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Over the last decades, rapid technological and scientific advances have led to a merge of molecular sciences and clinical medicine, resulting in a better understanding of disease mechanisms and the development of novel therapies that exploit specific molecular lesions or profiles driving disease. Precision oncology is here used as an example, illustrating the potential of precision/personalized medicine that also holds great promise in other medical fields. Real-world implementation can only be achieved by dedicated healthcare connected centers which amass and build up interdisciplinary expertise reflecting the complexity of precision medicine. Networks of such centers are ideally suited for a nation-wide outreach offering access to precision medicine to patients independent of their place of residence. Two of these multicentric initiatives, Genomic Medicine Sweden (GMS) and the Centers for Personalized Medicine (ZPM) initiative in Germany have teamed up to present and share their views on core concepts, potentials, challenges, and future developments in precision medicine. Together with other initiatives worldwide, GMS and ZPM aim at providing a robust and sustainable framework, covering all components from technology development to clinical trials, ethical and legal aspects as well as involvement of all relevant stakeholders, including patients and policymakers in the field.

This study evaluated two DNA-based next-generation sequencing approaches for detection of single-nucleotide variants (SNVs) and fusions in formalin-fixed, paraffin-embedded (FFPE) tissue specimens and liquid biopsies (AVENIO Targeted and Surveillance Panels). Reference standards (n = 7 with SNVs and structural variants) and real-world FFPE tissue specimens (n = 26 lung, colorectal, pancreas, ovary, breast, prostate, melanoma, and soft tissue cancer cases with n = 27 samples), liquid biopsies [n = 29 cases with n = 40 plasma/cell-free DNA (cfDNA) samples], and one pleural effusion (lung cancer) were analyzed by the AVENIO workflow for known SNVs (BRAF, BRCA1/2, CTNNB1, EGFR, KRAS, MET exon 14 skipping, NRAS, PIK3CA, and TP53), insertions and deletions (ERBB2 and KIT), and fusions (ALK and ROS1). Detection of SNVs, insertions and deletions, and fusions was reliable in 24 of 26 FFPE tissue specimen cases and at 1% allele frequency in 5 of 5 cfDNA reference standards and 37 of 40 plasma/cfDNA samples. Pitfalls were identified for the AVENIO workflow in calling and listing of clinically relevant variants, requiring additional manual inspection. Moreover, laboratory workflows are distinct for FFPE tissue specimens and liquid biopsies as well as time-consuming for sample quality control assays. In summary, the DNA-based next-generation sequencing approaches may be suitable for routine molecular pathology diagnostics on careful data interpretation and further optimization of the technical and laboratory workflows.

Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non‐small‐cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted‐capture high‐throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early‐stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative‐intent tumor resection (median follow‐up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1–2 weeks postsurgery, and during follow‐up. Deep next‐generation sequencing using unique molecular identifiers was performed to identify and quantify tumor‐specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL−1, respectively). Four patients (19%) remained ctDNA‐positive at 1–2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA‐negative patients (33%) after surgery experienced relapse during follow‐up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression‐free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early‐stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse., ctDNA profiling in plasma revealed higher levels and detection rates of ctDNA during tumor resection as compared to pretreatment time points in patients with early‐stage or locally advanced NSCLC. Moreover, patients testing positive for ctDNA immediately after tumor resection had worse clinical outcomes than patients with undetectable ctDNA. Our research highlights the role of ctDNA assessment for guiding treatment in patients with respectable NSCLC.

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Supplementary Figure and Table Legend. Legend for Supplementary Figures S1-S8 and Supplementary Tables S1-S4.

Supplementary Table S4. Loss of oncogenic B-Raf signaling induces transcripts associated with intestinal differentiation.

Supplementary Figures S1-S8. Effects of oncogenic B-Raf signaling on the phenotype of HT29 and Caco2-tet cells in conventional tissue culture (S1); Analysis of the loss of oncogenic B-Raf signaling on the phenotype of HT29 and Colo-205 cells (S2); Effects of B-RafV600E knockdown on Colo-205 cells (S3); Additional information on the transcriptome analysis of HT29 and Colo-205 spheroids, including further validation at the protein level (S4); Additional data supporting the (inverse) relationship between B-RafV600E signaling and the expression of Cdx-2, KIAA1199 and Claudins in HT29 and Colo-205 cells (S5); Additional data addressing the effects of B-RafV600E signaling on transcripts associated with invasion and stemness, on xenograft growth and tight junction proteins (S6); Supplementary data showing the single-channel images corresponding to Fig. 2C and 3D, respectively (S7); Inhibition of B-RafV600E modulates transcripts associated with intestinal differentiation (S8).

