The Swedish childhood tumor biobank: systematic collection and molecular characterization of all pediatric CNS and other solid tumors in Sweden.

The Swedish Childhood Tumor Biobank (BTB) is a nonprofit national infrastructure for collecting tissue samples and genomic data from pediatric patients diagnosed with central nervous system (CNS) and other solid tumors. The BTB is built on a multidisciplinary network established to provide the scientific community with standardized biospecimens and genomic data, thereby improving knowledge of the biology, treatment and outcome of childhood tumors. As of 2022, over 1100 fresh-frozen tumor samples are available for researchers. We present the workflow of the BTB from sample collection and processing to the generation of genomic data and services offered. To determine the research and clinical utility of the data, we performed bioinformatics analyses on next-generation sequencing (NGS) data obtained from a subset of 82 brain tumors and patient blood-derived DNA combined with methylation profiling to enhance the diagnostic accuracy and identified germline and somatic alterations with potential biological or clinical significance. The BTB procedures for collection, processing, sequencing, and bioinformatics deliver high-quality data. We observed that the findings could impact patient management by confirming or clarifying the diagnosis in 79 of the 82 tumors and detecting known or likely driver mutations in 68 of 79 patients. In addition to revealing known mutations in a broad spectrum of genes implicated in pediatric cancer, we discovered numerous alterations that may represent novel driver events and specific tumor entities. In summary, these examples reveal the power of NGS to identify a wide number of actionable gene alterations. Making the power of NGS available in healthcare is a challenging task requiring the integration of the work of clinical specialists and cancer biologists; this approach requires a dedicated infrastructure, as exemplified here by the BTB.

Amplification of ERBB2 (HER2) in embryonal rhabdomyosarcoma: A potential treatment target in rare cases?

The ERBB2 gene encodes a receptor tyrosine kinase also known as HER2. The gene is amplified and overexpressed in one-fifth of breast carcinomas; patients with such tumors benefit from targeted treatment with trastuzumab or other drugs blocking the receptor. In addition, ERBB2 has been shown to be amplified and/or overexpressed in a variety of other malignancies. Notably, both alveolar and embryonal rhabdomyosarcoma (RMS), especially in children, often show increased expression of ERBB2. Although high-level amplification of the gene has not been described in RMS, its frequent expression at the cell surface of RMS cells has been exploited for chimeric antigen receptor T-cell (CAR T)-based treatment strategies. We here describe two cases of pediatric, fusion-negative embryonal RMS with high-level amplification of the ERBB2 gene. One patient is currently treated with conventional chemotherapy for a recently detected standard risk RMS, whereas the other patient died from metastatic disease. Both tumors displayed focal amplicons (210 and 274 Kb, respectively) in chromosome band 17q12, with proximal and distal borders corresponding to those typically seen in breast cancer. In both tumors, the ERBB2 amplicon correlated with high expression at the RNA and protein levels. Thus, breast cancer-like ERBB2 amplification is a very rare, but recurrent feature of pediatric RMS, and should be exploited as an alternative treatment target.

Implementing data on targeted therapy from the INFORM registry platform for children with relapsed cancer in Sweden.

Background: Advances in treatment of childhood malignancies have improved overall cure rates to 80%. Nevertheless, cancer is still the most common cause of childhood mortality in Sweden. The prognosis is particularly poor for relapse of high-risk malignancies. In the international INFORM registry, tumor tissue from patients with relapsed, refractory, or progressive pediatric cancer as well as from very-high risk primary tumors is biologically characterized using next-generation sequencing to identify possible therapeutic targets. We analyzed data from Swedish children included in the INFORM registry concerning patient characteristics, survival, sequencing results and whether targeted treatment was administered to the children based on the molecular findings. Methods: A registry-based descriptive analysis of 184 patients included in the INFORM registry in Sweden during 2016-2021. Results: The most common diagnoses were soft tissue and bone sarcomas followed by high grade gliomas [including diffuse intrinsic pontine glioma (DIPG)]. Complete molecular analysis was successful for 203/212 samples originating from 184 patients. In 88% of the samples, at least one actionable target was identified. Highly prioritized targets, according to a preset scale, were identified in 48 (24%) samples from 40 patients and 24 of these patients received matched targeted treatment but only six children within a clinical trial. No statistically significant benefit in terms of overall survival or progression free survival was observed between children treated with matched targeted treatment compared to all others. Conclusion: This international collaborative study demonstrate feasibility regarding sequencing of pediatric high-risk tumors providing molecular data regarding potential actionable targets to clinicians. For a few individuals the INFORM analysis was of utmost importance and should be regarded as a new standard of care with the potential to guide targeted therapy.

