[Ligandrol-induced liver injury - Case Report]. / Ligandrol-induzierte toxische Hepatopathie ­ ein Fallbericht.

A 37-year-old man presented with painless jaundice and pruritus. Total Bilirubin was 30-fold the upper limit of normal (ULN), while ALT and further cholestasis parameters were found to be only slightly elevated. As comprehensive diagnostics showed no abnormal findings and ruled out frequent causes of elevated cholestasis markers, we performed a liver biopsy. The biopsy revealed canalicular cholestasis with ductopenia and periportal fibrosis. Only after a further and intensive anamnesis a ligandrol-abuse could be determined as cause for the symptoms. Ligandrol is misused as a selective androgen receptor modulator to promote muscle building. This case represents a typical case of abuse of anabolic substances in amateur sports.

Genetics and epigenetics in conventional chondrosarcoma with focus on non-coding RNAs.

The detection of mutations of isocitrate dehydrogenase 1 and 2 (IDH 1/2) as tumor driver genes in chondromas and chondrosarcomas more than ten years ago was a first major step for better understanding the molecular carcinogenenesis of these rare mesenchymal tumors. Within the TCA cycle, wild-typ IDH1/2 catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH mutations catalyze the production of a non-physiological metabolite, D-2hydroxyglutarate (D-2HG) from α-KG. D-2HG can inhibit the class of α-KG-dependent enzymes by binding competitively to its receptor. Important enzyme families, such as the Ten-Eleven Translocation (TET) family of 5-methylcytosine hydroxylases and the Jumonji family of histone lysine demethylases are α-KG dependent. Many of the TET and Jumonji family-dependent enzymes regulate epigenetic factors, such as DNA methylation, histone modification, and nucleosome remodeling, underscoring the central role of the epigenome in cancer development. When D-2HG acts with these enzymes instead α-KG their functions will be in disarray with heavily hypermethylated DNA and dysregulations in histone metylation. NcRNAs have increasingly been described as a cornerstone of cancer development. Therefore this review describes exemplarily the oncogenic functions of miRNAs in chondrosarcoma in more detail. Particularly in chondrosarcomas additional carcinogenic features are aquired by interactions of ncRNAs with α-KG-dependent epigenetic regulators. Distinct ncRNAs, miRNAs and lncRNAs alike, are involved in deregulating important cellular signalling pathways and thus contributing further to malignant transformation and development of malignant cellular traits in these rare mesenchymal tumors. This review specially empasizes the complex interactions between the world of ncRNAs and genetics and epigenetics.

Differential Expression of Long Noncoding RNA HOTAIR in Intestinal Metaplasia and Gastric Cancer.

INTRODUCTION: High expression of HOTAIR promotes tumor growth and carries a dismal prognosis for the patient. We investigated the prognostic value of HOTAIR expression in gastric cancer (GC) and systematically delineate the expression in relation to Helicobacter pylori infection and preneoplastic changes. METHODS: HOTAIR expression was analyzed in surgical paired tissue samples of patients with GC and biopsy samples from patients with atrophic gastritis and/or intestinal metaplasia (AG ± -IM), chronic nonatrophic gastritis, and controls. The cancer genome atlas (TCGA) data were used for validation. HOTAIR expression was evaluated in sera and ascites of patients with GC. Quantitative HOTAIR expression analysis was performed using quantitative polymerase chain reaction, and LINE-1 methylation was assessed by bisulfite pyrosequencing. RESULTS: HOTAIR was more frequently detected in tumor tissues compared with adjacent gastric mucosa (65.4% vs 8.6%). HOTAIR expression was associated with depth of tumor invasion and tumor location and with shorter overall survival in patients with diffuse-type GC as confirmed in the TCGA cohort. HOTAIR was not detectable in controls but was found in 2.2% of patients with chronic nonatrophic gastritis and 18.3% of patients with AG ± IM, which was further associated with IM, grade of IM, and H. pylori positivity. DISCUSSION: HOTAIR expression was associated with GC and preneoplastic changes of stomach mucosa. Although HOTAIR expression was strongly linked to IM, HOTAIR expression was only associated with worse prognosis in Lauren diffuse and not intestinal type of GC. Further studies are needed to evaluate the value of HOTAIR as diagnostic and predictive biomarker in IM and translational therapeutic relevance of HOTAIR in diffuse-type GC.

