Gene Fusions In Cancer Research Articles

Statistical algorithms improve accuracy of gene fusion detection.

Gene fusions are known to play critical roles in tumor pathogenesis. Yet, sensitive and specific algorithms to detect gene fusions in cancer do not currently exist. In this paper, we present a new statistical algorithm, MACHETE (Mismatched Alignment CHimEra Tracking Engine), which achieves highly sensitive and specific detection of gene fusions from RNA-Seq data, including the highest Positive Predictive Value (PPV) compared to the current state-of-the-art, as assessed in simulated data. We show that the best performing published algorithms either find large numbers of fusions in negative control data or suffer from low sensitivity detecting known driving fusions in gold standard settings, such as EWSR1-FLI1. As proof of principle that MACHETE discovers novel gene fusions with high accuracy in vivo, we mined public data to discover and subsequently PCR validate novel gene fusions missed by other algorithms in the ovarian cancer cell line OVCAR3. These results highlight the gains in accuracy achieved by introducing statistical models into fusion detection, and pave the way for unbiased discovery of potentially driving and druggable gene fusions in primary tumors.

RNA-Seq to reveal mitochondrial gene fusions in breast cancer.

e13019 Background: Several mitochondrial DNA (mtDNA) mutations have been identified in various types of human cancer. While gene fusions encoded by the nuclear genome have an important role in tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. mtDNA has limited protective mechanisms compared to that for nuclear DNA (nDNA), and hence more susceptible to genomic aberrations. Methods: We sequenced RNA from pre and post treatment breast cancer samples obtained from neoadjuvant clinical trials BrUOG 211A/211B. Seventy-three of these samples were considered baseline and 61 were from post-treatment samples, 54 samples at both time points. Patients were given a run-in dose of bevacizumab (B), nab- paclitaxel (N) or trastuzumab (T), followed by combination biologic/chemotherapy (HER2- with B/carboplatin/N; HER2+ with T/carboplatin/N). RNA was derived from biopsy pairs obtained pre/post 10-day exposure to run-in monotherapy. We used TopHat-Fusion and deFuse for fusion detection. Results: On average, in each patient 4.6 mtDNA gene fusions were detected at the baseline and 4 in the post treatment samples. In this cohort we detected in total 385 mtDNA fusions across the baseline and 344 across the post-treatment samples. A number of fusions had recurrently shown drastic increase from baseline to post-treatment, such as MT-ATP6:MT-ND2 ( N = 9), MT-ND6:MT-CO2 ( N = 6), MT-ND6:MT-CO1 ( N = 10), and MT-ND4:MT-ATP6 ( N = 9). We observe that some mitochondrial genes such as MT-ND6 have as many as 7 gene partners. MT-ND6 appeared in fusions that were most abundant among both baseline and post-treatment samples with MT:ND4 ( N = 39), MT:ND2 ( N = 37) and MD:ATP6 ( N = 36). Interestingly, gene fusions MT-ND6:J01415.25, MT-CYB:MT-ND2 and MT-ND6:MT-ND1 were observed respectively in 9, 5 and 4 baseline samples but not in their post treatment equivalent. Conclusions: While several studies have examined the presence of mtDNA mutations in breast cancer, the role of mitochondrial gene fusions is unknown in cancer. In our breast cancer cohort we observed quite a high number of mitochondrial fusions, and some of them seem to be recurrent in multiple samples. This could potentially have clinical implications as knowledge of fusions in solid tumours gains.

Use of Anchored Multiplex PCR Enrichment for Detection of Gene Fusions in Solid Tumors by Next Generation Sequencing

