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.

[Malignant round cell tumors : The Ewing sarcoma and beyond]. / Maligne Rundzelltumoren : Das Ewing-Sarkom und jenseits davon.

Ewing sarcomas are highly malignant tumors that are mainly found in children and adolescents. In addition to early clinical diagnosis, correct histopathological and molecular genetic classification is the most important step. Although EWSR1-FLI1 fusion is by far the most common detectable change, there are also other representatives of the Ewing sarcoma family that cannot be distinguished histopathologically and immunohistochemically from classical Ewing sarcomas and that have different molecular genetic profiles. Although a precise molecular genetic differentiation of the various representatives of small round blue cell tumors does not yet lead to any change in the standard chemotherapy and surgical treatment applied, it does allow an estimation of the prognosis and will probably contribute in the future to an even more individualized treatment of Ewing sarcomas within the framework of personalized medicine.

[Giant cell tumor of bone : Morphology, molecular pathogenesis, and differential diagnosis]. / Riesenzelltumor des Knochens : Morphologie, molekulare Pathogenese und Differenzialdiagnose.

The histological picture of giant cell tumor of bone is characterized by numerous osteoclast-like giant cells. However, these are not the actual tumor cells, but constitute a reactive infiltrate. Rather, the tumor cells are mononuclear mesenchymal cells, which even reveal an osteoblastic line of differentiation. The CD68-positive macrophages form the second group of mononuclear cells. The receptor activator of nuclear factor kappa-B/ligand (RANK/RANKL) system, which belongs to the tumor necrosis factor (TNF) cytokine family, is decisively involved in the activation of the giant cells. It is generally accepted that a RANKL expression of mononuclear stromal cells is responsible for the development and differentiation of osteoclast-like giant cells. Therefore, the RANKL inhibitor denosumab constituted an essential element for giant cell tumor therapy over the last several years, as it blocks the maturation of osteoclasts and thus the osteolytic activity and the spread of tumor. However, with time it became evident that the not risk-free therapy with denosumab may lead to extensive recurrences upon withdrawal, so this therapy is applied with caution today.At the molecular genetic level, the giant cell tumors of bone are characterized by point mutations in the H3F3A gene. The detection of this mutation is used for the diagnostic differentiation from other bone lesions containing giant cells. Giant cell osteosarcomas rarely contain H3F3A mutations. Chondroblastoma is characterized by mutations in the H3F3B gene.

[Cartilage tumors: morphology, genetics, and current aspects of target therapy]. / Knorpeltumoren: Morphologie, Genetik und Basisaspekte der Targettherapie.

Cartilage tumors are a heterogeneous group of mesenchymal tumors whose common characteristic is the formation of a chondroblastic differentiated groundsubstance by the tumor cells. The basic features of their histological classification were already developed in the 1940s and supplemented by further entities in the following decades. Only in the past 10-15 years have fundamental new insights been gained through molecular genetic analysis. So, osteochondromas are characterized by alterations in the EXT1 and EXT2 genes. The description of mutations of isocitrate dehydrogenase 1 and 2 (IDH 1 and 2) in chondromas and chondrosarcomas is particularly important. The mesenchymal chondrosarcoma is characterized by a fusion of the HEY1-NCOA2 genes. The molecular genetic alterations characteristic for the individual tumor entities are first of all an essential supplement for the differential diagnosis of radiologically and histologically difficult cases. They also provide the basis for the establishment of molecular target therapies for malignant chondrogenic tumors. This applies in particular to conventional chondrosarcoma, for which all approaches to chemo- and radiotherapy have proven to be ineffective. However, the use of target therapies is still in its beginnings.

[Osteoid-forming bone tumors : Morphology and current translational cell biology]. / Osteoidbildende Knochentumoren : Morphologie und aktuelle translationale Zellbiologie.

