Interchromosomal Translocations Research Articles

Different Tumor Types Share a Common Nuclear Map of Chromosome Territories.

Different tumor types are characterized by unique histopathological patterns including distinctive nuclear architectures. I hypothesized that the difference in nuclear appearance is reflected in different nuclear maps of chromosome territories, the discrete regions occupied by individual chromosomes in the interphase nucleus. To test this hypothesis, I used interchromosomal translocations (ITLs) as an analytical tool to map chromosome territories in 11 different tumor types from the TCGA PanCancer database encompassing 6003 tumors with 5295 ITLs. For each chromosome I determined the number and percentage of all ITLs for any given tumor type. Chromosomes were ranked according to the frequency and percentage of ITLs per chromosome. The ranking showed similar patterns for all tumor types. Chromosomes 1, 8, 11, 17, and 19 were ranked in the top quarter, accounting for 35.2% of 5295 ITLs, whereas chromosomes 13, 15, 18, 21, and X were in the bottom quarter, accounting for only 10.5% ITLs. The correlation between the chromosome ranking in the total group of 6003 tumors and the ranking in individual tumor types was significant, ranging from P < .0001 to .0033. Thus, contrary to my hypothesis, different tumor types share a common nuclear map of chromosome territories. Based on the large number of ITLs in 11 different types of malignancy one can discern a shared pattern of chromosome territories in cancer and propose a probabilistic model of chromosomes 1, 8, 11, 17, 19 in the center of the nucleus and chromosomes 13, 15, 18, 21, X at the periphery.

Interchromosomal translocation in neural progenitor cells exposed to L1 retrotransposition

LINE-1 (L1) elements are a class of transposons, comprising approximately 19% and 21% of the mouse and human genomes, respectively. L1 retrotransposons can reverse transcribe their own RNA sequence into a de novo DNA copy integrated into a new genomic location. This activity, known as retrotransposition, may induce genomic alterations, such as insertions and deletions. Interestingly, L1s can retrotranspose and generate more de novo L1 copies in brains than in other somatic tissues. Here, we describe for the first time interchromosomal translocation triggered by ectopic L1 retrotransposition in neural progenitor cells. Such an observation adds to the studies in neurological and psychiatric diseases that exhibited variation in L1 activity between diseased brains compared with controls, suggesting that L1 activity could be detrimental when de-regulated.

Novel Non-Coding, Coding and Structural Variants in Hairy Cell Leukemia from Whole Genome Transcriptome Sequencing