BackgroundThe known genomic complexities of human cancer are being used to combat the disease with novel targeted therapies. Next generation sequencing (NGS) technologies have been introduced to determine a human cancer profile that is then used to develop a therapeutic management strategy. To date most NGS assays assess SNVs, Indels and some CNVs in various cancer related genes. Gene fusions are another type of mutation that have recently been implicated in the diagnosis and treatment of human cancers. In this study we assessed the ability to detect gene fusions using an anchored multiplex PCR‐enriched NGS assay.MethodsRNA was extracted from 15 formalin fixed, paraffin embedded tissue samples and 10 cell lines using the AllPrep DNA/RNA FFPE kit. cDNA synthesis and library preparation were performed using 200 ng of RNA. RNA quantity and quality were evaluated using the Qubit HS RNA Assay and the Archer PreSeq RNA QC Kit, respectively. Libraries were quantified using the KAPA Library Quantification Kit and sequenced on the Illumina MiSeq System using the Archer FusionPlexTM Solid Tumor panel which detects fusions in 53 genes. Data analysis was performed using the Archer Analysis platform (v4.0.11). We examined the limit of detection (LOD) by varying the amount of total RNA input (200‐2.5 ng), precision in two sequencing runs using two different operators, and accuracy by comparison of results to results from independent FISH and NGS assays.ResultsWe established QC metrics that included: (1) tumor content, (2) RNA quantification, (3) RNA quality, (4) library quantification, and (5) post‐sequencing analysis. While NGS results could be obtained using 10 ng of input RNA, more robust results were obtained when we used higher RNA inputs. Accuracy was determined to be 100% with detection of for all known fusions in the samples used. The following fusions were present in these samples: EML4‐ALK, ETV6‐NTRK3, EWSR1‐FLI1, TPM3‐NTRK1, TNM4‐NRG1, FGFR2‐COL14A1, MBNL1‐RAF1, ETV6‐RUNX1, BRD4‐NUTM1, and TMPRSS2‐ERG. All positive (CCD6‐RET, EML4‐ALK, SLC34A2‐ROS1) and negative controls gave the expected results.ConclusionsThis study demonstrates the feasibility of using the Archer Solid Tumor FusionPlex assay for detection of gene fusions in human cancers. The assay detects fusions of any partner gene with the 53 genes in the panel and thus makes novel fusion discovery possible.

Identification of novel cancer fusion genes using chromosome breakpoint screening.

Gene fusion due to rearrangement or translocation of chromosomes is a powerful mutational mechanism during tumorigenesis. Several new high-resolution technologies have recently been developed to evaluate large numbers of small aberrations as candidate loci for fusion gene screening. In our previous whole-genome screening study using 500K SNP arrays, we identified more than 700homozygous deletions (HDs) and amplicons in 23cancer cell lines. To explore novel fusion genes in cancer, we established stringent criteria for defining HD and amplicon breakpoints. Then genomic PCR and sequencing analyses identified a fusion gene, FNDC3B-PRKCI, that resulted from chromosome intra-rearrangement. Western blotting and 3'-RACE analyses revealed that the chimeric transcript was an in-frame fusion between FNDC3B and PRKCI. Finally, cell migration and colony formation assays suggested that FNDC3B-PRKCI is a potential oncogene.

Abstract P3-07-02: Are we missing actionable targets in breast cancer? Novel insights into recurrent Ret alterations