Osteoid osteoma and osteoblastoma are the most important benign osteoid-forming tumors. They grow slowly and are well differentiated. Histologically, the tumor cells show no atypia and no increased mitoses. In typical cases, they can be clearly diagnosed. However, the rare cases on the dividing line between osteoblastoma and osteosarcoma are extremely problematic. In these cases, molecular genetic investigations should contribute to finding the correct diagnosis in the future.Juvenile highly malignant osteosarcoma is the most important malignant osteoid-forming tumor. About 40 years ago, neoadjuvant chemotherapy was introduced for the mostly young patients. This therapy highly significantly improved prognosis. However, a plateau phase was quickly reached and the last several decades have seen no further progress in conventional therapeutic approaches. There is no doubt that further progress can only be achieved on the basis of new molecular genetic and cell biological findings. The target-therapeutic strategies derived from these findings will be discussed in this review.The rare parosteal osteosarcoma and the even rarer periosteal osteosarcoma are mostly not highly malignant tumors that are located on the surface of bone. The parosteal osteosarcoma is usually G1 and the periosteal osteosarcoma G2. Occasionally, the differential diagnosis between a parosteal osteosarcoma and a fibrous dysplasia is difficult. In such rare cases, the detection of GNAS mutations in fibrous dysplasia can prove useful. In contrast to chondromas and chondrosarcomas, periosteal osteosarcomas do not contain IDH1 and IDH2 mutations.

Combined Gastric and Colorectal Cancer Screening-A New Strategy.

BACKGROUND: Our aim was to evaluate the feasibility of a serological assessment of gastric cancer risk in patients undergoing colonoscopy in countries with low-to-moderate incidence rates. METHODS: Serum samples were prospectively collected from 453 patients (>50 years old) undergoing colonoscopies. Of these, 279 (61.6%) also underwent gastroscopy to correlate the results for serum pepsinogen I and II (sPG-I and sPG-II), sPG-I/II ratio, and anti-H. pylori antibodies with gastric histopathology findings (graded according to the updated Sydney classification and the Operative Link of Gastritis Assessment (OLGA) and the Operative Link for Gastric Intestinal Metaplasia assessment (OLGIM) systems). RESULTS: H. pylori was found in 85 patients (30.5%). Chronic atrophic gastritis was diagnosed in 89 (31.9%) patients. High-risk OLGA (III⁻IV) stages were present in 24 patients, and high-risk OLGIM stages were present in 14 patients. There was an inverse correlation of sPG-I with the degree of atrophy and intestinal metaplasia (IM), as well as with the respective OLGA (r = -0.425; p < 0.001) and OLGIM (r = -0.303; p < 0.001) stages. A pathological sPG-I result was associated with a relative risk (RR) of 12.2 (95% confidence interval: 6.29⁻23.54; p < 0.001) for gastric preneoplastic changes. CONCLUSIONS: The assessment of serum pepsinogen allows the identification of patients at increased risk of gastric cancer. A prevention strategy of combining a screening colonoscopy with a serological screening for preneoplastic gastric changes should be considered in the general population.

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.

Oxidative stress and glyoxalase I activity mediate dicarbonyl toxicity in MCF-7 mamma carcinoma cells and a tamoxifen resistant derivative.

BACKGROUND: Acquired tamoxifen resistance is a significant problem in estrogen receptor positive breast cancer. In a cellular model, tamoxifen resistance was associated with increased sensitivity towards toxic dicarbonyls and reduced free sulfhydryl group content. We here analyzed the role of oxidative stress and glyoxalase I activity on dicarbonyl resistance and the significance of glyoxalase I expression for survival. METHODS: Reactive oxygen species were determined by 2,7-dihydrochlorofluorescein diacetate. Inhibitors for NADPH-oxidase (diphenyleneiodonium), p38 MAPK (SB203580) and ERK1/2 (UO126) were applied to investigate interactions of these signaling molecules. N-acetyl cysteine was used to evaluate the effect of oxidative stress on cell viability, which was assessed by the resazurin assay. Gene expression was analyzed by real time qRT-PCR. Glyoxalase activity was inhibited by the specific inhibitor CS-0683 and siRNA. The relevance of glyoxalase 1 mRNA abundance on survival of breast cancer patients was evaluated by the KM-plotter web interface. RESULTS: α-Oxo-aldehydes caused an immediate increase in reactive oxygen species where the tamoxifen resistant cell line (TamR) responded at lower concentrations than the MCF-7 parental cell line. Inhibitor studies placed ROS production by NADPH-oxidase downstream of p38 MAPK. The antioxidant N-acetyl cysteine (NAC) increased survival, whereas glyoxalase (GLO1) inhibition increased dicarbonyl toxicity. GLO1 mRNA abundance was correlated with unfavorable prognosis of breast cancer patients. CONCLUSIONS: Dicarbonyl toxicity was mediated by oxidative stress and GLO1 activity determines aldehyde toxicity in tamoxifen resistant cells. GENERAL SIGNIFICANCE: Glyoxalases might be predictive biomarkers for tamoxifen resistance and a putative target for the treatment of tamoxifen resistant breast cancer patients.

