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Background One targeted treatment option for isocitrate dehydrogenase (IDH)-wild-type glioblastoma focuses on tumors with fibroblast growth factor receptor 3::transforming acidic coiled-coil-containing protein 3 (FGFR3::TACC3) fusions. FGFR3::TACC3 fusion detection can be challenging, as targeted RNA next-generation sequencing (NGS) is not routinely performed, and immunohistochemistry is an imperfect surrogate marker. Fusion status can be determined using reverse transcription polymerase chain reaction (RT-PCR) on fresh frozen (FF) material, but sometimes only formalin-fixed, paraffin-embedded (FFPE) tissue is available. Aim To develop an RT-PCR assay to determine FGFR3::TACC3 status in FFPE glioblastoma samples. Methods Twelve tissue microarrays with 353 historical glioblastoma samples were immunohistochemically stained for FGFR3. Samples with overexpression of FGFR3 (n  = 13) were subjected to FGFR3::TACC3 RT-PCR on FFPE, using 5 primer sets for the detection of 5 common fusion variants. Fusion-negative samples were additionally analyzed with NGS (n  = 6), FGFR3 Fluorescence In Situ Hybridization (n  = 6), and RNA sequencing (n  = 5). Results Using RT-PCR on FFPE material of the 13 samples with FGFR3 overexpression, we detected an FGFR3::TACC3 fusion in 7 samples, covering 3 different fusion variants. For 5 of these FF was available, and the presence of the fusion was confirmed through RT-PCR on FF. With RNA sequencing, 1 additional sample was found to harbor an FGFR3::TACC3 fusion (variant not covered by current RT-PCR for FFPE). The frequency of FGFR3::TACC3 fusion in this cohort was 9/353 (2.5%). Conclusions RT-PCR for FGFR3::TACC3 fusions can successfully be performed on FFPE material, with a specificity of 100% and (due to limited primer sets) a sensitivity of 83.3%. This assay allows for the identification of potential targeted treatment options when only formalin-fixed tissue is available. [ABSTRACT FROM AUTHOR]

Correction to: Amplification of the PLAG-family genes - PLAGL1 and PLAGL2 - is a key feature of the novel tumor type CNS embryonal tumor with PLAGL amplification 6 Clinical outcomes of patients with CNS embryonal tumor with PLAGL gene amplification. a Kaplan-Meier plots showing OS and PFS stratified by subgroup and sex. Information about chemotherapy (CT) and radiotherapy (RT) treatment regarding the entire follow-up time is displayed in the squares on the left where available The original article has been corrected. [Extracted from the article]

Simple Summary: Chromosomal alterations and other structural variants have been recurrently identified in Wilms tumors (WT) and are promising biomarkers for risk stratification. Chromosome 1q gain occurs in one in three WTs and is associated with poor prognosis, but its impact on tumor biology remains unknown. Here, we investigated the mutational mechanisms and functional effects of chromosomal alterations in WTs, and in particular 1q gain. We identified subgroups of tumors with typical activated biological processes: muscle differentiation, immune system, kidney development and proliferation. Combining these subgroups with genomic data showed that tumors with 1q gain occur in all subgroups and can be associated with different functional effects. Also, 1q gain tumors differ in mutational mechanisms and co-occurring tumor-specific mutations. In conclusion, we identified subgroups of tumors with 1q gain and therefore propose that incorporating expression data in risk stratification could improve the clinical utility of 1q gain. Chromosomal alterations have recurrently been identified in Wilms tumors (WTs) and some are associated with poor prognosis. Gain of 1q (1q+) is of special interest given its high prevalence and is currently actively studied for its prognostic value. However, the underlying mutational mechanisms and functional effects remain unknown. In a national unbiased cohort of 30 primary WTs, we integrated somatic SNVs, CNs and SVs with expression data and distinguished four clusters characterized by affected biological processes: muscle differentiation, immune system, kidney development and proliferation. Combined genome-wide CN and SV profiles showed that tumors profoundly differ in both their types of 1q+ and genomic stability and can be grouped into WTs with co-occurring 1p−/1q+, multiple chromosomal gains or CN neutral tumors. We identified 1q+ in eight tumors that differ in mutational mechanisms, subsequent rearrangements and genomic contexts. Moreover, 1q+ tumors were present in all four expression clusters reflecting activation of various biological processes, and individual tumors overexpress different genes on 1q. In conclusion, by integrating CNs, SVs and gene expression, we identified subgroups of 1q+ tumors reflecting differences in the functional effect of 1q gain, indicating that expression data is likely needed for further risk stratification of 1q+ WTs. [ABSTRACT FROM AUTHOR]