Abstract Background: Recurrent gene fusions in breast cancer have been rarely reported suggesting that they either are not present or are not easily detected by standard sequencing methods. Comprehensive genomic profiling (CGP) by hybrid capture-based, high depth next-generation sequencing approaches, can be used to detect recurrent rearrangements and other genomic alterations involving target genes. We found that CGP can identify recurrent alterations involving RET, a known oncogenic tyrosine receptor kinase, in a subset of breast cancer. Methods: CGP using FoundationOne platform was performed interrogating the entire coding region for up to 315 cancer-related genes and introns of up to 28 genes involved in rearrangements at a depth of 500-1000X in formalin-fixed, paraffin embedded tumor tissue (Foundation Medicine, MA). Engineered representative RET fusion vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts), and cells were evaluated for RET kinase signaling, drug response, and tumorigenicity. Patient-derived xenografts (PDX) generated from two triple negative breast cancers (TNBCs) were used in an ex vivo assay (Response3DXTM, Molecular Response LLC, San Diego, CA). Results: Twenty-two RET rearrangements were identified in 8119 (0.27%) breast cancer cases. Of these, 5 rearrangements were activating RET fusions including CCDC6-RET (n=4) and NCOA4-RET (n=1), that have been described in other cancer types. Five other cases had clear evidence of genomic rearrangement involving RET, but the 5' partners could not be definitively identified. The remaining twelve cases had complex rearrangements of RET including internal duplications. RET amplification was also observed, both in TNBC and in a HER2+ breast cancer at onset of resistance to HER2-targeted therapy. Both NCOA4-RET and a novel RASGEF1A-RET fusion were characterized in vitro. Non-tumorigenic cells engineered to stably overexpress either RET fusions demonstrated transformed phenotypes. The fusions were constitutively active, as shown by endogenous phosphorylation of the kinase domain, and drove activation of downstream signaling as shown by increased phosphorylation of ERK and AKT. Cells transformed by RET-fusions were exquisitely sensitive to treatment with RET inhibitors. Interestingly, a PDX model of RET-amplified TNBC was sensitive to treatment with a PIK3CA inhibitor. An index case of ER+/PR-/HER2+, metastatic breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole was found to have a NCOA4-RET fusion by CGP. Subsequent treatment with with cabozantinib plus anastrazole led a rapid clinical and radiographic response. Conclusions: CGP can identify recurrent RET rearrangements in breast cancer that act as primary oncogenic drivers and can be therapeutically targeted. RET alterations may also play a role in acquired resistance to HER2-targeted therapies, suggesting a role for combined RET and HER2-targeted therapy in this setting. Our data demonstrate that RET alterations can be identified by clinical-grade CGP and are promising candidates as therapeutic targets in selected breast cancer patients. Citation Format: Hirshfield KM, Paratala BS, Hindoyan A, Dolfi SC, Yilmazel B, Schrock A, Gay L, Ali SM, Ross JS, Williams CB, Nair P, Ganesan S, Leyland-Jones B. Are we missing actionable targets in breast cancer? Novel insights into recurrent Ret alterations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-02.

Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in high-throughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86–96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof–of–principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting.

ChimPipe: accurate detection of fusion genes and transcription-induced chimeras from RNA-seq data

BackgroundChimeric transcripts are commonly defined as transcripts linking two or more different genes in the genome, and can be explained by various biological mechanisms such as genomic rearrangement, read-through or trans-splicing, but also by technical or biological artefacts. Several studies have shown their importance in cancer, cell pluripotency and motility. Many programs have recently been developed to identify chimeras from Illumina RNA-seq data (mostly fusion genes in cancer). However outputs of different programs on the same dataset can be widely inconsistent, and tend to include many false positives. Other issues relate to simulated datasets restricted to fusion genes, real datasets with limited numbers of validated cases, result inconsistencies between simulated and real datasets, and gene rather than junction level assessment.ResultsHere we present ChimPipe, a modular and easy-to-use method to reliably identify fusion genes and transcription-induced chimeras from paired-end Illumina RNA-seq data. We have also produced realistic simulated datasets for three different read lengths, and enhanced two gold-standard cancer datasets by associating exact junction points to validated gene fusions. Benchmarking ChimPipe together with four other state-of-the-art tools on this data showed ChimPipe to be the top program at identifying exact junction coordinates for both kinds of datasets, and the one showing the best trade-off between sensitivity and precision. Applied to 106 ENCODE human RNA-seq datasets, ChimPipe identified 137 high confidence chimeras connecting the protein coding sequence of their parent genes. In subsequent experiments, three out of four predicted chimeras, two of which recurrently expressed in a large majority of the samples, could be validated. Cloning and sequencing of the three cases revealed several new chimeric transcript structures, 3 of which with the potential to encode a chimeric protein for which we hypothesized a new role. Applying ChimPipe to human and mouse ENCODE RNA-seq data led to the identification of 131 recurrent chimeras common to both species, and therefore potentially conserved.ConclusionsChimPipe combines discordant paired-end reads and split-reads to detect any kind of chimeras, including those originating from polymerase read-through, and shows an excellent trade-off between sensitivity and precision. The chimeras found by ChimPipe can be validated in-vitro with high accuracy.

