EGAGenomicsBauke Ylstrahttps://ega-archive.org/dacs/EGAC00001000181EGAEGA DAC EGAC00001000181EGAb.ylstra@amsterdamumc.nlAggressive genomic features in clinically indolent primary HHV8-negative effusion-based lymphoma.MiR expression profiles of paired primary colorectal cancer and metastases by next-generation sequencing.DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly.Prognostic relevance of CD163 and CD8 combined with EZH2 and gain of chromosome 18 in follicular lymphoma: a study by the Lunenburg Lymphoma Biomarker Consortium.Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing.Evolution of DNA repair defects during malignant progression of low-grade gliomas after temozolomide treatment.Expression of let-7i and miR-192 is associated with resistance to cisplatin-based chemoradiotherapy in patients with larynx and hypopharynx cancer.Mendeville Matias M, Roemer Margaretha G M MGM, van den Hout Mari F C M MFCM, Los-de Vries G Tjitske GT, Bladergroen Reno R, Stathi Phylicia P, Hijmering Nathalie J NJ, Rosenwald Andreas A, Ylstra Bauke B, de Jong Daphne DStevens Wendy B C WBC, Mendeville Matias M, Redd Robert R, Clear Andrew J AJ, Bladergroen Reno R, Calaminici Maria M, Rosenwald Andreas A, Hoster Eva E, Hiddemann Wolfgang W, Gaulard Philippe P, Xerri Luc L, Salles Gilles G, Klapper Wolfram W, Pfreundschuh Michael M, Jack Andrew A, Gascoyne Randy D RD, Natkunam Yasodha Y, Advani Ranjana R, Kimby Eva E, Sander Birgitta B, Sehn Laurie H LH, Hagenbeek Anton A, Raemaekers John J, Gribben John J, Kersten Marie José MJ, Ylstra Bauke B, Weller Edie E, de Jong Daphne DNeerincx M M, Sie D L S DL, van de Wiel M A MA, van Grieken N C T NC, Burggraaf J D JD, Dekker H H, Eijk P P PP, Ylstra B B, Verhoef C C, Meijer G A GA, Buffart T E TE, Verheul H M W HMPoel Dennis D, Rustenburg François F, Sie Daoud D, van Essen Hendrik F HF, Eijk Paul P PP, Bloemena Elisabeth E, Elhorst Benites Teresita T, van den Berg Madeleine C MC, Vergeer Marije R MR, Leemans René C RC, Buffart Tineke E TE, Ylstra Bauke B, Brakenhoff Ruud H RH, Verheul Henk M HM, Voortman Jens JScheinin Ilari I, Sie Daoud D, Bengtsson Henrik H, van de Wiel Mark A MA, Olshen Adam B AB, van Thuijl Hinke F HF, van Essen Hendrik F HF, Eijk Paul P PP, Rustenburg François F, Meijer Gerrit A GA, Reijneveld Jaap C JC, Wesseling Pieter P, Pinkel Daniel D, Albertson Donna G DG, Ylstra Bauke Bvan Thuijl Hinke F HF, Mazor Tali T, Johnson Brett E BE, Fouse Shaun D SD, Aihara Koki K, Hong Chibo C, Malmström Annika A, Hallbeck Martin M, Heimans Jan J JJ, Kloezeman Jenneke J JJ, Stenmark-Askmalm Marie M, Lamfers Martine L M ML, Saito Nobuhito N, Aburatani Hiroyuki H, Mukasa Akitake A, Berger Mitchell S MS, Söderkvist Peter P, Taylor Barry S BS, Molinaro Annette M AM, Wesseling Pieter P, Reijneveld Jaap C JC, Chang Susan M SM, Ylstra Bauke B, Costello Joseph F JFAllahyar Amin A, Pieterse Mark M, Swennenhuis Joost J, Los-de Vries G Tjitske GT, Yilmaz Mehmet M, Leguit Roos R, Meijers Ruud W J RWJ, van der Geize Robert R, Vermaat Joost J, Cleven Arjen A, van Wezel Tom T, Diepstra Arjan A, van Kempen Léon C LC, Hijmering Nathalie J NJ, Stathi Phylicia P, Sharma Milan M, Melquiond Adrien S J ASJ, de Vree Paula J P PJP, Verstegen Marjon J A M MJAM, Krijger Peter H L PHL, Hajo Karima K, Simonis Marieke M, Rakszewska Agata A, van Min Max M, de Jong Daphne D, Ylstra Bauke B, Feitsma Harma H, Splinter Erik E, de Laat Wouter W<h4>Objectives</h4>The majority of patients with locally advanced larynx or hypopharynx squamous cell carcinoma are treated with organ-preserving chemoradiotherapy (CRT). Clinical outcome following CRT varies greatly. We hypothesized that tumor microRNA (miRNA) expression is predictive for outcome following CRT.<h4>Methods</h4>Next-generation sequencing (NGS) miRNA profiling was performed on 37 formalin-fixed paraffin-embedded (FFPE) tumor samples. Patients with a recurrence-free survival (RFS) of less than 2 years and patients with late/no recurrence within 2 years were compared by differential expression analysis. Tumor-specific miRNAs were selected based on normal mucosa miRNA expression data from The Cancer Genome Atlas database. A model was constructed to predict outcome using group-regularized penalized logistic ridge regression. Candidate miRNAs were validated by RT-qPCR in the initial sample set as well as in 46 additional samples.<h4>Results</h4>Thirteen miRNAs were differentially expressed (p < 0.05, FDR < 0.1) according to outcome group. Initial class prediction in the NGS cohort (n = 37) resulted in a model combining five miRNAs and disease stage, able to predict CRT outcome with an area under the curve (AUC) of 0.82. In the RT-qPCR cohort (n = 83), 25 patients (30%) experienced early recurrence (median RFS 8 months; median follow-up 42 months). Class prediction resulted in a model combining let-7i-5p, miR-192-5p and disease stage, able to discriminate patients with good versus poor clinical outcome (AUC:0.80).<h4>Conclusion</h4>The combined miRNA expression and disease stage prediction model for CRT outcome is superior to using either factor alone. This study indicates NGS miRNA profiling using FFPE specimens is feasible, resulting in clinically relevant biomarkers.Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures. In practice, very useful profiles can be obtained with ∼0.1× genome coverage. We improve on previous methods by first implementing a combined correction for sequence mappability and GC content, and second, by applying this procedure to sequence data from the 1000 Genomes Project in order to develop a blacklist of problematic genome regions. A small subset of these blacklisted regions was previously identified by ENCODE, but the vast majority are novel unappreciated problematic regions. Our procedures are implemented in a pipeline called QDNAseq. We have analyzed over 1000 samples, most of which were obtained from the fixed tissue archives of more than 25 institutions. We demonstrate that for most samples our sequencing and analysis procedures yield genome profiles with noise levels near the statistical limit imposed by read counting. The described procedures also provide better correction of artifacts introduced by low DNA quality than prior approaches and better copy number data than high-resolution microarrays at a substantially lower cost.In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.MicroRNAs (miRs) have been recognized as promising biomarkers. It is unknown to what extent tumor-derived miRs are differentially expressed between primary colorectal cancers (pCRCs) and metastatic lesions, and to what extent the expression profiles of tumor tissue differ from the surrounding normal tissue. Next-generation sequencing (NGS) of 220 fresh-frozen samples, including paired primary and metastatic tumor tissue and non-tumorous tissue from 38 patients, revealed expression of 2245 known unique mature miRs and 515 novel candidate miRs. Unsupervised clustering of miR expression profiles of pCRC tissue with paired metastases did not separate the two entities, whereas unsupervised clustering of miR expression profiles of pCRC with normal colorectal mucosa demonstrated complete separation of the tumor samples from their paired normal mucosa. Two hundred and twenty-two miRs differentiated both pCRC and metastases from normal tissue samples (false discovery rate (FDR) <0.05). The highest expressed tumor-specific miRs were miR-21 and miR-92a, both previously described to be involved in CRC with potential as circulating biomarker for early detection. Only eight miRs, 0.5% of the analysed miR transcriptome, were differentially expressed between pCRC and the corresponding metastases (FDR <0.1), consisting of five known miRs (miR-320b, miR-320d, miR-3117, miR-1246 and miR-663b) and three novel candidate miRs (chr 1-2552-5p, chr 8-20656-5p and chr 10-25333-3p). These results indicate that previously unrecognized candidate miRs expressed in advanced CRC were identified using NGS. In addition, miR expression profiles of pCRC and metastatic lesions are highly comparable and may be of similar predictive value for prognosis or response to treatment in patients with advanced CRC.In follicular lymphoma, studies addressing the prognostic value of microenvironment-related immunohistochemical markers and tumor cell-related genetic markers have yielded conflicting results, precluding implementation in practice. Therefore, the Lunenburg Lymphoma Biomarker Consortium performed a validation study evaluating published markers. To maximize sensitivity, an end of spectrum design was applied for 122 uniformly immunochemotherapy-treated follicular lymphoma patients retrieved from international trials and registries. The criteria were: early failure, progression or lymphoma-related death <2 years <i>versus</i> long remission, response duration of >5 years. Immunohistochemical staining for T cells and macrophages was performed on tissue microarrays from initial biopsies and scored with a validated computer-assisted protocol. Shallow whole-genome and deep targeted sequencing was performed on the same samples. The 96/122 cases with complete molecular and immunohistochemical data were included in the analysis. <i>EZH2</i> wild-type (<i>P</i>=0.006), gain of chromosome 18 (<i>P</i>=0.002), low percentages of CD8+ cells (<i>P</i>=0.011) and CD163+ areas (<i>P</i>=0.038) were associated with early failure. No significant differences in other markers were observed, thereby refuting previous claims of their prognostic significance. Using an optimized study design, this Lunenburg Lymphoma Biomarker Consortium study substantiates wild-type <i>EZH2</i> status, gain of chromosome 18, low percentages of CD8+ cells and CD163+ area as predictors of early failure to immunochemotherapy in follicular lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP [-like]), while refuting the prognostic impact of various other markers.Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naïve LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency.falseThe CCA Genome Core is responsible for archiving the sequencing data generated by the Cancer Centre Amsterdam (CCA).Data Access Committee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