Die rasanten Entwicklungen molekularer Technologien und gezielter Therapien haben in den vergangenen Jahren auch die Implementierung spezialisierter Tumorkonferenzen bzw. molekulare Tumorboards (MTB) bewirkt. MTB werden insbesondere dann wichtig, wenn Therapieempfehlungen benotigt werden, die auf molekularen Veranderungen beruhen, die uber die zugelassenen zielgerichteten Therapien hinausgehen. Wahrend das Ziel solcher MTB uberwiegend in der optimierten Versorgung von Krebspatienten durch individualisierte Diagnostik und Therapie unter Nutzung neuster Technologien und Biomarker liegt, sind die Vorgehensweisen von MTB jedoch noch heterogen. Dies betriff klinische Einschlusskriterien fur Krebspatienten, Zusammensetzung des MTB, durchgefuhrte diagnostische Tests und deren Aus- und Bewertung bzw. deren klinische Wertigkeit und Umsetzung in eine Behandlungsstrategie, die damit verbundene Qualitatssicherung sowie auch wissensgenerierende, okonomische, rechtliche und ethische Aspekte. Dieser Beitrag bietet eine Ubersicht uber das Spektrum von MTB sowie deren Herausforderungen und Moglichkeiten fur die individuelle Krebsmedizin.

Precision cancer medicine (PCM) holds great promises to offer more effective therapies to patients based on molecular profiling of their individual tumours. Although the PCM approach seems intuitive, multiple conceptional and structural challenges interfere with the broad implementation of PCM into clinical practice. Accordingly, concerted national and international efforts are needed to guide the further development and broad adoption of PCM in Germany. With support of the 'German Cancer Aid' (Deutsche Krebshilfe [DKH]) a task force 'Molecular Diagnostics and Therapy' was implemented. In two workshops supported by the DKH, delegates from the fourteen comprehensive cancer centresidentified key topics essential to implement quality-guided, harmonized and adaptable PCM. Based on an online questionnaire and using a modified Delphi approach, nine statements were drafted and evaluated within the group. These statements could serve as a basis to define a collaborative strategy for PCM in the future with the aim to sustain and further improve its quality.

Round robin testing is an important instrument for quality assurance. Increasingly, this also applies to the results of molecular diagnostics in pathology, which directly influence therapy decisions in precision oncology. In metastatic colorectal carcinoma (mCRC), the focus has been on detecting KRAS and NRAS mutations, whose absence allows therapy with EGFR blocking antibodies. Recently, BRAF has been added as another predictive marker, since mCRC patients with BRAF V600E mutation benefit significantly from treatment with encorafenib (a BRAF inhibitor) in combination with cetuximab (anti-EGFR antibody) after systemic therapy. Due to the approval of this treatment in 2020, it is a pre-requisite that BRAF V600E mutation detection in diagnostic pathologies is reliably performed. Therefore, this round robin test with BRAF V600E testing either by immunohistochemistry or molecular methods was performed. The round robin test results demonstrate that molecular BRAF V600E detection is currently clearly superior to immunohistochemical detection.Ringversuche sind ein wichtiges Instrument zur Qualitätssicherung. Dies betrifft in zunehmendem Maße auch die molekulare Diagnostik in der Pathologie, von deren Ergebnissen Therapieentscheidungen in der Präzisionsonkologie direkt abhängen. Beim metastasierten kolorektalen Karzinom (mKRK) stand bisher der Nachweis von KRAS-und NRAS-Mutationen im Vordergrund, deren Abwesenheit eine Therapie mit EGFR-blockierenden Antikörpern ermöglicht. Nun ist BRAF als weiterer prädiktiver Marker hinzugekommen, da mKRK Patienten mit einer BRAF-V600E-Mutation nach systemischer Vortherapie von einer Behandlung mit Encorafenib (einem BRAF-Inhibitor) in Kombination mit Cetuximab (Anti-EGFR-Antikörper) profitieren. Aufgrund der 2020 erfolgten Zulassung für diese Behandlung ist es wichtig, dass der diagnostische Nachweis einer BRAF-V600E-Mutation zuverlässig in den Pathologien durchgeführt werden kann. Daher wurde dieser Ringversuch durchgeführt, bei dem der Nachweis der BRAF-V600E-Mutation entweder mittels Immunhistochemie oder molekularer Verfahren erfolgen konnte. Die Ergebnisse des Ringversuchs belegen eindeutig, dass derzeit die molekulare BRAF-V600E-Bestimmung dem immunhistologischen Nachweis überlegen ist.