Author(s): Amy A. Caudy [1, 3]; René F. Ketting [2, 3]; Scott M. Hammond [1, 4]; Ahmet M. Denli [1]; Anja M. P. Bathoorn [1, 2]; Bastiaan B. J. Tops [...]

Six of the samples grouped with the DNA methylation class infantile hemispheric glioma, other tumors clustered with various reference classes of glioma from low- to high-grade (Fig. To identify gliomas with structural alterations affecting chromosome 6q (around the I ROS1 i locus), we systematically evaluated copy-number data of our DNA methylation dataset encompassing 20,723 gliomas, irrespective of specific entity and WHO grade (Supplementary Fig. Similarly, no data exist to determine whether I ROS1 i fusion-positive gliomas, irrespective of histology, may share further biological features, potentially supporting a "ROS1-subtype" of gliomas. [Extracted from the article]

Introduction: The role of copy number variants (CNVs) in disease is now well established. In parallel NGS technologies, such as long-read technologies, there is continual development and data analysis methods continue to be refined. Clinical exome sequencing data is now a reality for many diagnostic laboratories in both congenital genetics and oncology. This provides the ability to detect and report both SNVs and structural variants, including CNVs, using a single assay for a wide range of patient cohorts. Areas covered: Currently, whole-genome sequencing is mainly restricted to research applications and clinical utility studies. Furthermore, detecting the full-size spectrum of CNVs as well as somatic events remains difficult for both exome and whole-genome sequencing. As a result, the full extent of genomic variants in an individual’s genome is still largely unknown. Recently, new sequencing technologies have been introduced which maintain the long-range genomic context, aiding the detection of CNVs and structural variants. Expert commentary: The development of long-read sequencing promises to resolve many CNV and SV detection issues but is yet to become established. The current challenge for clinical CNV detection is how to fully exploit all the data which is generated by high throughput sequencing technologies. [ABSTRACT FROM AUTHOR]

Endometrial carcinomas are histologically classified as endometrioid, assumed to originate from hyperplastic endometrium, or non-endometrioid carcinomas, assumed to originate from atrophic endometrium. However, both on a histological and a molecular level there are indications that there are more carcinoma types and carcinogenetic pathways. This study aims to analyze endometrial carcinogenesis on a molecular level. The presence of known KRAS, PIK3CA, AKT1, CTNNB1, BRAF, EGFR and NRAS mutations was studied in proliferative, atrophic and hyperplastic endometrium, endometrioid and serous carcinomas, and the endometrium next to these carcinomas, using single molecule Molecular Inversion Probes. Mutations were found in 9 (15%) of the 62 non atypical, and in 6 (18%) of the 34 atypical hyperplasia cases. In comparison, mutations were found in 1 (3%) of the simple, and 8 (30%) of the 27 complex hyperplasia cases. In 12/22 (55%) endometrioid carcinomas, a mutation was found. The KRAS gene was most often mutated in carcinomas next to hyperplastic endometrium, whereas PIK3CA and CTNNB1 mutations were found in endometrioid carcinomas with adjacent atrophic endometrium. Complex hyperplasia rather than atypical hyperplasia appears to be the most important lesion in the carcinogenesis of endometrioid carcinomas, and KRAS, PIK3CA and CTNNB1 mutations appear to play an important role in this process. Carcinogenesis of endometrioid carcinomas next to hyperplasia seems to be different to that of those next to atrophia. The value of these findings in managing endometrial hyperplasia and carcinoma should be studied. [ABSTRACT FROM AUTHOR]

