Several different gene expression signatures have been proposed to predict response to therapy and clinical outcome in lung adenocarcinoma. Herein, we investigate if elements of published gene sets can be reproduced in a small dataset, and how gene expression profiles based on limited sample size relate to clinical parameters including histopathological grade and EGFR protein expression.

Background. Chronic pancreatitis is one of the main risk factors for pancreatic cancer. In acute and chronic pancreatitis, oxidative stress is thought to play a key role. In this respect, the recently described mitochondria-targeted antioxidant SkQ1 effectively scavenges reactive oxygen species at nanomolar concentrations. Therefore, we aimed to characterize the influence of SkQ1 on tissue injury and pain in acute and chronic pancreatitis. Methods. Both acute and chronic pancreatitis were induced in C57BL/6 mice by intraperitoneal cerulein injections and treatment with SkQ1 was carried out by peroral applications. Hyperalgesia was assessed by behavioral observation and measurement of abdominal mechanical sensitivity. Blood serum and pancreatic tissue were harvested for analysis of lipase and histology. Results. SkQ1 did not influence pain, serological, or histological parameters of tissue injury in acute pancreatitis. In chronic pancreatitis, a highly significant reduction of pain-related behavior (p < 0.0001) was evident, but histological grading revealed increased tissue injury in SkQ1-treated animals (p = 0.03). Conclusion. After SkQ1 treatment, tissue injury is not ameliorated in acute pancreatitis and increased in chronic pancreatitis. However, we show an analgesic effect in chronic pancreatitis. Further studies will need to elucidate the risks and benefits of mitochondria-targeted antioxidants as an analgesic.

Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.

Liver transplantation (LT) presents a curative treatment option in patients with early stage hepatocellular carcinoma (HCC) who are not eligible for resection or ablation therapy. Due to a risk of up 30% for waitlist drop-out upon tumor progression, bridging therapies are used to halt tumor growth. Transarterial chemoembolization (TACE) and less commonly stereotactic body radiation therapy (SBRT) or a combination of TACE and SBRT, are used as bridging therapies in LT. However, it remains unclear if one of those treatment options is superior. The analysis of explant livers after transplantation provides the unique opportunity to investigate treatment response by histopathology.

Colorectal cancer (CRC) is the third most common malignancy worldwide, but the key driver to distant metastases is still unknown. This study aimed to elucidate the link between immunosurveillance and organotropism of metastases in CRC by evaluating different gene signatures and pathways.

With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ' kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples.

Progressive respiratory failure and hyperinflammatory response is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. Despite mounting evidence of disruption of the hypothalamus-pituitary-adrenal axis in COVID-19, relatively little is known about the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to adrenal glands and associated changes. Here we demonstrate adrenal viral tropism and replication in COVID-19 patients. Adrenal glands showed inflammation accompanied by inflammatory cell death. Histopathologic analysis revealed widespread microthrombosis and severe adrenal injury. In addition, activation of the glycerophospholipid metabolism and reduction of cortisone intensities were characteristic for COVID-19 specimens. In conclusion, our autopsy series suggests that SARS-CoV-2 facilitates the induction of adrenalitis. Given the central role of adrenal glands in immunoregulation and taking into account the significant adrenal injury observed, monitoring of developing adrenal insufficiency might be essential in acute SARS-CoV-2 infection and during recovery.

Tumor stem cells play a pivotal role in carcinogenesis and metastatic spread in colorectal cancer (CRC). Olfactomedin 4 (OLFM4) is co-expressed with the established stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 at the bottom of intestinal crypts and has been suggested as a surrogate for cancer stemness and a biomarker in gastrointestinal tumors associated with prognosis. Therefore, it was the aim of the present study to clarify whether OLFM4 is involved in carcinogenesis and metastatic spread in CRC. We used a combined approach of functional assays using forced OLFM4 overexpression in human CRC cell lines, xenograft mice, and an immunohistochemical approach using patient tissues to investigate the impact of OLFM4 on stemness, canonical Wnt signaling, properties of metastasis and differentiation as well as prognosis. OLFM4 expression correlated weakly with tumor grade in one patient cohort (metastasis collection: p = 0.05; pooled analysis of metastasis collection and survival collection: p = 0.19) and paralleled the expression of differentiation markers (FABP2, MUC2, and CK20) (p = 0.002) but did not correlate with stemness-associated markers. Further analyses in CRC cells lines as well as xenograft mice including forced overexpression of OLFM4 revealed that OLFM4 neither altered the expression of markers of stemness nor epithelial-mesenchymal transition, nor did OLFM4 itself drive proliferation, migration, or colony formation, which are all prerequisites of carcinogenesis and tumor progression. In line with this, we found no significant correlation between OLFM4 expression, metastasis, and patient survival. In summary, expression of OLFM4 in human CRC seems to be characteristic of differentiation marker expression in CRC but is not a driver of carcinogenesis nor metastatic spread.