The rapid development of molecular technologies and targeted therapies has fostered the implementation of specialized tumor conferences, known as molecular tumor boards (MTBs). MTBs become particularly important when treatment recommendations are needed based on molecular alterations beyond the approved targeted therapies. While an MTB's goals are based on individualized diagnostics and therapies of tumor patients using innovative technologies and biomarkers, the procedures of MTBs are still quite heterogeneous. This applies to the primary inclusion criteria for tumor patients, the composition of MTBs, the applied diagnostic tests and their assessment and reporting, the evaluation of their clinical value and implementation in a therapeutic strategy, and the associated quality assurance measurements as well as knowledge-gaining, economical, legal, and ethical aspects.This article provides an overview of the spectrum of MTBs, their challenges, and the potential for individualized cancer medicine.Die rasanten Entwicklungen molekularer Technologien und gezielter Therapien haben in den vergangenen Jahren auch die Implementierung spezialisierter Tumorkonferenzen bzw. molekulare Tumorboards (MTB) bewirkt. MTB werden insbesondere dann wichtig, wenn Therapieempfehlungen benötigt werden, die auf molekularen Veränderungen beruhen, die über die zugelassenen zielgerichteten Therapien hinausgehen. Während das Ziel solcher MTB überwiegend in der optimierten Versorgung von Krebspatienten durch individualisierte Diagnostik und Therapie unter Nutzung neuster Technologien und Biomarker liegt, sind die Vorgehensweisen von MTB jedoch noch heterogen. Dies betriff klinische Einschlusskriterien für Krebspatienten, Zusammensetzung des MTB, durchgeführte diagnostische Tests und deren Aus- und Bewertung bzw. deren klinische Wertigkeit und Umsetzung in eine Behandlungsstrategie, die damit verbundene Qualitätssicherung sowie auch wissensgenerierende, ökonomische, rechtliche und ethische Aspekte.Dieser Beitrag bietet eine Übersicht über das Spektrum von MTB sowie deren Herausforderungen und Möglichkeiten für die individuelle Krebsmedizin.

Simple Summary Access to molecular cancer treatments outside of clinical trials is limited and the benefit of molecular-guided, individualized patient care in patients with cancer progression after standard treatment is unclear. We here present the four-year experience of one of Europe’s first Molecular Tumor Boards and show that precision oncology in the era of affordable, extended genetic and phenotypic tumor profiling is feasible and effective for a small but relevant proportion of advanced cancer patients. We performed a comprehensive analysis of clinical follow-up data and report our workflow optimizations and upscaling processes. These could help other centers to establish similar structures to support molecular-guided treatment for patients with limited therapy options. Abstract Molecular precision oncology faces two major challenges: first, to identify relevant and actionable molecular variants in a rapidly changing field and second, to provide access to a broad patient population. Here, we report a four-year experience of the Molecular Tumor Board (MTB) of the Comprehensive Cancer Center Freiburg (Germany) including workflows and process optimizations. This retrospective single-center study includes data on 488 patients enrolled in the MTB from February 2015 through December 2018. Recommendations include individual molecular diagnostics, molecular stratified therapies, assessment of treatment adherence and patient outcomes including overall survival. The majority of MTB patients presented with stage IV oncologic malignancies (90.6%) and underwent an average of 2.1 previous lines of therapy. Individual diagnostic recommendations were given to 487 patients (99.8%). A treatment recommendation was given in 264 of all cases (54.1%) which included a molecularly matched treatment in 212 patients (43.4%). The 264 treatment recommendations were implemented in 76 patients (28.8%). Stable disease was observed in 19 patients (25.0%), 17 had partial response (22.4%) and five showed a complete remission (6.6%). An objective response was achieved in 28.9% of cases with implemented recommendations and for 4.5% of the total population (22 of 488 patients). By optimizing the MTB workflow, case-discussions per session increased significantly while treatment adherence and outcome remained stable over time. Our data demonstrate the feasibility and effectiveness of molecular-guided personalized therapy for cancer patients in a clinical routine setting showing a low but robust and durable disease control rate over time.

Introduction In contrast to other driver mutations, no targeted therapies have yet been approved in ERBB2-mutated NSCLC (HER2mu NSCLC). Nevertheless, several compounds have revealed promising early efficacy data, which need to be evaluated in the context of current standard approaches. Although data on the efficacy of immune checkpoint inhibitors (ICIs) in second or subsequent lines of treatment remain limited and conflicting, there are virtually no data on patient outcome under ICI/platinum-doublet combinations in the first-line setting. Methods We retrospectively evaluated outcomes of patients with HER2mu NSCLC treated with ICI alone or in combination with chemotherapy within the German National Network Genomic Medicine Lung Cancer consortium by means of overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Results ICI either in combination with chemotherapy or as monotherapy was applied as first-line treatment in 27 patients, whereas 34 received single-agent ICI in second or subsequent lines. Patient characteristics were in line with previously published data. In treatment-naive patients receiving ICI in combination with chemotherapy, the ORR, median PFS, and OS rate at 1 year were 52%, 6 months, and 88%, respectively. In second or subsequent lines, ICI monotherapy was associated with an ORR of 16%, a median PFS of 4 months, and a median OS of 10 months. Conclusions ICIs are effective as monotherapy and in combination with platinum-doublet chemotherapy. Therefore, ICI-based treatments may be found as the current standard of care and benchmark for targeted therapies in HER2mu NSCLC.