Aims To investigate the spectrum of mutations in 20 genes involved in B-cell receptor and/or Toll-like receptor signalling resulting in activation of nuclear factor-κB (NF-κB) in 20 nodal marginal zone lymphomas (NMZLs), 20 follicular lymphomas (FLs), and 11 cases of B-cell lymphoma, unclassifiable (BCL-u). Methods and results Nodal marginal zone lymphomas were diagnosed according to strict criteria, including the expression of at least one putative marginal zone marker (MNDA and/or IRTA1). Cases that showed features of NMZL but did not fulfil all criteria were included as BCL-u. All FLs were required to have a BCL2 rearrangement. Mutations were found in: nine NMZLs, with recurrent mutations in TNFAIP3 and CD79B; 12 FLs, with recurrent mutations in TNFRSF14, TNFAIP3, and CARD11; and five cases of BCL-u, with recurrent mutations in TNFRSF14. TNFRSF14 mutations were present in FL and BCL-u, but not in any of the NMZLs. In the BCL-u group, TNFRSF14 mutations clustered with a FL immunophenotype. Conclusions These results suggest that TNFRSF14 mutations point towards a diagnosis of FL, and can be used in the sometimes difficult distinction between NMZL and FL, but to apply this in diagnostics would require confirmation in an independent cohort. In addition, the presence or absence of specific mutations in pathways converging on NF-κB could be important for decisions regarding targeted treatment. [ABSTRACT FROM AUTHOR]

The genetic cause underlying the development of multiple colonic adenomas, the premalignant precursors of colorectal cancer (CRC), frequently remains unresolved in patients with adenomatous polyposis. Here we applied whole-exome sequencing to 51 individuals with multiple colonic adenomas from 48 families. In seven affected individuals from three unrelated families, we identified a homozygous germline nonsense mutation in the base-excision repair (BER) gene NTHL1. This mutation was exclusively found in a heterozygous state in controls (minor allele frequency of 0.0036; n = 2,329). All three families showed recessive inheritance of the adenomatous polyposis phenotype and progression to CRC in at least one member. All three affected women developed an endometrial malignancy or premalignancy. Genetic analysis of three carcinomas and five adenomas from different affected individuals showed a non-hypermutated profile enriched for cytosine-to-thymine transitions. We conclude that a homozygous loss-of-function germline mutation in the NTHL1 gene predisposes to a new subtype of BER-associated adenomatous polyposis and CRC. [ABSTRACT FROM AUTHOR]

Background: The number of predictive biomarkers that will be necessary to assess in clinical practice will increase with the availability of drugs that target specific molecular alterations. Therefore, diagnostic laboratories are confronted with new challenges: costs, turn-around-time and the amount of material required for testing will increase with the number of tests performed on a sample. Our consortium of European clinical research laboratories set out to test if semi-conductor sequencing provides a solution for these challenges. Methods: We designed a multiplex PCR targeting 87 hotspot regions in 22 genes that are of clinical interest for lung and/ or colorectal cancer. The gene-panel was tested by 7 different labs in their own clinical setting using ion-semiconductor sequencing. Results: We analyzed 155 samples containing 112 previously identified mutations in the KRAS, EGFR en BRAF genes. Only 1 sample failed analysis due to poor quality of the DNA. All other samples were correctly genotyped for the known mutations, even as low as 2%, but also revealed other mutations. Optimization of the primers used in the multiplex PCR resulted in a uniform coverage distribution over the amplicons that allows for efficient pooling of samples in a sequencing run. Conclusions: We show that a semi-conductor based sequencing approach to stratify colon and lung cancer patients is feasible in a clinical setting. [ABSTRACT FROM AUTHOR]