Diagnostic value of syndecan-4 protein expression in colorectal cancer.

The prognosis of patients with colorectal cancer (CRC) is highly dependent on the disease stage at diagnosis. Therefore, it is crucial to study molecules involved in the progression of colorectal cancer tumorigenesis and to shed light on their potential use as targetable proteins in diagnostics and therapy. As syndecan-4 (SDC4) is a transmembrane proteoglycan with important functions in cell adhesion, migration, cytoskeleton organization, and gene expression through the binding of extracellular matrix molecules, it might play a role in local tumor cell invasion. To clarify its impact on the progression of CRC, we analyzed 177 patients for SDC4 expression in colon carcinoma tissue, lymph node and liver metastasis under consideration of specific morphological features and cellular elements of CRC. Highly upregulated SDC4 was particularly expressed at the tumor invasion front. Expression was strongest in tumor cell buds appearing as membranous expression polarized to peritumoral stromal cells. Increased SDC4 expression directed to the tumor-stromal- or tumor-endothelial-interface was also confirmed for metastasis and angioinvasive tumor cell clusters. Furthermore, strong immunoreactivity of SDC4 in fibroblasts and macrophages being in contact with invasive tumor cells suggests a cooperation between the different types of cells in tumor progression at the cell-matrix interface and a role for SDC4 in tumor cells attached to the extracellular matrix. Overexpression of SDC4 in tumor cells at the invasion front was significantly associated with progressive pathological features and inversely related to disease-free and overall survival. Therefore, overexpression of SDC4 may be a predictor for poor prognosis in patients with CRC and might prove useful in clinical practice, thus identifying patients with potential disease progression. Further investigations will have to reveal the functional role of SDC4 in tumor cell buds, fibroblasts and macrophages at the tumor stromal interface to confirm that SDC4 might also be a possible therapeutic target for the treatment of patients with advanced CRC.

Translational cell biology of highly malignant osteosarcoma.

Highly malignant osteosarcoma (HMO) is the most frequent malignant bone tumor preferentially occurring in adolescents and children with a second more flat peak in patients over the age of 60. The younger patients benefit from combined neoadjuvant chemotherapy with 65-70% 5-year survival rate. In patients with metastatic HMO the 5-year survival rate is consistently poor with approximately 30%. In the last several years strategies for target therapies have been developed by using next generation sequencing (NGS) for defining targetable molecular factors. However, it has so far been challenging to establish an effective target therapy for so-called 'orphan tumors' without recognizable driver mutations, including HMO. The molecular genetic studies using NGS have shown that HMOs are genomically unstable tumors with highly complex chaotic karyotypes. Before the background of this genetic complexity more investigations should be performed in the future for defining targetable biological factors. As the prognosis could not be improved for 40 years one may expect improvements for patients only by gaining a deeper understanding of the cell and molecular biology of HMO. The cell of origin of HMO is being clarified now. The majority of studies indicate that an osteoblastic progenitor cell is probably the cell of origin of HMO and not an undifferentiated mesenchymal stem cell. This means that the established histopathological definition of HMO through verification of osteoid production by the osteoblastic cells is well justified and will probably be the cornerstone for a precise differential diagnosis of HMO also in the years to come.

Inactivation of JNK2 as carcinogenic factor in colitis-associated and sporadic colorectal carcinogenesis.

We recently reported that dysregulated c-Jun N-terminal kinases (JNK) activity causes defective cell cycle checkpoint control, inducing neoplastic transformation in a cellular ulcerative colitis (UC) model. In the quiescent chronic phase of UC, p-p54 JNK was down-regulated and p-p46 JNK was up-regulated. Both were up-regulated in the acute phase. Consequently, increased p21WAF1 and γ-H2AX, two JNK-regulated proteins, induced cell cycle arrest. Their down-regulation led to checkpoint override, causing increased proliferation and undetected DNA damage in quiescent chronic phase, all characteristics of tumorigenesis. We investigated expression of p-JNK2, p-JNK1-3, p21WAF1, γ-H2AX and Ki67 by immunohistochemistry in cases of quiescent UC (QUC), active UC (AUC), UC-dysplasia and UC-related colorectal carcinoma (UC-CRC). Comparison was made to normal healthy colorectal mucosa, sporadic adenoma and colorectal carcinoma (CRC), diverticulitis and Crohns disease (CD). We found p-JNK2 up-regulation in AUC and its early down-regulation in UC-CRC and CRC carcinogenesis. With down-regulated p-JNK2, p21WAF1 was also decreased. Ki67 was inversely expressed, showing increased proliferation early in UC-CRC and CRC carcinogenesis. p-JNK1-3 was increased in AUC and QUC. Less increased γ-H2AX in UC-CRC compared to CRC gave evidence that colitis-triggered inflammation masks DNA damage, thus contributing to neoplastic transformation. We hypothesize that JNK-dependent cell cycle arrest is important in AUC, while chronic inflammation causes dysregulated JNK activity in quiescent phase that may contribute to checkpoint override, promoting UC carcinogenesis. We suggest restoring p-JNK2 expression as a novel therapeutic strategy to early prevent the development of UC-related cancer.