Gene Fusions in Human Cancers: A Review Focused on Diagnostic Biomarkers, Method Selections, and Treatments.

Recurrent gene fusions in cancers have been successfully applied in clinical diagnoses and treatments. Specific gene rearrangements or other specific cytogenetic translocations may be helpful in separating cancers from benign lesions. Also, the detection of gene fusions has brought great benefits to distinguish molecular subclassifications of cancers. Numerous approaches have been used to identify cancer-specific abnormalities, including FISH, RT-PCR, next-generation sequencing, etc. In addition to diagnostic and genetic values, molecular testing has been becoming a valuable tool in the therapeutic research. Recent clinical trials involving gene fusions in cancers have also been developing under a rapid speed. Generally, we review gene fusions in cancers, emphasizing on relevant diagnostic biomarkers, method selections, and treatments.

Novel Application of Loop-mediated Isothermal Amplification for Rapid Detection of Gene Translocation.

Identification of fusion genes in cancer is essential for pathological diagnosis and clinical therapy. Although methods for detection of fusion genes, such as fluorescence in situ hybridization (FISH) and real-time polymerase chain reaction (PCR), have been developed in last two decades, these methods are not ideal for detection of these genetic alterations owing to their high cost and time-consuming procedures. In this study, we developed novel application for detection of gene translocations using loop-mediated isothermal amplification (LAMP). We verified the amplified DNA products of echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK), synaptotagmin and synovial sarcoma, X breakpoint (SYT-SSX), and immunoglobulin heavy chain gene and B cell leukemia/lymphoma 2 (IgH/BCL2) by real-time PCR, agarose-gel electrophoresis, and the naked eye after the LAMP procedure. Fusion genes were detected in samples diluted 103 times within 60 min. Because of the advantages of rapid amplification, simple operation, and easy detection without requiring sophisticated equipment or technical skill, LAMP may have potential applications as an on-site analytical approach in hospitals for pathological diagnosis and decision making regarding appropriate therapeutic approachs.

MA12.01 Next Generation Sequencing Based Clinical Framework for Analyses of Treatment Predictive Mutations and Gene Fusions in Lung Cancer

The use of new, emerging techniques in the search of tailored patient therapies is rapidly becoming a reality. Here we describe the optimization and implementation of next generation sequencing for treatment predictive mutation screening in parallel with gene fusion status of ALK, RET and ROS1 in non-small cell lung cancer (NSCLC) patients. The Illumina TruSight tumor 26-gene NGS panel was validated in 81 clinical routine FFPE or cytology specimens and implemented in 533 diagnostic NSCLCs during one year of clinical analysis. In parallel, a RNA-based NanoString method was evaluated in 169 cases for gene fusion status of ALK, RET and ROS1. We have successfully established a streamlined workflow with a 5-day turnaround time from specimen arrival to mutation report. The concordance in the validation cohort was 99% for comparable variants. In the 533 diagnostic samples, 1-2 variants were detected in 79% of the cases. Most frequently mutated genes included TP53, KRAS, EGFR, STK11, and BRAF, all with differences in mutational patterns between histological subgroups. The RNA-based NanoString assay was successfully established and validated. The success rate in the 169 cases was 80% and 10 gene fusions were found (five ALK fusions, three RET fusions and two ROS1 fusions) all in adenocarcinomas. Integration of mutation and gene fusion status revealed that 68% of adenocarcinomas, 13% of SqCCs and 56% of NSCLC-NOS harbored ≥1 actionable alteration ALK, RET, ROS1, EGFR, KRAS, PIK3CA, BRAF, NRAS, MAP2K1, ERBB2 or AKT1. Specifically, in 13.2% of the adenocarcinomas where no EGFR or ALK alteration was detected emerging targeted therapy may be considered in addition to the 15.3% of patients that was eligible for EGFR or ALK inhibitors. The corresponding proportions for SqCCs were 5.5% in addition to the 2.2%, and for NSCLC-NOS 2.5% in addition to the 11.2% eligible for EGFR or ALK inhibitors. Next generation sequencing in combination with the NanoString technology is time- and cost efficient in the diagnostic routine for treatment predictive mutation screening and gene fusion status detection. The techniques represent valuable tools for pinpointing patients eligible to standard targeted therapies in addition to new emerging therapies.