The potential of liquid biopsy for detection of the KIAA1549-BRAF fusion in circulating tumor DNA from children with pilocytic astrocytoma.

Background: Low-grade gliomas (LGGs) represent children's most prevalent central nervous system tumor, necessitating molecular profiling to diagnose and determine the most suitable treatment. Developing highly sensitive screening techniques for liquid biopsy samples is particularly beneficial, as it enables the early detection and molecular characterization of tumors with minimally invasive samples. Methods: We examined CSF and plasma samples from patients with pilocytic astrocytoma (PA) using custom multiplexed droplet digital polymerase chain reaction (ddPCR) assays based on whole genome sequencing data. These assays included a screening test to analyze BRAF duplication and a targeted assay for the detection of patient-specific KIAA1549::BRAF fusion junction sequences or single nucleotide variants. Results: Our findings revealed that 5 out of 13 individual cerebrospinal fluid (CSF) samples tested positive for circulating tumor DNA (ctDNA). Among these cases, 3 exhibited the KIAA1549::BRAF fusion, which was detected through copy number variation (CNV) analysis (n = 1) or a fusion-specific probe (n = 2), while 1 case each displayed the BRAF V600E mutation and the FGFR1 N577K mutation. Additionally, a quantitative analysis of cell-free DNA (cfDNA) concentrations in PA CSF samples showed that most cases had low cfDNA levels, below the limit of detection of our assay (<1.9 ng). Conclusions: While CNV analysis of CSF samples from LGGs still has some limitations, it has the potential to serve as a valuable complementary tool. Furthermore, it can also be multiplexed with other aberrations, for example, to the BRAF V600 test, to provide important insights into the molecular characteristics of LGGs.

Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors-a nationwide, prospective Swedish study.

Background: Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors. Methods: gWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients. Findings: The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35). Interpretation: Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients. Funding: The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.

Age dependence of tumor genetics in unfavorable neuroblastoma: arrayCGH profiles of 34 consecutive cases, using a Swedish 25-year neuroblastoma cohort for validation.

BACKGROUND: Aggressive neuroblastoma remains a significant cause of childhood cancer death despite current intensive multimodal treatment protocols. The purpose of the present work was to characterize the genetic and clinical diversity of such tumors by high resolution arrayCGH profiling. METHODS: Based on a 32K BAC whole-genome tiling path array and using 50-250K Affymetrix SNP array platforms for verification, DNA copy number profiles were generated for 34 consecutive high-risk or lethal outcome neuroblastomas. In addition, age and MYCN amplification (MNA) status were retrieved for 112 unfavorable neuroblastomas of the Swedish Childhood Cancer Registry, representing a 25-year neuroblastoma cohort of Sweden, here used for validation of the findings. Statistical tests used were: Fisher's exact test, Bayes moderated t-test, independent samples t-test, and correlation analysis. RESULTS: MNA or segmental 11q loss (11q-) was found in 28/34 tumors. With two exceptions, these aberrations were mutually exclusive. Children with MNA tumors were diagnosed at significantly younger ages than those with 11q- tumors (mean: 27.4 vs. 69.5 months; p=0.008; n=14/12), and MNA tumors had significantly fewer segmental chromosomal aberrations (mean: 5.5 vs. 12.0; p<0.001). Furthermore, in the 11q- tumor group a positive correlation was seen between the number of segmental aberrations and the age at diagnosis (Pearson Correlation 0.606; p=0.037). Among nonMNA/non11q- tumors (n=6), one tumor displayed amplicons on 11q and 12q and three others bore evidence of progression from low-risk tumors due to retrospective evidence of disease six years before diagnosis, or due to tumor profiles with high proportions of numerical chromosomal aberrations. An early age at diagnosis of MNA neuroblastomas was verified by registry data, with an average of 29.2 months for 43 cases that were not included in the present study. CONCLUSION: MNA and segmental 11q loss define two major genetic variants of unfavorable neuroblastoma with apparent differences in their pace of tumor evolution and in genomic integrity. Other possible, but less common, routes in the development of aggressive tumors are progression of low-risk infant-type lesions, and gene amplifications other than MYCN. Knowledge on such nosological diversity of aggressive neuroblastoma might influence future strategies for therapy.