Background and Aims Hairy cell leukemia (HCL) is characterized by the BRAF V600E mutation in almost all cases. Other genomic abnormalities include co-mutations in KLF2 and CDKN1B in a proportion of patients (pts) as well as recurrent copy number (CN) abnormalities of 7q and chromosome 5. Targeted genomic approaches to date have not covered the full spectrum of genomic and transcriptomic abnormalities that may be present in HCL, therefore we aimed to comprehensively characterize HCL using whole genome transcriptome sequencing (WGTS) to detect novel genomic abnormalities. Methods WGTS was performed on 59 pts with HCL (peripheral blood [PB] n=36, bone marrow aspirates [BMA] n=23). WGTS libraries were sequenced on a NovaSeq and variant calling of sequence, CN and structural variants (SV) was performed using multiple tools including Strelka, Manta, STAR-Fusion, Arriba and FusionCatcher. ResultsBRAF V600E mutations were detected in all 59 pts. Co-mutations were detected in the known genes KLF2 (8%, 5/59), CDKN1B (8%, 5/59) KMT2C (5%, 3/59) and ARID1A (5%, 3/59). In addition we detected novel recurrently co-mutated genes including the JAK/STAT regulator PTPN6 (7%, 4/59), MAPK pathway regulator DUSP6 (5%, 3/59), SGK1 (5%, 3/59) and ACTB (5%, 3/59). CN abnormalities were detected in 32% (19/59) of pts. The most frequent CN change detected was loss of 7q (n=10) with a minimally deleted region (MDR) of approximately 6.2MB involving the telomere maintenance gene POT1. POT1 gene expression was significantly lower in patients harboring 7q deletion compared to those without (median log2CPM 2.41 vs 2.97, p<0.05). The MDR also involved the putative MAPK pathway inhibitor TMEM229A. Other recurrent CN changes included trisomy 5 (n=3) and monosomy 21 (n=3). Recurrent SVs were observed involving the TERT locus in 5/59 pts (8%) including two pts with chromosomal inversions and three with inter-chromosomal translocations. TERT SVs juxtaposed the TERT locus to known super-enhancers near genes overexpressed in HCL including MEF2C, PLEKHA2 and MTSS1. TERT expression was markedly elevated in pts with TERT SVs compared to those without (median log2CPM 2.2 vs -2.7, p<0.05). Non-coding TERT promoter mutations (TERTpmut) were identified in two patients (chr5:g.1295228G>A). Overall, 16/59 (27%) pts had genomic abnormalities affecting genes involved in telomere biology (including TERT promoter mutations, TERT SVs and deletions involving POT1). In order to exclude low variant allele frequency (VAF) TERTpmut we performed UMI-based error corrected sequencing (sensitivity 0.5% VAF) on an extension cohort of 19 pts with HCL and did not detect any low-level TERTpmut in this group. To understand the specificity of these findings in TERT for HCL, a focused evaluation of WGTS was performed on other patients with chronic B-cell neoplasms for both TERTpmut and TERT SVs resulting in locus transposition to regulatory elements. TERT SVs and/or TERTpmut were detected in 38% (24/64), 18% (7/40), 2% (6/349) and 24% (23/94) of patients with SBLPN (HCLv), SMZL, CLL and MCL respectively. TERT was over-expressed in patients with TERT SVs regardless of histological subtype. Finally, we assessed non-coding variants in pts with HCL by performing an enrichment analysis using bipartite graph models. Using this approach we detected a high frequency of activation-induced cytidine deaminase (AID) induced promoter/enhancer hypermutation of highly expressed genes including RHOH, MTSS1, ZFP36L1, TMSB4X and KLF2. Conclusion WGTS in this large cohort of pts with HCL has revealed a higher degree of genomic complexity than previously elucidated, with novel diverse coding and non-coding changes involving intracellular pathway regulators (DUSP6, PTPN6, TMEM229A), cytoskeletal proteins (ACTB, MTSS1) and recurrent genomic abnormalities affecting genes involved in telomere maintenance (TERT, POT1). Furthermore, our data reveals HCL has a high incidence of superenhancer juxtaposition and hypermutation associated with increased gene expression. These findings provide multiple future directions to investigate the unique biology of HCL and related neoplasms, in particular the specific consequences of regulatory element hypermutation and the avoidance of oncogene-induced senescence through aberrant telomere regulation.

Genomic Panorama and Clonal Evolutionary Trajectory of Classical Hairy Cell Leukemia

