Recurrent Fusion Transcripts Research Articles

Vulvar angiomyofibroblastoma is molecularly defined by recurrent MTG1-CYP2E1 fusions.

Angiomyofibroblastoma (AMF), a rare benign vulvovaginal mesenchymal tumour, poses a diagnostic challenge due to histologic and immunohistochemical overlap with other vulvar mesenchymal tumours. Recently, MTG1-CYP2E1 fusion transcripts were reported in 5/5 AMFs; no other genetic alterations have been described to date. Herein, we sought to investigate the frequency of the MTG1-CYP2E1 fusion and the presence of other potential genetic alterations in a cohort of AMFs (n = 7, patient age range: 28-49 years). Tumours demonstrated classic morphologic features including alternating hypo/hypercellular areas, capillary channels surrounded by epithelioid/spindled tumour cells, and variable amounts of mature adipose tissue. reverse transcription-polymerase chain reaction (RT-PCR) for MTG1-CYP2E1 fusion, performed in all seven cases, showed the fusion transcript in five of six cases (one case with technical failure). Two tumours, including the one lacking the fusion, were subjected to targeted next-generation sequencing (104 genes) and a sarcoma fusion assay (28 genes); the fusion negative AMF also underwent RNA sequencing. No additional mutations, copy number alterations, or fusion genes were identified with the assays employed. We conclude that the majority of AMFs harbour recurrent MTG1-CYP2E1 fusion transcripts and identification of this fusion may aid in the diagnosis.

ALL-061 Delineating the Genomic and Proteomic Landscape of BCR-ABL1-like Acute Lymphoblastic Leukemia Subtype: Bench-to-Bedside Translational Research

Baseline detection of Ph/BCR-ABL1-like-ALL cases is extremely beneficial for hematologists/physicians to design personalized medicine and to treat these patients efficiently. Worldwide, researchers use patented TaqMan low-density array (TLDA) gene expression profiling (GEP) to identify these cases. In developing countries, its detection is very challenging and expensive due to the unavailability of patented TLDA and there is a paucity of literature on the identification and characterization of Ph-like ALL cases. Stepwise identification followed by genomic and proteomic characterization of Ph-like ALL cases. This study was conducted at PGIMER, India (2016-2021). Flowcytometric-immunophenotyping and multiplex RT-PCR was performed to detect four recurrent fusion transcripts (n=1021). Transcriptome GEP was performed on 12 Ph-positive ALL cases. Ph-like-ALL cases identified using nCounter Platform (n=96) and RQ-PCR (n=536) were characterized using FISH, DD-PCR, RQ-PCR and MRD-positivity. Nine HKGs' expression was evaluated using 6 algorithms. Furthermore, we built and validated PHi-RACE classifier (n=629 cases) for its rapid detection. Lastly, we delineated the proteomic landscape of Ph-like ALL cases using LC-MS/MS. We studied 1021 treatment-naive and newly diagnosed B-ALL cases in this study cohort. Multiplex assay revealed 18.66% BCR-ABL1, 3.85% TCF3-PBX1, 5.27% ETV6-RUNX1, and 1.21% KMT2A-AFF1. Using transcriptome GEP, we identified 32.05% Ph-like-ALL cases using NanoString and 26.67% using RQ-PCR. These cases were substantially older and represented higher MRD positivity. In CRLF2 subgroup of Ph-like ALLs, we identified chimeric gene fusions including 10% JAK2 rearrangement, 8.33% CRLF2-IGH and CRLF2-P2RY8 translocation with concomitant R683S mutation. We found 8.33% IKZF1 (Δ4-7) deletion using DD-PCR. Furthermore, we developed an online logistic classifier entitled PHi-RACE (sensitivity=95.20%, specificity=83.70%, cut-off >0.28) for its detection using logistic regression. We have validated PHi-RACE classifier (n=93) and identified 56 Ph-like ALL cases. Six algorithms' findings revealed that EEF2, GAPDH and PGK1 represent the best HKGs for its detection. Lastly, from LC-MS/MS findings, we identified 3079 proteins, spliceosomal mediated mRNA splicing pathway and four miRNAs were significantly expressed in Ph-like ALL cases. The authors created and published the PHi-RACE classifier to detect Ph-like ALL cases, which has direct clinical implications. Further, we have delineated the genomic and proteomic landscape of Ph-like-ALL for the first time from developing countries.

The SH3PXD2A-HTRA1 fusion transcript is extremely rare in Norwegian sporadic vestibular schwannoma patients

IntroductionVestibular schwannoma (VS) is a benign intracranial tumor in which the underlying genetics is largely uncertain, apart from mutations in the tumor suppressor gene NF2. Alternative tumorigenic mechanisms have been proposed, including a recurrent in-frame fusion transcript of the HTRA1 and SH3PXD2A genes. The gene product of the SH3PXD2A-HTRA1 fusion has been shown to promote proliferation, invasion and resistance to cell death in vitro and tumor growth in vivo. The aim of this study was to replicate the findings and to investigate the frequency of this fusion gene in another cohort of vestibular schwannoma patients.MethodsThe SH3PXD2A-HTRA1 transcript was synthesized in vitro using PCR and used as a positive control to assess the sensitivity of a real-time PCR assay. This real-time PCR assay was used to search for the presence of the fusion transcript in 121 Norwegian sporadic VS patients.ResultsThe real-time PCR assay showed a high sensitivity and was able to detect as low as ~ 5 copies of the fusion transcript. Out of the 121 investigated tumors, only 1 harbored the SH3PXD2A-HTRA1 fusion.ConclusionEven though the SH3PXD2A-HTRA1 fusion has been shown to be a driver of tumorigenesis, our results suggest that it is a rare event in our VS patients. Further investigation is warranted in order to elucidate whether our results represent an extreme, and if the fusion is present also in other neoplasms.