Simultaneous Ultra-Sensitive Detection of Structural and Single Nucleotide Variants Using Multiplex Droplet Digital PCR in Liquid Biopsies from Children with Medulloblastoma.

Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.

Diagnostic Yield From a Nationwide Implementation of Precision Medicine for all Children With Cancer.

PURPOSE: Several studies have indicated that broad genomic characterization of childhood cancer provides diagnostically and/or therapeutically relevant information in selected high-risk cases. However, the extent to which such characterization offers clinically actionable data in a prospective broadly inclusive setting remains largely unexplored. METHODS: We implemented prospective whole-genome sequencing (WGS) of tumor and germline, complemented by whole-transcriptome sequencing (RNA-Seq) for all children diagnosed with a primary or relapsed solid malignancy in Sweden. Multidisciplinary molecular tumor boards were set up to integrate genomic data in the clinical decision process along with a medicolegal framework enabling secondary use of sequencing data for research purposes. RESULTS: During the study's first 14 months, 118 solid tumors from 117 patients were subjected to WGS, with complementary RNA-Seq for fusion gene detection in 52 tumors. There was no significant geographic bias in patient enrollment, and the included tumor types reflected the annual national incidence of pediatric solid tumor types. Of the 112 tumors with somatic mutations, 106 (95%) exhibited alterations with a clear clinical correlation. In 46 of 118 tumors (39%), sequencing only corroborated histopathological diagnoses, while in 59 cases (50%), it contributed to additional subclassification or detection of prognostic markers. Potential treatment targets were found in 31 patients (26%), most commonly ALK mutations/fusions (n = 4), RAS/RAF/MEK/ERK pathway mutations (n = 14), FGFR1 mutations/fusions (n = 5), IDH1 mutations (n = 2), and NTRK2 gene fusions (n = 2). In one patient, the tumor diagnosis was revised based on sequencing. Clinically relevant germline variants were detected in 8 of 94 patients (8.5%). CONCLUSION: Up-front, large-scale genomic characterization of pediatric solid malignancies provides diagnostically valuable data in the majority of patients also in a largely unselected cohort.

Novel amplifications in pediatric medulloblastoma identified by genome-wide copy number profiling.

Medulloblastoma (MB) is a WHO grade IV, invasive embryonal CNS tumor that mainly affects children. The aggressiveness and response to therapy can vary considerably between cases, and despite treatment, ~30% of patients die within 2 years from diagnosis. Furthermore, the majority of survivors suffer long-term side-effects due to severe management modalities. Several distinct morphological features have been associated with differences in biological behavior, but improved molecular-based criteria that better reflect the underlying tumor biology are in great demand. In this study, we profiled a series of 25 MB with a 32K BAC array covering 99% of the current assembly of the human genome for the identification of genetic copy number alterations possibly important in MB. Previously known aberrations as well as several novel focally amplified loci could be identified. As expected, the most frequently observed alteration was the combination of 17p loss and 17q gain, which was detected in both high- and standard-risk patients. We also defined minimal overlapping regions of aberrations, including 16 regions of gain and 18 regions of loss in various chromosomes. A few noteworthy narrow amplified loci were identified on autosomes 1 (38.89-41.97 and 84.89-90.76 Mb), 3 (27.64-28.20 and 35.80-43.50 Mb), and 8 (119.66-139.79 Mb), aberrations that were verified with an alternative platform (Illumina 610Q chips). Gene expression levels were also established for these samples using Affymetrix U133Plus2.0 arrays. Several interesting genes encompassed within the amplified regions and presenting with transcript upregulation were identified. These data contribute to the characterization of this malignant childhood brain tumor and confirm its genetic heterogeneity.

