32742DOI Listing
June 2020

Combined Treatment with MEK and mTOR Inhibitors is Effective in In Vitro and In Vivo Models of Hepatocellular Carcinoma.

Cancers (Basel) 2019 Jul 3;11(7). Epub 2019 Jul 3.

Pharmacy Faculty, Hubei University of Chinese Medicine Wuhan, Wuhan 430065, China.

: Hepatocellular carcinoma (HCC) is the most common primary liver cancer histotype, characterized by high biological aggressiveness and scarce treatment options. Recently, we have established a clinically relevant murine HCC model by co-expressing activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and oncogene c-mesenchymal-epithelial transition (c-Met) proto-oncogenes in the mouse liver via hydrodynamic tail vein injection (AKT/c-MET mice). Tumor cells from these mice demonstrated high activity of the AKT/ mammalian target of rapamycin (mTOR) and Ras/ Mitogen-activated protein kinase (MAPK) signaling cascades, two pathways frequently co-induced in human HCC. Methods: Here, we investigated the therapeutic efficacy of sorafenib, regorafenib, the MEK inhibitor PD901 as well as the pan-mTOR inhibitor MLN0128 in the AKT/c-Met preclinical HCC model. : In these mice, neither sorafenib nor regorafenib demonstrated any efficacy. In contrast, administration of PD901 inhibited cell cycle progression of HCC cells in vitro. Combined PD901 and MLN0128 administration resulted in a pronounced growth constraint of HCC cell lines. In vivo, treatment with PD901 or MLN0128 alone moderately slowed HCC growth in AKT/c-MET mice. Importantly, the simultaneous administration of the two drugs led to a stable disease with limited tumor progression in mice. Mechanistically, combined mitogen-activated extracellular signal-regulated kinase (MEK) and mTOR inhibition resulted in a stronger cell cycle inhibition and growth arrest both in vitro and in vivo. : Our study indicates that combination of MEK and mTOR inhibitors might represent an effective therapeutic approach against human HCC.
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http://dx.doi.org/10.3390/cancers11070930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679026PMC
July 2019

Loss of Fbxw7 synergizes with activated Akt signaling to promote c-Myc dependent cholangiocarcinogenesis.

J Hepatol 2019 10 11;71(4):742-752. Epub 2019 Jun 11.

Institute of Pathology, University of Greifswald, Greifswald, Germany; Institute of Pathology, University of Regensburg, Regensburg, Germany. Electronic address:

Background & Aims: The ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7) is recognized as a tumor suppressor in many cancer types due to its ability to promote the degradation of numerous oncogenic target proteins. Herein, we aimed to elucidate its role in intrahepatic cholangiocarcinoma (iCCA).

Methods: Herein, we first confirmed that FBXW7 gene expression was reduced in human iCCA specimens. To identify the molecular mechanisms by which FBXW7 dysfunction promotes cholangiocarcinogenesis, we generated a mouse model by hydrodynamic tail vein injection of Fbxw7ΔF, a dominant negative form of Fbxw7, either alone or in association with an activated/myristylated form of AKT (myr-AKT). We then confirmed the role of c-MYC in human iCCA cell lines and its relationship to FBXW7 expression in human iCCA specimens.

Results: FBXW7 mRNA expression is almost ubiquitously downregulated in human iCCA specimens. While forced overexpression of Fbxw7ΔF alone did not induce any appreciable abnormality in the mouse liver, co-expression with AKT triggered cholangiocarcinogenesis and mice had to be euthanized by 15 weeks post-injection. At the molecular level, a strong induction of Fbxw7 canonical targets, including Yap, Notch2, and c-Myc oncoproteins, was detected. However, only c-MYC was consistently confirmed as a FBXW7 target in human CCA cell lines. Most importantly, selected ablation of c-Myc completely impaired iCCA formation in AKT/Fbxw7ΔF mice, whereas deletion of either Yap or Notch2 only delayed tumorigenesis in the same model. In human iCCA specimens, an inverse correlation between the expression levels of FBXW7 and c-MYC transcriptional activity was observed.