Fusion Transcripts without Corresponding Cytogenetic Abnormalities in Acute Myeloid Leukemia: Implications for AML Pathogenesis

Chromosomal translocations and inversions that create fusion genes are important causal events in AML. 7 recurrent fusion genes define AML entities in the 2016 revision of the WHO classification of hematologic malignancies. We have developed a comprehensive diagnostic AML platform based on next-generation mRNA sequencing (RNAseq). The adaptable integrated bioinformatic pipeline HAMLET (Human AML Expedited Transcriptomics) analyses the raw data simultaneously for fusion transcripts and for sequence variants and expression levels of genes with relevance to AML pathogenesis, prognosis, or actionability.Poly(A)+ RNA from 100 cryopreserved AML samples with a blast count of 10-99% (median: 75%) from 96 patients (3 diagnosis-relapse pairs, 1 de novo AML and subsequent tAML) was sequenced at a depth of 59x106 paired-end reads/sample with median read length of 126 bp in an ISO17025-accredited Illumina HiSeq 2500 pipeline. Fusion transcripts were called by STAR-Fusion and FusionCatcher. HAMLET detected the recurrent fusion transcripts RUNX1-RUNX1T1, CBFB-MYH11, PML-RARA, ERG-FUS, DEK-NUP214, and KMT2A-MLLT3 plus the variants KMT2A-MLLT4, KMT2A-MLLT1, and KMT2A-MLLT6 with 100% sensitivity and 100% specificity compared to metaphase cytogenetics, FISH, and RT-PCR (Griffioen, ASH 2016).In addition, HAMLET identified fusion transcripts from 54 combinations of genes in 57 AML samples that lacked corresponding chromosomal aberrations by metaphase cytogenetics. 14 “unusual” fusions involved genes located on different chromosomes; 40 fusions represented intrachromosomal events. 8 fusions were detected recurrently, i.e. in at least 2 cases (Fig. A). 32 AML carried at least 2 unusual fusions, with a maximum of 9 different fusions in an AML with a complex karyotype (Fig. B). Unusual fusions were not correlated with classical recurrent AML fusion genes. The 5 most prevalent fusions and an ETV6-LYN fusion were successfully validated in a total of 18 AML by RT-PCR. The only fusion that could not be validated was a YWHAZ-KLF10 fusion (2 cases).The most prevalent RT-PCR-validated fusion (seen in 30 AML cases) has been described in ALL (Roberts, 2012; Safavi, 2015) and was an intrachromosomal TPM4-KLF2 fusion between the tropomyosin 4 and an alternative reading frame (ARF) of the transcription factor Kruppel-like factor 2 genes. Unexpectedly, we detected this fusion in all 3 tested normal bone marrows (BM) but not in EBV-transformed B cells. The hitherto unvalidated C15orf57-CBX3 fusion may be generated by retrotransposition (Schrider, 2013) and was present in 18 AML as well as in 2/3 normal BM and 2/3 EBV B-cells with CBX3 in ARF. The intrachromosomal NCOR2-UBC fusion (9 AML) is listed in the database ChimerDB without further characterization and involves the genes of Nuclear corepressor 2 and Ubiquitin C in the normal reading frame (NRF). The NUP98-NSD1 fusion (3 AML) with NSD1 in NRF is frequently found in hematological malignancies including AML and originates from a cryptic t(5;11) due to telomeric positions of both genes. The PIM3-SCO2 fusion between the serine/threonine kinase PIM3 and an ARF of Cytochrome C oxidase assembly protein can originate from a 0.6 Mb inv(22) in chronic neutrophilic leukemia (Menezes, 2013) and occurs frequently in pediatric AML (Kuhn, 2013). The well-characterized ETV6-LYN fusion (1 AML) fusing the TEL1 oncogene to the SRC family kinase LYN in NRF originates from a complex translocation with inversion involving chromosomes 8 and 12 (Telford, 2016) and has oncogenic activity by activation of STAT5 (Takeda, 2011).Fusion transcripts without recognizable chromosomal abnormalities on metaphase cytogenetics occur frequently in AML and involve actionable targets. The upstream sequence can be fused to the normal or alternative reading frame of the downstream coding sequences and involve potential or validated proto-oncogenes. Apart from cryptic translocations or inversions, trans-splicing is suggested as an alternative mechanism by the presence of some fusion genes in non-malignant hematopoietic cells. Trans-splicing without DNA rearrangement can occur physiologically and alter cellular development towards potential premalignant differentiation (Yuan, 2013). Increasing use of RNAseq technology in routine AML diagnostics, e.g. by HAMLET, will permit to unravel the prognostic significance of unexpected fusion transcripts and to guide novel targeted therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Detection of Gene Fusion Transcripts in Peripheral T-Cell Lymphoma Using a Multiplexed Targeted Sequencing Assay