The prevalence of gastric heterotopia of the proximal esophagus is underestimated, but preneoplasia is rare - correlation with Barrett's esophagus.

BACKGROUND: The previously reported prevalence of gastric heterotopia in the cervical esophagus, also termed inlet patch (IP), varies substantially, ranging from 0.18 to 14%. Regarding cases with adenocarcinoma within IP, some experts recommend to routinely obtain biopsies from IP for histopathology. Another concern is the reported relation to Barrett's esophagus. The objectives of the study were to prospectively determine the prevalence of IP and of preneoplasia within IP, and to investigate the association between IP and Barrett's esophagus. METHODS: 372 consecutive patients undergoing esophagogastroduodenoscopy were carefully searched for the presence of IP. Biopsies for histopathology were targeted to the IP, columnar metaplasia of the lower esophagus, gastric corpus and antrum. Different definitions of Barrett's esophagus were tested for an association with IP. RESULTS: At least one IP was endoscopically identified in 53 patients (14.5%). Histopathology, performed in 46 patients, confirmed columnar epithelium in 87% of cases, which essentially presented corpus and/or cardia-type mucosa. Intestinal metaplasia was detected in two cases, but no neoplasia. A previously reported association of IP with Barrett's esophagus was weak, statistically significant only when short segments of cardia-type mucosa of the lower esophagus were included in the definition of Barrett's esophagus. CONCLUSIONS: The prevalence of IP seems to be underestimated, but preneoplasia within IP is rare, which does not support the recommendation to regularly obtain biopsies for histopathology. Biopsies should be targeted to any irregularities within the heterotopic mucosa. The correlation of IP with Barrett's esophagus hints to a partly common pathogenesis.

Recurrent diffuse gastric bleeding as a leading symptom of gastrointestinal AL amyloidosis.

Amyloidosis is a rare disease (incidence about 0.8/100 000) characterized by extracellular tissue deposition of fibrils composed of low molecular weight subunits of a variety of serum proteins. Clinical manifestations are largely determined by the type of precursor protein, the tissue distribution and the amount of amyloid deposition. Gastrointestinal (GI) manifestations of amyloidosis are even more uncommon (3 % of all amyloidosis patients). Symptoms of GI amyloidosis are nonspecific, heterogeneous, and include weight loss, GI bleeding, heartburn, early satiety, diarrhea and abdominal pain. The histopathological examination of biopsy specimens from the GI tract leads to the diagnosis.Herein we report the case of a 63-year-old woman with recurrent diffuse gastric bleeding. Endoscopic biopsies revealed distinct amyloid deposits in the mucosa of the stomach. Further histochemical assessment confirmed systemic light chain (AL) amyloidosis with clinically predominant gastrointestinal manifestation. An induction therapy with bortezomib and dexamethasone was initiated.Our report illustrates the importance of the multidisciplinary approach for diagnosis and management of AL amyloidosis. Current treatment of systemic AL amyloidosis is based on cytostatic targeting of immunoglobulin producing plasma cells. Therapeutic options are limited and highly toxic, making the development of novel treatment approaches an urgent need.

Ballooning osteolysis in 71 failed total ankle arthroplasties.