Conclusions: Downregulation of FBXW7 is ubiquitous in human iCCA and cooperates with AKT to induce cholangiocarcinogenesis in mice via c-Myc-dependent mechanisms. Targeting c-MYC might represent an innovative therapy against iCCA exhibiting low FBXW7 expression.

Lay Summary: There is mounting evidence that FBXW7 functions as a tumor suppressor in many cancer types, including intrahepatic cholangiocarcinoma, through its ability to promote the degradation of numerous oncoproteins. Herein, we have shown that the low expression of FBXW7 is ubiquitous in human cholangiocarcinoma specimens. This low expression is correlated with increased c-MYC activity, leading to tumorigenesis. Our findings suggest that targeting c-MYC might be an effective treatment for intrahepatic cholangiocarcinoma.
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http://dx.doi.org/10.1016/j.jhep.2019.05.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773530PMC
October 2019

Metabolic, Anti-apoptotic and Immune Evasion Strategies of Primary Human Myeloma Cells Indicate Adaptations to Hypoxia.

Mol Cell Proteomics 2019 05 21;18(5):936-953. Epub 2019 Feb 21.

Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria;. Electronic address:

Multiple Myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). It develops from a premalignant stage, monoclonal gammopathy of undetermined significance (MGUS), often via an intermediate stage, smoldering MM (SMM). The mechanisms of MM progression have not yet been fully understood, all the more because patients with MGUS and SMM already carry similar initial mutations as found in MM cells. Over the last years, increased importance has been attributed to the tumor microenvironment and its role in the pathophysiology of the disease. Adaptations of MM cells to hypoxic conditions in the BM have been shown to contribute significantly to MM progression, independently from the genetic predispositions of the tumor cells. Searching for consequences of hypoxia-induced adaptations in primary human MM cells, CD138-positive plasma cells freshly isolated from BM of patients with different disease stages, comprising MGUS, SMM, and MM, were analyzed by proteome profiling, which resulted in the identification of 6218 proteins. Results have been made fully accessible via ProteomeXchange with identifier PXD010600. Data previously obtained from normal primary B cells were included for comparative purposes. A principle component analysis revealed three clusters, differentiating B cells as well as MM cells corresponding to less and more advanced disease stages. Comparing these three clusters pointed to the alteration of pathways indicating adaptations to hypoxic stress in MM cells on disease progression. Protein regulations indicating immune evasion strategies of MM cells were determined, supported by immunohistochemical staining, as well as transcription factors involved in MM development and progression. Protein regulatory networks related to metabolic adaptations of the cells became apparent. Results were strengthened by targeted analyses of a selected panel of metabolites in MM cells and MM-associated fibroblasts. Based on our data, new opportunities may arise for developing therapeutic strategies targeting myeloma disease progression.
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http://dx.doi.org/10.1074/mcp.RA119.001390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6495257PMC
May 2019

MEK inhibition suppresses K-Ras wild-type cholangiocarcinoma in vitro and in vivo via inhibiting cell proliferation and modulating tumor microenvironment.

Cell Death Dis 2019 02 11;10(2):120. Epub 2019 Feb 11.

Department of Bioengineering and Therapeutic Sciences, and Liver Center, University of California, San Francisco, CA, USA.

