The majority of β-catenin mutations in colorectal cancer is homozygous

Background: β-catenin activation plays a crucial role for tumourigenesis in the large intestine but except for Lynch syndrome (LS) associated cancers stabilizing mutations of β-catenin gene (CTNNB1) are rare in colorectal cancer (CRC). Previous animal studies provide an explanation for this observation. They showed that CTNNB1 mutations induced transformation in the colon only when CTNNB1 was homozygously mutated or when membranous β-catenin binding was hampered by E-cadherin haploinsufficiency. We were interested, if these mechanisms are also found in human CTNNB1 mutated CRCs. Results: Among 869 CRCs stabilizing CTNNB1 mutations were found in 27 cases. Homo- or hemizygous CTNNB1 mutations were detected in 74% of CTNNB1 mutated CRCs (13 microsatellite instabile (MSI-H), 7 microsatellite stabile (MSS)) but only in 3% (1/33) of extracolonic CTNNB1 mutated cancers. In contrast to MSS CRC, CTNNB1 mutations at codon 41 or 45 were highly selected in MSI-H CRC. Of the examined three CRC cell lines, β-catenin and E-cadherin expression was similar in cell lines without or with hetereozygous CTNNB1 mutations (DLD1 and HCT116), while a reduced E-cadherin expression combined with cytoplasmic accumulation of β-catenin was found in a cell line with homozygous CTNNB1 mutation (LS180). Reduced expression of E-cadherin in human MSI-H CRC tissue was identified in 60% of investigated cancers, but no association with the CTNNB1 mutational status was found. Conclusions: In conclusion, this study shows that in contrast to extracolonic cancers stabilizing CTNNB1 mutations in CRC are commonly homo- or hemizygous indicating a higher threshold of β-catenin stabilization to be required for transformation in the colon as compared to extracolonic sites. Moreover, we found different mutational hotspots in CTNNB1 for MSI-H and MSS CRCs suggesting a selection of different effects on β-catenin stabilization according to the molecular pathway of tumourigenesis. Reduced E-cadherin expression in CRC may further contribute to higher levels of transcriptionally active β-catenin, but it is not directly linked to the CTNNB1 mutational status

Genome-Wide Analysis of Interchromosomal Interaction Probabilities Reveals Chained Translocations and Overrepresentation of Translocation Breakpoints in Genes in a Cutaneous T-Cell Lymphoma Cell Line

In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models

BACKGROUND/AIMS The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110\textgreeka inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. METHODS Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. RESULTS BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. CONCLUSION Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus

Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect.

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes

Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS

One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics

Genomic loss of the putative tumor suppressor gene E2A in human lymphoma

The transcription factor E2A is essential for lymphocyte development. In this study, we describe a recurrent E2A gene deletion in at least 70% of patients with Sézary syndrome (SS), a subtype of T cell lymphoma. Loss of E2A results in enhanced proliferation and cell cycle progression via derepression of the protooncogene MYC and the cell cycle regulator CDK6. Furthermore, by examining the gene expression profile of SS cells after restoration of E2A expression, we identify several E2A-regulated genes that interfere with oncogenic signaling pathways, including the Ras pathway. Several of these genes are down-regulated or lost in primary SS tumor cells. These data demonstrate a tumor suppressor function of E2A in human lymphoid cells and could help to develop new treatment strategies for human lymphomas with altered E2A activity

Differenzierung embryonaler Stammzellen der Maus zu Hepatozyten � Etablierung eines in vitro-Systems zur Analyse des Differenzierungsprozesses auf der Basis transgener Reporterzelllinien