Expression of aurora kinase A correlates with the Wnt-modulator RACGAP1 in gastric cancer.

Canonical Wnt signaling is involved in gastric carcinogenesis. The aim of this study was to identify the link between Wnt signaling and aurora kinase A (AURKA), a target for the treatment of gastrointestinal cancers. Publicly available microarray data were used to identify phenotype-specific protein-protein interaction (PPI) subnetworks. The in silico analysis revealed a gastric cancer-specific PPI subnetwork consisting of 2745 proteins and 50,935 interactions. We focused on the link of AURKA to a Wnt-specific interaction module consisting of 92 proteins. There was a direct association of AURKA with Rac GTPase-activating protein 1 (RACGAP1), as well as with CTNBB1 (ß-catenin) and CDKN1A as second-order interactors. Differential expression analysis revealed a significant downregulation of both AURKA and RACGAP1 in gastric cancer compared to noncancer controls. Biopsies from a prospective cohort of 56 patients with gastric cancer (32 intestinal type, 24 diffuse type) and 20 noncancer controls were used for validation of the identified targets. The RT-PCR data confirmed a strong correlation of AURKA and RACGAP1 gene expression both in the tumor, the tumor-adjacent and the tumor-distant mucosa. RACGAP1 in the tumor was also associated with CTNBB1 expression, and inversely associated with CDKN1A gene expression. Immunohistochemistry confirmed expression of the RACGAP1 protein in gastric cancer and the tumor-adjacent mucosa. RACGAP1 expression was not associated with tumor stage, grading, Lauren type, Helicobacter pylori infection, or age. In conclusion, AURKA is directly associated with the expression of RACGAP1, a modulator of the canonical Wnt signaling pathway.

Oxyntic gastric atrophy in Helicobacter pylori gastritis is distinct from autoimmune gastritis.

AIM: To assess characteristics of oxyntic gastric atrophy (OGA) in autoimmune gastritis (AIG) compared with OGA as a consequence of Helicobacter pylori infection. METHODS: Patients undergoing oesophagogastroduodenoscopy from July 2011 to October 2014 were prospectively included (N=452). Gastric biopsies were obtained for histology and H. pylori testing. Serum gastrin-17 (G17), pepsinogen (PG) I, PGII and antibodies against H. pylori and cytotoxin-associated gene A protein were determined in all patients. Antibodies against parietal cells and intrinsic factor were determined in patients with advanced (moderate to severe) OGA. Areas under the receiver operating characteristic curves (AUCs) were calculated for serum biomarkers and compared with histology. RESULTS: Overall, 34 patients (8.9%) had advanced OGA by histology (22 women, age 61±15 years). Current or past H. pylori infection and AIG were present in 14/34 and 22/34 patients, respectively. H. pylori-negative AIG patients (N=18) were more likely to have another autoimmune disease (OR 6.3; 95% CI 1.3 to 29.8), severe corpus atrophy (OR 10.1; 95% CI 1.9 to 54.1) and corpus intestinal metaplasia (OR 26.9; 95% CI 5.3 to 136.5) compared with H. pylori-positive patients with advanced OGA. Antrum atrophy was present in 39% of H. pylori-negative AIG patients. The diagnostic performance of G17, PG I and PGI/II was excellent for AIG patients (AUC=0.83, 0.95 and 0.97, respectively), but limited for H. pylori-positive patients with advanced OGA (AUC=0.62, 0.75 and 0.67, respectively). CONCLUSIONS: H. pylori-negative AIG has a distinct clinical, morphological and serological phenotype compared with advanced OGA in H. pylori gastritis.