Classical Hairy Cell Leukemia (HCLc) is a rare B-cell malignancy characterized by hairy-like cell membrane projections. The cell of origin and pathway of malignant transformation in HCLc remain unknown. Early work using whole exome sequencing identified BRAFV600E as a genetic hallmark in HCLc (Tiacci et al 2011 and Weston-Bell et al 2016). However, no other recurrent coding mutations appeared, marking it an uncommon monogenic cancer. As BRAFV600E appears insufficient to drive tumorigenesis in other cancers (Dankort et al 2009; Rad et al 2013), it suggested that other driver alterations may be implicated in HCLc pathogenesis, perhaps in the non-coding genome, overlooked in exome studies alone. To probe this, we carried out whole genome sequencing (WGS) in HCLc. We established the genomic panorama, and importantly leveraged data to derive tumor phylogenies to determine the relative order of early driver mutational events that associate with the most recent common ancestor (MRCA) of all tumor cells. WGS was performed in 10 typical HCLc cases each paired with germline in purified cell sorted populations of disaggregated splenocytes. Sequencing involved 150 bp paired ends on Illumina HiSeq4000. Data analyses included identification of somatic single-nucleotide variants (SNVs) and small indels using CaVEMan and Pindel, respectively; copy-number aberrations (CNA) with Battenberg; and structural variants (SVs) with BRASS. The fitting of COSMIC mutational signatures was performed using mmsig. Mutational SNVs and CNA drivers were used for the analysis of tumor phylogenesis of individual tumors. We mapped the whole genome landscape in HCLc. Somatic coding variants emerged as previously reported. BRAFV600E was present as a clonal mutation in all HCLc tumors. Missense mutations in KLF2 (p.S275N and p.S275I) were identified in 2 tumors. Two somatic SNVs were detected in PRSS3 (p.D95G and p.R94S) and ANKRD30B (p.K388E and p.N392K) in 2 different cases respectively. In the noncoding genome, BCL6 and ZFP36L1 promoters were recurrently mutated in 5/10 cases. Driver mutations in BCL6 promoters have been reported in other B-cell malignancies to disrupt negative autoregulatory circuits (Pasqualucci et al 2003). Analysis of CNA showed a relatively stable genome (ploidy ~2). A large CNA chr5 gain was observed in a single tumor. Two additional focal CNA were spotlighted at chr17q25 (Gain) and chr20q11 (LOH) in two individual tumors. A recurrent interchromosomal translocation at chr12q14.3 locus was identified in 5 individual tumors, and an identical translocation at 14q21.3 with 15q11.2 observed in 2 tumors. Interestingly, novel kataegis-like mutations were observed at TRB loci across all HCLc cohort samples. We further identified somatic hypermutation (SHM) imprints in non-Ig loci, notably in BCL6. The mutational rate of AID targets at TRB and non-Ig loci were comparable to SHM occurring in Ig IGV genes. Deciphering the mutational signatures in HCLc genomes highlighted SBS9, a mutational signature associated with SHM in B-cell leukemia as one of the most relevant mechanisms, allied to the clock-like signatures of SBS1 and SBS5. Next, we utilized estimates of the cancer cell fraction to define which mutations are clonal and associate with the MRCA, and which mutations occured subclonally to derive individual tumor phylogenetic trees using methodology we have reported previously (Rabbie et al 2020; Ansari-Pour 2021). Here, analyses of clonal trajectories revealed that BRAFV600E occurred prior to subclonal diversification in MRCA in all cases. Significantly however, 1 or 2 additional driver mutations were conjointly detected with BRAFV600E in the MRCA in 6/10 HCLc cases prior to any subclonal evolution. Our data provide a comprehensive map of the whole genome in HCLc for the first time. Clonal evolutionary trajectories reveal heterogeneous genetic origins in HCLc, in which BRAFV600E is the sole driver mutation in one subset of disease, but requires 1 or 2 additional mutational drivers in the other subset to promote malignant transformation. Thus, unlike other cancers, BRAFV600E alone appears to suffice to induce malignant transformation in some HCLc. Additionally, SHM in non-Ig loci including BCL6 may implicate early traffic of the tumor cell of origin through the germinal center and early exit prior to acquisition of CD27, or mutational events at ectopic sites.

Optical genome mapping identifies clinically relevant genomic rearrangements in prostate cancer biopsy sample

BackgroundProstate cancer (PCa) is characterized by complex genomic rearrangements such as the ETS oncogene family fusions, yet the clinical relevance is not well established. While paneled genetic tests of DNA repair genes are recommended in advanced PCa, conventional genomic or cytogenetic tools are not ideal for genome-wide screening of structural variations (SVs) such as balanced translocation due to cost and/or resolution issues.MethodsIn this study, we tested the feasibility of whole-genome optical genomic mapping (OGM), a newly developed platform for genome-wide SV analysis to detect complex genomic rearrangements in consecutive unselected PCa samples from MRI/US-fusion targeted biopsy.ResultsWe tested ten samples, and nine (90%) passed quality check. Average mapping rate and coverage depth were 58.1 ± 23.7% and 157.3 ± 97.7×, respectively (mean ± SD). OGM detected copy number alterations such as chr6q13 loss and chr8q12-24 gain. Two adjacent tumor samples were distinguished by inter/intra-chromosomal translocations, revealing that they’re from the same ancestor. Furthermore, OGM detected large deletion of chr13q13.1 accompanied by inter-chromosomal translocation t(13;20)(q13.1;p13) occurring within BRCA2 gene, suggesting complete loss of function.ConclusionIn conclusion, clinically relevant genomic SVs were successfully detected in PCa samples by OGM. We suggest that OGM can complement panel sequencing of DNA repair genes BRCA1/2 or ATM in high-risk PCa.

