In Situ Analysis of mTORC1/C2 and Metabolism-Related Proteins in Pediatric Osteosarcoma.

Activation of the mTOR pathway has been observed in osteosarcoma, however the inhibition of mammalian target of rapamycin (mTOR) complex 1 has had limited results in osteosarcoma treatment. Certain metabolic pathways can be altered by mTOR activation, which can affect survival. Our aim was to characterize the mTOR profile and certain metabolic alterations in pediatric osteosarcoma to determine the interactions between the mTOR pathway and metabolic pathways. We performed immunohistochemistry on 28 samples to analyze the expression of mTOR complexes such as phospho-mTOR (pmTOR), phosphorylated ribosomal S6 (pS6), and rapamycin-insensitive companion of mTOR (rictor). To characterize metabolic pathway markers, we investigated the expression of phosphofructokinase (PFK), lactate dehydrogenase-A (LDHA), ß-F1-ATPase (ATPB), glucose-6-phosphate dehydrogenase (G6PDH), glutaminase (GLS), fatty acid synthetase (FASN), and carnitin-O-palmitoyltransferase-1 (CPT1A). In total, 61% of the cases showed low mTOR activity, but higher pmTOR expression was associated with poor histological response to chemotherapy and osteoblastic subtype. Rictor expression was higher in metastatic disease and older age at the time of diagnosis. Our findings suggest the importance of the Warburg-effect, pentose-phosphate pathway, glutamine demand, and fatty-acid beta oxidation in osteosarcoma cells. mTOR activation is linked to several metabolic pathways. We suggest performing a detailed investigation of the mTOR profile before considering mTORC1 inhibitor therapy. Our findings highlight that targeting certain metabolic pathways could be an alternative therapeutic approach.

Adhesion stabilization in the early postoperative period in a rat model / Az adhézió stabilizálódásának meghatározása a korai posztoperatív idoszakban patkány modellen

Introduction: Adhesion formation is a complex series of events that results from cellular and molecular processes where, in contrast to the normal case, events that support adhesion genesis dominate over adhesion lysis. Tissue injury, haemorrhage, tissue desiccation and inflammatory processes, among others, play a role in its induction. Since the presence of adhesions can be associated with a number of negative complications, the primary aim is to prevent their development. There are several preventive targets for the process, but in many cases therapy is only provided immediately after the procedure. In this study, we present an experimental rat model of adhesion, where the aim is to understand the stabilization period of adhesion. All animals underwent the same surgical procedure, inducing tissue injury, minor haemorrhage and tissue desiccation, differing only in the timing of reoperations and sampling. On postoperative days 1­7, we assessed macroscopically and histopathologically the type of adhesions formed, the adhesive tissue, the stability of the adhesion. We found that stabilization was a process lasting several days, with unstable and moderately stable adhesions predominating by postoperative day 4. Knowing this allows to broaden the therapeutic window, targeting the most appropriate period in the early postoperative period, possibly combining treatments, to make adhesion prevention even more effective.

[Survival of pediatric patients with Ewing sarcoma treated at Semmelweis University]. / A Semmelweis Egyetemen kezelt Ewing-szarkómás gyermekek kezelési eredményeinek vizsgálata.

The survival of children treated with Ewing sarcoma at Semmelweis University were investigated. Pediatric patients with Ewing sarcoma treated at Semmelweis University from 2001 through 2013 were analyzed in terms of overall survival and clinical factors (age, primary localization and extent of the tumor, time interval from primary complaints to diagnosis). For statistical analysis Kaplan-Meier estimated survival and log rank test were applied. Mean age and follow-up time of the 78 patients were 11.16 and 6.29 years, respectively. In 57% of patients time interval from primary symptoms to diagnosis was less than half year. In 53.8% of the patients the disease was metastatic at primary diagnosis (pulmonary only: 29.5%, any other: 24.3%). 5- and 10-year overall survival of patients were 68.1% and 60.4%, respectively. Among the analyzed factors, the presence of metastasis impaired 5-year overall survival significantly (88.5% for localized disease, 63.5% for pulmonary only and 40.9% for any other metastasis). The survival rate of pediatric patients with Ewing sarcoma treated at Semmelweis University is similar to the result in Western European countries.

