Identification of a Novel Oncogenic Fusion Gene SPON1-TRIM29 in Clinical Ovarian Cancer That Promotes Cell and Tumor Growth and Enhances Chemoresistance in A2780 Cells

Gene structure alterations, such as chromosomal rearrangements that develop fusion genes, often contribute to tumorigenesis. It has been shown that the fusion genes identified in public RNA-sequencing datasets are mainly derived from intrachromosomal rearrangements. In this study, we explored fusion transcripts in clinical ovarian cancer specimens based on our RNA-sequencing data. We successfully identified an in-frame fusion transcript SPON1-TRIM29 in chromosome 11 from a recurrent tumor specimen of high-grade serous carcinoma (HGSC), which was not detected in the corresponding primary carcinoma, and validated the expression of the identical fusion transcript in another tumor from a distinct HGSC patient. Ovarian cancer A2780 cells stably expressing SPON1-TRIM29 exhibited an increase in cell growth, whereas a decrease in apoptosis was observed, even in the presence of anticancer drugs. The siRNA-mediated silencing of SPON1-TRIM29 fusion transcript substantially impaired the enhanced growth of A2780 cells expressing the chimeric gene treated with anticancer drugs. Moreover, a subcutaneous xenograft model using athymic mice indicated that SPON1-TRIM29-expressing A2780 cells rapidly generated tumors in vivo compared to control cells, whose growth was significantly repressed by the fusion-specific siRNA administration. Overall, the SPON1-TRIM29 fusion gene could be involved in carcinogenesis and chemotherapy resistance in ovarian cancer, and offers potential use as a diagnostic and therapeutic target for the disease with the fusion transcript.

MLL-SEPT5 Fusion Transcript in Myelodysplastic Syndrome Patient With t(11;22)(q23;q11)

Objectives: This study aimed to identify unknown mixed lineage leukemia (MLL) translocation partner genes in a de novo patient with myelodysplastic syndrome (MDS) with t(11;22)(q23;q11) and investigate the clinical and molecular features of this patient.Methods: Bone marrow cells were assessed by karyotype analysis to reveal chromosomal abnormalities. Fluorescence in situ hybridization (FISH) was performed to detect MLL gene rearrangement using an MLL-specific break-apart probe. LDI-PCR and RT-PCR were performed, and the PCR products were sequenced using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). The sequence data of the PCR products were analyzed using bioinformatics tools. Meanwhile, clinical data were collected to evaluate the prognosis of the patient.Results: Chromosomal karyotype analysis showed that the karyotype of the patient was 46, XX, t(11;22)(q23;q11)[10]/46, XX[1]. Subsequently, FISH data confirmed MLL gene rearrangement in the patient. LDI-PCR precisely showed that SEPT5 was the MLL translocation partner gene. RT-PCR and sequencing analysis disclosed the presence of MLL-SEPT5 fusion transcript and confirmed the fusion between MLL exon 8 and SEPT5 exon 3. Moreover, the patient had a recurrence shortly after allogeneic hematopoietic stem cell transplantation.Conclusion: Although the MLL-SEPT5 fusion transcript was occasionally described in acute myeloid leukemia, it was first identified in MDS. Patients with MLL-SEPT5 fusion gene exhibited a poor prognosis even with an aggressive treatment.

Rare Case of Pericentric Inversion, Inv(9)(p13q34) of the Der(9)t(9;22)(q34;q11) in A Patient with Chronic Myeloid Leukemia – A Case Report

As we know, the Philadelphia chromosome (Ph) is a highly specific marker for chronic myeloid leukemia (CML). This hematological disease is characterised by the formation of the BCR/ABL1 fusion gene, usually with typical translocation pattern including 9q34 and 22q11. In this paper we describe a 55 years old female patient with typical clinical and haematological signs of CML and a chromosome 9 differing from that which normally participates in translocation t(9;22). The karyotype of this Ph positive patient is characterised by pericentric inv(9)(p13q34) of the der(9)t(9;22)(q34;q11). Reverse transcriptase-polymerase chain reaction revealed a e14a2 type of BCR/ABL1 fusion transcript. As a consequence of this unusual translocation, FISH also found the separation of the ABL1/BCR1 fusion gene on chromosome 9. On reviewing the literature, to date only 10 Ph-positive leukemia patients have been noticed to have pericentric inversion inv(9)(p22q34)der(9)t(9;22)(q34;q11). No one case has been described with pericentric inversion inv(9)(p13q34) of the der(9)t(9;22)(q34;q11). This indicate that pericentric inv(9)(p13q34) of the der(9)t(9;22)(q34;q11) is a novel, rare, chromosomal abnormality in Ph-positive CML.

