Chromosomal instability and a deregulated cell cycle are intrinsic features of high-risk gastrointestinal stromal tumours with a metastatic potential.

Patients with localised, high-risk gastrointestinal stromal tumours (GIST) benefit from adjuvant imatinib treatment. Still, approximately 40% of patients relapse within 3 years after adjuvant therapy and the clinical and histopathological features currently used for risk classification cannot precisely predict poor outcomes after standard treatment. This study aimed to identify genomic and transcriptomic profiles that could be associated with disease relapse and thus a more aggressive phenotype. Using a multi-omics approach, we analysed a cohort of primary tumours from patients with untreated, resectable high-risk GISTs. We compared patients who developed metastatic disease within 3 years after finishing adjuvant imatinib treatment and patients without disease relapse after more than 5 years of follow-up. Combining genomics and transcriptomics data, we identified somatic mutations and deregulated mRNA and miRNA genes intrinsic to each group. Our study shows that increased chromosomal instability (CIN), including chromothripsis and deregulated kinetochore and cell cycle signalling, separates high-risk samples according to metastatic potential. The increased CIN seems to be an intrinsic feature for tumours that metastasise and should be further validated as a novel prognostic biomarker for high-risk GIST.

Mutational analysis and protein profiling predict drug sensitivity in multiple myeloma cell lines.

Introduction: Multiple myeloma (MM) is a heterogeneous disease where cancer-driver mutations and aberrant signaling may lead to disease progression and drug resistance. Drug responses vary greatly, and there is an unmet need for biomarkers that can guide precision cancer medicine in this disease. Methods: To identify potential predictors of drug sensitivity, we applied integrated data from drug sensitivity screening, mutational analysis and functional signaling pathway profiling in 9 cell line models of MM. We studied the sensitivity to 33 targeted drugs and their association with the mutational status of cancer-driver genes and activity level of signaling proteins. Results: We found that sensitivity to mitogen-activated protein kinase kinase 1 (MEK1) and phosphatidylinositol-3 kinase (PI3K) inhibitors correlated with mutations in NRAS/KRAS, and PI3K family genes, respectively. Phosphorylation status of MEK1 and protein kinase B (AKT) correlated with sensitivity to MEK and PI3K inhibition, respectively. In addition, we found that enhanced phosphorylation of proteins, including Tank-binding kinase 1 (TBK1), as well as high expression of B cell lymphoma 2 (Bcl-2), correlated with low sensitivity to MEK inhibitors. Discussion: Taken together, this study shows that mutational status and signaling protein profiling might be used in further studies to predict drug sensitivities and identify resistance markers in MM.

Clonal evolution in primary breast cancers under sequential epirubicin and docetaxel monotherapy.

BACKGROUND: Subclonal evolution during primary breast cancer treatment is largely unexplored. We aimed to assess the dynamic changes in subclonal composition of treatment-naïve breast cancers during neoadjuvant chemotherapy. METHODS: We performed whole exome sequencing of tumor biopsies collected before, at therapy switch, and after treatment with sequential epirubicin and docetaxel monotherapy in 51 out of 109 patients with primary breast cancer, who were included in a prospectively registered, neoadjuvant single-arm phase II trial. RESULTS: There was a profound and differential redistribution of subclones during epirubicin and docetaxel treatment, regardless of therapy response. While truncal mutations and main subclones persisted, smaller subclones frequently appeared or disappeared. Reassessment of raw data, beyond formal mutation calling, indicated that the majority of subclones seemingly appearing during treatment were in fact present in pretreatment breast cancers, below conventional detection limits. Likewise, subclones which seemingly disappeared were still present, below detection limits, in most cases where tumor tissue remained. Tumor mutational burden (TMB) dropped during neoadjuvant therapy, and copy number analysis demonstrated specific genomic regions to be systematically lost or gained for each of the two chemotherapeutics. CONCLUSIONS: Sequential epirubicin and docetaxel monotherapy caused profound redistribution of smaller subclones in primary breast cancer, while early truncal mutations and major subclones generally persisted through treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00496795 , registered on July 4, 2007.

Combining a Universal Telomerase Based Cancer Vaccine With Ipilimumab in Patients With Metastatic Melanoma - Five-Year Follow Up of a Phase I/IIa Trial.