The genetic basis of peripheral T-cell lymphoma (PTCL) is complex and encompasses several recurrent fusion transcripts discovered over the past years by means of massive parallel sequencing. However, there is currently no affordable and rapid technology for their simultaneous detection in clinical samples. Herein, we developed a multiplex ligation-dependent RT-PCR-based assay, followed by high-throughput sequencing, to detect 33 known PTCL-associated fusion transcripts. Anaplastic lymphoma kinase (ALK) fusion transcripts were detected in 15 of 16 ALK-positive anaplastic large-cell lymphomas. The latter case was further characterized by a novel SATB1_ALK fusion transcript. Among 239 other PTCLs, representative of nine entities, non-ALK fusion transcripts were detected in 24 samples, mostly of follicular helper T-cell (TFH) derivation. The most frequent non-ALK fusion transcript was ICOS_CD28 in nine TFH-PTCLs, one PTCL not otherwise specified, and one adult T-cell leukemia/lymphoma, followed by VAV1 rearrangements with multiple partners (STAP2, THAP4, MYO1F, and CD28) in five samples (three PTCL not otherwise specified and two TFH-PTCLs). The other rearrangements were CTLA4_CD28 (one TFH-PTCL), ITK_SYK (two TFH-PTCLs), ITK_FER (one TFH-PTCL), IKZF2_ERBB4 (one TFH-PTCL and one adult T-cell leukemia/lymphoma), and TP63_TBL1XR1 (one ALK-negative anaplastic large-cell lymphoma). All fusions detected by our assay were validated by conventional RT-PCR and Sanger sequencing in 30 samples with adequate material. The simplicity and robustness of this targeted multiplex assay make itan attractive tool for the characterization of these heterogeneous diseases.

The CTSC-RAB38 Fusion Transcript Is Associated With the Risk of Hemorrhage in Brain Arteriovenous Malformations.

Brain arteriovenous malformations (bAVMs) are congenital anomalies of blood vessels that cause intracranial hemorrhage in children and young adults. Chromosomal rearrangements and fusion genes play an important role in tumor pathogenesis, though the role of fusion genes in bAVM pathophysiological processes is unclear. The aim of this study was to identify fusion transcripts in bAVMs and analyze their effects. To identify fusion transcripts associated with bAVM, RNA sequencing was performed on 73 samples, including 66 bAVM and 7 normal cerebrovascular samples, followed by STAR-Fusion analysis. Reverse transcription polymerase chain reaction and Sanger sequencing were applied to verify fusion transcripts. Functional pathway analysis was performed to identify potential effects of different fusion types. A total of 21 fusion transcripts were detected. Cathepsin C (CTSC)-Ras-Related Protein Rab-38 (RAB38) was the most common fusion and was detected in 10 of 66 (15%) bAVM samples. In CTSC-RAB38 fusion-positive samples, CTSC and RAB38 expression was significantly increased and activated immune/inflammatory signaling. Clinically, CTSC-RAB38 fusion bAVM cases had a higher hemorrhage rate than non-CTSC-RAB38 bAVM cases (p < 0.05). Our study identified recurrent CTSC-RAB38 fusion transcripts in bAVMs, which may be associated with bAVM hemorrhage by promoting immune/inflammatory signaling.

Measurable residual disease assessment by qPCR in peripheral blood is an informative tool for disease surveillance in childhood acute myeloid leukaemia.

Serial assessments of measurable (or minimal) residual disease (MRD) by qPCR may identify nascent relapse in children with acute myeloid leukaemia (AML) and enable pre-emptive therapy. We investigated the kinetics and prognostic impact of recurrent fusion transcripts (RUNX1-RUNX1T1, CBFB-MYH11, KMT2A-MLLT3 or KMT2A-ELL) in 774 post-induction samples from bone marrow (BM, 347) and peripheral blood (PB, 427) from 75 children with AML. BM MRD persistence during consolidation did not increase the risk of relapse, and MRD at therapy completion did not correlate to outcome (HR=0·64/MRD log reduction (CI: 0·32-1·26), P=0·19). In contrast, 8/8 patients with detectable MRD in PB after first consolidation relapsed. Persistence (n=4) and shifting from negative to positive (n=10) in PB during follow-up predicted relapse in 14/14 patients. All 253PB samples collected during follow-up from 36 patients in continuous complete remission were MRD negative. In core-binding factor AML, persistent low-level MRD positivity in BM during follow-up was frequent but an increment to above 5×10-4 heralded subsequent haematological relapse in 12/12 patients. We demonstrate that MRD monitoring in PB after induction therapy is highly informative and propose an MRD increment above 5×10-4 in PB and BM as a definition of molecular relapse since it always leads to haematological relapse.