PD901, a MEK inhibitor, has been demonstrated of therapeutic efficacy against cholangiocarcinoma (CCA) harboring K-Ras oncogenic mutations. However, most CCA exhibit no K-Ras mutations. In the current study, we investigated the therapeutic potential of PD901, either alone or in combination with the pan-mTOR inhibitor MLN0128, for the treatment of K-Ras wild-type CCA in vitro using human CCA cell lines, and in vivo using AKT/YapS127A CCA mouse model. We discovered that in vitro, PD901 treatment strongly inhibited CCA cell proliferation, and combined PD901 and MLN0128 therapy further increased growth inhibition. In vivo, treatment of PD901 alone triggered tumor regression, which was not further increased when the two drugs were administered simultaneously. Mechanistically, PD901 efficiently hampered ERK activation in vitro and in vivo, leading to strong inhibition of CCA tumor cell cycle progression. Intriguingly, we discovered that PD901, but not MLN0128 treatment resulted in changes affecting the vasculature and cancer-associated fibroblasts in AKT/YapS127A mouse lesions. It led to the decreased hypoxia within tumor lesions, which may further enhance the anti-cell proliferation activities of PD901. Altogether, our study demonstrates that MEK inhibitors could be effective for the treatment of K-Ras wild-type CCA via inhibiting cell proliferation and modulating tumor microenvironment.
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http://dx.doi.org/10.1038/s41419-019-1389-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370758PMC
February 2019

Diagnostic performance of Gd-EOB-DTPA-enhanced MRI for evaluation of liver dysfunction: a multivariable analysis of 3T MRI sequences.

Oncotarget 2018 Nov 20;9(91):36371-36378. Epub 2018 Nov 20.

Department of Radiology, University Hospital Regensburg, Regensburg, Germany.

Objective: The aim of this study was to evaluate the diagnostic performance of a multiparametric gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA)-enhanced MRI examination for the estimation of liver dysfunction classified by the Model for End-Stage Liver Disease (MELD) score.

Results: Liver dysfunction can be assessed by different methods. In a logistic regression analysis, T1- and T2-weighted images were affected by impaired liver function. In the assessment of liver dysfunction, the reduction rate in T1 mapping sequences showed a significant correlation in simple and multiple logistic regression.

Conclusion: Changes in Gd-EOB-DTPA-enhanced MRI between plain images and images obtained during the hepatobiliary phase allowed good prediction of liver dysfunction, especially when using T1 mapping sequences.

Materials And Methods: A total of 199 patients underwent contrast-enhanced MRI with a hepatocyte-specific contrast agent at 3T. In the multivariable analysis, the full range of available MRI sequences was used to estimate the liver dysfunction of patients with various MELD scores.
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http://dx.doi.org/10.18632/oncotarget.26368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284745PMC
November 2018

In vivo confirmation of altered hepatic glucose metabolism in patients with liver fibrosis/cirrhosis by F-FDG PET/CT.

EJNMMI Res 2018 Nov 9;8(1):98. Epub 2018 Nov 9.

Department of Nuclear Medicine, University Hospital Regensburg, Regensburg, Germany.

Objective: The aim of this study was to assess the value of F-FDG PET/CT for quantitative assessment of hepatic metabolism in patients with different stages of liver fibrosis/cirrhosis.

Materials And Methods: F-FDG PET/CT scans of 37 patients either with or without liver fibrosis/cirrhosis, classified according to the METAVIR score (F0-F4) obtained from histopathological analysis of liver specimen, were analyzed retrospectively and classified as follows: no liver fibrosis (F0, n = 6), mild liver fibrosis (F1, n = 11), advanced liver fibrosis (F2, n = 6), severe liver fibrosis (F3, n = 5), and liver cirrhosis (F4, n = 11). The liver-to-blood ratio (LBR, scan time corrected for a reference time of 75 min) was compared between patient groups.

Results: Patients with liver fibrosis or cirrhosis (≥ F1; LBR 1.53 ± 0.35) showed a significant higher LBR than patients with normal liver parenchyma (F0, 1.08 ± 0.23; P = 0.004). In direct comparison, LBR increased up to the advanced stage of liver fibrosis (F2; 2.00 ± 0.40) and decreased until liver cirrhosis is reached (F4, 1.32 ± 0.14).