Background: Ipilimumab improves survival for patients with metastatic malignant melanoma. Combining a therapeutic cancer vaccine with ipilimumab may increase efficacy by providing enhanced anti-tumor immune responses. UV1 consists of three synthetic long peptides from human telomerase reverse transcriptase (hTERT). These peptides comprise epitopes recognized by T cells from cancer patients experiencing long-term survival following treatment with a first-generation hTERT vaccine, and generate long-lasting immune responses in cancer patients when used as monotherapy. The objective of this trial was to investigate the safety and efficacy of combining UV1 with ipilimumab in metastatic melanoma. Patients and Methods: In this phase I/IIa, single center trial [NCT02275416], patients with metastatic melanoma received repeated UV1 vaccinations, with GM-CSF as an adjuvant, in combination with ipilimumab. Patients were evaluated for safety, efficacy and immune response. Immune responses against vaccine peptides were monitored in peripheral blood by measuring antigen-specific proliferation and IFN-γ production. Results: Twelve patients were recruited. Adverse events were mainly diarrhea, injection site reaction, pruritus, rash, nausea and fatigue. Ten patients showed a Th1 immune response to UV1 peptides, occurring early and after few vaccinations. Three patients obtained a partial response and one patient a complete response. Overall survival was 50% at 5 years. Conclusion: Treatment was well tolerated. The rapid expansion of UV1-specific Th1 cells in the majority of patients indicates synergy between UV1 vaccine and CTLA-4 blockade. This may have translated into clinical benefit, encouraging the combination of UV1 vaccination with standard of care treatment regimes containing ipilimumab/CTLA-4 blocking antibodies.

Results of multigene panel testing in familial cancer cases without genetic cause demonstrated by single gene testing.

We have surveyed 191 prospectively sampled familial cancer patients with no previously detected pathogenic variant in the BRCA1/2, PTEN, TP53 or DNA mismatch repair genes. In all, 138 breast cancer (BC) cases, 34 colorectal cancer (CRC) and 19 multiple early-onset cancers were included. A panel of 44 cancer-predisposing genes identified 5% (9/191) pathogenic or likely pathogenic variants and 87 variants of uncertain significance (VUS). Pathogenic or likely pathogenic variants were identified mostly in familial BC individuals (7/9) and were located in 5 genes: ATM (3), BRCA2 (1), CHEK2 (1), MSH6 (1) and MUTYH (1), followed by multiple early-onset (2/9) individuals, affecting the CHEK2 and ATM genes. Eleven of the 87 VUS were tested, and 4/11 were found to have an impact on splicing by using a minigene splicing assay. We here report for the first time the splicing anomalies using this assay for the variants ATM c.3806A > G and BUB1 c.677C > T, whereas CHEK1 c.61G > A did not result in any detectable splicing anomaly. Our study confirms the presence of pathogenic or likely pathogenic variants in genes that are not routinely tested in the context of the above-mentioned clinical phenotypes. Interestingly, more than half of the pathogenic germline variants were found in the moderately penetrant ATM and CHEK2 genes, where only truncating variants from these genes are recommended to be reported in clinical genetic testing practice.

Androgen-dependent alternative mRNA isoform expression in prostate cancer cells.

Background: Androgen steroid hormones are key drivers of prostate cancer. Previous work has shown that androgens can drive the expression of alternative mRNA isoforms as well as transcriptional changes in prostate cancer cells. Yet to what extent androgens control alternative mRNA isoforms and how these are expressed and differentially regulated in prostate tumours is unknown. Methods: Here we have used RNA-Seq data to globally identify alternative mRNA isoform expression under androgen control in prostate cancer cells, and profiled the expression of these mRNA isoforms in clinical tissue. Results: Our data indicate androgens primarily switch mRNA isoforms through alternative promoter selection. We detected 73 androgen regulated alternative transcription events, including utilisation of 56 androgen-dependent alternative promoters, 13 androgen-regulated alternative splicing events, and selection of 4 androgen-regulated alternative 3' mRNA ends. 64 of these events are novel to this study, and 26 involve previously unannotated isoforms. We validated androgen dependent regulation of 17 alternative isoforms by quantitative PCR in an independent sample set. Some of the identified mRNA isoforms are in genes already implicated in prostate cancer (including LIG4, FDFT1 and RELAXIN), or in genes important in other cancers (e.g. NUP93 and MAT2A). Importantly, analysis of transcriptome data from 497 tumour samples in the TGCA prostate adenocarcinoma (PRAD) cohort identified 13 mRNA isoforms (including TPD52, TACC2 and NDUFV3) that are differentially regulated in localised prostate cancer relative to normal tissue, and 3 ( OSBPL1A, CLK3 and TSC22D3) which change significantly with Gleason grade and  tumour stage. Conclusions: Our findings dramatically increase the number of known androgen regulated isoforms in prostate cancer, and indicate a highly complex response to androgens in prostate cancer cells that could be clinically important.