Comparison of Predictive Immunohistochemical Marker Expression of Primary Breast Cancer and Paired Distant Metastasis using Surgical Material: A Practice-Based Study.

Parallel studies of primary breast carcinomas and corresponding distant metastases samples reveal considerable differences. Our aim was to highlight this issue from another perspective and provide further data based on 98 patient samples: 69 primary breast carcinoma and 85 distant metastases from bone, central nervous system (CNS) and lung (56 paired). Two independent series of immunohistochemical reactions with different antibodies for estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (Her2), along with HER2 fluroscence in situ hybridization (FISH) were performed on tissue microarrays to classify breast carcinoma and distant metastases samples into Luminal A, Luminal B-proliferating, Luminal B-HER2+, HER2+ and triple negative (TNBC) surrogate breast cancer groups. Correlation and agreement between the two assessments of ER and PgR were fair-to-moderate, and almost perfect for HER2 and Ki67. There was 40% discordance concerning immunophenotype between breast carcinomas and distant metastases. Most common metastatic site of ER+ breast carcinoma was the skeletal system (59.2%), whereas that of TNBCs was the CNS (58.8%) and lungs (23.5%). Distant metastases in bones were mostly luminal (54.3%), in the CNS, Luminal B (53.2%), and in the lung, TNBC (37.5%). The change of drugable properties of primary breast cancers in the respective bone and CNS metastases suggests that characterization of the metastasis is necessary for appropriate treatment planning.

Expression of proliferation markers Ki67, cyclin A, geminin and aurora-kinase A in primary breast carcinomas and corresponding distant metastases.

AIMS: To assess the expression of the following cell cycle regulatory proteins in primary metastatic breast carcinomas (MBCs) and on availability in matched distant metastases (DMs): Ki67, cyclin A, geminin and aurora-kinase A (aurkA); and to compare the expression of these markers in early MBC (EMBC) and late MBC separated into groups according to median time point on metastatic event occurred (28 months). METHODS: The expression of the above mentioned markers was analysed in a total of 47 primary MBCs and 59 DMs (out of which 37 were pairs) by immunohistochemistry. Fourteen breast carcinomas with no relapse over a 10-year follow-up period were utilised as control cases (CBC). RESULTS: Among the MBCs, 22 metastasised to the bone, 4 to the lung and 21 to the central nervous system (CNS). Geminin (p<0.001) and Ki67 (p=0.001) were increased in the MBCs while aurkA and cyclin A showed no difference when compared with CBCs. There were no differences between aurkA, cyclin A and geminin expression in MBCs and DMs in general. Expression of Ki67 was, however, elevated (p=0.027) in DMs. In CNS metastases all markers showed elevated expression as compared to MBCs. In bone metastases, geminin was lower (p<0.001) compared with primary MBCs. In the metastases of the lung, the evaluated markers did not show different expression. According to the median follow-up until the metastatic event, Ki67 was found to be significantly elevated in EMBC (p=0.018). CONCLUSIONS: Ki67 index and geminin distinguish a fraction of MBC with worse prognosis, showing increased levels in the latter in comparison to CBC being tumour-free over a 10-year follow-up period. Ki67 could possibly identify a group of MBCs that develop early DMs.

Multiple splice variants of EWSR1-ETS fusion transcripts co-existing in the Ewing sarcoma family of tumors.

