Andreas M. Hoff

Transforming Pluripotency: An Exon-Level Study of Malignancy-Specific Transcripts in Human Embryonal Carcinoma and Embryonic Stem Cells

To circumvent difficulties of isolating pure populations of cancer stem cells (CSCs) for the purpose of identifying malignancy-specific gene expression, we have compared exon-resolution transcriptomic profiles of 5 embryonal carcinoma (EC) cell lines, a histological subtype of germ cell tumor (GCT), to their nonmalignant caricature, specifically 6 human embryonic stem (ES) cell lines. Both cell types are readily accessible, and were purified for undifferentiated cells only. We identified a set of 28 differentially expressed genes, many of which had cancer and stemness roles. Overexpression of the recently discovered pluripotency gene NR5A2 in malignant EC cells revealed an intriguing indication of how WNT-mediated dysregulation of pluripotency is involved with malignancy. Expression of these 28 genes was further explored within 2 publically available data sets of primary EC tumors and normal testis. At the exon-level, alternative splicing events were detected in ZNF195, DNMT3B, and PMF1, and alternative promoters were detected for ASH2L and ETV5. These events were validated by reverse transcriptase-polymerase chain reaction-based methods in EC and ES lines, where the alternative splicing event in the de novo DNA methyltransferase DNMT3B may have functional consequences. In conclusion, we have identified malignancy-specific gene expression differences within a rigorous pluripotent stem cell context. These findings are of particular interest for both GCT and ES cell biology, and, in general, to the concept of CSCs.

High Frequency of Fusion Transcripts Involving TCF7L2 in Colorectal Cancer: Novel Fusion Partner and Splice Variants

VTI1A-TCF7L2 was reported as a recurrent fusion gene in colorectal cancer (CRC), found to be expressed in three out of 97 primary cancers, and one cell line, NCI-H508, where a genomic deletion joins the two genes [1]. To investigate this fusion further, we analyzed high-throughput DNA and RNA sequencing data from seven CRC cell lines, and identified the gene RP11-57H14.3 (ENSG00000225292) as a novel fusion partner for TCF7L2. The fusion was discovered from both genome and transcriptome data in the HCT116 cell line. By triplicate nested RT-PCR, we tested both the novel fusion transcript and VTI1A-TCF7L2 for expression in a series of 106 CRC tissues, 21 CRC cell lines, 14 normal colonic mucosa, and 20 normal tissues from miscellaneous anatomical sites. Altogether, 42% and 45% of the CRC samples expressed VTI1A-TCF7L2 and TCF7L2-RP11-57H14.3 fusion transcripts, respectively. The fusion transcripts were both seen in 29% of the normal colonic mucosa samples, and in 25% and 75% of the tested normal tissues from other organs, revealing that the TCF7L2 fusion transcripts are neither specific to cancer nor to the colon and rectum. Seven different splice variants were detected for the VTI1A-TCF7L2 fusion, of which three are novel. Four different splice variants were detected for the TCF7L2-RP11-57H14.3 fusion. In conclusion, we have identified novel variants of VTI1A-TCF7L2 fusion transcripts, including a novel fusion partner gene, RP11-57H14.3, and demonstrated detectable levels in a large fraction of CRC samples, as well as in normal colonic mucosa and other tissue types. We suggest that the fusion transcripts observed in a high frequency of samples are transcription induced chimeras that are expressed at low levels in most samples. The similar fusion transcripts induced by genomic rearrangements observed in individual cancer cell lines may yet have oncogenic potential as suggested in the original study by Bass et al.

Novel RNA variants in colorectal cancers

With an annual estimated incidence of 1.4 million, and a five-year survival rate of 60%, colorectal cancer (CRC) is a major clinical burden. To identify novel RNA variants in CRC, we analyzed exon-level microarray expression data from a cohort of 202 CRCs. We nominated 25 genes with increased expression of their 3′ parts in at least one cancer sample each. To efficiently investigate underlying transcript structures, we developed an approach using rapid amplification of cDNA ends followed by high throughput sequencing (RACE-seq). RACE products from the targeted genes in 23 CRC samples were pooled together and sequenced. We identified VWA2-TCF7L2, DHX35-BPIFA2 and CASZ1-MASP2 as private fusion events, and novel transcript structures for 17 of the 23 other candidate genes. The high-throughput approach facilitated identification of CRC specific RNA variants. These include a recurrent read-through fusion transcript between KLK8 and KLK7, and a splice variant of S100A2. Both of these were overrepresented in CRC tissue and cell lines from external RNA-seq datasets.