Translocation detection from Hi-C data via scan statistics.

Recent Hi-C technology enables more comprehensive chromosomal conformation research, including the detection of structural variations, especially translocations. In this paper, we formulate the interchromosomal translocation detection as a problem of scan clustering in a spatial point process. We then develop TranScan, a new translocation detection method through scan statistics with the control of false discovery. The simulation shows that TranScan is more powerful than an existing sophisticated scan clustering method, especially under strong signal situations. Evaluation of TranScan against current translocation detection methods on realistic breakpoint simulations generated from real data suggests better discriminative power under the receiver-operating characteristic curve. Power analysis also highlights TranScan's consistent outperformance when sequencing depth and heterozygosity rate is varied. Comparatively, Type I error rate is lowest when evaluated using a karyotypically normal cell line. Both the simulation and real data analysis indicate that TranScan has great potentials in interchromosomal translocation detection using Hi-Cdata.

EagleC: A deep-learning framework for detecting a full range of structural variations from bulk and single-cell contact maps.

The Hi-C technique has been shown to be a promising method to detect structural variations (SVs) in human genomes. However, algorithms that can use Hi-C data for a full-range SV detection have been severely lacking. Current methods can only identify interchromosomal translocations and long-range intrachromosomal SVs (>1 Mb) at less-than-optimal resolution. Therefore, we develop EagleC, a framework that combines deep-learning and ensemble-learning strategies to predict a full range of SVs at high resolution. We show that EagleC can uniquely capture a set of fusion genes that are missed by whole-genome sequencing or nanopore. Furthermore, EagleC also effectively captures SVs in other chromatin interaction platforms, such as HiChIP, Chromatin interaction analysis with paired-end tag sequencing (ChIA-PET), and capture Hi-C. We apply EagleC in more than 100 cancer cell lines and primary tumors and identify a valuable set of high-quality SVs. Last, we demonstrate that EagleC can be applied to single-cell Hi-C and used to study the SV heterogeneity in primary tumors.

Dissection of the expressed actionable fusions’ repertoire in solid tumors tested serially in the clinical setting across 14 cancer types.

e18804 Background: More frequently, select gene fusions have been tested in clinical practice as more approvals of very effective therapies targeting oncogenic chimeric fusion proteins. Large studies based on DNA-based NGS testing alongside clinical trials have established the prevalence of clinically actionable gene fusions such as NTRK1/2/3. However, much less is known about the broader repertoire and prevalence of bonafide expressed gene fusions. Methods: 6009 tumors across 14 cancer types were tested in our clinical laboratory between January and October 2021. Enrichment was performed using a custom hybridization-based capture after RNA to cDNA conversion with custom designed probes targeting &gt; 1104 different genes and enriched for 2230 specific RNA fusions to maximize sensitivity and breadth before sequencing ̃50 million 150x150 reads/sample on a NovaSeq 6000. Data was deidentified and aggregated according an approved IRB. Statistical significance was calculated by Fisher’s exact test. Results: We found that 37% of tumors tested carry &gt; 1 expressed chimeric transcript and that 7.2% of tumors (n = 431) express a gene fusion involving one of 19 actionable gene families/pathways targeted by an approved therapy. The most frequent involved FGFR1/2/3/4 (15%), ALK (14%), NTRK1/2/3 (12%), BRAF, ERBB2, RET and MET (6% each), RAS/RAF/MAPK and CDK4/6 pathway (5%). These fusions imply intra- and inter-chromosomal translocations on all chromosomes, especially on chromosomes 1, 3, 6, 7, 11, 12 and 17. NTRK1/2/3 fusions prevalence (0.9%) is significantly higher (p = 1.1e-10) than the 0.3% reported on a recent study from a large dataset (296K samples) based on DNA-sequencing testing (1). Moreover, we found that 32% of the NTRK partners were unique vs. 10% (p = 6.5e-08) from the Westphalen et al. study (1). 14 of 25 unique NTRK partners have not been described on large datasets (1) and only 3 have been reported on the literature as NTRK partners. Among them, we identified the hypoxia-induced neovascularization gene HIF1A, as a novel in frame 3’ fusion partner of NTRK3 [t(15;14)(15q25.3; 14q23.1). Finally, NTRK fusions were detected across 14 cancer types with higher frequency on brain tumors (17%), followed by lung cancers, colorectal and head and neck cancer (13% each) and sarcomas (12%). Conclusions: Gene fusions involving common actionable genes showed a high prevalence in the clinical setting. This study confirms that the number and breadth of gene fusions can present with common actionable information in &gt; 7% of clinical cases. We report here 14 new unique NTRK partners. Despite a 50 times smaller dataset, 3-fold more actionable expressed NTRK fusions involving novel partners were detected with a targeted RNA based hybridization approach in lieu of DNA based testing supporting the benefit of this strategy. Overexpression and translation can be confirmed only by this approach.