Conclusion: Functional changes in liver parenchyma during liver fibrosis/cirrhosis affect hepatic glucose metabolism and significantly differ between stages of liver fibrosis/cirrhosis, classified according to the METAVIR scoring system, as demonstrated by LBR quantification by F-FDG PET/CT.
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http://dx.doi.org/10.1186/s13550-018-0452-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226405PMC
November 2018

PI3K: A master regulator of brain metastasis-promoting macrophages/microglia.

Glia 2018 11 25;66(11):2438-2455. Epub 2018 Oct 25.

Department of Internal Medicine III, University Hospital Regensburg, Regensburg, Germany.

Mutations and activation of the PI3K signaling pathway in breast cancer cells have been linked to brain metastases. However, here we describe that in some breast cancer brain metastases samples the protein expression of PI3K signaling components is restricted to the metastatic microenvironment. In contrast to the therapeutic effects of PI3K inhibition on the breast cancer cells, the reaction of the brain microenvironment is less understood. Therefore we aimed to quantify the PI3K pathway activity in breast cancer brain metastasis and investigate the effects of PI3K inhibition on the central nervous system (CNS) microenvironment. First, to systematically quantify the PI3K pathway activity in breast cancer brain metastases, we performed a prospective biomarker study using a reverse phase protein array (RPPA). The majority, namely 30 out of 48 (62.5%) brain metastatic tissues examined, revealed high PI3K signaling activity that was associated with a median overall survival (OS) of 9.41 months, while that of patients, whose brain metastases showed only moderate or low PI3K activity, amounted to only 1.93 and 6.71 months, respectively. Second, we identified PI3K as a master regulator of metastasis-promoting macrophages/microglia during CNS colonization; and treatment with buparlisib (BKM120), a pan-PI3K Class I inhibitor with a good blood-brain-barrier penetrance, reduced their metastasis-promoting features. In conclusion, PI3K signaling is active in the majority of breast cancer brain metastases. Since PI3K inhibition does not only affect the metastatic cells but also re-educates the metastasis-promoting macrophages/microglia, PI3K inhibition may hold considerable promise in the treatment of brain metastasis and the respective microenvironment.
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http://dx.doi.org/10.1002/glia.23485DOI Listing
November 2018

Integration of "omics" techniques: Dronedarone affects cardiac remodeling in the infarction border zone.

Exp Biol Med (Maywood) 2018 07;243(11):895-910

1 Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, Greifswald D-17475, Germany.

Dronedarone improves microvascular flow during atrial fibrillation and reduces the infarct size in acute models of myocardial infarction. However, dronedarone might be harmful in patients with recent decompensated heart failure and increases mortality in patients with permanent atrial fibrillation. A pathophysiological explanation for these discrepant data is lacking. This study investigated the effects of dronedarone on gene and protein expression in the infarcted area and border zone in pigs subjected to anterior ischemia/reperfusion myocardial infarction. The ischemia/reperfusion myocardial infarction was induced in 16 pigs. Eight pigs were treated with dronedarone for 28 days after myocardial infarction, the remaining pigs served as control. Microarray-based transcriptome profiling and 2D-DIGE-based proteome analysis were used to assess the effects of dronedarone on left ventricular gene expression in healthy (LV), infarcted (MI), and border zone tissue. Selected targets were validated by RT-qPCR or immunoblot analyses, with special emphasize given to the transcriptome/proteome overlap. Combined "omics" analysis was performed to identify most significant disease and function charts affected by dronedarone and to establish an integrated network. The levels of 879 (BZ) or 7 (MI) transcripts and 51 (LV) or 15 (BZ) proteins were significantly altered by dronedarone, pointing to a substantial efficacy of dronedarone in the border zone. Transcriptome and proteome data indicate that dronedarone influences post-infarction remodeling processes and identify matricellular proteins as major targets of dronedarone in this setting. This finding is fully supported by the disease and function charts as well as by the integrated network established by combined "omics". Dronedarone therapy alters myocardial gene expression after acute myocardial infarction with pronounced effects in the border zone. Dronedarone promotes infarct healing via regulation of periostin and might contribute to the limitation of its expansion as well as cardiac rupture. Thus, there are no experimental hints that dronedarone per se has direct harmful effects after MI in ventricular tissue. Impact statement Dronedarone reduced the infarct size in models of acute myocardial infarction (MI). Here, we show that dronedarone attenuates many of the substantial changes in gene expression that are provoked by acute myocardial infarction (AMI) in pigs. Dronedarone modifies the expression of gene panels related to post-infarction cardiac healing and remodeling processes and, most remarkably, this occurs predominantly in the infarction border-zone and much less so in the vital or infarcted myocardium. Combined "omics" identified matricellular proteins and ECM as major dronedarone-regulated targets and emphasizes their relevance for Disease Charts and Tox Function Charts associated with tissue remodeling and cellular movement. The results demonstrate dronedarone's capability of regulating cardiac repair and remodeling processes specifically in the infarction border zone and identify underlying mechanisms and pathways that might be employed in future therapeutic strategies to improve long-term cardiac tissue function and stability.
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http://dx.doi.org/10.1177/1535370218788517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108048PMC
July 2018