chimeraviz: a tool for visualizing chimeric RNA

Summary: Advances in high‐throughput RNA sequencing have enabled more efficient detection of fusion transcripts, but the technology and associated software used for fusion detection from sequencing data often yield a high false discovery rate. Good prioritization of the results is important, and this can be helped by a visualization framework that automatically integrates RNA data with known genomic features. Here we present chimeraviz, a Bioconductor package that automates the creation of chimeric RNA visualizations. The package supports input from nine different fusion‐finder tools: deFuse, EricScript, InFusion, JAFFA, FusionCatcher, FusionMap, PRADA, SOAPfuse and STAR‐FUSION. Availability and implementation: chimeraviz is an R package available via Bioconductor (https://bioconductor.org/packages/release/bioc/html/chimeraviz.html) under Artistic‐2.0. Source code and support is available at GitHub (https://github.com/stianlagstad/chimeraviz). Contact: rolf.i.skotheim@rr‐research.no Supplementary information: Supplementary data are available at Bioinformatics online.

Identification of novel fusion genes in testicular germ cell tumors

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men ages 15 to 44 years. Embryonal carcinomas (EC) comprise a subset of TGCTs that exhibit pluripotent characteristics similar to embryonic stem (ES) cells, but the genetic drivers underlying malignant transformation of ECs are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the existence of fusion genes or aberrant fusion transcript expression, we performed RNA sequencing of EC cell lines and their nonmalignant ES cell line counterparts. We identified eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. Four out of nine transcripts were found recurrently expressed in an extended panel of primary TGCTs and additional EC cell lines, but not in normal parenchyma of the testis, implying tumor-specific expression. Two of the recurrent transcripts involved an intrachromosomal fusion between RCC1 and HENMT1 located 80 Mbp apart and an interchromosomal fusion between RCC1 and ABHD12B. RCC1-ABHD12B and the ETV6 transcript variant were found to be preferentially expressed in the more undifferentiated TGCT subtypes. In vitro differentiation of the NTERA2 EC cell line resulted in significantly reduced expression of both fusion transcripts involving RCC1 and the ETV6 transcript variant, indicating that they are markers of pluripotency in a malignant setting. In conclusion, we identified eight novel fusion transcripts that, to our knowledge, are the first fusion genes described in TGCT and may therefore potentially serve as genomic biomarkers of malignant progression.

Multifocal Primary Prostate Cancer Exhibits High Degree of Genomic Heterogeneity

BACKGROUND Most primary prostate cancers are multifocal with individual tumors harboring different aggressiveness; however, the genomic heterogeneity among these tumors is poorly understood. OBJECTIVE To better understand the biological basis for clinical variability among different lesions, we sought to comprehensively characterize the heterogeneity of somatic gene mutations in multifocal prostate cancer. DESIGN, SETTING, AND PARTICIPANTS High-coverage whole-exome sequencing of 153 frozen tissue samples, taken from two to three distinct tumor foci and one non-cancerous area from each of 41 patients, covering a total of 89 tumor foci. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS State-of-the-art bioinformatics tools for mutation calling and copy number determination from whole-exome sequencing data. RESULTS AND LIMITATIONS We found a very high degree of interfocal heterogeneity among tumors, that is, 76% of pairwise-compared tumor foci from the same prostatectomy specimen had no point mutations in common and DNA copy number changes were rarely shared across cancer foci. The few point mutations shared across tumor foci were seldom in cancer-critical genes. CONCLUSIONS In this first large genomic heterogeneity study of primary prostate cancer, we observe that different tumor foci within the same patient are genetically distinct, only rarely sharing any somatic gene mutations, including those in cancer driver genes. This heterogeneity affects how genomics-based management of prostate cancer can be implemented, as information from all tumor foci is necessary to draw valid conclusions about the cancers genomic alterations. PATIENT SUMMARY Most primary prostate cancers consist of multiple tumors within the same organ, but little is known about their relationships. We have compared the sets of gene mutations among such tumors and found that they only exceptionally have any in common. This will influence treatment decisions in the future as each tumors mutations will render it unique and have to be considered to gain the best treatment results.