Growth and metastasis of colorectal cancer is closely connected to the biosynthetic capacity of tumor cells, and colorectal cancer stem cells that reside at the top of the intratumoral hierarchy are especially dependent on this feature. By performing disease modeling on patient-derived tumor organoids, we found that elevated expression of the ribosome biogenesis factor NLE1 occurs upon SMAD4 loss in TGFβ1-exposed colorectal cancer organoids. TGFβ signaling-mediated downregulation of NLE1 was prevented by ectopic expression of c-MYC, which occupied an E-box-containing region within the NLE1 promoter. Elevated levels of NLE1 were found in colorectal cancer cohorts compared with normal tissues and in colorectal cancer subtypes characterized by Wnt/MYC and intestinal stem cell gene expression. In colorectal cancer cells and organoids, NLE1 was limiting for de novo protein biosynthesis. Upon NLE1 ablation, colorectal cancer cell lines activated p38/MAPK signaling, accumulated p62- and LC3-positive structures indicative of impaired autophagy, and displayed more reactive oxygen species. Phenotypically, knockout of NLE1 inhibit.ed proliferation, migration and invasion, clonogenicity, and anchorage-independent growth. NLE1 loss also increased the fraction of apoptotic tumor cells, and deletion of TP53 further sensitized NLE1-deficient colorectal cancer cells to apoptosis. In an endoscopy-guided orthotopic mouse transplantation model, ablation of NLE1 impaired tumor growth in the colon and reduced primary tumor-derived liver metastasis. In patients with colorectal cancer, NLE1 mRNA levels predicted overall and relapse-free survival. Taken together, these data reveal a critical role of NLE1 in colorectal cancer growth and progression and suggest that NLE1 represents a potential therapeutic target in colorectal cancer patients.

Catecholamines stimulate epithelial proliferation, but the role of sympathetic nerve signaling in pancreatic ductal adenocarcinoma (PDAC) is poorly understood. Catecholamines promoted ADRB2-dependent PDAC development, nerve growth factor (NGF) secretion, and pancreatic nerve density. Pancreatic Ngf overexpression accelerated tumor development in LSL-Kras+/G12D;Pdx1-Cre (KC) mice. ADRB2 blockade together with gemcitabine reduced NGF expression and nerve density, and increased survival of LSL-Kras+/G12D;LSL-Trp53+/R172H;Pdx1-Cre (KPC) mice. Therapy with a Trk inhibitor together with gemcitabine also increased survival of KPC mice. Analysis of PDAC patient cohorts revealed a correlation between brain-derived neurotrophic factor (BDNF) expression, nerve density, and increased survival of patients on nonselective β-blockers. These findings suggest that catecholamines drive a feedforward loop, whereby upregulation of neurotrophins increases sympathetic innervation and local norepinephrine accumulation.

Wnt signaling drives epithelial self-renewal and disease progression in human colonic epithelium and colorectal cancer (CRC). Characterization of Wnt effector pathways is key for our understanding of these processes and for developing therapeutic strategies that aim to preserve tissue homeostasis. O-glycosylated cell surface proteins, such as α-dystroglycan (α-DG), mediate cellular adhesion to extracellular matrix components. We revealed a Wnt/LARGE2/α-DG signaling pathway which triggers this mode of colonic epithelial cell-to-matrix interaction in health and disease.

The basic helix-loop-helix transcription factor AP4/TFAP4/AP-4 is encoded by a c-MYC target gene and displays up-regulation concomitantly with c-MYC in colorectal cancer (CRC) and numerous other tumor types. Here a genome-wide characterization of AP4 DNA binding and mRNA expression was performed using a combination of microarray, genome-wide chromatin immunoprecipitation, next-generation sequencing, and bioinformatic analyses. Thereby, hundreds of induced and repressed AP4 target genes were identified. Besides many genes involved in the control of proliferation, the AP4 target genes included markers of stemness (LGR5 and CD44) and epithelial-mesenchymal transition (EMT) such as SNAIL, E-cadherin/CDH1, OCLN, VIM, FN1, and the Claudins 1, 4, and 7. Accordingly, activation of AP4 induced EMT and enhanced migration and invasion of CRC cells. Conversely, down-regulation of AP4 resulted in mesenchymal-epithelial transition and inhibited migration and invasion. In addition, AP4 induction was required for EMT, migration, and invasion caused by ectopic expression of c-MYC. Inhibition of AP4 in CRC cells resulted in decreased lung metastasis in mice. Elevated AP4 expression in primary CRC significantly correlated with liver metastasis and poor patient survival. These findings imply AP4 as a new regulator of EMT that contributes to metastatic processes in CRC and presumably other carcinomas.