Application of RNA Sequencing Detects a Large Number of Novel Fusion Transcripts in Patients with AML and MDS

Background: Chimeric transcripts are frequent genetic abnormalities in hematological malignancies that often contribute to leukemogenesis and play crucial roles for risk stratification, MRD monitoring and targeted therapy. However, current standard techniques for detection of gene fusions (chromosome banding analysis (CBA) and fluorescence in situ hydridization (FISH)) are only able to predict known and/or non-cryptic gene fusions. RNA sequencing (RNAseq) has successfully been applied for determination of genomic gene fusions. Aim: Comprehensive analysis of fusion genes in AML (acute myeloid leukemia) and MDS (myelodysplastic syndrome) patients and evaluation of the beneficial use of RNAseq in detecting recurrent and novel fusions. Methods: RNAseq was performed for 579 AML and 630 MDS patients for the detection of recurrent and novel fusion transcripts. WGS (whole genome sequencing) and cytogenetics (CBA, FISH) were applied for validation of the respective transcripts. 151 bp paired-end reads were produced on a NovaSeq 6000 system (Illumina, San Diego, CA) with a yield between 35 and 125 million paired reads per sample. Reciprocal fusion transcripts were counted as one fusion event. All reported p-values are two-sided and were considered significant at p&amp;lt;0.05. Results: After stringent filtering and validation by WGS and/or cytogenetics, for AML, 279 fusion events (corresponding to 147 unique gene fusions) were detected in 208 cases (36% of patients). 213/279 (76%) were confirmed by WGS and cytogenetics, 54/279 cases (19%) by WGS only and 12/279 (4%) cases by cytogenetics only. As a proof of principle, entity-defining rearrangements were detected most frequently: RUNX1-RUNX1T1 (n=42), CBFB-MYH11 (n=39) and PML-RARA (n=39). Other recurrent fusions included KMT2A-MLLT3 (n=6), KMT2A-MLLT10 (n=3) and DEK-NUP214 (n=4) (Fig 1A). However, in addition a high number (130/279, 47%) of so far unreported gene fusions were detected, including 49/130 inter-chromosomal and 81/130 intra-chromosomal fusions. These comprised 92/130 cases for which both fusion partners have not been reported before and 38/130 cases with a novel partner of a gene previously reported in hematological malignancies, including novel partners for NUP98 (XRN1), ETV6 (FAAP100, ARNTL2, SMCO2), RUNX1 (THOC6, EIF3E, OPHN1, TMEM50B), RARA (CCDC33), CBFB (TTC3, HMGB1) and KMT2A (NCBP1, ARHGEF12) (Fig 1B) (all validated by WGS and/or cytogenetics (see above)). Most of the novel fusions were detected only once, however two of them were observed in two patients each (NRIP2-ITFG2, CTDSP1-CFLAR). Moreover, cases with novel fusions were characterized by a very high frequency of TP53 mutations (59% vs. 1% in cases with known fusions and 9% in cases with no detected fusions, p&amp;lt;0.001), whereas FLT3-ITD and NPM1 mutations were rather rare (NPM1: 8% (novel) vs. 0% (known) vs. 35% (no fusion); FLT3-ITD: 3% (novel) vs. 14% (known) vs. 24% (no fusion). Consequently, a large number of cases with novel fusions depicted a complex karyotype (62% (novel) vs. 1% (known) vs. 9% (no fusion)). Regarding age, patients with known fusions were significantly younger than patients with novel fusions or without detected fusions (median age: 54 years (known) vs. 72 (novel) vs. 70 (no fusion), p&amp;lt;0.001). For MDS, 30 fusions (29 unique transcripts) were observed in 28/630 cases (4% of all patients). 4/30 (13%) of detected gene fusions were inter-chromosomal, while the majority (26/30, 87%) was intra-chromosomal. 27/30 (90%) fusions were validated by WGS, 9/30 (30%) by CBA, comprising 6/30 (20%) cases that were validated by both methods. Only one of the detected fusions was detected in &amp;gt;1 patient (n=2) and was described previously (MECOM-NRIP1), all others (n=28) are so far unreported. Of these, 23/28 comprise both so far undescribed fusion partners, whereas for 5/28 fusions one partner was previously reported to function in hematological neoplasms (e.g. MYB-PEX7, RABEP1-NUP88). Conclusions: (1) A large number of novel fusions were detected by RNAseq and validated by WGS and cytogenetics, especially in AML. (2) These novel fusions correlate with a very high frequency of TP53 mutations, their pathogenic role has to be evaluated further. (3) This data may provide the basis for identifying potential new actionable targets (e.g. for personalized vaccine or adoptive cell-based therapy development) and developing markers for patient sensitive MRD monitoring. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

317P - Novel fusion genes identified from matched primary and recurrent breast cancers by RNA-sequencing

BackgroundBreast cancers display substantial inter/intra-tumor heterogeneity. While numerous fusion genes have been identified, most are found to be subclonal, passenger alterations. To discover fusion genes which drive tumor progression and metastasis, we performed RNA-sequencing of matched primary and metastatic breast cancer samples. MethodsRNA-sequencing was performed from sixteen patients matched primary-recurrent tumor tissue and RNA-sequencing data was successfully achieved from sixteen primary tumors and eight recurrent tumors. DeFuse program was used to identify fusion transcripts (FT). ResultsAmong the sixteen patients, six were hormone receptor positive, three were HER2 positive and seven were triple negative tumors. Three cases displayed loco-regional recurrence only and other patients had distant metastases. Overall, 516 fusion transcripts were identified. Mean numbers of fusions in primary and recurred tumors were 28 and 14.6 FTs per sample. Numbers of fusions were greater in two cases with BRCA1 pathogenic germline mutations while no significant differences were observed across subtypes. Novel inter-chromosomal fusion transcript, BCL2-ESR1, CSMD1-ESR1 and HPGDS-ESR1 were found in one hormone receptor positive patient’s metastasis and/or primary tumor. All fusions resulted in preservation of the DNA-binding domain and ligand-binding domain (exon4-10) of the ESR1 gene, with high ESR1 FPKM expression value. Fusions of ERBB2-, MALAT1- and CDK6- genes were found. Among the identified FTs, three cases harbored a previously reported recurrent fusion transcript EEF1DP3-FRY. ConclusionsRNA sequencing revealed numerous fusion transcripts. Among them we found novel fusions including ESR1 fusions which need further validation and functional annotation to confirm their role in tumor progression and metastasis. Legal entity responsible for the studyThe authors. FundingAsan Medical Center. DisclosureAll authors have declared no conflicts of interest.