Combined CDK4/6 and Pan-mTOR Inhibition Is Synergistic Against Intrahepatic Cholangiocarcinoma.

Clin Cancer Res 2019 01 3;25(1):403-413. Epub 2018 Jul 3.

Department of Bioengineering and Therapeutic Sciences and Liver Center, University of California, San Francisco, California.

Purpose: Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer type, lacking effective therapies and associated with a dismal prognosis. Palbociclib is a selective CDK4/6 inhibitor, which has been shown to suppress cell proliferation in many experimental cancer models. Recently, we demonstrated that pan-mTOR inhibitors, such as MLN0128, effectively induce apoptosis, although have limited efficacy in restraining proliferation of ICC cells. Here, we tested the hypothesis that palbociclib, due to its antproliferative properties in many cancer types, might synergize with MLN0128 to impair ICC growth.

Experimental Design: Human ICC cell lines and the AKT/YapS127A ICC mouse model were used to test the therapeutic efficacy of palbociclib and MLN0128, either alone or in combination.

Results: Administration of palbociclib suppressed ICC cell growth by inhibiting cell-cycle progression. Concomitant administration of palbociclib and MLN0128 led to a pronounced, synergistic growth constraint of ICC cell lines. Furthermore, while treatment with palbociclib or MLN0128 alone resulted in tumor growth reduction in AKT/YapS127A mice, a remarkable tumor regression was achieved when the two drugs were administered simultaneously. Mechanistically, palbociclib was found to potentiate MLN0128 mTOR inhibition activity, whereas MLN0128 prevented the upregulation of cyclin D1 induced by palbociclib treatment.

Conclusions: Our study indicates the synergistic activity of palbociclib and MLN0128 in inhibiting ICC cell proliferation. Thus, combination of CDK4/6 and mTOR inhibitors might represent a novel, promising, and effective therapeutic approach against human ICC..
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423983PMC
January 2019

DWI - histology: a possible means of determining degree of liver fibrosis?

Oncotarget 2018 Apr 13;9(28):20112-20118. Epub 2018 Apr 13.

Department of Radiology, University Hospital Regensburg, Regensburg, Germany.

Objectives: The aim of this study was to determine the diagnostic value of diffusion-weighted MRI of the liver at 3T to classify liver fibrosis/cirrhosis.

Methods: 62 patients who underwent both histopathological examination and diffusion-weighted imaging of the liver via 3T MRI within a period of 3 months were included in the study. The Ishak score (1-6) was used to determine the degree of fibrosis: No liver fibrosis (NLF; Ishak 0, n = 16), mild liver fibrosis (MLF; Ishak 1-2, n = 23), advanced liver fibrosis (ALF; Ishak 3-5, n = 12), and liver cirrhosis (LC; Ishak 6, n = 11).