PURPOSE: The Ewing sarcoma family of tumors (EFT) is characterized by fusions of the EWSR1 gene on chromosome 22q12 with either one of the genes encoding members of the ETS family of transcription factors, in the majority of cases FLI1 or ERG. Many alternative EWSR1-ETS gene fusions have been encountered, due to variations in the locations of the EWSR1 and ETS genomic breakpoints. The resulting heterogeneity in EWSR1-ETS fusion transcripts may further be increased by the occurrence of multiple splice variants within the same tumor. Here we present a retrospective study designed to detect all of the EWSR1-FLI1 and EWSR1-ERG fusion transcripts in a series of 23 fresh frozen EFT tissues. METHODS: RT-PCR and nested fluorescent multiplex PCR were used to amplify EWSR1-FLI1 and EWSR1-ERG transcripts from EFT tissues. Fusion transcripts were identified by laser-induced fluorescent capillary electrophoresis and confirmed by sequence analysis. RESULTS: Nine different EWSR1-FLI1 fusion transcripts and one EWSR1-ERG fusion transcript were identified in 21 out of 23 fresh frozen EFT tissue samples. In five cases multiple fusion transcripts were found to coexist in the same tumor sample. We additionally reviewed previous reports on twelve cases with multiple EWSR1-ETS fusion transcripts. CONCLUSIONS: Alternative splicing may frequently affect the process of EFT-associated fusion gene transcription and, as such, may significantly contribute to the pathogenic role of EFT-associated chromosome translocations. In a considerable number of cases this may result in multiple splice variants within the same tumor.

Utilisation of fluorescent multiplex PCR and laser-induced capillary electrophoresis for the diagnosis of Ewing family of tumours in formalin-fixed paraffin-embedded tissues.

AIMS: The localisation of the translocation breakpoint of the Ewing sarcoma family of tumours shows significant variability on relatively large regions of fusion partner genes. As a consequence, many alternative forms of EWSR1-ETS translocation exist which make the RNA-based molecular diagnostics of Ewing sarcoma family of tumours complicated. In addition to the heterogeneity of fusion transcripts, the degradation of RNA also presents a significant difficulty in the molecular analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Our aim was to establish a sensitive method which is able to identify all combinatorially possible EWSR1-FLI1 and EWSR1-ERG translocation transcripts in FFPE tissue samples despite significant RNA-degradation. METHODS: The combination of fluorescent multiplex PCR with laser-induced capillary electrophoresis was used to detect and identify EWSR1-FLI1 and EWSR1-ERG chimeric transcripts on the basis of amplicon size, and forward primers labelled by distinct fluorophores. RESULTS: Using this method, we processed 60 FFPE samples of Ewing sarcoma family of tumours, and identified six types EWSR1-FLI1 and one type EWSR1-ERG chimeric transcripts acceptable for RT-PCR analysis in 27 out of 45 samples. This result shows 60% sensitivity for detecting the most frequent Ewing family of tumour (EFT)-related fusion transcripts. CONCLUSIONS: The utilisation of fluorescent multiplex PCR and laser-induced fluorescent capillary electrophoresis is effective for the diagnosis of EFT in FFPE tissue, and after the defined modifications it can offer a sensitive method to overcome the diagnostic difficulties connected with heterogeneity of the variant translocations in EFT.

Adamantinoma of long bones: a long-term follow-up study of 11 cases.

The aim of this study was to evaluate the clinicopathological features and prognostic significances of 11 histologically proven adamantinoma cases based on an average 12,7 year long follow-up. The male: female ratio was 8:3, aged between 4 and 80 years (mean 29,3 years). The initial diagnosis at referral was other than adamantinoma in six patients (fibrous dysplasia, carcinoma metastasis, osteofibrous dysplasia, bone cyst, non-ossifying fibroma), referring to the differential diagnostic problems. All tumors were localized to the mid part of tibia. By histological evaluation, basaloid pattern on a background of fibrotic stroma dominated in six patients, while spindle and squamous features were less frequently seen. All adamantinoma were positive for cytokeratins often in coexpression with vimentin. No correlation was experienced between histology and clinical outcome. Intralesional curettage (2 pts) was followed by recurrence of the tumor. Wide resection was performed in eight patients with reconstruction using intercalary fibula autografts in seven patients. Reconstruction-related complications occurred in two third of the cases, all of them could however be controlled by repeated surgery. Six recurrences occurred in four patients, two of these recurrences occurred 20 and 16 years after initial surgery. One patient died 9 years after recognition of the tumor of pulmonary metastases. Adamantinoma of the long bones is a low grade malignant tumor, which clinical outcome is difficult to predict based on histology or surgical stage of the tumor. Wide surgical margin, e.g. resection the tumor reduces the rate of recurrence. This study underlines that recurrences do occur even decades after recognition the tumor, therefore a life-long follow-up of the patient is necessary.

25 years' experience in the treatment of gestational trophoblastic neoplasia in Hungary.