PS922 BENEFITS OF RNA SEQUENCING IN DETECTING RECURRENT AND NOVEL FUSION TRANSCRIPTS IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Chimeric transcripts are frequent genetic abnormalities in hematological malignancies often contributing to leukemogenesis. So far, analysis of chromosomal translocations giving rise to gene fusions is based on cytogenetic techniques such as chromosome banding analysis (CBA) and fluorescence in situ hybridization (FISH). However, these techniques only detect known gene fusions, while cryptic or novel translocations remain elusive. The impact of gene fusions is increasing as they are not only entity defining and important for risk stratification, but also potentially useful biomarkers for MRD monitoring or targeted therapy. Recently, RNA sequencing (RNAseq) has become a powerful tool to identify disease associated transcriptome profiles and determine recurrent and novel genomic gene fusions underlying the disease.Aims:Comprehensive analysis of the fusion gene landscape in ALL (acute lymphoblastic leukemia) patients and evaluation of the benefits of RNAseq in detecting recurrent and novel fusion transcripts.Methods:We screened the whole transcriptome of 288 patients with ALL (B‐ and T‐lineage) for the detection of recurrent and novel fusion transcripts and applied a combination of whole genome sequencing (WGS) and cytogenetics (CBA, FISH, and genomic arrays) for validation. Gene expression profiling was performed to determine expression signatures of emerging ALL subtypes.Results:The analysis included 288 patients that were screened for chimeric transcripts by RNAseq. After stringent filtering and validation by WGS data and/or cytogenetic analysis a total number of 122 fusion transcripts, corresponding to 61 unique gene fusions in 102 ALL patients, was identified with high confidence. These included 93/122 (76%) gene fusions comprising breakpoints located on different chromosomes while the remaining 29/122 (24%) fusion genes were located on the same chromosome. Among detected fusion events, 90/122 (74%) gene fusions were confirmed by WGS and cytogenetic data, 25/122 (20%) cases were confirmed by WGS only and 7/122 (6%) cases by cytogenetics only. Besides recurrent entity‐defining fusion gene families such as BCR‐ABL1 (n = 30), KMT2A/MLL‐r (n = 22), ETV6‐RUNX1 (n = 4), and TCF3‐PBX1 (n = 4), RNAseq detected so far unreported gene fusions or novel fusion partners of known genes in 38 cases including novel partners for RUNX1, ABL1, and MLLT10. Among patients lacking canonical chromosomal translocations, rearrangements involving 3’ ZNF384 were identified in 8/92 patients (9%) with either EP300 (n = 7) or EWSR1 (n = 1) as 5’ fusion partner, constituting the largest subtype in this subgroup. All ZNF384‐rearranged cases were validated by RT‐PCR. Notably, in none of the cases involving EP300 as fusion partner, standard cytogenetic techniques were able to detect the respective rearrangement as all cases displayed a normal karyotype. ZNF384‐rearrangements were recently defined as novel ALL subtype, based on gene expression signature and clinical features. As they are furthermore potentially targetable by small molecule inhibitors, accurate identification of patients harboring these and other cryptic aberrations by RNAseq is pivotal.Summary/Conclusion:Our data determine the capability of RNAseq to detect known as well as cytogenetically cryptic and novel fusion transcripts in ALL leading to a better characterization of patients with unresolved rearrangements and highlight its immediate importance as a valuable tool in routine diagnostics.

Molecular mechanisms and pathobiology of oncogenic fusion transcripts in epithelial tumors.

Recurrent fusion transcripts, which are one of the characteristic hallmarks of cancer, arise either from chromosomal rearrangements or from transcriptional errors in splicing. DNA rearrangements include intrachromosomal or interchromosomal translocation, tandem duplication, deletion, inversion, or result from chromothripsis, which causes complex rearrangements. In addition, fusion proteins can be created through transcriptional read-through. Fusion genes can be transcribed to fusion transcripts and translated to chimeric proteins, with many having demonstrated transforming activities through multiple mechanisms in cells. Fusion proteins represent novel therapeutic targets and diagnostic biomarkers of diagnosis, disease status, or progression. This review focuses on the mechanisms underlying the formation of oncogenic fusion genes and transcripts and their impact on the pathobiology of epithelial tumors.