P1.02-013 Clinicopathological Characteristics and Survival of ALK, ROS1 and RET Arrangements in Non-Adenocarcinoma Non-Small Cell Lung Cancer Patients

ALK, ROS1 and RET rearrangements represent three most frequency of fusion genes in non-small cell lung cancer (NSCLC). Rearrangements of the three genes are predominantly found in lung adenocarcinoma，while,rare in non-adenocarcinoma. The aim of this study was to investigate the frequency, clinicopathological characteristics and survival of ALK, ROS1 and RET arrangements in non-adenocarcinoma NSCLC patients. We screened ALK, ROS1, and RET arrangements in patients with completely resected non-adenocarcinoma NSCLC using reverse transcriptase polymerase chain reaction (PCR). All positive samples were confirmed with fluorescence in situ hybridization (FISH). Survival analysis was performed with Kaplan-Meier method and log-rank for comparison. Totally, 385 patients, who underwent complete resection, including 245 with squamous cell carcinoma, 85 with adenosquamous carcinoma and 55 with large cell carcinoma were enrolled. Twelve patients were identified as harboring fusion genes, including seven with ALK, three with ROS1 and two with RET rearrangements. The frequencies of fusions in adenosquamous carcinoma, squamous cell carcinoma, and large cell carcinoma were 8.2%, 1.6% and 1.8%, respectively. The median age of 12 patients was 49.5 years and three patients had smoking history. No survival difference existed between fusion genes positive and negative patients (36.7 vs.50.2 months, P=0.21). The frequencies of ALK, ROS1 and RET rearrangements are low in non-adenocarcinoma NSCLC patients, and the clinical characteristics are similar with those in lung adenocarcinoma. Fusions of the three genes are not prognostic marker for non-adnocarcinoma NSCLC patients.

Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study.

Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5'-kinase fusion genes, combinatorial effects between 3'-KDR kinases and their 5'-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3'-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of 'effective' (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3'-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs' clinical implications.

Cytogenetic Resources and Information.

The main databases devoted stricto sensu to cancer cytogenetics are the "Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer" ( http://cgap.nci.nih.gov/Chromosomes/Mitelman ), the "Atlas of Genetics and Cytogenetics in Oncology and Haematology" ( http://atlasgeneticsoncology.org ), and COSMIC ( http://cancer.sanger.ac.uk/cosmic ).However, being a complex multistep process, cancer cytogenetics are broadened to "cytogenomics," with complementary resources on: general databases (nucleic acid and protein sequences databases; cartography browsers: GenBank, RefSeq, UCSC, Ensembl, UniProtKB, and Entrez Gene), cancer genomic portals associated with recent international integrated programs, such as TCGA or ICGC, other fusion genes databases, array CGH databases, copy number variation databases, and mutation databases. Other resources such as the International System for Human Cytogenomic Nomenclature (ISCN), the International Classification of Diseases for Oncology (ICD-O), and the Human Gene Nomenclature Database (HGNC) allow a common language.Data within the scientific/medical community should be freely available. However, most of the institutional stakeholders are now gradually disengaging, and well-known databases are forced to beg or to disappear (which may happen!).