OBJECTIVE: To review our clinical experience in the treatment of gestational trophoblastic neoplasia (GTN) over the past 25 years in our national trophoblastic disease center. STUDY DESIGN: Between January 1, 1977, and December 31, 2001, we treated 355 patients with GTN. The patients were between 14 and 53 years of age, with an average of 28.3. Primary chemotherapy was selected based on the patient's stage of gestational trophoblastic tumor (GTT) and prognostic score. RESULTS: We found metastases in 49.3% (175 of 355) of our patients. Of 173 patients, 162 (93.2%) achieved remission as a result of methotrexate therapy. In 11 patients (6.8%) complete remission was achieved by combination chemotherapy, in some cases assisted by operation. Of 68 patients, 63 (92.6%) achieved remission as a result of actinomycin D therapy, and 5 (7.4%) achieved complete remission by combination chemotherapy. Chemotherapy, surgical intervention or other supplementary treatments resulted in 100% successful therapy in cases of nonmetastatic and low-risk metastatic disease. CONCLUSION: According to our experience, methotrexate/folinic acid or actinomycin D should be the primary treatment in patients with nonmetastatic or low-risk metastatic GTN. Patients with resistance to single-agent chemotherapy regularly achieve remission with combination chemotherapy.

Morphological changes in the lower esophageal sphincter influencing the result of antireflux surgical interventions in chronic gastroesophageal reflux disease.

BACKGROUND/AIMS: It is still unclear whether long-term reflux episodes result in morphological changes in the lower esophageal sphincter or not. If the answer is supposedly yes, do these changes influence the postoperative functional results following antireflux surgery? METHODOLOGY: Between 1 January 2002 and 2004, we performed antireflux surgery on 85 patients. Muscle samples were taken from the lower esophageal sphincter (LES) in 57 patients on operation. Patients with endoscopic findings of moderate or severe reflux esophagitis--Los Angeles B, C, D--were excluded. Control samples were obtained from muscle tissue at the gastroesophageal junction that had been removed from 16 patients undergoing gastric or esophageal resection. Histologic (hematoxylin and eosin and Giemsa), and immunohistologic (S-100 Protein, NCL-SERCA2, alpha-SMA) and electronmicroscopic analysis were used to evaluate the specimens. The number of smooth muscle cell nuclei in these intraoperative biopsies was used to compare the results of antireflux operations (Visick I and II-III). RESULTS: In 19% (11/57) of the reflux-type LES muscle samples perivascular inflammatory infiltration has been noted and in 6 of these cases (6/57 = 11%) this has incorporated marked intramuscular and adventitial granulocyte infiltration. In one patient (1/57 = 2%) eosinophil infiltration of the myenteric plexus and the ganglion has been revealed. Significantly lower Schwann and smooth muscle cell count could be detected in LES muscle samples taken from patients with GERD (p < 0.05). The analysis of the values of the 9 patients in Visick groups II and III at two months after surgery, has shown a significant decrease in the number of smooth muscle cell nuclei as compared to those patients in Visick group I (p < 0.01). CONCLUSIONS: Our results draw attention to the morphological changes occurring in the LES muscles of reflux patients. The enteric ganglionitis induced by GERD may result in various functional esophageal diseases. The histologic changes--that very much resemble hypertrophy--developing in LES muscles may serve as a reason for symptoms after antireflux surgery, presumably for the most common complaint of dysphagia.

Synchronous multiple glomus tumors of the esophagus and lung.

A case of synchronous esophageal and multiple pulmonary tumors presenting complex diagnostic problems is described. In the course of routine pulmonary screening a pulmonary coin lesion of the right lung and three very small foci on the left side, under the pleura falling just within the range of diagnostic parameters was identified in a symptom-free patient. In addition to the multiple lung lesions a single sub-mucosal esophageal tumor was detected. Following minimally invasive surgical excision of the tumors the precise nature of the neoplasms was determined by means of comparative histological, light-and electro-microscopic as well as immunohistochemical studies. The earlier diagnosis of carcinoid was reviewed, rejected and glomus tumor was confirmed. Multiple glomus tumors of the above localizations have not been previously described in the literature.