Long-read sequencing unveils high-resolution HPV integration and its oncogenic progression in cervical cancer

Integration of human papillomavirus (HPV) DNA into the human genome is considered as a key event in cervical carcinogenesis. Here, we perform comprehensive characterization of large-range virus-human integration events in 16 HPV16-positive cervical tumors using the Nanopore long-read sequencing technology. Four distinct integration types characterized by the integrated HPV DNA segments are identified with Type B being particularly notable as lacking E6/E7 genes. We further demonstrate that multiple clonal integration events are involved in the use of shared breakpoints, the induction of inter-chromosomal translocations and the formation of extrachromosomal circular virus-human hybrid structures. Combined with the corresponding RNA-seq data, we highlight LINC00290, LINC02500 and LENG9 as potential driver genes in cervical cancer. Finally, we reveal the spatial relationship of HPV integration and its various structural variations as well as their functional consequences in cervical cancer. These findings provide insight into HPV integration and its oncogenic progression in cervical cancer.

Comprehensive Analysis of CRISPR-Cas9 Editing Outcomes in Yeast Xanthophyllomyces dendrorhous.

DNA repair after Cas9 cutting can result in deletions/insertions, genomic rearrangements, and rare nucleotide substitutions. However, most work has only focused on deletions/insertions resulting from repair after CRISPR-Cas9 action. Here, we comprehensively analyzed the editing outcomes induced by CRISPR-Cas9 treatment in yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing and identified diverse DNA repair patterns, including DNA deletions, interchromosomal translocations, and on-target nucleotide substitutions (point mutations). Some deletions were observed repeatedly, and others, especially large deletions, varied in size. Genome sequencing and structural variation analysis showed that the interchromosomal translocations happened between Cas9 target sites and the endogenous ADH4 promoter. In contrast to previous studies, analysis revealed that the on-target point mutations were not random. Importantly, these point mutations showed strong sequence dependence that is not consistent with previous work in Hela cells, where CRISPR-mediated substitutions were found to lack sequence dependence and conversion preferences. Finally, we found that the non-homologous end joining components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the production of both point mutations and deletions. This work expands our knowledge of CRISPR-Cas9 mediated DNA repair.

Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Comparison of structural variants in the whole genome sequences of two Medicago truncatula ecotypes: Jemalong A17 and R108

BackgroundStructural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes–Jemalong A17 and R108– that have been extensively used in various legume biology studies.ResultsTo catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis.ConclusionsOur results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

Background & AimsHBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.MethodsDNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.ResultsHBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.ConclusionsTargeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.Lay summaryFresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

BCL6-SPECC1L: A Novel Fusion Gene in Nasopharyngeal Carcinoma.