Phenotypically concordant and discordant monozygotic twins display different DNA copy-number-variation profiles.

The exploration of copy-number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic makeup between twins derived from the same zygote represent an irrefutable example of somatic mosaicism. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype by using two platforms for genome-wide CNV analyses and showed that CNVs exist within pairs in both groups. These findings have an impact on our views of genotypic and phenotypic diversity in monozygotic twins and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool for identifying disease-predisposition loci. Our results also imply that caution should be exercised when interpreting disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics.

Focal amplifications are associated with high grade and recurrences in stage Ta bladder carcinoma.

Urinary bladder cancer is a heterogeneous disease with tumors ranging from papillary noninvasive (stage Ta) to solid muscle infiltrating tumors (stage T2+). The risk of progression and death for the most frequent diagnosed type, Ta, is low, but the high incidence of recurrences has a significant effect on the patients' quality of life and poses substantial costs for health care systems. Consequently, the purpose of this study was to search for predictive factors of recurrence on the basis of genetic profiling. A clinically well characterized cohort of Ta bladder carcinomas, selected by the presence or absence of recurrences, was evaluated by an integrated analysis of DNA copy number changes and gene expression (clone-based 32K, respectively, U133Plus2.0 arrays). Only a few chromosomal aberrations have previously been defined in superficial bladder cancer. Surprisingly, the profiling of Ta tumors with a high-resolution array showed that DNA copy alterations are relatively common in this tumor type. Furthermore, we observed an overrepresentation of focal amplifications within high-grade and recurrent cases. Known (FGFR3, CCND1, MYC, MDM2) and novel candidate genes were identified within the loci. For example, MYBL2, a nuclear transcription factor involved in cell-cycle progression; YWHAB, an antiapoptotic protein; and SDC4, an important component of focal adhesions represent interesting candidates detected within two amplicons on chromosome 20, for which DNA amplification correlated with transcript up-regulation. The observed overrepresentation of amplicons within high-grade and recurrent cases may be clinically useful for the identification of patients who will benefit from a more aggressive therapy.

Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants.

Whole-genome sequencing (WGS) is a fundamental technology for research to advance precision medicine, but the limited availability of portable and user-friendly workflows for WGS analyses poses a major challenge for many research groups and hampers scientific progress. Here we present Sarek, an open-source workflow to detect germline variants and somatic mutations based on sequencing data from WGS, whole-exome sequencing (WES), or gene panels. Sarek features (i) easy installation, (ii) robust portability across different computer environments, (iii) comprehensive documentation, (iv) transparent and easy-to-read code, and (v) extensive quality metrics reporting. Sarek is implemented in the Nextflow workflow language and supports both Docker and Singularity containers as well as Conda environments, making it ideal for easy deployment on any POSIX-compatible computers and cloud compute environments. Sarek follows the GATK best-practice recommendations for read alignment and pre-processing, and includes a wide range of software for the identification and annotation of germline and somatic single-nucleotide variants, insertion and deletion variants, structural variants, tumour sample purity, and variations in ploidy and copy number. Sarek offers easy, efficient, and reproducible WGS analyses, and can readily be used both as a production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups. The Sarek source code, documentation and installation instructions are freely available at https://github.com/nf-core/sarek and at https://nf-co.re/sarek/.

A segmental maximum a posteriori approach to genome-wide copy number profiling.