Background and purpose - Aseptic loosening is a major cause of failure in total ankle arthroplasty (TAA). In contrast to other total joint replacements, large periarticular cysts (ballooning osteolysis) have frequently been observed in this context. We investigated periprosthetic tissue responses in failed TAA, and performed an element analysis of retrieved tissues in failed TAA. Patients and methods - The study cohort consisted of 71 patients undergoing revision surgery for failed TAA, all with hydroxyapatite-coated implants. In addition, 5 patients undergoing primary TAA served as a control group. Radiologically, patients were classified into those with ballooning osteolysis and those without, according to defined criteria. Histomorphometric, immunohistochemical, and elemental analysis of tissues was performed. Von Kossa staining and digital microscopy was performed on all tissue samples. Results - Patients without ballooning osteolysis showed a generally higher expression of lymphocytes, and CD3+, CD11c+, CD20+, and CD68+ cells in a perivascular distribution, compared to diffuse expression. The odds of having ballooning osteolysis was 300 times higher in patients with calcium content >0.5 mg/g in periprosthetic tissue than in patients with calcium content ≤0.5 mg/g (p < 0.001). Interpretation - There have been very few studies investigating the pathomechanisms of failed TAA and the cause-effect nature of ballooning osteolysis in this context. Our data suggest that the hydroxyapatite coating of the implant may be a contributory factor.

Helicobacter pylori but not gastrin is associated with the development of colonic neoplasms.

Recent studies have suggested that Helicobacter pylori (H. pylori) constitutes a risk for the development of colonic neoplasia. Hypergastrinemia can be induced by H. pylori infection, and gastrin can act as putative promoter of colorectal carcinogenesis. Aim of our study was to assess whether H. pylori infection and/or increased serum gastrin levels are associated with the occurrence of colonic neoplasms. For this, we reviewed prospectively collected data of 377 patients with a minimum age of 50 years who underwent colonoscopy. H. pylori and CagA status were determined by serology. Serum gastrin levels were measured in fasting state by commercially available assay. In H. pylori infected patients (n = 138; 36.6%), the overall prevalence of colonic neoplasms was more frequent compared to H. pylori negative patients (n = 239; 63.4%) (OR = 2.73, 95% CI: 1.76-4.24). H. pylori infection occurred more frequently in patients with hyperplastic polyps (OR = 2.66, 95% CI: 1.23-5.74) and adenomas presenting with low grade intraepithelial neoplasia (IEN) (OR = 1.85, 95% CI: 1.14-2.99). Attributable risk for adenomas with high grade IEN and colorectal adenocarcinoma (n = 14) was not assessed due to the low number of cases. The expression of CagA was also associated with an increased risk for colonic neoplasms (OR = 2.25, 95% CI: 1.29-3.94). Hypergastrinemia did not increase the risk for any colonic neoplasms and there was no difference in basal serum gastrin levels between H. pylori positive and negative patients. In conclusion, H. pylori infection, including CagA expression is associated with an increased risk for the development of colonic neoplasm.

Repeated H2 O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis.

The production of hydrogen peroxide (H2 O2 ) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H2 O2 activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c-Jun N-terminal Kinases (JNK) that induces p21(WAF1) . Moreover, caspases circumvented the G1/S and intra-S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H2 O2 exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H2 O2 -exposed HCEC cells (C-cell cultures) was associated with (i) increased phospho-p46 JNK, (ii) decreased total JNK and phospho-p54 JNK and (iii) p21(WAF1) down-regulation. Altered JNK activation and p21(WAF1) down-regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H2 O2 exposure. This may cause increased proliferation of C-cell cultures, a defining initiating feature in the inflammation-carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non-apoptotic function of caspases, causing checkpoint adaptation in C-cell cultures. Additionally, loss of cell-contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell-contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H2 O2 -induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21(WAF1) , c-Fos, c-Jun/phospho-c-Jun, ATF2/phospho-ATF2, ß-catenin/TCF4-signalling, c-Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.

Non-apoptotic function of caspases in a cellular model of hydrogen peroxide-associated colitis.