CRABP1, C1QL1 and LCN2 are biomarkers of differentiated thyroid carcinoma, and predict extrathyroidal extension

BackgroundThe prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis.MethodsWe used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers.ResultsWe identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC.ConclusionsWe conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.

Abstract 2700: Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men. Embryonal carcinoma (EC) comprises a histological subgroup of TGCT that exhibits pluripotent characteristics similar to embryonic stem (ES) cells. The genetic drivers underlying malignant transformation of TGCT, including ECs, are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the formation of fusion genes or expression of aberrant transcript variants, we have previously performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. From this dataset, we identified and characterized eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. These were the first fusion genes to be shown in TGCT (Hoff et al., Cancer Research, in press). To search for recurrence of the originally nominated TGCT specific fusion transcripts and other transcript variants in an external sample series, we have here parsed raw RNA sequencing data from 102 primary TGCTs obtained from The Cancer Genome Atlas (TCGA; unpublished). In this process, we have evaluated multiple fusion finder algorithms and approaches to determine the optimal method, on the basis of sensitivity and specificity, to query specific transcript variants and breakpoints in RNA sequencing datasets. We were able to detect the recurrent fusion transcripts; RCC1-ABHD12B, RCC1-HENMT1, CLEC6A-CLEC4D and the transcript variant of ETV6, which was previously detected from our in-house RNA sequencing dataset from EC cell lines. We also demonstrate that expression of several fusion transcripts correlate significantly with the histological subtype. In conclusion, the novel fusion transcripts and ETV6 transcript variant are expressed from a high fraction of primary TGCTs with low differentiation grade, as detected across independent sample series. Citation Format: Andreas M. Hoff, Sen Zhao, Bjarne Johannessen, Rolf I. Skotheim. Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2700.

Abstract 2235: High frequency of fusion transcripts involving TCF7L2 in colorectal cancer: Novel fusion partner and splice variants

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA VTI1A-TCF7L2 was reported as a recurrent fusion gene in colorectal cancer (CRC), found to be expressed in three out of 97 primary cancers, and one cell line, NCI-H508, where a genomic deletion joins the two genes (Bass et al. Nature Genetics, 2011). To investigate this fusion further, we analyzed high-throughput DNA and RNA sequencing data from seven CRC cell lines in addition to publicly available data from the NCI-H508 cell line. From both genome and transcriptome data, the gene RP11-57H14.3 (ENSG00000225292) was identified as a novel fusion partner for TCF7L2 in the HCT 116 cell line. The originally reported fusion VTI1A-TCF7L2 was identified as expected from the analysis of NCI-H508 data. By triplicate nested RT-PCR, we tested both the novel fusion transcript and VTI1A-TCF7L2 for expression in a series of 106 CRC tissues, 21 colon cancer cell lines, 14 normal colonic mucosa, and 20 normal tissues from miscellaneous anatomical sites. Altogether, 42% and 45% of the CRC samples expressed VTI1A-TCF7L2 and TCF7L2-RP11-57H14.3 fusion transcripts, respectively. The two fusion transcripts were both seen in 29% of the normal colonic mucosa samples, and in 25% and 75% of the tested normal tissues from other organs. Seven different splice variants were detected for the VTI1A-TCF7L2 fusion, of which three are novel. Four different splice variants were detected for the TCF7L2-RP11-57H14.3 fusion. In conclusion, we have identified novel variants of VTI1A-TCF7L2 fusion transcripts, including a novel fusion partner gene, RP11-57H14.3. We demonstrated detectable levels of TCF7L2 involving fusion transcripts in a large fraction of CRC samples, as well as in normal colonic mucosa and other normal tissue types, revealing that these fusions, at transcript level, are neither specific to cancer nor to the colon and rectum. Citation Format: Andreas M. Hoff, Torfinn Nome, Anne Cathrine Bakken, Torleiv O. Rognum, Arild Nesbakken, Rolf I. Skotheim. High frequency of fusion transcripts involving TCF7L2 in colorectal cancer: Novel fusion partner and splice variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2235. doi:10.1158/1538-7445.AM2014-2235