Measurable Residual Disease Assessment By qPCR in Peripheral Blood Is an Informative Tool for Disease Surveillance in Childhood Acute Myeloid Leukemia

Background: Relapse remains a serious event and main obstacle to permanent cure in childhood acute myeloid leukemia (AML). Reduction of measurable/minimal residual disease (MRD) assessed by real-time quantitative PCR (qPCR) during therapy is predictive of outcome in adults but persistent MRD positivity may be observed despite long-term remission and hamper accurate risk assignment. Sequential MRD determinations, rather than analysis at single landmark time points, may better capture the dynamics of leukemia eradication and identify relapse at molecular levels when applied in the post-therapy setting. We investigated the post-induction qPCR MRD kinetics in peripheral blood (PB) and bone marrow (BM) in a large cohort of childhood AML patients and demonstrate the utility of serial assessments for disease surveillance after therapy completion.Methods: We collected the results from qPCR MRD analyses of 774 samples (BM 347, PB 427) from 75 children with AML harboring recurrent fusion transcripts (32 RUNX1-RUNX1T1, 24 CBFB-MYH11, 16 KMT2A-MLLT3 and 3 KMT2A-ELL). Patients were treated according to The Nordic Society for Paediatric Haematology and Oncology (NOPHO)-AML 2004 or NOPHO-DBH AML 2012 protocols (2004 - 2016), or AML-BFM 2012 protocol (2014 - 2016). Only patients who achieved complete remission (CR) during induction therapy and received standard-risk consolidation without allografting were eligible for the study.Risk of relapse as a function of time of MRD positivity in sequential samples from BM or PB during consolidation therapy was modeled using restricted cubic splines. Considering shifting from MRD negative to positive in PB and MRD increments in BM above 5x10-4 as time-dependent variables, the Mantel-Byar method was applied to evaluate the prognostic impact of MRD kinetics during follow-up.Results: Risk of relapse was independent of MRD persistence in BM during therapy, but showed a strong correlation with time of MRD positivity in PB where 8/8 patients with detectable MRD after first consolidation course relapsed (Figure 1A and 1B).At therapy completion, MRD level in BM did not correlate to outcome, HR=0.64/MRD log reduction, 95% confidence interval (CI):0.32 - 1.26 (P=0.19), and there was no difference in 3-year cumulative incidence of relapse (CIR) according to MRD status (P=0.51, Figure 2A). Four patients remained MRD positive throughout consolidation therapy and all subsequently relapsed, whereas 7/28 patients who were MRD negative in PB at the end of therapy suffered relapse, 3-year CIR=27%, CI:14% - 49% (P<0.001, Figure 2B).Shifting from negative to positive in PB during follow-up predicted subsequent relapse in 10/10 patients. All 253 PB samples collected during follow-up from 36 patients in continuous CR were MRD negative. In core binding factor (CBF) AML, persistent low-level MRD positivity in BM was frequent (detected in 38% of patients tested within six months of therapy completion) but an increment to above 5x10-4 heralded subsequent relapse in 12/12 patients. Pre-relapse MRD kinetics were delineated in 16 patients and revealed a median log increment/30 days of 0.8 (range:0.4 - 1.5) in CBF patients versus 2.2 (range:1.1 - 3.7) in patients with KMT2A-MLLT3 (P=0.008).Perspectives: This study demonstrates that qPCR MRD monitoring in PB, rather than BM, represents an accurate discriminator of prognosis and is a highly informative tool for disease surveillance in childhood AML. Early relapse detection during follow-up may facilitate preemptive therapy strategies of molecular relapse, possibly improving relapse treatment outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MRI Phenotype of RELA-fused Pediatric Supratentorial Ependymoma.

Epigenetic profiling has recently identified clinically and molecularly distinct subgroups of ependymoma. The 2016 World Health Organization (WHO) classification recognized supratentorial ependymomas (ST-EPN) with REL-associated protein/p65 (RELA) fusion as aclinicopathological entity. These tumors represent 70% of pediatric ST-EPN characterized by recurrent C11orf95-RELA fusion transcripts, which lead to pathological activation of the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) signaling pathway. Cyclin-dependent kinase inhibitor 2A (CDKN2A) inactivation has also been reported to correlate with poor prognosis. Here, we systematically describe magnetic resonance imaging (MRI) characteristics of RELA-fused ST-EPN, with respect to CDKN2A deletion status. Our cohort of patients with ST-EPN (n = 57) was obtained from the database of the German Brain Tumor Reference Center of the German Society for Neuropathology and Neuroanatomy (DGNN), and tumors were diagnosed according to the 2016 WHO classification. Molecular characterization identified 47RELA-fused tumors. We analyzed the preoperative MRI according to standardized criteria, and comparison was performed between CDKN2A altered (n = 21) and CDKN2A wild type (n = 26) tumors. The RELA-fused ST-EPN showed aspectrum of predominantly hemispheric tumors with cysts and necrosis. Statistical analysis on CDKN2A status revealed significant differences in terms of younger manifestation age (p =0.002) and more intratumoral hemorrhage in T2-weighted imaging (T2WI) (p =0.010) in wild type tumors; however, the location was not aparameter for differentiation. This study first provides comprehensive MRI data for RELA-fused ST-EPN as adistinct entity, with further interest on CDKN2A genomic status. Patient stratification by morphological MRI alone seems difficult at present. The results may support ongoing research in ST-EPN within the framework of the radiogenomics concept.

Identification of novel cyclin gene fusion transcripts in endometrioid ovarian carcinomas.