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)-associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non-tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H2 O2 ). A population of HCEC survived H2 O2 -induced oxidative stress via JNK-dependent cell cycle arrests. Caspases, p21(WAF1) and γ-H2AX were identified as JNK-regulated proteins. Up-regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan-caspase inhibitor Z-VAD-FMK caused up-regulation of γ-H2AX, a DNA-damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G1 -phase as first response to oxidative stress and increased S-phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non-apoptotic function by promoting cells through G1 - and S-phase by overriding the G1 /S- and intra-S checkpoints despite DNA-damage. This led to the accumulation of cells in the G2 /M-phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ-H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase-dependent proteolytic degradation of the DNA-damage checkpoint protein ATM that is upstream of γ-H2AX. As a consequence, undetected DNA-damage and increased proliferation were found in repeatedly H2 O2 -exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non-apoptotic function of caspases. Overexpression of upstream p-JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.

ATF2 knockdown reinforces oxidative stress-induced apoptosis in TE7 cancer cells.

Cancer cells showing low apoptotic effects following oxidative stress-induced DNA damage are mainly affected by growth arrest. Thus, recent studies focus on improving anti-cancer therapies by increasing apoptosis sensitivity. We aimed at identifying a universal molecule as potential target to enhance oxidative stress-based anti-cancer therapy through a switch from cell cycle arrest to apoptosis. A cDNA microarray was performed with hydrogen peroxide-treated oesophageal squamous epithelial cancer cells TE7. This cell line showed checkpoint activation via p21(WAF1) , but low apoptotic response following DNA damage. The potential target molecule was chosen depended on the following demands: it should regulate DNA damage response, cell cycle and apoptosis. As the transcription factor ATF2 is implicated in all these processes, we focused on this protein. We investigated checkpoint activation via ATF2. Indeed, ATF2 knockdown revealed ATF2-triggered p21(WAF1) protein expression, suggesting p21(WAF1) transactivation through ATF2. Using chromatin immunoprecipitation (ChIP), we identified a hitherto unknown ATF2-binding sequence in the p21(WAF1) promoter. p-ATF2 was found to interact with p-c-Jun, creating the AP-1 complex. Moreover, ATF2 knockdown led to c-Jun downregulation. This suggests ATF2-driven induction of c-Jun expression, thereby enhancing ATF2 transcriptional activity via c-Jun-ATF2 heterodimerization. Notably, downregulation of ATF2 caused a switch from cell cycle arrest to reinforced apoptosis, presumably via p21(WAF1) downregulation, confirming the importance of ATF2 in the establishment of cell cycle arrest. 1-Chloro-2,4-dinitrobenzene also led to ATF2-dependent G2/M arrest, suggesting that this is a general feature induced by oxidative stress. As ATF2 knockdown also increased apoptosis, we propose ATF2 as a target for combined oxidative stress-based anti-cancer therapies.

GPER-1 expression decreases during breast cancer tumorigenesis.

GPER-1 protein expression was immunohistochemically examined in 164 primary breast cancer specimens and their matched normal breast epithelium. GPER-1 down-regulation correlated significantly with increased histological grading (p = .015), lymph node metastases (p = .032), and negative estrogen receptor status (p = .018). The decrease of GPER-1 expression in breast cancer tissue, relative to normal tissue, was associated with poor overall survival (p = .043) and disease-free survival (p = .037) and remained a significant unfavorable factor in multivariate analysis for DFS (HR = 1.569; 95% CI, 1.024-2.797; p = .041) and OS (HR = 2.082; 95% CI, 1.248-4.773; p = .039). Thus aberrant GPER-1 expression seems to be an important factor in breast cancer progression.

Comparative analysis of miRNA expression in dedifferentiated and well-differentiated components of dedifferentiated chondrosarcoma.