Background: Nasopharyngeal carcinomas (NPCs) are malignant tumors originating from the lining epithelium of the nasopharynx. Fusion genes have been confirmed to play important roles in the occurrence and development of various malignant tumors, but the role of fusion genes in NPC is poorly understood. We aimed to explore new fusion genes that promote the occurrence and development of NPC. Methods: RNA-seq was used to search for interchromosomal translocations in 18 NPC tissues. Polymerase chain reaction (PCR) and Sanger sequencing were applied to verify the presence of BCL6-SPECC1L (BS); quantitative PCR (qPCR) and Western blotting were used to measure the expression level of BCL-6 in NPC cells; MTT and in vivo tumorigenesis assays were applied to evaluate the cell proliferation ability; immunofluorescence assays were used to determine the cellular localization of BCL6 and BS; and a luciferase reporter assay was performed to evaluate the ability of BCL6 and BS to inhibit transcription. Results: BS was present in 5.34% (11/206) of primary NPC biopsies and 2.13% (1/47) of head and neck cancer biopsies. The expression of BCL6 was downregulated in NPC, and silencing of endogenous BCL6 promoted NPC cell proliferation in vitro. Overexpression of BCL6 but not BS inhibited the growth of NPC cells in vivo and in vitro. Mechanistically, BCL6 localized in the nucleus can inhibit the G1/S transition to suppress the growth of NPC cells. However, after the fusion of BCL6 and SPECC1L, the product cannot localize to the nucleus, and the transcriptional inhibitory function of BCL6 is abolished, eventually abolishing its tumor suppressor effect and leading to the development of NPC. Conclusion: BS is a novel fusion gene in NPC that may play an important role in the occurrence and development of this cancer. The clinical significance of the BS fusion gene needs further elucidation.

Whole-genome optical mapping of bone-marrow myeloma cells reveals association of extramedullary multiple myeloma with chromosome 1 abnormalities

Extramedullary disease (EMM) represents a rare, aggressive and mostly resistant phenotype of multiple myeloma (MM). EMM is frequently associated with high-risk cytogenetics, but their complex genomic architecture is largely unexplored. We used whole-genome optical mapping (Saphyr, Bionano Genomics) to analyse the genomic architecture of CD138+ cells isolated from bone-marrow aspirates from an unselected cohort of newly diagnosed patients with EMM (n = 4) and intramedullary MM (n = 7). Large intrachromosomal rearrangements (> 5 Mbp) within chromosome 1 were detected in all EMM samples. These rearrangements, predominantly deletions with/without inversions, encompassed hundreds of genes and led to changes in the gene copy number on large regions of chromosome 1. Compared with intramedullary MM, EMM was characterised by more deletions (size range of 500 bp–50 kbp) and fewer interchromosomal translocations, and two EMM samples had copy number loss in the 17p13 region. Widespread genomic heterogeneity and novel aberrations in the high-risk IGH/IGK/IGL, 8q24 and 13q14 regions were detected in individual patients but were not specific to EMM/MM. Our pilot study revealed an association of chromosome 1 abnormalities in bone marrow myeloma cells with extramedullary progression. Optical mapping showed the potential for refining the complex genomic architecture in MM and its phenotypes.

Abstract 742: The integration of a structure-function rule and a transcriptional signature to assign ESR1 fusion activity in metastatic breast cancer

