Andreas Bräuninger

Differentially expressed miRNAs in Ewing sarcoma compared to mesenchymal stem cells: low miR-31 expression with effects on proliferation and invasion.

Ewing sarcoma, the second most common bone tumor in children and young adults, is an aggressive malignancy with a strong potential to metastasize. Ewing sarcoma is characterised by translocations encoding fusion transcription factors with an EWSR1 transactivation domain fused to an ETS family DNA binding domain. microRNAs are post-transcriptional regulators of gene expression and aberrantly expressed microRNAs have been identified as tumor suppressors or oncogenes in most cancer types. To identify potential oncogenic and tumor suppressor microRNAs in Ewing sarcoma, we determined and compared the expression of 377 microRNAs in 40 Ewing sarcoma biopsies, 6 Ewing sarcoma cell lines and mesenchymal stem cells, the putative cellular origin of Ewing sarcoma, from 6 healthy donors. Of the 35 differentially expressed microRNAs identified (fold change >4 and q<0.05), 19 were higher and 16 lower expressed in Ewing sarcoma. In comparisons between Ewing sarcoma samples with EWS-FLI or EWS-ERG translocations, with differing dissemination characteristics and of primary samples and metastases no significantly differential expressed microRNAs were detected using various stringency criteria. For miR-31, the microRNA with lowest expression in comparison to mesenchymal stem cells, functional analyses were performed to determine its potential as a tumor suppressor in Ewing sarcoma. Two of four miR-31 transfected Ewing sarcoma cell lines showed a significantly reduced proliferation (19% and 33% reduction) due to increased apoptosis in one and increased length of G1-phase in the other cell line. All three tested miR-31 transfected Ewing sarcoma cell lines showed significantly reduced invasiveness (56% to 71% reduction). In summary, we identified 35 microRNAs differentially expressed in Ewing sarcoma and demonstrate that miR-31 affects proliferation and invasion of Ewing sarcoma cell lines in ex vivo assays.

A Recurrent Activating PLCG1 Mutation in Cardiac Angiosarcomas Increases Apoptosis Resistance and Invasiveness of Endothelial Cells

Primary cardiac angiosarcomas are rare tumors with unfavorable prognosis. Pathogenic driver mutations are largely unknown. We therefore analyzed a collection of cases for genomic aberrations using SNP arrays and targeted next-generation sequencing (tNGS) of oncogenes and tumor-suppressor genes. Recurrent gains of chromosome 1q and a small region of chromosome 4 encompassing KDR and KIT were identified by SNP array analysis. Repeatedly mutated genes identified by tNGS were KDR with different nonsynonymous mutations, MLL2 with different nonsense mutations, and PLCG1 with a recurrent nonsynonymous mutation (R707Q) in the highly conserved autoinhibitory SH2 domain in three of 10 cases. PLCγ1 is usually activated by Y783 phosphorylation and activates protein kinase C and Ca(2+)-dependent second messengers, with effects on cellular proliferation, migration, and invasiveness. Ectopic expression of the PLCγ1-R707Q mutant in endothelial cells revealed reduced PLCγ1-Y783 phosphorylation with concomitant increased c-RAF/MEK/ERK1/2 phosphorylation, increased IP3 amounts, and increased Ca(2+)-dependent calcineurin activation compared with ectopic expressed PLCγ1-wild-type. Furthermore, cofilin, whose activation is associated with actin skeleton reorganization, showed decreased phosphorylation, and thus activation after expression of PLCγ1-R707Q compared with PLCγ1-wild-type. At the cellular level, expression of PLCγ1-R707Q in endothelial cells had no influence on proliferation rate, but increased apoptosis resistance and migration and invasiveness in in vitro assays. Together, these findings indicate that the PLCγ1-R707Q mutation causes constitutive activation of PLCγ1 and may represent an alternative way of activation of KDR/PLCγ1 signaling besides KDR activation in angiosarcomas, with implications for VEGF/KDR targeted therapies.

Noninvasive fetal genotyping of human platelet antigen-1a using targeted massively parallel sequencing

Fetal human platelet antigen (HPA) genotyping is required to determine whether the fetus is at risk and whether prenatal interventions to prevent fetal bleeding are required in pregnant women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Methods for noninvasive genotyping of HPA alleles with the use of maternal plasma cell‐free DNA were published recently but do lack internal controls to exclude false‐negative results.