MMP2 and MMP7 at the invasive front of gastric cancer are not associated with mTOR expression.

BACKGROUND: Regulation of MMP expression by activation of mTOR signalling has been demonstrated for several tumor types, but has thus far not been confirmed in gastric cancer. FINDINGS: The study compromised 128 patients who underwent gastric resection for cancer (66.4 % male; 86 intestinal, 42 diffuse type). Immunohistochemical staining of MMPs was performed to analyse the topographical pattern of MMP expression at the tumor center and the invasive front, respectively. MMP2 showed higher expression at the invasive front compared to the tumor center, whereas MMP7 staining scores were higher in the tumor center, and there was no difference for MMP9. The expression of p-mTOR was higher in the tumor center than at the invasive front, with a similar trend for mTOR. For intestinal type gastric cancer there was a weak correlation of MMP9 with expression of mTOR in the tumor center. Otherwise, there was no correlation of the MMPs with mTOR. By treatment of MKN45 gastric cancer cells with rapamycin, a reduction of p-mTOR in the Western blot was achieved; however, expression of MMPs remained unaffected. CONCLUSIONS: Expression of MMP2 and MMP7 in gastric cancer is not associated with mTOR, MMP9 expression might be related to mTOR signalling in a subset of tumors.

Possible role of TGF ß1 in inflammatory pseudotumor associated with multiple neuroendocrine tumors of the small intestine.

INTRODUCTION: Inflammatory myofibroblastic tumors (IMTs), a rare condition of unknown etiology, have often been reported to be associated with specific infections or malignant tumors. The question of whether IMT themselves are an inflammatory or a neoplastic process is still going on. CASE REPORT: A 57-year-old female patient was transferred to our hospital with ileus caused by a mesenterial tumor. Intraoperatively, the mesenteric mass and the dependent small intestine segment, as well as a suspect hepatic lesion, were resected. The histopathological investigation revealed 8 malignant neuroendocrine tumors (NET) of the small intestine with lymphatic and hepatic metastasis and a mesenteric IMT. The postoperative course was uneventful, and the patient was discharged on the 18th postoperative day. The last follow-up after 30 months showed no recurrence of the IMT but clinical and radiological evidence of a persistent hepatic metastasis of the NETs. While plasma Chromogranin A remained suppressed by Sandostatin, the TGF ß1 level was markedly elevated. DISCUSSION: Based on the current literature and our previous experiences, we can state that IPT are an aberrant secondary immunological process possibly induced by excessive TGF ß1 and not a neoplasia. Nevertheless, the tumorous behavior points to a continuity between inflammation and neoplasia. Differential diagnoses and the potential molecular pathogenesis are further discussed.

Dysregulation of apoptotic signaling pathways by interaction of RPLP0 and cathepsin X/Z in gastric cancer.

Cathepsin X (CTSX, also called cathepsin Z/P) is a cysteine protease that still plays an unknown role in human cancer. It has been shown to bind cell surface heparin sulphate proteoglycans and integrins, indicating possible functions of CTSX in cellular adhesion, phagocytosis, and immune response. Our previous studies have shown an association between Helicobacter pylori (H. pylori) infection, a strong up-regulation of CTSX, and development of gastric cancer. In this study, yeast two-hybrid analysis revealed that RPLP0, a ribosomal protein P0, interacts with the human CTSX protein in gastric cancer. The CTSX/RPLP0 interaction was confirmed by co-immunoprecipitation assays. In addition, co-localization studies in cancer cell line N87 and gastric cancer tissue samples were performed. Laserscan microscopy revealed a shuttling of RPLP0 (and CTSX) from cytoplasm to the nucleus after CTSX knockdown. Down-regulation of RPLP0 resulted in G1 arrest of gastric cancer cells, whereas knockdown of CTSX led to G1 arrest and apoptosis after 48 h. Knockdown of both proteins caused increased apoptosis. RPLP0 deficiency could suppress cell growth and cell cycle progression by down-regulating CDK2. It was further demonstrated that RPLP0 affected p21 expression, but did not change the expression of Cyclin E. Down-regulation of both proteins at least through CDK2 suggests an anti-apoptotic effect on gastric cancer cells and opens up new possibilities for apoptotic immune modulation and gastric cancer therapy.