Dedifferentiated chondrosarcoma (DDCS) is a rare malignant cartilage tumor arising out of a low-grade chondrosarcoma, whereby the well-differentiated and the dedifferentiated components coexist in the same localization. DDCS has a massively increased metastatic potential in comparison to low-grade chondrosarcoma. So far, the underlying mechanisms of DDCS development and the increased malignancy are widely unknown. Targeted DNA sequencing revealed no genetic differences between both tissue components. Besides genetic events, alterations in epigenetic control may play a role in DDCS development. In this preliminary study, we have analyzed the differential miRNA expression in paired samples of both components of four primary DDCS cases and a rare lung metastasis with both components using the nCounter MAX analysis system from NanoString technologies. We identified 21 upregulated and two downregulated miRNAs in the dedifferentiated components of the primary cases. Moreover, three miRNAs were also significantly deregulated in the dedifferentiated component of the lung metastasis, supporting their possible role in DDCS development. Additionally, validated targets of the 23 deregulated miRNAs are involved in signaling pathways, like PI3K/Akt, Wnt/ß-catenin, and TGF-ß, as well as in cellular processes, like cell cycle regulation, apoptosis, and dedifferentiation. Further investigations are necessary to confirm and understand the role of the identified miRNAs in DDCS development.

Gastro-oesophageal reflux disease is associated with up-regulation of desmosomal components in oesophageal mucosa.

AIMS: Gastro-oesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. This study was aimed at investigating the role of desmosomal proteins in relation to GERD. METHODS AND RESULTS: Ninety-five patients with GERD-related symptoms (erosive, n = 51; non-erosive, n = 44) and 27 patients lacking those symptoms were included. Endoscopic and histological characterization of oesophagitis was performed according to the Los Angeles and Ismeil-Beigi criteria, respectively. Multiple biopsies were taken from the oesophageal mucosa of each patient. Gene expression analysis of plakoglobin, desmoglein-1, desmoglein-2 and desmoglein-3 was performed by quantitative real time (RT)-polymerase chain reaction and immunohistochemistry in the oesophageal mucosa. Routine histology revealed specific GERD-related alterations, such as dilatation of intercellular spaces (DIS), basal cell hyperplasia (BCH), and elongation of the papillae, in the oesophageal mucosa of patients with GERD, as compared with controls (all parameters: P < 0.05). All four genes and corresponding proteins were found to be up-regulated by between 1.7 and 8.1-fold (transcript level, P < 0.05; protein level, P < 0.05). Induced gene expression levels of plakoglobin, desmoglein-1 and desmoglein-2 correlated significantly with DIS and BCH. CONCLUSIONS: Taken together, the uniform up-regulation of desmosomal genes/proteins in the oesophageal mucosa of patients with GERD supports the concept of architectural and molecular changes in the desmosomal compartment in the pathogenesis of GERD.

Role of tight junction proteins in gastroesophageal reflux disease.

BACKGROUND: Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function that is regulated by cell-cell contacts. The aim of the study was to investigate the expression pattern of selected components involved in the formation of tight junctions in relation to GERD. METHODS: Eighty-four patients with GERD-related symptoms with endoscopic signs (erosive: n = 47) or without them (non-erosive: n = 37) as well as 26 patients lacking GERD-specific symptoms as controls were included. Endoscopic and histological characterization of esophagitis was performed according to the Los Angeles and adapted Ismeil-Beigi criteria, respectively. Mucosal biopsies from distal esophagus were taken for analysis by histopathology, immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (RT-PCR) of five genes encoding tight junction components [Occludin, Claudin-1, -2, Zona occludens (ZO-1, -2)]. RESULTS: Histopathology confirmed GERD-specific alterations as dilated intercellular spaces in the esophageal mucosa of patients with GERD compared to controls (P < 0.05). Claudin-1 and -2 were 2- to 6-fold upregulation on transcript (P < 0.01) and in part on protein level (P < 0.015) in GERD, while subgroup analysis of revealed this upregulation for ERD only. In both erosive and non-erosive reflux disease, expression levels of Occludin and ZO-1,-2 were not significantly affected. Notably, the induced expression of both claudins did not correlate with histopathological parameters (basal cell hyperplasia, dilated intercellular spaces) in patients with GERD. CONCLUSIONS: Taken together, the missing correlation between the expression of tight junction-related components and histomorphological GERD-specific alterations does not support a major role of the five proteins studied in the pathogenesis of GERD.

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.