Die Entwicklung der Organe höherer Lebewesen ist ein komplexer Vorgang, bei dem es zu zahlreichen Wechselwirkungen zwischen Zellen aus unterschiedlichen Geweben kommt. Die Isolierung embryonaler Stammzellen (ES-Zellen) und die Entdeckung, dass diese auch in vitro zu verschiedenen reifen somatischen Zelltypen differenzieren können, bietet ein neues System, um einen Einblick in die Entstehung unterschiedlicher Zelltypen während der Entwicklung der Säugetiere zu erhalten. Die Veröffentlichung mehrerer Protokolle, die eine Generierung hepatozytenähnlicher Zellen aus murinen ES-Zellen in vitro beschreiben, eröffnet nun die Möglichkeit, auch die Organogenese der Leber in diesem System genauer zu studieren. Neben der Untersuchung des Einflusses externer Faktoren auf den Differenzierungsprozess könnte die Durchführung differentieller Genexpressionsanalysen neue Gene identifizieren, die für die Entwicklung der Hepatozyten relevant sind. Voraussetzung hierfür sind neben effizienten Differenzierungsprotokollen Methoden, die eine unkomplizierte Analyse des Differenzierungsvorgangs ermöglichen. Zudem bedarf es Verfahren, die eine Isolierung erfolgreich differenzierter Zellen zu unterschiedlichen Zeitpunkten des Differenzierungsprozesses gestatten. Ziel der Arbeit war es zunächst ein optimiertes Differenzierungsprotokoll zu etablieren und ein Reportersystem zur Analyse des Differenzierungsvorgangs zu generieren. Im Zuge dieser Arbeit wurde deshalb ein Differenzierungsprotokoll etabliert, das auf dem im Jahr 2001 von Takashi Hamazaki et al. publizierten Protokoll beruht und auf der Generierung dreidimensionaler Aggregate, so genannten embryoid bodies (EBs), und anschließender Stimulierung durch externe Faktoren beruht. Im Zuge der Optimierung des Protokolls konnte durch eine Anpassung des Zeitpunktes der Zugabe der einzelnen Faktoren an das spezifische Differenzierungsverhalten der verwendeten Zelllinie eine gesteigerte Expression hepatozytenspezifischer Markergene erzielt werden. Zudem wurde versucht, neue Erkenntnisse über die in vitro-Differenzierung embryonaler Stammzellen zu Hepatozyten im Differenzierungsprotokoll zu berücksichtigen. Der von einer anderen Gruppe beschriebene positive Einfluss eines zeitlich begrenzten Serumentzugs auf die Entstehung von Hepatozyten-Vorläufern während der Differenzierung in EBs konnte dabei nicht bestätigt werden. Auch fibroblast growth factor-4, der in einem auf der Differenzierung in Monolayer-Kultur basierenden Protokoll zu einer vermehrten Entstehung hepatozytenähnlicher Zellen führt, zeigte im hier verwendeten System nicht den erhofften Effekt. Durch den Einsatz unterschiedlicher Serumpräparationen zeigt die Arbeit jedoch, dass nicht nur die An- oder Abwesenheit von im Serum enthaltenen Faktoren einen entscheidenden Einfluss auf den Differenzierungsprozesses hat, sondern bereits geringe Unterschiede in deren Zusammensetzung und Konzentration, wie sie bei verschiedenen Chargen vorliegen, die Differenzierung der Zellen deutlich beeinflusst. Dies verdeutlicht die Probleme bei der Vergleichbarkeit der Ergebnisse unterschiedlicher Arbeitsgruppen und somit die Notwendigkeit definierter Bedingungen, wie sie nur bei einer vollständig serumfreien Kultivierung der Zellen vorliegen. Da weder reife Hepatozyten noch ihre Vorläuferzellen spezifische Oberflächenmarker besitzen, die eine Isolierung mittels Durchflusszytometrie ohne weiteres ermöglichen, wurde versucht, ein Reportersystem zu schaffen, das eine Identifizierung und Isolierung der Zellen zu verschiedenen Zeitpunkten des Differenzierungsprotokolls ermöglicht. Dafür wurden für verschiedene Fluoreszenzproteine kodierende Sequenzen hinter ein Transthyretin-, ein Albumin- bzw. ein Glukose-6-Phosphatase-Promotor-Konstrukt kloniert. Diese Promotoren werden im Zuge der hepatozytenspezifischen Differenzierung aktiviert. Die Konstrukte sollten stabil in das Genom undifferenzierter ES-Zelllinien integriert werden, um so eine zell- bzw. entwicklungsspezifische Expression der Reportergene während des Differenzierungsprozesses zu ermöglichen. Hierfür wurden zwei Verfahren getestet: Zum einen wurde versucht, die transgenen ES-Zellen mittels retroviralem Gentransfer zu erzeugen. Die Infektion der ES-Zellen mit den rekombinanten Viren führte jedoch zu keiner funktionalen Zelllinie. Zum anderen wurde versucht, die Reporterkonstrukte über homologe Rekombination gezielt in den während der Embryonalentwicklung ubiquitär exprimierten ROSA26-Lokus zu integrieren. Hierdurch wurden mit hoher Effizienz Zelllinien generiert, welche die Reporterkonstrukte stabil integriert haben. Im Falle des Albumin-Promotor-Konstruktes wurde so eine Zelllinie erzeugt, in der eine differenzierungsspezifische Synthese des enhanced green fluorescent protein beobachtet werden kann. Dies zeigt, dass diese Methode grundsätzlich geeignet ist, ein solches Reportersystem zügig zu erzeugen, um so den Differenzierungsverlauf analysieren zu können