Sample-Index Misassignment Impacts Tumour Exome Sequencing.

Sample pooling enabled by dedicated indexes is a common strategy for cost-effective and robust high-throughput sequencing. Index misassignment leading to mutual contamination between pooled samples has however been described as a general problem of the latest Illumina sequencing instruments utilizing exclusion amplification. Using real-life data from multiple tumour sequencing projects, we demonstrate that index misassignment can induce artefactual variant calls closely resembling true, high-quality somatic variants. These artefactual calls potentially impact cancer applications utilizing low allelic frequencies, such as in clonal analysis of tumours. We discuss the available countermeasures with an emphasis on improved library indexing methods, and provide software that can assist in the identification of variants that may be consequences of index misassignment.

Identification of genetic variants for clinical management of familial colorectal tumors.

BACKGROUND: The genetic mechanisms for families who meet the clinical criteria for Lynch syndrome (LS) but do not carry pathogenic variants in the mismatch repair (MMR) genes are still undetermined. We aimed to study the potential contribution of genes other than MMR genes to the biological and clinical characteristics of Norwegian families fulfilling Amsterdam (AMS) criteria or revised Bethesda guidelines. METHODS: The Hereditary Cancer Biobank of the Norwegian Radium Hospital was interrogated to identify individuals with a high risk of developing colorectal cancer (CRC) for whom no pathogenic variants in MMR genes had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by using our in-house designed TruSeq amplicon-based assay for targeted sequencing. RNA splicing- and protein-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as likely to affect splicing were experimentally analyzed by resorting to minigene assays. RESULTS: We identified a patient who met the revised Bethesda guidelines and carried a likely pathogenic variant in CHEK2 (c.470 T > C, p.I157T). In addition, 25 unique VUS were identified in 18 individuals, of which 2 exonic variants (MAP3K1 c.764A > G and NOTCH3 c.5854G >A) were analyzed in the minigene splicing assay and found not to have an effect on RNA splicing. CONCLUSIONS: Among high-risk CRC patients that fulfill the AMS criteria or revised Bethesda guidelines, targeted gene sequencing identified likely pathogenic variant and VUS in other genes than the MMR genes (CHEK2, NOTCH3 and MAP3K1). Our study suggests that the analysis of genes currently excluded from routine molecular diagnostic screens may confer cancer susceptibility.

Genetic variants of prospectively demonstrated phenocopies in BRCA1/2 kindreds.

BACKGROUND: In kindreds carrying path_BRCA1/2 variants, some women in these families will develop cancer despite testing negative for the family's pathogenic variant. These families may have additional genetic variants, which not only may increase the susceptibility of the families' path_BRCA1/2, but also be capable of causing cancer in the absence of the path_BRCA1/2 variants. We aimed to identify novel genetic variants in prospectively detected breast cancer (BC) or gynecological cancer cases tested negative for their families' pathogenic BRCA1/2 variant (path_BRCA1 or path_BRCA2). METHODS: Women with BC or gynecological cancer who had tested negative for path_BRCA1 or path_BRCA2 variants were included. Forty-four cancer susceptibility genes were screened for genetic variation through a targeted amplicon-based sequencing assay. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the ones most likely affecting pre-mRNA splicing were experimentally analyzed in a minigene assay. RESULTS: We identified 48 women who were tested negative for their family's path_BRCA1 (n = 13) or path_BRCA2 (n = 35) variants. Pathogenic variants in the ATM, BRCA2, MSH6 and MUTYH genes were found in 10% (5/48) of the cases, of whom 15% (2/13) were from path_BRCA1 and 9% (3/35) from path_BRCA2 families. Out of the 26 unique VUS, 3 (12%) were predicted to affect RNA splicing (APC c.721G > A, MAP3K1 c.764A > G and MSH2 c.815C > T). However, by using a minigene, assay we here show that APC c.721G > A does not cause a splicing defect, similarly to what has been recently reported for the MAP3K1 c.764A > G. The MSH2 c.815C > T was previously described as causing partial exon skipping and it was identified in this work together with the path_BRCA2 c.9382C > T (p.R3128X). CONCLUSION: All women in breast or breast/ovarian cancer kindreds would benefit from being offered genetic testing irrespective of which causative genetic variants have been demonstrated in their relatives.