We investigated corticospinal tract (CST) integrity in the absence of white matter (WM) lesions using diffusion tensor imaging (DTI) in early MS disease stages.

Liver resection is a curative treatment for hepatocellular carcinoma (HCC). Tumor-infiltrating leukocytes (TILs) are important players in predicting HCC recurrence. However, the invasive margin could not be confirmed as relevant for HCC. The migration of immune cells into HCC may originate from intratumoral vessels. No previous study has examined perivascular (PV) infiltration. Tumors from 60 patients were examined. Immunohistochemistry was performed against CD3, CD8, CD20, and CD66b. TILs were counted in the PV regions using an algorithm for quantification of the tumor immune stroma (QTiS). The results were correlated with overall (OS) and disease-free survival (DFS), clinical parameters, and laboratory values. PV infiltration of TILs was predominant in resected HCC. Higher PV infiltration of CD3⁺ (p = 0.016) and CD8⁺ (p = 0.028) independently predicted better OS and DFS, respectively. CD20⁺ showed a trend towards better DFS (p = 0.076). Scoring of CD3⁺, CD8⁺, and CD20⁺ independently predicted OS and DFS (p < 0.01). The amount of perivascular-infiltrating CD3⁺ cells is an independent predictor of better OS, and CD8⁺ cells independently predict prolonged DFS. Our novel perivascular infiltration scoring (PVIS) can independently predict both DFS and OS in resected HCC patients.

Evaluating patients and treatment decisions in a multidisciplinary tumor board has led to better quality of care and longer survival in cancer patients. The aim of this study was to evaluate tumor board recommendations for thoracic oncology patients regarding guideline adherence and transferal of recommendations into clinical practice.

Currently, there is no consensus about the use of adjuvant chemotherapy for patients with stage II colorectal cancer. Here, we aimed to identify and validate a prognostic mRNA expression signature for the stratification of patients with stage II colorectal cancer according to their risk for relapse. First, publicly available mRNA expression profiling datasets from 792 primary, stage II colorectal cancers from six different training cohorts were analyzed to identify genes that are consistently associated with patient relapse-free survival (RFS). Second, the identified gene expression signature was experimentally validated using NanoString technology and computationally refined on primary colorectal cancer samples from 205 patients with stage II colorectal cancer. Third, the refined signature was validated in two independent publicly available cohorts of 166 patients with stage II colorectal cancer. Bioinformatics analysis of training cohorts identified a 61-gene signature that was highly significantly associated with RFS (HR = 37.08, P = 2.68*10-106, sensitivity = 89.29%, specificity = 89.61%, and AUC = 0.937). The experimental validation and refinement revealed a 15-gene signature that robustly predicted relapse in three independent cohorts: an in-house cohort (HR = 20.4, P = 8.73*10-23, sensitivity = 90.32%, specificity = 80.99%, AUC = 0.812), GSE161158 (HR = 5.81, P = 3.57*10-4, sensitivity = 64.29%, specificity = 81.67%, AUC = 0.796), and GSE26906 (HR = 7.698, P = 7.26*10-8, sensitivity = 61.54%, specificity = 78.33%, AUC = 0.752). In the pooled training cohort, the 15-gene signature (HR = 4.72, P = 7.76*10-25, sensitivity = 75%, specificity = 67.44%, AUC = 0.784) was superior to the Oncotype DX colon 7-gene signature (HR = 2.698, P = 6.3*10-8, sensitivity = 62.16%, specificity = 55.5%, AUC = 0.633). We report the identification and validation of a novel mRNA expression signature for robust prognostication and stratification of patients with stage II colorectal cancer, with superior performance in the analyzed validation cohorts when compared with clinicopathologic biomarkers and signatures currently used for stage II colorectal cancer prognostication.