Purpose: Predictive diagnostics play an increasingly important role in personalized medicine for cancer treatment. Whole-genome sequencing (WGS)-based treatment selection is expected to rapidly increase worldwide. This study aimed to calculate and compare the total cost of currently used diagnostic techniques and of WGS in treatment of non-small cell lung carcinoma (NSCLC), melanoma, colorectal cancer (CRC), and gastrointestinal stromal tumor (GIST) in the Netherlands.Methods: The activity-based costing (ABC) method was conducted to calculate total cost of included diagnostic techniques based on data provided by Dutch pathology laboratories and the Dutch-centralized cancer WGS facility. Costs were allocated to four categories: capital costs, maintenance costs, software costs, and operational costs.Results: The total cost per cancer patient per technique varied from € 58 (Sanger sequencing, three amplicons) to € 2925 (paired tumor-normal WGS). The operational costs accounted for the vast majority (over 90%) of the total per cancer patient technique costs.Conclusion: This study outlined in detail all costing aspects and cost prices of current and new diagnostic modalities used in treatment of NSCLC, melanoma, CRC, and GIST in the Netherlands. Detailed cost differences and value comparisons between these diagnostic techniques enable future economic evaluations to support decision-making. [ABSTRACT FROM AUTHOR]

Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically generated for this purpose, but also to extract insights through comparative analysis of the ever-increasing wealth of publically available phosphorylation data. [ABSTRACT FROM AUTHOR]

Comparative proteomics has emerged as a powerful approach to determine differences in protein abundance between biological samples. The introduction of stable-isotopes as internal standards especially paved the road for quantitative proteomics for comprehensive approaches to accurately determine protein dynamics. Metabolic labeling with 15N isotopes is applied to an increasing number of organisms, including Drosophila, C. elegans, and rats. However, 15N-enrichment is often suboptimal (<98%), which may hamper identification and quantitation of proteins. Here, we systematically investigated two independent 15N-Iabeled data sets to explore the influence of heavy nitrogen enrichment on the number of identifications as well as on the error in protein quantitation. We show that specifically larger. 15N-labeled peptides are under-represented when compared to their 14N counterparts and propose a correction method, which significantly increases the number of identifications. In addition, we developed a method that corrects for inaccurate peptide ratios introduced by incomplete 15N enrichment. This results in improved accuracy and precision of protein quantitation. Altogether, this study provides insight into the process of protein identification and quantitation, and the methods described here can be used to improve both qualitative and quantitative data obtained by labeling with heavy nitrogen with enrichment less than 100%. [ABSTRACT FROM AUTHOR]

EWSR1 belongs to the FET family of RNA-binding proteins including also Fused in Sarcoma (FUS), and TATA-box binding protein Associated Factor 15 (TAF15). As consequence of the multifunctional role of EWSR1 leading to a high frequency of transcription of the chromosomal region where the gene is located, EWSR1 is exposed to aberrations such as rearrangements. Consecutive binding to other genes leads to chimeric proteins inducing oncogenesis. The other TET family members are homologous. With the advent of widely used modern molecular techniques during the last decades, it has become obvious that EWSR1 is involved in the development of diverse benign and malignant tumors with mesenchymal, neuroectodermal, and epithelial/myoepithelial features. As oncogenic transformation mediated by EWSR1-fusion proteins leads to such diverse tumor types, there must be a selection on the multipotent stem cell level. In this review, we will focus on the wide variety of soft tissue and bone entities, including benign and malignant lesions, harboring EWSR1 rearrangement. Fusion gene analysis is the diagnostic gold standard in most of these tumors. We present clinicopathologic, immunohistochemical, and molecular features and discuss differential diagnoses. [ABSTRACT FROM AUTHOR]