Personal Cancer Genome Reporter: variant interpretation report for precision oncology.

Summary: Individual tumor genomes pose a major challenge for clinical interpretation due to their unique sets of acquired mutations. There is a general scarcity of tools that can (i) systematically interrogate cancer genomes in the context of diagnostic, prognostic, and therapeutic biomarkers, (ii) prioritize and highlight the most important findings and (iii) present the results in a format accessible to clinical experts. We have developed a stand-alone, open-source software package for somatic variant annotation that integrates a comprehensive set of knowledge resources related to tumor biology and therapeutic biomarkers, both at the gene and variant level. Our application generates a tiered report that will aid the interpretation of individual cancer genomes in a clinical setting. Availability and implementation: The software is implemented in Python/R, and is freely available through Docker technology. Documentation, example reports, and installation instructions are accessible via the project GitHub page: https://github.com/sigven/pcgr. Contact: sigven@ifi.uio.no. Supplementary information: Supplementary data are available at Bioinformatics online.

Potentially pathogenic germline CHEK2 c.319+2T>A among multiple early-onset cancer families.

To study the potential contribution of genes other than BRCA1/2, PTEN, and TP53 to the biological and clinical characteristics of multiple early-onset cancers in Norwegian families, including early-onset breast cancer, Cowden-like and Li-Fraumeni-like syndromes (BC, CSL and LFL, respectively). The Hereditary Cancer Biobank from the Norwegian Radium Hospital was used to identify early-onset BC, CSL or LFL for whom no pathogenic variants in BRCA1/2, PTEN, or TP53 had been found in routine diagnostic DNA sequencing. Forty-four cancer susceptibility genes were selected and analyzed by our in-house designed TruSeq amplicon-based assay for targeted sequencing. Protein- and RNA splicing-dedicated in silico analyses were performed for all variants of unknown significance (VUS). Variants predicted as the more likely to affect splicing were experimentally analyzed by minigene assay. We identified a CSL individual carrying a variant in CHEK2 (c.319+2T>A, IVS2), here considered as likely pathogenic. Out of the five VUS (BRCA2, CDH1, CHEK2, MAP3K1, NOTCH3) tested in the minigene splicing assay, only NOTCH3 c.14090C>T (p.Ser497Leu) showed a significant effect on RNA splicing, notably by inducing partial skipping of exon 9. Among 13 early-onset BC, CSL and LFL patients, gene panel sequencing identified a potentially pathogenic variant in CHEK2 that affects a canonical RNA splicing signal. Our study provides new information on genetic loci that may affect the risk of developing cancer in these patients and their families, demonstrating that genes presently not routinely tested in molecular diagnostic settings may be important for capturing cancer predisposition in these families.

Androgen Receptor Deregulation Drives Bromodomain-Mediated Chromatin Alterations in Prostate Cancer.

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

GSuite HyperBrowser: integrative analysis of dataset collections across the genome and epigenome.

Background: Recent large-scale undertakings such as ENCODE and Roadmap Epigenomics have generated experimental data mapped to the human reference genome (as genomic tracks) representing a variety of functional elements across a large number of cell types. Despite the high potential value of these publicly available data for a broad variety of investigations, little attention has been given to the analytical methodology necessary for their widespread utilisation. Findings: We here present a first principled treatment of the analysis of collections of genomic tracks. We have developed novel computational and statistical methodology to permit comparative and confirmatory analyses across multiple and disparate data sources. We delineate a set of generic questions that are useful across a broad range of investigations and discuss the implications of choosing different statistical measures and null models. Examples include contrasting analyses across different tissues or diseases. The methodology has been implemented in a comprehensive open-source software system, the GSuite HyperBrowser. To make the functionality accessible to biologists, and to facilitate reproducible analysis, we have also developed a web-based interface providing an expertly guided and customizable way of utilizing the methodology. With this system, many novel biological questions can flexibly be posed and rapidly answered. Conclusions: Through a combination of streamlined data acquisition, interoperable representation of dataset collections, and customizable statistical analysis with guided setup and interpretation, the GSuite HyperBrowser represents a first comprehensive solution for integrative analysis of track collections across the genome and epigenome. The software is available at: https://hyperbrowser.uio.no.