Induction of Endoplasmic Reticulum Stress by Sorafenib and Activation of NF-κB by Lestaurtinib as a Novel Resistance Mechanism in Hodgkin Lymphoma Cell Lines

Hodgkin-Reed/Sternberg (HRS) cells of classical Hodgkin lymphoma show aberrant expression and activation of several receptor tyrosine kinases (RTK) in the majority of cases. Therefore, we tested whether tyrosine kinase inhibitors (TKI) already in clinical use or late stages of clinical trials have antiproliferative effects on HRS cell lines and evaluated the targets, affected signaling pathways, and mechanisms of cell death and resistance. Sorafenib and lestaurtinib had antiproliferative effects on HRS cell lines at concentrations achievable in patients. Sorafenib inhibited platelet-derived growth factor receptor (PDGFR) α, TRKA and RON, caused decreases in total and phosphorylated amounts of several signaling molecules, and provoked caspase-3–independent cell death, most likely due to endoplasmic reticulum stress as indicated by upregulation of GADD34 and GADD153 and phosphorylation of PERK. Lestaurtinib inhibited TRKA, PDGFRα, RON, and JAK2 and had only a cytostatic effect. Besides deactivation, lestaurtinib also caused activation of signaling pathways. It caused increases in CD30L and TRAIL expression, and CD30L/CD30 signaling likely led to the observed concomitant activation of extracellular signal–regulated kinase 1/2 and the alternative NF-κB pathway. These data disclose the possible use of sorafenib for the treatment of Hodgkin lymphoma and highlight NF-κB activation as a potential novel mechanism of resistance toward TKIs. Mol Cancer Ther; 12(2); 173–83. ©2012 AACR.

Detection of an activated JAK3 variant and a Xq26.3 microdeletion causing loss of PHF6 and miR-424 expression in myelodysplastic syndromes by combined targeted next generation sequencing and SNP array analysis

Myelodysplastic syndromes (MDS) are hematopoietic disorders characterized by ineffective hematopoiesis and progression to acute leukemia. In patients ineligible for hematopoietic stem cell transplantation, azacitidine is the only treatment shown to prolong survival. However, with the availability of a growing compendium of cancer biomarkers and related drugs, analysis of relevant genetic alterations for individual MDS patients might become part of routine evaluation. Therefore and in order to cover the entire bone marrow microenvironment involved in the pathogenesis of MDS, SNP array analysis and targeted next generation sequencing (tNGS) for the mostly therapy relevant 46 onco- and tumor-suppressor genes were performed on bone marrow biopsies from 29 MDS patients. In addition to the detection of mutations known to be associated with MDS in NRAS, KRAS, MPL, NPM1, IDH1, PTPN11, APC and MET, single nucleotide variants so far unrelated to MDS in STK11 (n=1), KDR (n=3), ATM (n=1) and JAK3 (n=2) were identified. Moreover, a recurrent microdeletion was detected in Xq26.3 (n=2), causing loss of PHF6 expression, a potential tumor suppressor gene, and the miR-424, which is involved in the development of acute myeloid leukemia. Finally, combined genetic aberrations affecting the VEGF/VEGFR pathway were found in the majority of cases demonstrating the diversity of mutations affecting different nodes of a particular signaling network as an intrinsic feature in MDS patients. We conclude that combined SNP array analyses and tNGS can identify established and novel therapy relevant genomic aberrations in MDS patients and track them in a clinical setting for individual therapy selection.

75-jährige Patientin mit Pleuraerguss und gastralen Metastasen eines wenig differenzierten Karzinoms

A 75-year-old woman was found to have left-sided pleural effusion and endoscopy revealed the rare entity of adenoid cystic carcinoma metastases in the gastric mucosa. Approximately 20% of patients with this carcinoma suffer from distant metastases. For the initial staging detection of adenoid cystic carcinoma metastasis with positron emission tomography (PET) or PET computed tomography (CT) is recommended. The recurrent t(6;9)(q22-23;p23-24) translocation that results in a fusion of the two transcription factor genes MYB and NFIB is detectable in half of the cases. As in our case molecular pathology can confirm the correct diagnosis and identification of the localization of the primary tumor.

[A 75-year-old female patient with pleural effusion and gastric metastases of a poorly differentiated carcinoma].

A 75-year-old woman was found to have left-sided pleural effusion and endoscopy revealed the rare entity of adenoid cystic carcinoma metastases in the gastric mucosa. Approximately 20% of patients with this carcinoma suffer from distant metastases. For the initial staging detection of adenoid cystic carcinoma metastasis with positron emission tomography (PET) or PET computed tomography (CT) is recommended. The recurrent t(6;9)(q22-23;p23-24) translocation that results in a fusion of the two transcription factor genes MYB and NFIB is detectable in half of the cases. As in our case molecular pathology can confirm the correct diagnosis and identification of the localization of the primary tumor.