Round robin testing is an important instrument for quality assurance. Increasingly, this also applies to the results of molecular diagnostics in pathology, which directly influence therapy decisions in precision oncology. In metastatic colorectal carcinoma (mCRC), the focus has been on detecting KRAS and NRAS mutations, whose absence allows therapy with EGFR blocking antibodies. Recently, BRAF has been added as another predictive marker, since mCRC patients with BRAF V600E mutation benefit significantly from treatment with encorafenib (a BRAF inhibitor) in combination with cetuximab (anti-EGFR antibody) after systemic therapy. Due to the approval of this treatment in 2020, it is a pre-requisite that BRAF V600E mutation detection in diagnostic pathologies is reliably performed. Therefore, this round robin test with BRAF V600E testing either by immunohistochemistry or molecular methods was performed. The round robin test results demonstrate that molecular BRAF V600E detection is currently clearly superior to immunohistochemical detection., Ringversuche sind ein wichtiges Instrument zur Qualitätssicherung. Dies betrifft in zunehmendem Maße auch die molekulare Diagnostik in der Pathologie, von deren Ergebnissen Therapieentscheidungen in der Präzisionsonkologie direkt abhängen. Beim metastasierten kolorektalen Karzinom (mKRK) stand bisher der Nachweis von KRAS-und NRAS-Mutationen im Vordergrund, deren Abwesenheit eine Therapie mit EGFR-blockierenden Antikörpern ermöglicht. Nun ist BRAF als weiterer prädiktiver Marker hinzugekommen, da mKRK Patienten mit einer BRAF-V600E-Mutation nach systemischer Vortherapie von einer Behandlung mit Encorafenib (einem BRAF-Inhibitor) in Kombination mit Cetuximab (Anti-EGFR-Antikörper) profitieren. Aufgrund der 2020 erfolgten Zulassung für diese Behandlung ist es wichtig, dass der diagnostische Nachweis einer BRAF-V600E-Mutation zuverlässig in den Pathologien durchgeführt werden kann. Daher wurde dieser Ringversuch durchgeführt, bei dem der Nachweis der BRAF-V600E-Mutation entweder mittels Immunhistochemie oder molekularer Verfahren erfolgen konnte. Die Ergebnisse des Ringversuchs belegen eindeutig, dass derzeit die molekulare BRAF-V600E-Bestimmung dem immunhistologischen Nachweis überlegen ist.

Purpose Dramatic advances in our understanding of the molecular pathophysiology of cancer, along with a rapidly expanding portfolio of molecular targeted drugs, have led to a paradigm shift toward personalized, biomarker-driven cancer treatment. Here, we report the 2-year experience of the Comprehensive Cancer Center Freiburg Molecular Tumor Board (MTB), one of the first interdisciplinary molecular tumor conferences established in Europe. The role of the MTB is to recommend personalized therapy for patients with cancer beyond standard-of-care treatment. Methods This retrospective case series includes 198 patients discussed from March 2015 through February 2017. The MTB guided individual molecular diagnostics, assessed evidence of actionability of molecular alterations, and provided therapy recommendations, including approved and off-label treatments as well as available matched clinical trials. Results The majority of patients had metastatic solid tumors (73.7%), mostly progressive (77.3%) after a mean of 2.0 lines of standard treatment. Diagnostic recommendations resulted in 867 molecular diagnostic tests for 172 patients (five per case), including exome analysis in 36 cases (18.2%). With a median turnaround time of 28 days, treatment recommendations were given to 104 patients (52.5%). These included single-agent targeted therapies (42.3%), checkpoint inhibitors (37.5%), and combination therapies (18.3%). Treatment recommendations were implemented in 33 of 104 patients (31.7%), of whom 19 (57.6%) showed stable disease or partial response, including 14 patients (7.1% of the entire population) receiving off-label treatments. Conclusion Personalized extended molecular-guided patient care is effective for a small but clinically meaningful proportion of patients in challenging clinical situations. Limited access to targeted drugs, lack of trials, and submission at late disease stage prevents broader applicability, whereas genome-wide analyses are not a strict requirement for predictive molecular testing.

The purpose of this study is to map spatial metabolite differences across three molecular subgroups of glial tumors, defined by the IDH1/2 mutation and 1p19q-co-deletion, using magnetic resonance spectroscopy. This work reports a new MR spectroscopy based classification algorithm by applying a radiomics analytics pipeline. 65 patients received anatomical and chemical shift imaging (5 × 5 × 20 mm voxel size). Tumor regions were segmented and registered to corresponding spectroscopic voxels. Spectroscopic features were computed (n = 860) in a radiomic approach and selected by a classification algorithm. Finally, a random forest machine-learning model was trained to predict the molecular subtypes. A cluster analysis identified three robust spectroscopic clusters based on the mean silhouette widths. Molecular subgroups were significantly associated with the computed spectroscopic clusters (Fisher’s Exact test p