The rainfall plot: its motivation, characteristics and pitfalls.

BACKGROUND: A visualization referred to as rainfall plot has recently gained popularity in genome data analysis. The plot is mostly used for illustrating the distribution of somatic cancer mutations along a reference genome, typically aiming to identify mutation hotspots. In general terms, the rainfall plot can be seen as a scatter plot showing the location of events on the x-axis versus the distance between consecutive events on the y-axis. Despite its frequent use, the motivation for applying this particular visualization and the appropriateness of its usage have never been critically addressed in detail. RESULTS: We show that the rainfall plot allows visual detection even for events occurring at high frequency over very short distances. In addition, event clustering at multiple scales may be detected as distinct horizontal bands in rainfall plots. At the same time, due to the limited size of standard figures, rainfall plots might suffer from inability to distinguish overlapping events, especially when multiple datasets are plotted in the same figure. We demonstrate the consequences of plot congestion, which results in obscured visual data interpretations. CONCLUSIONS: This work provides the first comprehensive survey of the characteristics and proper usage of rainfall plots. We find that the rainfall plot is able to convey a large amount of information without any need for parameterization or tuning. However, we also demonstrate how plot congestion and the use of a logarithmic y-axis may result in obscured visual data interpretations. To aid the productive utilization of rainfall plots, we demonstrate their characteristics and potential pitfalls using both simulated and real data, and provide a set of practical guidelines for their proper interpretation and usage.

Use of liquid biopsies to monitor disease progression in a sarcoma patient: a case report.

BACKGROUND: Many patients experience local recurrence or metastases after receiving potentially curative treatment, and early detection of these events is important for disease control. Recent technological advances make it possible to use blood plasma containing circulating cell-free tumour DNA (ctDNA) as a liquid biopsy. In this case report we show how serial liquid biopsies can be used to monitor the disease course and detect disease recurrence in a sarcoma patient. CASE PRESENTATION: A 55-year-old male presented with a rapidly growing, painful palpable mass in the left groin region, and a biopsy revealed a high-grade malignant spindle cell sarcoma. No metastases were detected on radiologic imaging scans. Using targeted resequencing with a custom 900 cancer gene panel, eight somatic mutations among them KRAS and NF1, were identified in the primary tumour. Targeted resequencing of plasma cell-free DNA (ctDNA) collected before and after surgery and at disease progression confirmed the presence of six of eight mutations at all three time points. The ctDNA level, estimated from the somatic allele frequencies of these six mutations, was high in plasma taken at the time of surgery, at levels similar to the primary tumour. Detection of low levels of ctDNA three days after surgery indicated persistent microscopic disease. Repeated radiologic imaging six weeks postoperatively showed widespread metastatic disease in the lungs, skeleton and the pelvic region. At this time point there was a dramatic increase in the ctDNA level, reflecting the disease progression of the patient. The patient had an unusually aggressive cancer, and succumbed to the disease 13 weeks after surgery. CONCLUSIONS: This case report demonstrated that targeted resequencing of ctDNA from longitudinal collected plasma can be used to monitor disease progression in a soft tissue sarcoma patient, including manifestation of metastatic disease. The ctDNA represented the genomic profile of the tumour, supporting clinical use of liquid biopsies to identify tumour-specific mutations as well as recurrent disease.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability.

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

TP53 Mutation Spectrum in Smokers and Never Smoking Lung Cancer Patients.