MOTIVATION: Copy number profiling methods aim at assigning DNA copy numbers to chromosomal regions using measurements from microarray-based comparative genomic hybridizations. Among the proposed methods to this end, Hidden Markov Model (HMM)-based approaches seem promising since DNA copy number transitions are naturally captured in the model. Current discrete-index HMM-based approaches do not, however, take into account heterogeneous information regarding the genomic overlap between clones. Moreover, the majority of existing methods are restricted to chromosome-wise analysis. RESULTS: We introduce a novel Segmental Maximum A Posteriori approach, SMAP, for DNA copy number profiling. Our method is based on discrete-index Hidden Markov Modeling and incorporates genomic distance and overlap between clones. We exploit a priori information through user-controllable parameterization that enables the identification of copy number deviations of various lengths and amplitudes. The model parameters may be inferred at a genome-wide scale to avoid overfitting of model parameters often resulting from chromosome-wise model inference. We report superior performances of SMAP on synthetic data when compared with two recent methods. When applied on our new experimental data, SMAP readily recognizes already known genetic aberrations including both large-scale regions with aberrant DNA copy number and changes affecting only single features on the array. We highlight the differences between the prediction of SMAP and the compared methods and show that SMAP accurately determines copy number changes and benefits from overlap consideration.

Somatic mosaicism for copy number variation in differentiated human tissues.

Two major types of genetic variation are known: single nucleotide polymorphisms (SNPs), and a more recently discovered structural variation, involving changes in copy number (CNVs) of kilobase- to megabase-sized chromosomal segments. It is unknown whether CNVs arise in somatic cells, but it is, however, generally assumed that normal cells are genetically identical. We tested 34 tissue samples from three subjects and, having analyzed for each tissue < or =10(-6) of all cells expected in an adult human, we observed at least six CNVs, affecting a single organ or one or more tissues of the same subject. The CNVs ranged from 82 to 176 kb, often encompassing known genes, potentially affecting gene function. Our results indicate that humans are commonly affected by somatic mosaicism for stochastic CNVs, which occur in a substantial fraction of cells. The majority of described CNVs were previously shown to be polymorphic between unrelated subjects, suggesting that some CNVs previously reported as germline might represent somatic events, since in most studies of this kind, only one tissue is typically examined and analysis of parents for the studied subjects is not routinely performed. A considerable number of human phenotypes are a consequence of a somatic process. Thus, our conclusions will be important for the delineation of genetic factors behind these phenotypes. Consequently, biobanks should consider sampling multiple tissues to better address mosaicism in the studies of somatic disorders.

Profiling of copy number variations (CNVs) in healthy individuals from three ethnic groups using a human genome 32 K BAC-clone-based array.

To further explore the extent of structural large-scale variation in the human genome, we assessed copy number variations (CNVs) in a series of 71 healthy subjects from three ethnic groups. CNVs were analyzed using comparative genomic hybridization (CGH) to a BAC array covering the human genome, using DNA extracted from peripheral blood, thus avoiding any culture-induced rearrangements. By applying a newly developed computational algorithm based on Hidden Markov modeling, we identified 1,078 autosomal CNVs, including at least two neighboring/overlapping BACs, which represent 315 distinct regions. The average size of the sequence polymorphisms was approximately 350 kb and involved in total approximately 117 Mb or approximately 3.5% of the genome. Gains were about four times more common than deletions, and segmental duplications (SDs) were overrepresented, especially in larger deletion variants. This strengthens the notion that SDs often define hotspots of chromosomal rearrangements. Over 60% of the identified autosomal rearrangements match previously reported CNVs, recognized with various platforms. However, results from chromosome X do not agree well with the previously annotated CNVs. Furthermore, data from single BACs deviating in copy number suggest that our above estimate of total variation is conservative. This report contributes to the establishment of the common baseline for CNV, which is an important resource in human genetics.

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells.

Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.

Erratum to: Identification of mutations, gene expression changes and fusion transcripts by whole transcriptome RNAseq in docetaxel resistant prostate cancer cells.

[This corrects the article DOI: 10.1186/s40064-016-3543-0.].