Oesophageal squamous cell carcinomas and oesophageal adenocarcinomas display distinct patterns of ErbB expression and dimers. The functional effects of specific ErbB homo- or heterodimers on oesophageal (cancer) cell behaviour, particularly invasion of early carcinogenesis remains unknown. Here, a new cellular model system for controlled activation of EGFR or HER2 and EGFR/HER2 or HER2/HER3 homo- and heterodimers was studied in non-neoplastic squamous oesophageal epithelial Het-1A cells. EGFR, HER2 and HER3 intracellular domains (ICDs) were fused to dimerization domains (DmrA / DmrA and DmrC), and transduced into Het-1A cells lacking ErbB expression. Dimerization of EGFR, HER2 or EGFR/HER2, HER2/HER3 ICDs was induced by synthetic ligands (A/A or A/C dimerizers). This was accompanied by phosphorylation of the respective EGFR, HER2 and HER3 ICDs and activation of distinct down-stream signalling pathways, such as PLCγ1, Akt, STAT and Src family kinases. Phenotypically, ErbB homo- and heterodimers caused cell rounding and non-apoptotic blebbing in EGFR/HER2 and HER2/HER3 heterodimer cells. In a Transwell assay, cell migration velocity was elevated in HER2-dimer as compared to empty vector cells. In addition, HER2-dimer cells showed in increased cell invasion, reaching significance for induced HER2/HER3 heterodimers (p=0.015). Importantly, in three-dimensional organotypic cultures, empty vector cells grew as a superficial cell layer, resembling oesophageal squamous epithelium. In contrast, induced HER2-dimer cells (HER2 homodimers) were highly invasive into the matrix and formed cell clusters. This was associated with partial loss of CK7 (when HER2 homodimers were modelled) and p63 (when EGFR/HER2 heterodimers were modelled), which suggests a change or loss of squamous cell differentiation. Controlled activation of specific EGFR, HER2 and HER3 homo- and heterodimers caused oesophageal squamous epithelial cell migration and/or invasion, especially in a three dimensional microenvironment, thereby functionally identifying ErbB homo- and heterodimers as important drivers of oesophageal carcinogenesis.

// Sarah Lang 1 , Hauke Busch 2, 3 , Melanie Boerries 3, 4 , Tilman Brummer 3, 4, 5 , Sylvia Timme 6 , Silke Lassmann 4, 5, 6 , Klaus Aktories 1 and Gudula Schmidt 1 1 Institute for Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, Albert-Ludwigs-University, Freiburg, Germany 2 Lubeck Institute of Experimental Dermatology, Institute for Cardiogenetics, University of Lubeck, Lubeck, Germany 3 Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Freiburg, Germany 4 German Cancer Consortium (DKTK), Freiburg, Germany, German Cancer Research Center (DKFZ), Heidelberg, Germany 5 Center for Biological Signalling Studies (BIOSS), University of Freiburg, Freiburg, Germany 6 Institute for Surgical Pathology, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany Correspondence to: Gudula Schmidt, email: Gudula.Schmidt@pharmakol.uni-freiburg.de Keywords: Rho GTPase, invasion, cyclooxygenase, breast cancer Received: June 14, 2017 Accepted: August 26, 2017 Published: September 16, 2017 ABSTRACT Rho GTPases are regulators of many cellular functions and are often dysregulated in cancer. However, the precise role of Rho proteins for tumor development is not well understood. In breast cancer, overexpression of RhoC is linked with poor prognosis. Here, we aim to compare the function of RhoC and its homolog family member RhoA in breast cancer progression. We established stable breast epithelial cell lines with inducible expression of RhoA and RhoC, respectively. Moreover, we made use of Rho-activating bacterial toxins (Cytotoxic Necrotizing Factors) to stimulate the endogenous pool of Rho GTPases in benign breast epithelial cells and simultaneously knocked down specific Rho proteins. Whereas activation of Rho GTPases was sufficient to induce an invasive phenotype in three-dimensional culture systems, overexpression of RhoA or RhoC were not. However, RhoC but not RhoA was required for invasion, whereas RhoA and RhoC equally regulated proliferation. We further identified downstream target genes of RhoC involved in invasion and identified PTGS2 (COX-2) being preferentially upregulated by RhoC. Consistently, the COX-2 inhibitor Celecoxib blocked the invasive phenotype induced by the Rho-activating toxins.

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is dynamically expressed in human and murine renal epithelia during development. The levels of EpCAM in the renal epithelium are upregulated both during regeneration after ischemia/reperfusion injury and in renal-derived carcinomas. The role of EpCAM in early kidney development, however, has remained unclear. The zebrafish pronephros shows a similar segmentation pattern to the mammalian metanephric nephron, and has recently emerged as a tractable model to study the regulatory programs governing early nephrogenesis. Since EpCAM shows persistent expression in the pronephros throughout early development, we developed a method to study the global changes in gene expression in specific pronephric segments of wild type and EpCAM-deficient zebrafish embryos. In epcam mutants, we found 379 differentially expressed genes. Gene ontology analysis revealed that EpCAM controls various developmental programs, including uretric bud development, morphogenesis of branching epithelium, regulation of cell differentiation and cilium morphogenesis.