Esophageal intraluminal baseline impedance differentiates gastroesophageal reflux disease from functional heartburn.

BACKGROUND & AIMS: Mucosal integrity can be assessed in patients with gastroesophageal reflux disease (GERD) by measuring intraluminal baseline impedance (BI). However, it is not clear whether BI is abnormal in patients with functional heartburn (FH), or can be used to distinguish them from patients with GERD. We compared differences in BI between patients with FH vs GERD. METHODS: We performed a prospective study of 52 patients (16 men; mean age, 55 y; range, 23-78 y) seen at a tertiary university hospital from February 2009 through December 2012. Thirty-five patients had GERD (19 had nonerosive reflux disease [NERD], 16 had erosive reflux disease [ERD]) and 17 had FH. All patients discontinued proton pump inhibitor therapy and then underwent esophagogastroduodenoscopy and multichannel intraluminal impedance and pH monitoring. BI was assessed at 3, 5, 7, 9, 15, and 17 cm proximal to the lower esophageal sphincter in recumbent patients. Biopsy specimens were taken from 3 cm above the gastroesophageal junction; histology analysis was performed to identify and semiquantitatively score (scale, 0-3) dilated intercellular spaces. RESULTS: Baseline impedance in the distal esophagus was significantly lower in patients with NERD or erosive reflux disease (ERD) than FH (P = .0006). At a cut-off value of less than 2100 Ω, BI measurements identified patients with GERD with 78% sensitivity and 71% specificity, with positive and negative predictive values of 75%. Also in the proximal esophagus, reduced levels of BI levels were found only in patients with ERD. There were negative correlations between level of BI and acid exposure time (r = -0.45; P = .0008), number of acidic reflux episodes (r = -0.45; P = .001), and proximal extent (r = -0.40; P = .004). Biopsy specimens from patients with NERD or ERD had significant increases in dilation of intercellular spaces, compared with those from patients with FH; there was an inverse association between dilated intercellular spaces and BI in the distal esophagus (r = -0.28; P = .06). CONCLUSIONS: Measurement of BI in the lower esophagus can differentiate patients with ERD or NERD from patients with FH (78% sensitivity and 71% specificity), and therefore should be considered as a diagnostic tool for patients with proton pump inhibitor-refractory reflux. Low levels of BI are associated with increased exposure to acid and dilation of intercellular spaces, indicating that BI is a marker of mucosal integrity.

Characterization of cathepsin X in colorectal cancer development and progression.

The lysosomal cysteine carboxypeptidase cathepsin X (CTSX), localized predominantly in immune cells, has been associated with the development and progression of cancer. To determine its specific role in colorectal carcinoma (CRC), we analyzed CTSX expression in non-malignant mucosa and carcinoma of 177 patients as well as in 111 adenomas and related it with clinicopathological parameters. Further, the role of CTSX in the adhesion and invasion of the colon carcinoma cell lines HT-29 and HCT116 was investigated in an in vitro culture cell system with fibroblasts and monocytes, reflecting the situation at the tumor invasion front. Epithelial CTSX expression significantly increased from normal mucosa to adenoma and carcinoma, with highest expression levels in high grade intraepithelial neoplasia and in early tumor stages. Loss of CTSX occurred with tumor progression, and correlated with advanced local invasion, lymph node and distal metastasis, lymphatic vessel and vein invasion, tumor cell budding and poorer overall survival of patients with CRC. The subcellular distribution of CTSX changed from vesicular paranuclear expression in the tumor center to submembranous expression in cells of the invasion front. Peritumoral macrophages showed highest expression of CTSX. In vitro assays identified CTSX as relevant factor for cell-cell adhesion and tumor cell anchorage to fibroblasts and basal membrane components, whereas inhibition of CTSX caused increased invasiveness of colon carcinoma cells in mono- and co-culture. In conclusion, CTSX is involved in early tumorigenesis and in the stabilization of tumor cell formation in CRC. The results suggest that loss of CTSX may be needed for tumor cell detachment, local invasion and tumor progression. In addition, CTSX in tumor-associated macrophages indicates a role for CTSX in the anti-tumor immune response.