Identification of mutations, gene expression changes and fusion transcripts by whole transcriptome RNAseq in docetaxel resistant prostate cancer cells.

Docetaxel has been the standard first-line therapy in metastatic castration resistant prostate cancer. The survival benefit is, however, limited by either primary or acquired resistance. In this study, Du145 prostate cancer cells were converted to docetaxel-resistant cells Du145-R and Du145-RB by in vitro culturing. Next generation RNAseq was employed to analyze these cell lines. Forty-two genes were identified to have acquired mutations after the resistance development, of which thirty-four were found to have mutations in published sequencing studies using prostate cancer samples from patients. Fourteen novel and 2 previously known fusion genes were inferred from the RNA-seq data, and 13 of these were validated by RT-PCR and/or re-sequencing. Four in-frame fusion transcripts could be transcribed into fusion proteins in stably transfected HEK293 cells, including MYH9-EIF3D and LDLR-RPL31P11, which were specific identified or up-regulated in the docetaxel resistant DU145 cells. A panel of 615 gene transcripts was identified to have significantly changed expression profile in the docetaxel resistant cells. These transcriptional changes have potential for further study as predictive biomarkers and as targets of docetaxel treatment.

Cytokines affect PDX-1 expression, insulin and proinsulin secretion from iNOS deficient murine islets.

In rodent islets, exposure to interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) induces expression of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) formation, which may inhibit islet function. However, cytokines may also induce NO-independent islet suppression. The present aim was to investigate the effect of cytokine exposure to iNOS deficient (iNOS-/-) mouse islets on various islet functions. Islets from iNOS-/- and wt mice exposed to IL-1beta or (IL-1beta + IFN-gamma) for 2-20 h showed different kinetics of glucose-stimulated insulin secretion. In iNOS-/- islets, IL-1beta at high glucose induced a delayed and prolonged stimulation of insulin secretion, and this was followed by an increase in phospholipase D mRNA expression. After 6 and 24 h, proinsulin convertase 1 and 2 (PC1 and PC2) mRNA expression was suppressed and proinsulin secretion increased from wt islets. In iNOS-/- islets, PC1 expression was recovered after 24 h, and there was no difference in proinsulin secretion. PDX-1 mRNA expression was suppressed independent of NO-formation. We conclude that cytokines induce both NO-dependent and NO-independent functional inhibition of murine beta-cells.

Whole Exome- and mRNA-Sequencing of an AT/RT Case Reveals Few Somatic Mutations and Several Deregulated Signalling Pathways in the Context of SMARCB1 Deficiency.

BACKGROUND: AT/RTs are rare aggressive brain tumours, mainly affecting young children. Most cases present with genetic inactivation of SMARCB1, a core member of the SWI/SNF chromatin-remodeling complex. We have performed whole exome- and mRNA-sequencing on an early onset AT/RT case for detection of genetic events potentially contributing to the disease. RESULTS: A de novo germline variant in SMARCB1, c.601C>T p.Arg201∗, in combination with somatic deletion of the healthy allele is likely the major tumour causing event. Only seven somatic small scale mutations were discovered (hitting SEPT03, H2BFM, ZIC4, HIST2H2AB, ZIK1, KRTAP6-3, and IFNA8). All were found with subclonal allele frequencies (range 5.7-17%) and none were expressed. However, besides SMARCB1, candidate genes affected by predicted damaging germline variants that were expressed were detected (KDM5C, NUMA1, and PCM1). Analysis of differently expressed genes revealed many dysregulated pathways in the tumour, such as cell cycle, CXCR4 pathway, GPCR-signalling, and neuronal system. FGFR1, CXCR4, and MDK were upregulated and may represent possible drug targets. CONCLUSION: The loss of SMARCB1 function leads to AT/RT development and deregulated genes and pathways. Additional predisposing events may however contribute. Studies utilizing NGS technologies in larger cohorts will probably identify recurrent genetic and epigenetic alterations and molecular subgroups with implications for clinical practice and development of targeted therapies.