Liquid biopsy offers the potential for non-invasive monitoring and mutation detection in cancer. Detection of mutations with very low allele frequencies using PCR based technologies is a technical challenge. Several quantitative PCR based systems are integrated in routine molecular diagnostics. The detection limit may not always suffice with these qPCR systems, but may be achieved by new digital PCR systems.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are epigenetic eraser enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) in vitro. Mode of action investigations revealed that one compound, deferasirox, is a bona fide active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to similarly inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox might provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are epigenetic eraser enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) in vitro. Mode of action investigations revealed that one compound, deferasirox, is a bona fide active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to similarly inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox may provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.

Next-generation sequencing has become a cornerstone of therapy guidance in cancer precision medicine and an indispensable research tool in translational oncology. Its rapidly increasing use during the last decade has expanded the options for targeted tumor therapies, and molecular tumor boards have grown accordingly. However, with increasing detection of genetic alterations, their interpretation has become more complex and error-prone, potentially introducing biases and reducing benefits in clinical practice. To facilitate interdisciplinary discussions of genetic alterations for treatment stratification between pathologists, oncologists, bioinformaticians, genetic counselors and medical scientists in specialized molecular tumor boards, several systems for the classification of variants detected by large-scale sequencing have been proposed. We review three recent and commonly applied classifications and discuss their individual strengths and weaknesses. Comparison of the classifications underlines the need for a clinically useful and universally applicable variant reporting system, which will be instrumental for efficient decision making based on sequencing analysis in oncology. Integrating these data, we propose a generalizable classification concept featuring a conservative and a more progressive scheme, which can be readily applied in a clinical setting.

Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method ‘greenCUT&RUN’ for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, ‘piggy-back’ DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique.

Barrett's esophagus (BE) is defined as metaplasia of the esophageal squamous epithelium with multiple cell layers into a single layer of intestinal columnar epithelial cells - or, in other words, skyscrapers are turned into town houses. The underlying pathomechanism(s) and the cell of origin of BE lesions have not been defined yet. However, four potential hypotheses for BE development have been suggested. The morphological changes during BE development are associated with rather well-described aberrant gene/protein expression patterns. However, the potential key regulators of this conversion process are still unclear. The process of metaplastic conversion is difficult to monitor in a spatiotemporal manner in vitro, and robust models are lacking. There is therefore a need for novel experimental systems. This review focuses on potential key regulators, microenvironmental influences, epigenetic alterations and experimental research systems related to BE.

According to the 2016 WHO classification lower-grade gliomas consist of three groups: IDH-mutated and 1p/19q co-deleted, IDH-mutated and IDH-wildtype tumors. The aim of this study was to evaluate the impact of surgical therapy for lower-grade gliomas with a particular focus on the molecular subgroups. This is a bi-centric retrospective analysis including 299 patients, who underwent treatment for lower-grade glioma between 1990 and 2016. All tumors were re-classified according to the 2016 WHO classification. Data concerning baseline and tumor characteristics, overall survival, different treatment modalities and functional outcome were analyzed. A total of 112 (37.5%) patients with IDH-mutation and 1p/19q co-deletetion, 86 (28.8%) patients with IDH-mutation and 101 (33.8%) patients with IDH-wildtype tumors were identified. The median overall survival (mOS) differed significantly between the groups (p

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered. Electronic supplementary material The online version of this article (10.1007/s00428-017-2288-7) contains supplementary material, which is available to authorized users.

Background: The EGF receptor is a therapeutic target in cancer cells, whereby mutations of EGFR and/or signalling members act as predictive markers. EGFR however also exhibits dynamic changes of subcellular localization, leading to STAT5 complex formation, nuclear translocation and induction of Aurora-A expression in squamous cancer cells. We previously described high EGFR and Aurora-A expression in esophageal cancer cells. Here, we investigated subcellular localization of EGFR and STAT5 in esophageal cancer cells. Results: Quantitative immunofluorescence analyses of four esophageal cancer cell lines reflecting esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) revealed that the subcellular localization of EGFR was shifted from a membranous to cytoplasmic localization upon EGF-stimulation in OE21 (ESCC) cells. Thereby, EGFR in part co-localized with E-Cadherin. In parallel, phosphorylated STAT5-Tyr694 appeared to increase in the nucleus and to decrease at the cell membrane. In three additional cell lines, EGFR was only marginally (Kyse-410/ESCC; OE19/EAC) and weakly (OE33, EAC) detectable at the cell membrane. Partial co-localization of EGFR and E-Cadherin occurred in OE33 cells. Post EGF-stimulation, EGFR was detected in the cytoplasm, resembling endosomal compartments. Furthermore, OE19 and OE33 exhibited nuclear STAT5-Tyr694 phosphorylation upon EGF-stimulation. None of the four cell lines showed nuclear EGFR expression and localization. Conclusion: In contrast to other (squamous) cancer cells, activation of EGFR in esophageal squamous cancer cells does not result in nuclear translocation of EGFR. Still, the subcellular localization of EGFR may influence STAT5-associated signaling pathways in esophageal cancer cells and hence possibly also the responses to ErbB, respective EGFR-targeted therapies.

