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Testing for genetic alterations in tumor tissue allows clinicians to identify patients who most likely will benefit from molecular targeted treatment. EXLIQUID – exploiting liquid biopsies to advance cancer precision medicine – investigates the potential of additional non-invasive tools for guiding therapy decisions and monitoring of advanced cancer patients. The term “liquid biopsy” (LB) refers to non-invasive analysis of tumor-derived circulating material such as cell-free DNA in blood samples from cancer patients. Although recent technological advances allow sensitive and specific detection of LB biomarkers, only few LB assays have entered clinical routine to date. EXLIQUID is a German Cancer Consortium (DKTK)-wide joint funding project that aims at establishing LBs as a minimally-invasive tool to analyze molecular changes in circulating tumor DNA (ctDNA). Here, we present the structure, clinical aim, and methodical approach of the new DKTK EXLIQUID consortium. Within EXLIQUID, we will set up a multicenter repository of high-quality LB samples from patients participating in DKTK MASTER and local molecular tumor boards, which use molecular profiles of tumor tissues to guide targeted therapies. We will develop LB assays for monitoring of therapy efficacy by the analysis of tumor mutant variants and tumor-specific DNA methylation patterns in ctDNA from these patients. By bringing together LB experts from all DKTK partner sites and exploiting the diversity of their particular expertise, complementary skills and technologies, the EXLIQUID consortium addresses the challenges of translating LBs into the clinic. The DKTK structure provides EXLIQUID a unique position for the identification of liquid biomarkers even in less common tumor types, thereby extending the group of patients benefitting from non-invasive LB testing. Besides its scientific aims, EXLIQUID is building a valuable precision oncology cohort and LB platform which will be available for future collaborative research studies within the DKTK and beyond.

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.

Micro (mi)RNAs are short, noncoding RNAs and deregulation of miRNAs and their targets are implicated in tumor generation and progression in many cancers. Meningiomas are mostly benign, slow growing tumors of the central nervous system with a small percentage showing a malignant phenotype. Following in silico prediction of potential targets of miR-34a-3p, SMAD4, FRAT1, and BCL2 have been confirmed as targets by dual luciferase assays with co-expression of miR-34a-3p and reporter gene constructs containing the respective 3'UTRs. Disruption of the miR-34a-3p binding sites in the 3'UTRs resulted in loss of responsiveness to miR-34a-3p overexpression. In meningioma cells, overexpression of miR-34a-3p resulted in decreased protein levels of SMAD4, FRAT1 and BCL2, while inhibition of miR-34a-3p led to increased levels of these proteins as confirmed by Western blotting. Furthermore, deregulation of miR-34a-3p altered cell proliferation and apoptosis of meningioma cells in vitro. We show that SMAD4, FRAT1 and BCL2 are direct targets of miR-34a-3p and that deregulation of miR-34a-3p alters proliferation and apoptosis of meningioma cells in vitro. As part of their respective signaling pathways, which are known to play a role in meningioma genesis and progression, deregulation of SMAD4, FRAT1 and BCL2 might contribute to the aberrant activation of these signaling pathways leading to increased proliferation and inhibition of apoptosis in meningiomas.

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.

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.

Wilms tumor (WT) is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA) processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA) differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT.

Micro (mi)RNAs are short, noncoding RNAs that downregulate gene expression by binding to specific sequences in the 3′ untranslated regions of their target genes and thereby either inhibiting the binding of the translation machinery or leading to degradation of the corresponding mRNAs.1 Recent research has attributed key roles to miRNAs in cancer initiation, progression, and metastasis as key regulators of gene expression, whose deregulation affects multiple cellular pathways, including angiogenesis, epithelial-to-mesenchymal transition (EMT), and signaling pathways crucial for proliferation.2 Meningiomas account for about 35% of all primary brain tumors and are classified into 3 malignancy grades and 15 histological subtypes.3 About 80% of meningiomas (World Health Organization [WHO] grade I) are slow-growing benign tumors, with most common histological subtypes being meningothelial, fibroblastic, and transitional meningioma. Meningiomas of WHO grades II and III represent 18%–20% and 1%–2%, respectively, of all meningiomas and are characterized by a more aggressive growth with increased recurrence rates and shorter disease-specific survival.4 Cytogenetic aberrations in meningioma include partial or complete loss of chromosome 22 and loss of chromosomes 1p, 6q, 9p, 10, 14q, and 18q and gains on chromosomes 1q, 9q, 12q, 15q, 17q, and 20q in high-grade meningiomas (reviewed by Perry et al5). Loss of expression of neurofibromatosis 2, a gene located on 22q, is more frequent in fibroblastic than in meningothelial meningioma, suggesting different drivers for tumorigenesis in the mesenchymal (fibroblastic) and epithelial (meningothelial) lineages of benign meningioma.6 Deregulated signaling pathways associated with meningioma include Wnt, hedgehog, transforming growth factor–β (TGFB), mitogen-activated protein kinase (MAPK)/phosphatidylinositol-3 kinase (PI3K), and vascular endothelial growth factor (VEGF) pathways,7 and a role for EMT in meningioma genesis has been recently proposed.8 Despite their undisputed importance in various cancer types, only a few miRNAs have been associated with meningiomas, including miR-200a, -145, -190, -29c-3p, and -219-5p.9–12 MiR-200a was found downregulated in benign meningioma, and its overexpression inhibited meningioma cell growth in vitro and in vivo. Targets of miR-200a include β-catenin, a major component of the Wnt signaling pathway frequently involved in meningioma genesis, and nonmuscle heavy chain IIb, a protein involved in cell migration and divison.10,12 Another study provided evidence that a combination of high expression of miR-190 and low expression of miR-29c-3p and miR-219-5p significantly correlates with increased recurrence rates in meningiomas.11 The only study on differential miRNA expression between low-grade and high-grade meningiomas focused on 5 known cancer-related miRNAs, showing that miR-145 is significantly downregulated in WHO grades II and III meningiomas.9 When reexpressed in higher-grade meningioma cell lines in vitro, miR-145 expression led to decreased proliferation and migratory potential and an increased sensitivity for apoptosis, suggesting an important antimigratory and antiproliferative role for miR-145 in higher-grade meningiomas. In the present study we aim at deepening the knowledge of miRNA deregulation in meningiomas of different histological subtypes and grades and exploring the clinical usability of miRNAs for prediction of higher-grade meningioma. Additionally, we try to link deregulated miRNAs to signaling pathways associated with meningioma initiation and progression using their validated and potential targets derived from an in silico analysis.

In a previous study, we identified thioredoxin domain containing 16 (TXNDC16) as a meningioma-associated Ag by protein macroarray screening. Serological screening detected autoantibodies against TXNDC16 exclusively in meningioma patients’ sera and not in sera of healthy controls. TXNDC16 was previously found to be an endoplasmic reticulum (ER)–luminal glycoprotein. In this study, we show an additional ER-associated localization of TXNDC16 in the cytosol by in vitro synthesis, molecular mass shift assay, and flow cytometry. We were able to show TXNDC16 secretion in different human cell lines due to masked and therefore nonfunctional ER retrieval motif. A previously indicated exosomal TXNDC16 secretion could not be confirmed in HEK293 cells. The secreted serum protein TXNDC16 is bound in circulating immune complexes, which were found both in meningioma and healthy blood donor sera. Employing a customized array with 163 overlapping TXNDC16 peptides and measuring autoantibody reactivity, we achieved discrimination of meningioma sera from healthy controls with an accuracy of 87.2% using a set of only five immunogenic TXNDC16 epitopes.