Results: The corresponding ADC values for the individual patient groups were as follows: NLF: 1123 (SD 95.8); MLF: 1032 (SD 77.6); ALF: 962 (SD 68.8); LC: 1015 (SD 60.2) mm2/s. There is a significant difference between NLF and MLF (p = 0.004) and between MLF and ALF (p = 0.022). A significant difference between patients with ALF and LC (p = 0.117) could not be found.

Conclusion: Liver fibrosis/cirrhosis lowers the ADC values of the liver parenchyma in 3T MRI. However, the degree of fibrosis can only be conditionally determined on the basis of ADC values.
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http://dx.doi.org/10.18632/oncotarget.24981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5929450PMC
April 2018

Detecting liver fibrosis with Gd-EOB-DTPA-enhanced MRI: A confirmatory study.

Sci Rep 2018 04 18;8(1):6207. Epub 2018 Apr 18.

Department of Radiology, University Hospital Regensburg, Regensburg, Germany.

Strong correlations between the grade of fibrosis and cirrhosis, classified using the Ishak scoring system, and the uptake characteristics of Gd-EOB-DTPA with the relative enhancement (RE) of the liver parenchyma have been reported. To confirm the results of a retrospective analysis, patients undergoing liver surgery were prospectively examined with Gd-EOB-DTPA-enhanced liver 3 Tesla MRI to determine the degree of liver fibrosis. Correlations between the grade of fibrosis and cirrhosis, classified using the Ishak scoring system, and RE were investigated and compared with those derived from an initial retrospective study. After validating the cut-off values in the retrospective study (Ishak ≥ 1, RE-cut-off 0.90; Ishak ≥ 2, RE-cut-off 0.79; Ishak ≥ 4, RE-cut-off 0.60; and Ishak = 6, RE-cut-off 0.47), we showed that Gd-EOB-DTPA has a high sensitivity (≥86%) and a high positive predictive value (≥86%). These results support the use of Gd-EOB-DTPA-enhanced liver MRI as a non-invasive method for determining the degree of liver fibrosis and cirrhosis.
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http://dx.doi.org/10.1038/s41598-018-24316-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906481PMC
April 2018

Analysis of Luminex-based Algorithms to Define Unacceptable HLA Antibodies in CDC-crossmatch Negative Kidney Transplant Recipients.

Transplantation 2018 06;102(6):969-977

Department of Nephrology, University Hospital Regensburg, Regensburg, Germany.

Background: HLA-specific antibodies detected by solid phase assays are increasingly used to define unacceptable HLA antigen mismatches (UAM) before renal transplantation. The accuracy of this approach is unclear.

Methods: Day of transplant sera from 211 complement-dependent cytotoxicity crossmatch-negative patients were retrospectively analyzed for donor-specific anti-HLA antibodies (DSA) using Luminex technology. HLA were defined as UAM if DSA had mean fluorescence intensity above (I) 3000 (patients retransplanted and those with DSA against HLA class I and II) or 5000 (all other patients), (II) 5000 for HLA-A, -B, and -DR and 10 000 for HLA DQ or (III) 10 000 (all HLA). We then studied the accuracy of these algorithms to identify patients with antibody-mediated rejection (AMR) and graft loss. UAM were also determined in 256 transplant candidates and vPRA levels calculated.

Results: At transplantation, 67 of 211 patients had DSA. Of these, 31 (algorithm I), 24 (II) and 17 (III) had UAM. Nine (I and II) and 8 (III) of 11 early AMR episodes and 7 (I), 6 (II) and 5 (III) of 9 graft losses occurred in UAM-positive patients during 4.9 years of follow-up. Algorithms I and II identified patients with persistently lower glomerular filtration rate even in the absence of overt AMR. Of the waiting list patients, 22-33% had UAM with median virtual panel reactive antibody of 69.2% to 79.1%.