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitro and in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach.

Experimental model systems identified phosphorylation of linker histone variant H1.4 at Ser 27 (H1.4S27p) as a novel mitotic mark set by Aurora B kinase. Here, we examined expression of Aurora B and H1.4S27p in colorectal carcinoma (CRC) cell lines (HCT116, DLD1, Caco-2, HT29) and tissue specimens (n = 36), in relation to microsatellite instability (MSI) status and ploidy. In vitro, Aurora B (pro-/meta-/anaphase) and H1.4S27p (pro-/metaphase) were localized in mitotic figures. The proportion of labeled mitoses was significantly different between cell lines for Aurora B (p = 0.019) but not for H1.4S27p (p = 0.879). For Aurora B, these differences were not associated with an altered Aurora B gene copy number (FISH) or messenger RNA (mRNA) expression level (qRT-PCR). Moreover, Aurora B expression and H1.4S27 phosphorylation were no longer coordinated during metaphase in aneuploid HT29 cells (p = 0.039). In CRCs, immunoreactivity for Aurora B or H1.4S27p did not correlate with T- or N-stage, grade, or MSI status. However, metaphase labeling of H1.4S27p was significantly higher in diploid than in aneuploid CRCs (p = 0.011). Aurora B was significantly correlated with H1.4S27p-positive metaphases in MSI (p = 0.010) or diploid (p = 0.003) CRCs. Finally, combined classification of MSI status and ploidy revealed a significant positive correlation of Aurora B with H1.4S27p in metaphases of diploid/MSI (p = 0.010) and diploid/microsatellite-stable (MSS; p = 0.031) but not of aneuploid/MSS (p = 0.458) CRCs. The present study underlines the functional link of Aurora B expression and H1.4S27p during specific phases of mitosis in diploid and/or MSI-positive CRCs in vitro and in situ. Importantly, the study shows that the coordination between Aurora B expression and phosphorylation of H1.4 at Ser 27 is lost in cycling aneuploid CRC cells.

Squamous cell carcinoma (SCC) is one of the most common skin cancers and causes significant morbidity. Although the expression of the epithelial adhesion molecule collagen XVII (ColXVII) has been linked to SCC invasion, only little is known about its mechanistic contribution. Here, we demonstrate that ColXVII expression is essential for SCC cell proliferation and motility. Moreover, it revealed that particularly the post-translational modification of ColXVII by ectodomain shedding is the major driver of SCC progression, because ectodomain-selective immunostaining was mainly localized at the invasive front of human cutaneous SCCs, and exclusive expression of a non-sheddable ColXVII mutant in SCC-25 cells inhibits their matrix-independent growth and invasiveness. This cell surface proteolysis, which is strongly elevated during SCC invasion and metastasis, releases soluble ectodomains and membrane-anchored endodomains. Both released ColXVII domains play distinct roles in tumor progression: the endodomain induces proliferation and survival, whereas the ectodomain accelerates invasiveness. Furthermore, specific blockage of shedding by monoclonal ColXVII antibodies repressed matrix-independent growth and invasion of SCC cells in organotypic co-cultures. Thus, selective inhibition of ColXVII shedding may offer a promising therapeutic strategy to prevent SCC progression.

Metastatic ovarian cancer has a dismal prognosis and current chemotherapeutic approaches have very limited success. Metadherin (MTDH) is expressed in human ovarian cancer tissue and its expression inversely correlates with patients overall survival. Consistent with these studies, we observed MTDH expression in tissue specimens of FIGO Stage III ovarian carcinomas (72/83 cases). However, we also observed this in normal human ovarian epithelial (OE) cells, which raised the question of whether MTDH-variants with functional differences exist. We identified a novel MTDH exon 11 skipping variant (MTDHdel) which was seen at higher levels in ovarian cancer compared to benign OE cells. We analyzed MTDH-binding partner interactions and found that 12 members of the small ribosomal subunit and several mRNA binding proteins bound stronger to MTDHdel than to wildtype MTDH which indicates differential effects on gene translation. Knockdown of MTDH in ovarian cancer cells reduced the amount of distant metastases and improved the survival of ovarian cancer-bearing mice. Selective overexpression of the MTDHdel enhanced murine and human ovarian cancer progression and caused a malignant phenotype in originally benign human OE cells. MTDHdel was detectable in microdissected ovarian cancer cells of some human tissue specimens of ovarian carcinomas. In summary, we have identified a novel MTDH exon 11 skipping variant that shows enhanced binding to small ribosomal subunit members and that caused reduced overall survival of ovarian cancer bearing mice. Based on the findings in the murine system and in human tissues, MTDHdel must be considered a major promalignant factor for ovarian cancer.