BACKGROUND: TP53 mutations are among the most common mutations found in lung cancers, identified as an independent prognostic factor in many types of cancers. The purpose of this study was to investigate the frequency and prognostic impact of TP53 mutations in never-smokers and in different histological subtypes of lung cancer. METHODS: We analyzed tumor tissue from 394 non-small cell carcinomas including adenocarcinomas (n = 229), squamous cell carcinomas (n = 112), large cell carcinomas (n = 30), and others (n = 23) for mutations in TP53 by the use of Sanger sequencing (n = 394) and next generation sequencing (n = 100). RESULTS: TP53 mutations were identified in 47.2% of the samples, with the highest frequency (65%) of mutations among squamous cell carcinomas. Among never-smokers, 36% carried a TP53 mutation, identified as a significant independent negative prognostic factor in this subgroup. For large cell carcinomas, a significantly prolonged progression free survival was found for those carrying a TP53 mutation. In addition, the frequency of frameshift mutations was doubled in squamous cell carcinomas (20.3%) compared to adenocarcinomas (9.1%). CONCLUSION: TP53 mutation patterns differ between the histological subgroups of lung cancers, and are also influenced by smoking history. This indicates that the histological subtypes in lung cancer are genetically different, and that smoking-induced TP53 mutations may have a different biological impact than TP53 mutations occurring in never-smokers.

Transcriptomic Profiling of Tumor Aggressiveness in Sporadic Nonfunctioning Pancreatic Neuroendocrine Neoplasms.

OBJECTIVES: The aim of the study was to compare RNA sequencing data of sporadic nonfunctioning pancreatic neuroendocrine neoplasms (PNENs) to identify gene expression patterns that may be important for molecular differentiation of tumor aggressiveness. METHODS: RNA sequencing was performed on samples of sporadic nonfunctioning PNENs, grouped as tumors with mild behavior (nonmetastatic and Ki67 < 5%) or aggressive behavior (metastatic and Ki67 ≥ 5%), on an Illumina Genome Analyzer II platform. Bioinformatic analyses were performed on the resulting data. RESULTS: Of 22,810 identified transcripts from protein-coding genes, a set of 309 genes were significantly differentially expressed between the 2 groups, of which 166 were upregulated and 143 downregulated in the aggressive disease group. Among the top protein-coding upregulated genes, we found genes encoding proteins involved in DNA packaging, ability to taste, chromosome structuring, cytoskeleton structuring, and cell-cell signaling. Among the top protein-coding downregulated genes, we found genes encoding proteins involved in neuronal differentiation, cytoskeleton structuring, cell-cell signaling, and immunological processes. CONCLUSIONS: A higher degree of tumor aggressiveness in sporadic nonfunctioning PNENs seems to be associated with upregulation of genes involved in regulation of the cell cycle and cell division. Small sample size and lack of a replication set are limitations of this study.

Unscrambling the genomic chaos of osteosarcoma reveals extensive transcript fusion, recurrent rearrangements and frequent novel TP53 aberrations.

In contrast to many other sarcoma subtypes, the chaotic karyotypes of osteosarcoma have precluded the identification of pathognomonic translocations. We here report hundreds of genomic rearrangements in osteosarcoma cell lines, showing clear characteristics of microhomology-mediated break-induced replication (MMBIR) and end-joining repair (MMEJ) mechanisms. However, at RNA level, the majority of the fused transcripts did not correspond to genomic rearrangements, suggesting the involvement of trans-splicing, which was further supported by typical trans-splicing characteristics. By combining genomic and transcriptomic analysis, certain recurrent rearrangements were identified and further validated in patient biopsies, including a PMP22-ELOVL5 gene fusion, genomic structural variations affecting RB1, MTAP/CDKN2A and MDM2, and, most frequently, rearrangements involving TP53. Most cell lines (7/11) and a large fraction of tumor samples (10/25) showed TP53 rearrangements, in addition to somatic point mutations (6 patient samples, 1 cell line) and MDM2 amplifications (2 patient samples, 2 cell lines). The resulting inactivation of p53 was demonstrated by a deficiency of the radiation-induced DNA damage response. Thus, TP53 rearrangements are the major mechanism of p53 inactivation in osteosarcoma. Together with active MMBIR and MMEJ, this inactivation probably contributes to the exceptional chromosomal instability in these tumors. Although rampant rearrangements appear to be a phenotype of osteosarcomas, we demonstrate that among the huge number of probable passenger rearrangements, specific recurrent, possibly oncogenic, events are present. For the first time the genomic chaos of osteosarcoma is characterized so thoroughly and delivered new insights in mechanisms involved in osteosarcoma development and may contribute to new diagnostic and therapeutic strategies.