Abstract Background: In-frame chimeric ESR1 fusion proteins can cause endocrine therapy (ET) resistance in estrogen receptor-positive (ER+) metastatic breast cancer (MBC) (PMC6171747). Here, we further investigated multiple new examples of ESR1 fusions in order to differentiate between transcriptionally active “driver” ESR1 fusions from inactive, likely “passenger” events. Methods: ESR1 fusions were identified by RNA-sequencing (RNA-seq) or whole genome sequencing in MBC (PMC5913625, PMC6872491 and unpublished). ESR1 fusion cDNA constructs were expressed in ER+ breast cancer cell lines. Cell proliferation and cell motility were assessed. RNA-seq followed by qPCR validation was conducted to examine individual transcriptional profiles. Results: All ESR1 fusions studied contained the first six exons of ESR1 (e6) fused in-frame to diverse partner gene sequences that replaced the ESR1 drug/ligand binding domain. Fusions with a transcription factor (TF) or coactivator (CoA) gene partner, for example ESR1-YAP1, ESR1-ARNT2-e18 and ESR1-SOX9 activated fulvestrant-resistant cell growth and hormone-independent cell motility. Other ESR1-e6 fusions, including ESR1-DAB2, ESR1-GYG1, ESR1-PCMT1 and ESR1-ARID1B did not show these phenotypes. We thus conclude that in-frame ESR1-e6 fusions arising from inter-chromosomal translocations with 3' TF/CoA partners are likely to be active. However, the active ESR1-PCDH11X fusion involves a proto-cadherin and is therefore an exception to this rule. This outlier emphasizes the need to establish additional approaches to determine ESR1 fusion activity. RNA-seq of T47D cells expressing active and inactive ESR1 fusions defined a gene signature associated with active ESR1 fusions, with activation of estrogen response and epithelial-to-mesenchymal transition (EMT) genes. This transcriptional signature was present in a patient-derived xenograft bearing the ESR1-YAP1 fusion, and thus potentially identifies the presence of an active ESR1 fusion protein. We subsequently identified two new ESR1 fusions involving recurrent TF/CoA partners, ESR1-ARNT2-e2 and ESR1-LPP. Both demonstrated tumor cell growth and motility activation, as predicted by the functional rule. The gene activation patterns were similar to the three other active fusions suggesting that despite marked diversity in the 3' partners, the transcriptional activities were similar and potentially diagnostic. Conclusion: The integration of the structure-function rule and the stereotypic transcriptional signature distinguishes pathogenic ESR1 fusions from non-active passenger events, thus prioritizing patients bearing ESR1 translocation-driven tumors for targeted therapeutic approaches. Citation Format: Xuxu Gou, Meenakshi Anurag, Jonathan T. Lei, Sinem Seker, Adrian V. Lee, Dan R. Robinson, Matthew J. Ellis. The integration of a structure-function rule and a transcriptional signature to assign ESR1 fusion activity in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 742.

Identification and characterization of inheritable structural variations induced by ion beam radiations in rice.

High energy ion beams are effective physical mutagens for mutation induction in plants. Due to their high linear energy transfer (LET) property, they are known to generate single nucleotide variations (SNVs) and insertion/deletions (InDels, <50 bp) as well as structural variations (SVs). However, due to the technical difficulties to identify SVs, studies on ion beam induced SVs by genome sequencing have so far been limited in numbers and inadequate in nature, and knowledge of SVs is scarce with regards to their characteristics. In the present study, we identified and validated SVs in six M4 plants (designated as Ar_50, Ar_100, C_150, C_200, Ne_50 and Ne_100 according to ion beam types and irradiation doses), two each induced by argon (40Ar18+), carbon (12C6+) and neon (20Ne10+) ion beams and performed in depth analyses of their characteristics. In total, 22 SVs were identified and validated, consisting of 11 deletions, 1 duplication, and 4 intra-chromosomal and 6 inter-chromosomal translocations. There were several SVs larger than 1 kbp. The SVs were distributed across the whole genome with an aggregation with SNVs and InDels only in the Ne_50 mutants. An enrichment of a 11-bp wide G-rich DNA motif 'GAAGGWGGRGG' was identified around the SV breakpoints. Three mechanisms might be involved in the SV formation, i.e., the expansion of tandem repeats, transposable element insertion, and non-allelic homologous recombination. Put together, the present study provides a preliminary view of SVs induced by Ar, C and Ne ion beam radiations, and as a pilot study, it contributes to our understanding of how SVs might form after ion beam irradiation in rice.

Genome-wide detection of enhancer-hijacking events from chromatin interaction data in rearranged genomes.

Recent efforts have shown that structural variations (SVs) can disrupt three-dimensional genome organization and induce enhancer hijacking, yet no computational tools exist to identify such events from chromatin interaction data. Here, we develop NeoLoopFinder, a computational framework to identify the chromatin interactions induced by SVs, including interchromosomal translocations, large deletions and inversions. Our framework can automatically resolve complex SVs, reconstruct local Hi-C maps surrounding the breakpoints, normalize copy number variation and allele effects and predict chromatin loops induced by SVs. We applied NeoLoopFinder in Hi-C data from 50 cancer cell lines and primary tumors and identified tens of recurrent genes associated with enhancer hijacking. To experimentally validate NeoLoopFinder, we deleted the hijacked enhancers in prostate adenocarcinoma cells using CRISPR-Cas9, which significantly reduced expression of the target oncogene. In summary, NeoLoopFinder enables identification of critical oncogenic regulatory elements that can potentially reveal therapeutic targets.