Formation of fusion genes is pathogenetically crucial in many solid tumors. They are particularly characteristic of several mesenchymal tumors, but may also be found in epithelial neoplasms. Ovarian carcinomas, too, may harbor fusion genes but only few of these were found to be recurrent with a rate ranging from 0.5 to 5%. Because most attempts to find specific and recurrent fusion transcripts in ovarian carcinomas focused exclusively on high‐grade serous carcinomas, the situation in the other carcinoma subgroups remains largely uninvestigated as far as fusion genes are concerned. We performed transcriptome sequencing on a series of 34 samples from ovarian tumors that included borderline, clear cell, mucinous, endometrioid, low‐grade and high‐grade serous carcinomas in search of fusion genes typical of these subtypes. We found a total of 24 novel fusion transcripts. The PCMTDI‐CCNL2 fusion transcript, which involves a member of the cyclin family, was found recurrently involved but only in endometrioid carcinomas (4 of 18 tumors; 22%). We also found three additional fusion transcripts involving genes belonging to the cyclin family: ANXA5‐CCNA2 and PDE4D‐CCNB1 were detected in two endometrioid carcinomas, whereas CCNY‐NRG4 was identified in a clear cell carcinoma. The recurrent involvement of CCNL2 in four fusions and of three other genes of the cyclin family in three additional transcripts hints that deregulation of cyclin genes is important in the pathogenesis of ovarian carcinomas in general but of endometrioid carcinomas particularly.

High frequency of intermediate and poor risk copy number abnormalities in pediatric cohort of B-ALL correlate with high MRD post induction

Copy number abnormalities (CNAs) and recurrent fusion transcripts are important genetic events which define and prognosticate B-Cell Acute Lymphoblastic Leukemia (B-ALL). We evaluated CNAs and fusion transcripts in 67 pediatric B-ALL cases and correlated the data with standard risk factors and early treatment outcome parameters. Common fusion transcripts ETV6-RUNX1, E2A-PBX, BCR-ABL1 and MLL-AF4 were examined by RT-PCR and noted in 15%, 15%, 13% and 1.4% of all cases respectively. CNAs in IKZF1, PAX5, EBF1, BTG1, RB1, CDKN2A/B and genes from PAR1 region viz., CSF2RA, IL3RA,P2RY8, SHOX region and CRLF2 were analyzed by multiplex ligation dependent probe amplification assay and were detected in 70% (47/67) of cases, with predominantly deletions in CDKN2A/B (36%), PAX5 (18%) and IKZF1 (16%). A statistically significant association of intermediate/poor risk CNAs was noted with high WBC count (p = 0.001), NCI group (p = 0.02) and minimal residual disease at Day35 (p < 0.0001). IKZF1 and CDKN2A/B deletion revealed poor EFS of 56% at 24 months as compared to EFS of 80% in rest of the cases (p = 0.05) suggesting their potential role in early risk stratification.

329 Primary and Secondary Glioblastomas are Driven by Distinct Forms of Fusion Oncoproteins

Abstract INTRODUCTION The role of chromosomal rearrangement in neoplastic transformation has been well-studied in number of cancers. However, the area remains under-studied in high-grade gliomas. METHODS We performed RNA-seq of 272 gliomas, identifying 214 fusion transcripts. Additional review of the literature identified an additional 772 fusion transcripts in the published literature. Analysis was performed pertaining to rearrangement hot-spots and recurrent fusion transcripts. RESULTS &gt;The most common form of fusion transcript arose from joining of sequences from the same chromosome (76%) rather than sequences from difference chromosomes. Frequency of fusion transcript increased with advanced tumor grade. More fusion transcripts are found in the classical subtype of glioblastoma (P = 0.012), particularly in tumors with amplification of EGFR. Fusion transcripts were most commonly mapped to chromosomes 7 and 12, suggesting these chromosomes contain hots-spots for chromosomal rearrangement. For primary glioblastomas, the most prevalent fusion transcripts involved 1) segments of the EGFR sequence fused to other segments of EGFR or to sequences derived from non-EGFR genes (5.6%) or 2) fusion between FGFR and TACC (FGFR3-TACC3 (3.8%) and FGFR1-TACC1 (0.5%)). PTPRZ1-MET fusions are unique in that they are predominantly found in secondary glioblastomas. All three classes of the encoded fusion proteins have been shown to modulate aspects of glioblastoma biology, including tumorigenesis and invasion. CONCLUSION While ∼50% of glioblastoma harbor fusion transcripts, the occurrence of “driver” fusion transcripts is a relative rarity (&lt;5%). Primary and secondary glioblastomas harbor distinct forms of “driver” fusion transcripts.