Multi-Database Description of Primary Splenic Diffuse Large B-Cell Lymphoma

Introduction:Although the spleen is involved in about a third of patients with Diffuse Large B-cell Lymphoma (DLBCL), stage I splenic DLBCL is rare and there is little data to guide management. Potential treatment modalities include combinations of splenectomy, radiation and immuno-chemotherapy, but there is no consensus on the optimal approach. Using multiple publically available and propriety databases, we sought to further define the prognosis and outcomes for stage I splenic DLBCL.Methods:We utilized the Surveillance Epidemiology and End Results (SEER) 18 registry to identify patients with stage I splenic DLBCL diagnosed during the years 1973-2013. Appropriate cases were identified using a combination of International Classification of Diseases for Oncology, 3rd edition code DLBCL (9680/3) and location code for spleen (C42.2). These patients were divided into two cohorts based on the year of diagnosis (1983-2005; 2006-2013) as rituximab (R) was approved by the FDA in 2006 for use in the first line treatment of DLBCL. Patient characteristics and survival outcomes were analyzed by GraphPad Prism 6 and Microsoft Excel. Disease specific survival (DSS) and Overall survival (OS) comparisons were made using the Gehan-Breslow-Wilcoxon test. All p-values were 2-sided and results ≤0.05 were considered significant.We queried the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer to identify patients with stage I splenic lymphoma during the years 1973-2013. We also searched the Caris Life Sciences database for patients profiled by Caris Molecular Intelligence using the keywords "splenic lymphoma " in the clinical history and diagnosis fields for cases of stage I splenic DLBCL from 2007-2015. All database searches were conducted with IRB approval.Results:107,550 patients with known stage and histologically confirmed DLBCL were identified within the SEER database. 30,455 patients had stage I disease with 470 of them diagnosed as stage I splenic DLBCL. The mean age was 66 years (range, 17-93 years) and the majority were Caucasian (89%). There was equal distribution of patients in the pre-rituximab (pre-R) era and post rituximab (R) era (53% vs 47%). 42% (470/1116) of splenic DLBCL were diagnosed as stage I compared to 28% (29,985/106,434) of all other types of DLBCL (p<0.01). Stage I splenic DLBCL treated in the R-era has better OS than all other stage I DLBCLs treated during the same period (HR = 0.62, 95% CI 0.53-0.86, p<0.01)The rate of splenectomy was lower after the approval of rituximab (82% in pre-R vs 72% in R era, p=0.03). Splenectomy was associated with an improvement in DSS (HR = 0.57, 0.28 - 0.96, p=0.04), but when stratified by pre-R and R era, improved DSS with splenectomy was only seen in the pre-R (HR = 0.51, 0.17-1.08, p =0.04), but not in the R-era (HR =0.62, 0.25 - 1.38 p=0.53). OS advantage with splenectomy was also seen only in the pre-R era (HR = 0.66, 0.32 - 1.14, 106m vs 137m, p=0.03) but splenectomy had no impact on OS in the R-era (HR = 0.74, 0.37 - 1.43, p=0.45). There was a trend toward improved OS with the introduction of rituximab (HR = 0.75, 0.48 - 1.14, p=0.054). There was no difference in the utilization of radiation between these cohorts (6% in pre-R vs 3% in R era, p=0.13).Our search of the Mitelman database found 1413 patients with DLBCL, but none of them had stage I splenic DLBCL. We also identified 20 patients with Non-Hodgkin lymphoma profiled by Caris Life Sciences, of which only one had primary splenic DLBCL. This patient was a 35 year old male who on immunohistochemistry was found to be positive for MGMT, MRP1, PTEN, TOP2A, TOPO1, and TS.Conclusion:The utilization of splenectomy for stage I splenic DLBCL has decreased with the introduction of rituximab without compromise of outcomes. Radiation is rarely used and does not appear to influence outcomes. Splenic DLBCL is more likely to present as stage I disease compared to other types of DLBCLs and Stage I splenic DLBCL appears to have a favorable prognosis relative to other stage I DLBCLs. Alternative databases added little to the understanding of the biology of stage I splenic DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Distribution of 9p24 Abnormalities in Hematologic Malignancies with an Emphasis on Diffuse Large B-Cell Lymphoma