Integrative structural variant and breakpoint detection using optical genome mapping in a patient with a transformed diffuse large B-cell lymphoma from chronic lymphocytic leukemia.

e19511 Background: The finding of complex karyotypes has been a clinically significant finding in cancers of advanced stage or during cancer progression. Conventional cytogenetic and FISH analyses have been limited by the low-resolution of chromosomes and the number of FISH probes which can be implemented in one assay. Recent study revealed the potential of using optical genome mapping to decipher the architecture of cancer genome at nucleotide level. Methods: Karyotyping, FISH and optical genome mapping of bone marrow specimen. Results: We reported on a 63-year-old female with chronic lymphocytic leukemia for nine years and transformed to diffuse large B-cell lymphoma (DLBCL). The karyotype revealed a hypodiploid chromosomal complement: 42,X,-X,der(2)t(2;?;8)(p25;?;q11.2),t(2;9)(q3?5;p2?2),der(4)t(4;?;8)(p16;?;q11.2)t(4;15)(q35;q15),-8,-15,dic(17;21)(p11.2;p11.2),der(18)t(8;18)(q21.2;p11.32)[12]/46,XX[8]. FISH analysis showed loss of TP53, monosomy 8 with 3-5 copies of the Myc genes. Optical genome mapping analysis revealed 12 insertions, 29 deletions, 8 duplications, 6 intrachromosomal translocations and 12 interchromosomal translocations. Whole genome analysis identified multiple gains of 2p, 8p and 8q, losses of 9p, 15q and 17p. The breakpoints of two unbalanced translocation and one complex chromosomal rearrangement have been narrowed down to nucleotide resolution: ogm[GRCh37] t(2;9)(q34;p21.2) g.[chr2:211,281,003::chr9:26,532,620], ogm t(2;8)(p25.3;p23.3) g.[chr2:15,924::chr8:215,177] and ogm der(4)t(8q24.3→8q21.13→2p24.2→2q34→4p15.2→4q34.3→15q15.1) g.[chr8:80,461,191::chr2:18,079,530→chr2:211,274,379::chr4:26,381,729→chr4:180,989,408::chr15:42,404,514]. Conclusions: Integrative analysis using conventional cytogenetic and molecular cytogenomic methodologies unveil the complex architectural alterations of caner genome. The resolution achieved by optical genome mapping would potentially lead to discovery of carcinogenic mechanisms, new fusion genes, prognostic and therapeutic markers.

Modified chromosome structure caused by phosphomimetic H2A modulates the DNA damage response by increasing chromatin mobility in yeast.

In budding yeast and mammals, double-strand breaks (DSBs) trigger global chromatin mobility together with rapid phosphorylation of histone H2A over an extensive region of the chromatin. To assess the role of H2A phosphorylation in this response to DNA damage, we have constructed strains where H2A has been mutated to the phosphomimetic H2A-S129E. We show that mimicking H2A phosphorylation leads to an increase in global chromatin mobility in the absence of DNA damage. The intrinsic chromatin mobility of H2A-S129E is not due to downstream checkpoint activation, histone degradation or kinetochore anchoring. Rather, the increased intrachromosomal distances observed in the H2A-S129E mutant are consistent with chromatin structural changes. Strikingly, in this context the Rad9-dependent checkpoint becomes dispensable. Moreover, increased chromatin dynamics in the H2A-S129E mutant correlates with improved DSB repair by non-homologous end joining and a sharp decrease in interchromosomal translocation rate. We propose that changes in chromosomal conformation due to H2A phosphorylation are sufficient to modulate the DNA damage response and maintain genome integrity.This article has an associated First Person interview with the first author of the paper.