Abstract 529: The significance of activated PI3K/AKT pathway in FGFR3-TACC3 fusion positive cervical cancer

Abstract Uterine cervical cancer is one of the most common cancer in women worldwide, and the prognosis in advanced or recurrent cases remains poor because of no effective molecular target therapies for this disease. The aim of our study is to identify and validate therapeutically targetable gene fusions in uterine cervical cancer, leading to the development of new therapeutic strategies. We have analyzed RNA sequencing data of 253 TCGA cervical cancer samples to search for gene fusions by using PRADA algorithm, and validated recurrent fusions in our Japanese dataset (n = 100) by RT-PCR and sanger sequencing. In TCGA samples, we detected 358 fusion transcripts and extracted 3 kinds of recurrent in-frame fusion transcripts (FGFR3-TACC3, ARL8B-ITPR1 and NXN-ABR). We focused on FGFR3-TACC3 fusion as a candidate of therapeutic kinase fusion and identified FGFR3-TACC3 fusion in 2 of 253 (0.8%) TCGA samples and 2 of 100 (2%) Japanese samples. All of fusion positive samples, which were histologically diagnosed as squamous cell carcinoma, showed high expression of both FGFR3 and TACC3 genes. To evaluate the possibility as therapeutically targetable fusions in uterine cervical cancer, we transfected FGFR3-TACC3, wild type FGFR3 and TACC3 transcripts to normal immortalized cervical keratinocytes derived from ectocervix (Ect1/E6E7) and four uterine cervical cancer cell lines (SiHa, ME180, HeLa, CaSki). Continuous expression of FGFR3-TACC3 fusion transcript led to anchorage-independent growth in Ect1/E6E7 and the dramatically alteration of cell proliferation in all cancer cell lines. On the other hand, no obvious phenotype change was observed in FGFR3 or TACC3 transfected cells. Western blotting analysis demonstrated that MAPK pathway was activated in all the fusion transfected cell lines but PI3K/AKT pathway was activated only in ME180 and CaSki harboring PIK3CA mutation. Fusion transfected cell lines exhibited high sensitivity to fusion specific siRNA and FGFR inhibitor compared to control. Although PI3K/AKT activated cell lines (ME180 and CaSki) showed less effective against FGFR inhibitor compared to PI3K/AKT neutral cell lines (SiHa and HeLa), combined inhibition of FGFR and AKT had a synergic effect in PI3K/AKT activated cell lines. Our findings suggest FGFR3-TACC3 fusion gene is an oncogenic driver event and therapeutic target in a fraction of cervical cancer. When FGFR3-TACC3 fusion positive cervical cancer is treated by FGFR inhibitor, genomic background such as PI3K/AKT status should be considered. Citation Format: Ryo Tamura, Kosuke Yoshihara, Tetsuya Saito, Ryosuke Ishimura, Emmanuel Martínez-Ledesma, Yutaro Mori, Kaoru Yamawaki, Kazuaki Suda, Tatsuya Ishiguro, Yoichi Aoki, Seiya Sato, Hiroaki Itamochi, Masaaki Komatsu, Roeland Verhaak, Takayuki Enomoto. The significance of activated PI3K/AKT pathway in FGFR3-TACC3 fusion positive cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2017-529

Recurrent fusion transcripts in squamous cell carcinomas of the vulva.

Juxtaposition of two different genes or gene parts due to chromosomal rearrangement is a well-known neoplasia-associated pathogenetic mechanism. The detection and characterization of such tumorigenic fusions is of great importance both research-wise, diagnostically because they may be specific for distinct tumor entities, and because they may serve as therapeutic targets for antioncogenic drugs that interact directly with the molecular changes responsible for neoplastic transformation.At present, more than 10,000 fusion transcripts have been reported in different types of neoplasia, with one tenth of them being identified in squamous cell carcinomas (SCC) of different locations. No recurrent fusion gene has to date been identified in SCC of the vulva.We performed high-throughput paired-end RNA-sequencing of 12 vulvar SCC and found two recurrent fusions with the STIP1-CREB3L1 and ZDHHC5-GPR137 being present in two tumors each. The transcripts were detected only in the tumor samples, not in normal vulvar tissue from healthy donors used as control. The CREB3L1 and ZDHHC5 genes encode proteins involved in transcription suggesting that the chimeras may alter downstream events in their respective pathways. Expression analysis of the CREB3L1 gene showed the presence of two distinct groups of tumors, one having fusion and downregulation of the gene and the other showing upregulation of CREB3L1.

Abstract 1504: Identification of novel kinase fusion transcripts in endometrial cancer

Abstract The prognosis of patients with advanced stage or recurrent endometrial cancer remains poor and there is at present no effective therapeutic molecular target for this disease. Our aim is to identify therapeutic targetable targets in endometrial cancer. We focused on fusion transcripts whose inhibitors show impressive activity in some tumors and have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts. We have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications but have not detected recurrent fusion transcripts. We performed Sanger sequencing of RT-PCR amplification products and validated 27 (93.1%) of the 29 fusion transcripts that were randomly selected. As targetable fusion candidates, we extracted three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. Western blotting did not show the detectable protein level of the CPQ-PRKDC similar to CAPZA2-MET, and VGLL4-PRKG1. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. In conclusion, we have described the landscape of fusion transcripts in endometrial cancer by using RNA-sequencing data. We could not identify therapeutically targetable kinase fusions in a small set of 25 endometrial cancer cell lines. Our findings suggest that the transcript allele fraction is a good predictor for determining bona fide targetable fusion transcripts when applying fusion detection methods that use RNA-sequencing data. Citation Format: Ryo Tamura, Kosuke Yoshihara, Kazuaki Suda, Kaoru Yamawaki, Tatsuya Ishiguro, Sosuke Adachi, Takayuki Enomoto. Identification of novel kinase fusion transcripts in endometrial cancer. [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 1504.

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

Abstract 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.