Introduction: 9p24 contains genes that are critical for immune evasion and propagating cell division. The loci for PD-L1, PD-L2, JAK2, and the histone demethylases KDM4C/JMJD2C are linked on 9p24 (Van Roosbroeck et al. Genes, Chromosomes and Cancer 2016). Amplification or rearrangements of this region have been described in the pathogenesis of classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (Ansell et al. New England Journal of Medicine 2015). Additionally, JAK2 amplification up-regulates PD-L1 and L2, which leads to increased T-cell inactivation and suggests synergy between these drug targets. The recent success of PD-1 blockade in numerous malignancies has led to the development and approval of PD-1 inhibitors in cHL as well as other cancers. Targeted therapies are approved for JAK2 inhibition, such as ruxolitinib, and are in development for histone demethylases, which illustrates the utility of identifying the 9p24 amplicon in hematologic malignancies (HM) (Van Roosbroeck et al. Genes, Chromsomes and Cancer 2016). The goal of this analysis is to better understand the distribution of 9p24 abnormalities across a broader range of leukemias and lymphomas in order to facilitate future studies of targeted therapy.Methods: The National Cancer Institute's Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer was queried for 9p24 breakpoint abnormalities within HM. Incidence of additions and rearrangements in chromosome 9p24 for all subtypes of HM listed in the Mitelman Database were calculated. Individual references were manually reviewed and pathologic data was extracted as available from the primary sources. Diffuse large B-cell lymphoma (DLBCL) cases were further assessed for co-incident rearrangement of MYC (8q24), BCL2 (18q21) and BCL6 (3q27) with 9p24. All subtypes with greater than 2% incidence of additions and/or rearrangements in chromosome 9p24 were reported.Results: 48,761 patients (pts) with HM across 74 lymphoid and myeloid subtypes were identified. 361 (<1%) pts had additions or rearrangements involving 9p24 across 53 subtypes of HM. 21 subtypes of HM had no 9p24 abnormalities. PTCL NOS had the highest incidence of aberrations (3.1% (6/193), 1.1% with additions and 2% with rearrangements) followed by cHL (2.7% (5/186), all additions) and DLBCL (2.7% (37/1413), 1.5% additions and 1.2% rearrangements) in malignancies with over 100 cases. Among DLBCL cases 9p24 was co-amplified/rearranged with MYC in 22% (8 cases), BCL6 27% (10), BCL2 14% (5) and in 3% (1) double-hit lymphomas. Minimal data was available on cell of origin; therefore, further analysis was not performed. No cases of DLBCL with 9p24 aberrations were T-cell rich DLBCL’s (0/37). Among the rarer lymphomas, extranodal NK/T cell lymphoma (11%, 2/18) and intravascular B cell lymphoma (7%, 1/14) had higher incidences of 9p24 additions or rearrangements (Table 1).Conclusion: Amplifications and rearrangement of 9p24 are rare in HM within the Mitelman database. Our study suggests that patients with DLBCL, adult T-cell leukemia/lymphoma, extranodal NK/T cell lymphoma, intravascular B cell lymphoma, peripheral T-cell lymphoma, and extranodal marginal zone B-cell may be considered for further studies with FISH and aCGH to further define the incidence of 9p24 alterations and potentially for targeted clinical trials. 9p24 abnormalities did not correlate with previously described phenotypic subtypes of DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic Analysis of B-Cell Lymphomas Associated with Hemophagocytic Lymphohistiocytosis (HLH)