Bedeutung der Schnellschnittdiagnostik für das Management laryngealer Tumoren

The frozen section procedure for immediate intraoperative pathological diagnosis represents a pivotal method in tumor diagnosis. In laryngeal tumors the most frequent indication for the use of this method is the documentation of the residual tumor status, while intraoperative consultation with the purpose of primary tumor diagnosis is less common. The specimen management employed in each case should be chosen depending on the clinical question: while the collection of a maximum amount of tissue is advisable for the determination of the residual tumor status, sparing a portion of the remaining tissue for possible future examinations is advisable in the case of primary tumor diagnosis. Moreover, intraoperative frozen section diagnosis with no immediate consequences should be avoided.ZusammenfassungDie Schnellschnittuntersuchung stellt eine elementare Methode in der Tumordiagnostik dar. Die häufigste Indikation im Kehlkopfbereich ist die Dokumentation des Residualtumorstatus. Seltener werden Schnellschnittuntersuchungen zur Tumorerstdiagnose erfragt. Die Untersuchung von Sentinellymphknoten ist im Kopf-Hals-Bereich bis heute kein Routineverfahren. Die Vorgehensweise sollte von der Indikation abhängig gemacht werden, die grundsätzlich klinisch und nicht durch den Pathologen zu stellen ist. Während zur alleinigen Bestimmung des Residualtumorstatus möglichst viel Gewebe im Schnellschnitt untersucht werden sollte, ist es ratsam im Fall einer Tumorerstdiagnose Restgewebe für eventuell später nötige Zusatzuntersuchungen zurückzuhalten. Schnellschnittuntersuchungen, die keine unmittelbare intraoperative Konsequenz haben, sollten vermieden werden.AbstractThe frozen section procedure for immediate intraoperative pathological diagnosis represents a pivotal method in tumor diagnosis. In laryngeal tumors the most frequent indication for the use of this method is the documentation of the residual tumor status, while intraoperative consultation with the purpose of primary tumor diagnosis is less common. The specimen management employed in each case should be chosen depending on the clinical question: while the collection of a maximum amount of tissue is advisable for the determination of the residual tumor status, sparing a portion of the remaining tissue for possible future examinations is advisable in the case of primary tumor diagnosis. Moreover, intraoperative frozen section diagnosis with no immediate consequences should be avoided.

Significance of frozen section diagnosis for the management of laryngeal tumors

The frozen section procedure for immediate intraoperative pathological diagnosis represents a pivotal method in tumor diagnosis. In laryngeal tumors the most frequent indication for the use of this method is the documentation of the residual tumor status, while intraoperative consultation with the purpose of primary tumor diagnosis is less common. The specimen management employed in each case should be chosen depending on the clinical question: while the collection of a maximum amount of tissue is advisable for the determination of the residual tumor status, sparing a portion of the remaining tissue for possible future examinations is advisable in the case of primary tumor diagnosis. Moreover, intraoperative frozen section diagnosis with no immediate consequences should be avoided.ZusammenfassungDie Schnellschnittuntersuchung stellt eine elementare Methode in der Tumordiagnostik dar. Die häufigste Indikation im Kehlkopfbereich ist die Dokumentation des Residualtumorstatus. Seltener werden Schnellschnittuntersuchungen zur Tumorerstdiagnose erfragt. Die Untersuchung von Sentinellymphknoten ist im Kopf-Hals-Bereich bis heute kein Routineverfahren. Die Vorgehensweise sollte von der Indikation abhängig gemacht werden, die grundsätzlich klinisch und nicht durch den Pathologen zu stellen ist. Während zur alleinigen Bestimmung des Residualtumorstatus möglichst viel Gewebe im Schnellschnitt untersucht werden sollte, ist es ratsam im Fall einer Tumorerstdiagnose Restgewebe für eventuell später nötige Zusatzuntersuchungen zurückzuhalten. Schnellschnittuntersuchungen, die keine unmittelbare intraoperative Konsequenz haben, sollten vermieden werden.AbstractThe frozen section procedure for immediate intraoperative pathological diagnosis represents a pivotal method in tumor diagnosis. In laryngeal tumors the most frequent indication for the use of this method is the documentation of the residual tumor status, while intraoperative consultation with the purpose of primary tumor diagnosis is less common. The specimen management employed in each case should be chosen depending on the clinical question: while the collection of a maximum amount of tissue is advisable for the determination of the residual tumor status, sparing a portion of the remaining tissue for possible future examinations is advisable in the case of primary tumor diagnosis. Moreover, intraoperative frozen section diagnosis with no immediate consequences should be avoided.

Targeted Next-Generation Sequencing Is a Sensitive Tool for Differential Diagnosis of Myelodysplastic Syndromes in Bone Marrow Trephines

Myelodysplastic syndromes are hematological neoplasias in which immunohistologic examination of bone marrow trephines is important for a definite diagnosis. Unequivocal distinction from reactive bone marrow changes is, however, sometimes difficult. Because neoplastic clones in myelodysplastic syndrome carry mutations in recurrent genes, mutation detection by targeted next-generation sequencing may be a useful support for differential diagnosis. To elucidate the accuracy of this approach in the clinical diagnostic setting, we analyzed single and consecutive bone marrow trephines processed for immunohistologic examination from 145 patients by targeted next-generation sequencing of 12 genes recurrently mutated in myelodysplastic syndromes. Of 110 patients with immunohistologic unequivocal diagnosis, 41 of 47 with myelodysplastic syndrome carried mutations. In 14 consecutive samples available from these patients, remissions were accompanied by loss of mutations and ongoing disease with persisting mutations. Of 35 samples with indefinite immunohistologic appearance, 22 developed clinical unequivocal myelodysplastic syndrome in the further course, and 19 carried mutations already in the initial biopsy, which persisted in consecutive samples available from 13 patients. No mutation was detected in any initial and consecutive sample of 13 patients with indefinite immunohistologic appearance without clinical unequivocal myelodysplastic syndrome in the further course. We conclude that targeted next-generation sequencing is an accurate tool for differential diagnosis of myelodysplastic syndrome in the clinical diagnostic setting.