The incidence of early-onset colorectal cancer (EO-CRC, in patients younger than 50) is increasing worldwide. The specific gene signatures in EO-CRC patients are largely unknown. Since EO-CRC with microsatellite instability is frequently associated with Lynch syndrome, we aimed to comprehensively characterize the tumor microenvironment (TME) and gene expression profiles of EO-CRC with microsatellite stable (MSS-EO-CRC). Here, we demonstrated that MSS-EO-CRC has a similar pattern of tumor-infiltrating immune cells, immunotherapeutic responses, consensus molecular subtypes, and prognosis as late-onset CRC with MSS (MSS-LO-CRC). 133 differential expressed genes were identified as unique gene signatures of MSS-EO-CRC. Moreover, we established a risk score, which was positively associated with PD-L1 expression and could reflect both the level of tumor-infiltrating immune cells and the prognosis of MSS-EO-CRC patients. Application of this score on the anti-PD-L1 treatment cohort demonstrated that the low-risk score group has significant therapeutic advantages and clinical benefits. In addition, candidate driver genes were identified in the different-sidedness of MSS-EO-CRC patients. Altogether, MSS-EO-CRC exhibits distinct molecular profiles that differ from MSS-LO-CRC even though they have a similar TME characterization and survival pattern. Our risk score appears to be robust enough to predict prognosis and immunotherapeutic response and therefore could help to optimize the treatment of MSS-EO-CRC.

Flow stagnation of peri-ischemic capillaries due to dynamic leukocyte stalls has been described to be a contributor to ongoing penumbral injury in transient brain ischemia, but has not been investigated in permanent experimental stroke so far. Moreover, it is discussed that obstructing neutrophils are involved in this process; however, their contribution has not yet been proven. Here, we characterize the dynamics of neutrophil granulocytes in two models of permanent stroke (photothrombosis and permanent middle cerebral artery occlusion) using intravital two-photon fluorescence microscopy. Different to previous studies on LysM-eGFP+ cells we additionally apply a transgenic mouse model with tdTomato-expressing neutrophils to avoid interference from additional immune cell subsets. We identify repetitively occurring capillary stalls of varying duration promoted by neutrophils in both models of permanent cerebral ischemia, validating the suitability of our new transgenic mouse model in determining neutrophil occlusion formation in vivo. Flow cytometric analysis of peripheral blood (PB) and brain tissue from mice subjected to photothrombosis reveal an increase in the total proportion of neutrophils, with selective upregulation of endothelial adherence markers in the PB. In conclusion, the dynamic microcirculatory stall phenomenon that is described after transient ischemia followed by reperfusion also occurs after permanent small- or large-vessel stroke and is clearly attributable to neutrophils.

In the treatment of patients with HCC awaiting liver transplantation (LT), local ablative treatments (LAT) are available either for downstaging or as bridging treatment. We present our clinical experience with both available radiation-based techniques, brachytherapy (BT), and stereotactic body radiotherapy (SBRT).

We determined whether active PI3K signaling together with nuclear accumulation of β-Catenin is necessary to fully activate canonical WNT signaling and examined the association of both signaling pathways with colon cancer progression. Using reporter gene assays we examined the activation of β-Catenin mediated transcription upon PI3K inhibition with or without β-Catenin nuclear accumulation. Ectopically induced as well as constitutively active WNT signaling strictly required PI3K activity whereas PI3K inhibition had no effect on β-Catenin subcellular localization but impaired β-Catenin binding to WNT target gene promoters and decreased WNT target gene expression. Transcriptional activity of nuclear β-Catenin depended on active PI3K signaling as nuclear accumulation of β-Catenin failed to induce WNT reporter gene transcription upon PI3K inhibition. PI3K dependend transcriptional transactivation of β-Catenin relies on events beyond phosphorylation at the AKT target site serine 552, as S552D-phosphomimetic β-Catenin mutants were unable to restore WNT signaling when inhibiting PI3K. To study the prognostic value of PI3K pathway activation (activating PIK3CA mutations or loss of PTEN expression) and nuclear β-Catenin expression, both variables were determined in 55 matched pairs of primary right sided colon cancer cases with or without distant metastasis. Activating mutations in the PIK3CA gene or loss of PTEN expression did not correlate with distant metastasis while high nuclear β-Catenin expression combined with activation of the PI3K pathway identified cases in which distant metastasis had occurred. Activation of the PI3K pathway was not associated with nuclear β-Catenin expression. We conclude that the transcriptional activity of nuclear β-Catenin depends on PI3K activity. However, PI3K on its own does not affect β-Catenin subcellular localization. Both factors synergize for full WNT signaling activity and are associated with distant metastasis in colon cancer. Thus, the detection of high nuclear β-Catenin expression and simultaneous PI3K pathway activation identifies colon cancer patients with a high risk for distant metastasis.