Conclusions: Algorithms I and II had comparable efficacy but were superior to Algorithm III in identifying at-risk patients at an acceptable false-positive rate. However, Luminex-defined UAM significantly restrict the donor pool of affected patients, which might prolong waiting time.
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http://dx.doi.org/10.1097/TP.0000000000002129DOI Listing
June 2018

Efficacy of MEK inhibition in a K-Ras-driven cholangiocarcinoma preclinical model.

Cell Death Dis 2018 01 18;9(2):31. Epub 2018 Jan 18.

Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly malignancy with limited treatment options. Gain-of-function mutations in K-Ras is a very frequent alteration, occurring in ~15 to 25% of human iCCA patients. Here, we established a new iCCA model by expressing activated forms of Notch1 (NICD) and K-Ras (K-Ras) in the mouse liver (K-Ras/NICD mice). Furthermore, we investigated the therapeutic potential of MEK inhibitors in vitro and in vivo using human CCA cell lines and K-Ras/NICD mice, respectively. Treatment with U0126, PD901, and Selumetinib MEK inhibitors triggered growth restraint in all CCA cell lines tested, with the most pronounced growth suppressive effects being observed in K-Ras mutant cells. Growth inhibition was due to reduction in proliferation and massive apoptosis. Furthermore, treatment of K-Ras/NICD tumor-bearing mice with PD901 resulted in stable disease. At the molecular level, PD901 efficiently inhibited ERK activation in K-Ras/NICD tumor cells, mainly leading to increased apoptosis. Altogether, our study demonstrates that K-Ras/NICD mice represent a novel and useful preclinical model to study K-Ras-driven iCCA development and the effectiveness of MEK inhibitors in counteracting this process. Our data support the usefulness of MEK inhibitors for the treatment of human iCCA.
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http://dx.doi.org/10.1038/s41419-017-0183-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833851PMC
January 2018

Preparation of A Spaceflight: Apoptosis Search in Sutured Wound Healing Models.

Int J Mol Sci 2017 Dec 3;18(12). Epub 2017 Dec 3.

Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.

To prepare the ESA (European Space Agency) spaceflight project "Wound healing and Sutures in Unloading Conditions", we studied mechanisms of apoptosis in wound healing models based on ex vivo skin tissue cultures, kept for 10 days alive in serum-free DMEM/F12 medium supplemented with bovine serum albumin, hydrocortisone, insulin, ascorbic acid and antibiotics at 32 °C. The overall goal is to test: (i) the viability of tissue specimens; (ii) the gene expression of activators and inhibitors of apoptosis and extracellular matrix components in wound and suture models; and (iii) to design analytical protocols for future tissue specimens after post-spaceflight download. Hematoxylin-Eosin and Elastica-van-Gieson staining showed a normal skin histology with no signs of necrosis in controls and showed a normal wound suture. TdT-mediated dUTP-biotin nick end labeling for detecting DNA fragmentation revealed no significant apoptosis. No activation of caspase-3 protein was detectable. , , , , , , , , , and mRNAs were not altered in epidermis and dermis samples with and without a wound compared to 0 day samples (specimens investigated directly post-surgery). , , and mRNAs were downregulated in epidermis/dermis samples with and/or without a wound compared to 0 day samples. , were upregulated in 10 day wound samples compared to 0 day samples in epidermis/dermis. mRNAs were elevated in 10 day wound and no wound samples compared to 0 day samples in dermis. In conclusion, we demonstrate that it is possible to maintain live skin tissue cultures for 10 days. The viability analysis showed no significant signs of cell death in wound and suture models. The gene expression analysis demonstrated the interplay of activators and inhibitors of apoptosis and extracellular matrix components, thereby describing important features in ex vivo sutured wound healing models. Collectively, the performed methods defining analytical protocols proved to be applicable for post-flight analyzes of tissue specimens after sample return.
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http://dx.doi.org/10.3390/ijms18122604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751207PMC
December 2017