In recent years, there has been a growing interest in molecular imaging markers of tumor-induced angiogenesis. Several radiolabeled RGD (arginine, glycine, aspartate) peptides have been developed for PET imaging of αvβ3 integrins in the tumor vasculature, but there are only limited data on how angiogenesis inhibitors affect the tumor uptake of these peptides. Methods: Changes in 68Ga-NODAGA-c(RGDfK) peptide uptake were measured using PET during bevacizumab therapy of 2 αvβ3-negative squamous cell carcinoma cell lines (A-431 and FaDu) that induce αvβ3-positive neovasculature when transplanted into nude mice. Tumor uptake of 68Ga-NODAGA-c(RGDfK) was correlated to microvascular density, vascular morphology, and permeability as well as αvβ3 integrin expression. Results: Bevacizumab significantly inhibited growth of A-431 tumors and caused a significant reduction in microvascular density and αvβ3 integrin expression within 7 d after start of therapy. Bevacizumab also caused a normalization of blood vessel morphology and decreased tumor necrosis. However, 68Ga-NODAGA-c(RGDfK) uptake was significantly increased at day 7 of therapy and did not decrease until after 3 wk of treatment. In Fadu xenografts, bevacizumab therapy caused only a minor inhibition of tumor growth and minor changes in 68Ga-NODAGA-c(RGDfK) uptake. Conclusion: Uptake of radiolabeled RGD peptides is not necessarily decreased by effective antiangiogenic therapy. Early in the course of therapy a decrease in the expression of αvβ3 integrins may not be reflected by a decrease in the uptake of RGD peptides.

EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma‐carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti‐correlated with inducers of epithelial‐mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E‐box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1‐induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes.

ErbB family receptor tyrosine kinases (ErbBs) play a role in cell adhesion and migration and are frequently overexpressed in esophageal squamous cell carcinomas (ESCCs) or esophageal adenocarcinomas (EACs). Targeting ErbBs by tyrosine kinase inhibitors (TKIs) may therefore limit esophageal cancer cell migration. Here, we studied the impact of TKIs on ErbB dimerization, cell signaling pathways, and cell migration in three esophageal cell lines: OE21 (ESCC), OE33 (EAC), and Het-1A (non-neoplastic esophageal epithelium). In OE21 cells, the TKIs erlotinib, gefitinib, and lapatinib slightly affected epidermal growth factor receptor EGFR/EGFR, but not EGFR/HER2 dimerization as detected by in situ proximity ligation assay (in situ PLA). Still, TKIs inhibited ERK1/2, Akt, STAT3, and RhoA activity in OE21 cells, as assessed by Western blot, antibody arrays, and Rho GTPase effector pull-down assays. This was accompanied by reduced OE21 cell migration, induction of focal adhesions, and actin cytoskeleton reorganization, as shown by Oris™ migration assay and focal adhesion kinase (FAK)/phalloidin staining. In contrast, in OE33 cells, only lapatinib decreased STAT5, Src family kinase (SFK), and FAK activity as well as β-catenin expression. This impeded cell migration and induced morphological changes in OE33 cells. No alterations were seen for the non-neoplastic Het-1A cells. Thus, we identified the ErbB signaling network as regulator of esophageal cancer cell’s actin cytoskeleton, focal adhesions, and cell migration. ErbB targeted TKIs therefore also limit ESCC and EAC cell motility and migration. • Clinical tyrosine kinase inhibitors (TKIs) reduce esophageal cancer cell migration. • Loss of cell migration is linked to reduced Akt, ERK1/2, STAT (3 or 5), FAK, SFKs, and RhoA activity. • Clinical TKIs act via distinct signaling in the two main histotypes of esophageal cancer.

The inhibitors of DNA binding (ID) inhibit basic helix-loop-helix transcription factors and thereby guide cellular differentiation and proliferation. To elucidate the involvement of IDs in hematopoiesis and acute leukemias (AL), we analyzed ID2 and ID3 expression in hematopoiesis and leukemic blasts in bone marrow biopsies (BMB). BMB of healthy stem cell donors (n = 19) and BMB of patients with acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MD; n = 19), de novo AML (n = 20), B-acute lymphoblastic leukemia (B-ALL) (n = 23), T-ALL (n = 19), were immunohistochemically stained for ID2 and ID3 expression. The expression patterns were evaluated and quantified for each hematopoietic lineage and each leukemia subtype. In normal BMB, immature granulopoiesis showed weak ID2 and strong ID3 expression, which was lost during maturation (p < 0.001). Erythropoiesis remained negative for ID2/3 (p < 0.001). ID2/3 expression differed between immature granulopoiesis and leukemic blasts (p < 0.001). Moreover, differential ID2/3 expression was seen between AL subgroups: AML, especially AML-MD, had more ID2- (p < 0.001) and ID3-positive (p < 0.001) blasts than ALL. We show a comprehensive in situ picture of ID2/3 expression in hematopoiesis and AL. Morphologically, ID2/3 proteins seem to be involved in the granulopoietic maturation. Importantly, the distinct ID2/3 expression patterns in AL indicate a specific deregulation of ID2/3 in the various types of AL and may support subtyping of AL.