Introduction:HLH is an aggressive and life-threatening syndrome of excessive immune activation. HLH can be familial or secondary. Secondary HLH is usually associated with malignancy, infections or rheumatologic disorders. 27% of the secondary HLH are related to malignancy, the vast majority of which are hematologic malignancies (HM), and are associated with a mortality rate of 80%. Chemotherapy may not salvage these patients due to end-organ dysfunction at presentation (Johnson 2016). We sought to further describe the genetic landscape of HM associated HLH to identify potential targets for clinical trials.Methods:We queried the National Cancer Institute’s Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer to identify patients (pts) with HM associated HLH. All identified references were reviewed and pt data was extracted regarding clinical and pathologic details as well as therapy and outcomes. Frequencies of gene rearrangements in HLH associated lymphomas were contrasted to non-HLH associated lymphomas within the Mitelman database. Data was analyzed using Microsoft Excel and GraphPad Prism. All p-values are two- sided and results ≤0.05 were considered significant.Results:16 pts with HLH associated B-cell lymphomas were identified. 8 pts had DLBCL associated HLH, 5 had EBV associated B-cell lymphomas and 3 had B-cell lymphomas NOS. The 8 pts without DLBCL were considered as a single group for further analysis. Pts with DLBCL-HLH were significantly older (mean 64.9 years, range 41-82) than the others (8.9, 0.3-33, t-test <0.0001) (Figure 1). 75% (6/8) of DLBCL-HLH pts were male vs 63% (5/8) of others. Median LDH, median ferritin, and median IL-2R were 1,117 (U/L), 1,073 (ng/mL), and 13,323 (pg/mL) respectively in the DLBCL-HLH pts and these were not reported in the others. 88% (7/8) pts with DLBCL-HLH received CHOP while 100% (8/8) of the others received etoposide and corticosteroids with or without other agents. DLBCL-HLH were more likely to harbor unbalanced rearrangements (75% v 13%, RR=3.9, 95%CI 1.1 - 13.6, p=0.04) and rearrangements in immune related loci (9p24 - PDL1/2 (2), 19q13 - IFN lambda 1,2,3 (2), and 12p11 - DDX11 (1)) versus none in the others (63% v 0%, RR=3.7, 95%CI 1.4 - 9.6, p=0.03). One pt each with DLBCL-HLH harbored rearrangements of MYC (1/8) and BCL2 (1/8) while none of the others had rearrangements in MYC or BCL2. No patient in either group had a rearrangement of BCL6. Median overall survival was 12.5m in DLBCL-HLH versus 4.5m in the others (log rank p=0.23) (Figure 2).Compared to non-HLH associated cases of DLBCL within Mitelman the incidence of rearrangements and additions in the immune related loci 9p24 (25% (2/8) v 3% (37/1413) (p=0.02)), 19q13 (25% (2/8) v 4% (61/1413) (p<0.05)) and 12p11 (13% (1/8) v <1% (6/1413) (p=0.04)) were significantly higher in DLBCL-HLH.Conclusion:DLBCL-HLH is a distinct entity with dismal outcomes with standard chemotherapy and frequent rearrangements of immune related genes. Novel treatment strategies are needed and may include immune modulatory agents such as PD-1 inhibitors. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

INTEGRATE-neo: a pipeline for personalized gene fusion neoantigen discovery.

MotivationWhile high-throughput sequencing (HTS) has been used successfully to discover tumor-specific mutant peptides (neoantigens) from somatic missense mutations, the field currently lacks a method for identifying which gene fusions may generate neoantigens.ResultsWe demonstrate the application of our gene fusion neoantigen discovery pipeline, called INTEGRATE-Neo, by identifying gene fusions in prostate cancers that may produce neoantigens.Availability and ImplementationINTEGRATE-Neo is implemented in C ++ and Python. Full source code and installation instructions are freely available from https://github.com/ChrisMaherLab/INTEGRATE-Neo.Supplementary information Supplementary data are available at Bioinformatics online.

High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine

Aberrant chromosal rearrangements, such as the multiple variants of TMPRSS2:ERG fusion gene mutations in prostate cancer (PCa), are promising diagnostic and prognostic biomarkers due to their specific expression in cancerous tissue only. Additionally, TMPRSS2:ERG variants are detectable in urine to provide non-invasive PCa diagnostic sampling as an attractive surrogate for needle biopsies. Therefore, rapid and simplistic assays for identifying multiple urinary TMPRSS2:ERG variants are potentially useful to aid in early cancer detection, immediate patient risk stratification, and prompt personalized treatment. However, current strategies for simultaneous detection of multiple gene fusions are limited by tedious and prolonged experimental protocols, thus limiting their use as rapid clinical screening tools. Herein, we report a simple and rapid gene fusion strategy which expliots the specificity of DNA ligase and the speed of isothermal amplification to simultaneously detect multiple fusion gene RNAs within a short sample-to-answer timeframe of 60min. The method has a low detection limit of 2 amol (1000 copies), and was successfully applied for non-invasive fusion gene profiling in patient urine samples with subsequent validation by a PCR-based gold standard approach.

Gene fusions in epithelial cancers

Gene fusions in epithelial cancers

Identification of RNA-Seq Gene Fusions in Ovarian Cancer

Identification of RNA-Seq Gene Fusions in Ovarian Cancer