The Prognostic and Predictive Value of ESR1 Fusion Gene Transcripts in Primary Breast Cancer

Background: In breast cancer (BC), recurrent fusion genes of estrogen receptor alpha (ESR1) and AKAP12, ARMT1 and CCDC170 have been reported. In these gene fusions the ligand binding domain of ESR1 has been replaced by the transactivation domain of the fusion partner constitutively activating the receptor. As a result, these gene fusions can drive tumor growth hormone independently as been shown in preclinical models, but the clinical value of these fusions have not been reported. Here, we studied the prognostic and predictive value of different frequently reported ESR1 fusion transcripts in primary BC. Methods: We evaluated 732 patients with primary BC (131 ESR1-negative and 601 ESR1-positive cases), including two ER-positive BC patient cohorts: one cohort of 322 patients with advanced disease who received first-line endocrine therapy (ET) (predictive cohort), and a second cohort of 279 patients with lymph node negative disease (LNN) who received no adjuvant systemic treatment (prognostic cohort). Fusion gene transcript levels were measured by reverse transcriptase quantitative PCR. The presence of the different fusion transcripts was associated, in uni- and multivariable Cox regression analysis taking along current clinic-pathological characteristics, to progression free survival (PFS) during first-line endocrine therapy in the predictive cohort, and disease- free survival (DFS) and overall survival (OS) in the prognostic cohort.Results: The ESR1-CCDC170 fusion transcript was present in 27.6% of the ESR1-positive BC subjects and in 2.3% of the ESR1-negative cases. In the predictive cohort, none of the fusion transcripts were associated with response to first-line ET. In the prognostic cohort, the median DFS and OS were respectively 37 and 93 months for patients with an ESR1-CCDC170 exon 8 gene fusion transcript and respectively 91 and 212 months for patients without this fusion transcript. In a multivariable analysis, this ESR1-CCDC170 fusion transcript was an independent prognostic factor for DFS (HR) (95% confidence interval (CI): 1.8 (1.2–2.8), P=0.005) and OS (HR (95% CI: 1.7 (1.1–2.7), P=0.023). Conclusions: Our study shows that in primary BC only ESR1-CCDC170 exon 8 gene fusion transcript carries prognostic value. None of the ESR1 fusion transcripts, which are considered to have constitutive ER activity, was predictive for outcome in BC with advanced disease treated with endocrine treatment.

Higher Incidence of Co-Expression of BCR-ABL Fusion Transcripts in an Eastern Indian Population

Background Chronic myeloid leukaemia (CML) is a hematopoietic stem cell disorder, caused by a balanced reciprocal translocation (t(9;22) (q34;q11))that lead to the formation of BCR (Break point Cluster Region)-ABL (Abelson) fusion transcripts known as Philadelphia (Ph) chromosome. Prevalence of BCR-ABL fusion transcripts in Indian CML population is poorly understood and few studies have been reported from India. The aim of present study was to determine the frequencies as well as prognostic effects of the three fusion transcripts i.e. b2a2, b3a2 and e1a2 in an Indian population. Methods RNA was isolated from total 123 sample 27 bone marrow (BM) sample and 96 Peripheral blood (PB) sample of CML patient followed by cDNA synthesis. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed using TaqMan→ assay (ABI, CA, USA) to monitor BCR-ABL transcript. Results Ph' chromosome was observed in 103 patients whereas it was not detected in 20 cases. qRT-PCR revealed that the b3a2 fusion transcripts was the most common transcript in CML patients (63.41%) while b2a2 fusion transcript was present in 16.26% cases. Co-expression of b3a2+b2a2 fusion transcript was observed in 0.81% cases whereas co-expression of b3a2+e1a2 fusion transcript was found in 1.63% cases. There was no co-relation observed between b3a2 fusion transcript and platelet count. The fusion transcript b2a2 was observed in relatively younger patients compared to b3a2 fusion transcript. Although this correlation was not statistically significant. Conclusion The co-expression of BCR-ABL fusion transcripts was higher (63.41% aggregate of b3a2) in the present population in contrast to other populations reported. This finding was consistent with the frequency data reported from Sudan.

FDJD: RNA-Seq Based Fusion Transcript Detection Using Jaccard Distance

Gene fusions events, which are the result of two genes fused together to create a hybrid gene, were first described in cancer cells in the early 1980s. These events are relatively common in many cancers including prostate, lymphoid, soft tissue, and breast. Recent advances in next-generation sequencing (NGS) provide a high volume of genomic data, including cancer genomes. The detection of possible gene fusions requires fast and accurate methods. However, current methods suffer from inefficiency, lack of sufficient accuracy, and a high false-positive rate. We present an RNA-Seq fusion detection method that uses dimensionality reduction and parallel computing to speed up the computation. We convert the RNA categorical space into a compact binary array called binary fingerprints, which enables us to reduce the memory usage and increase efficiency. The search and detection of fusion candidates are done using the Jaccard distance. The detection of candidates is followed by refinement. We benchmarked our fusion prediction accuracy using both simulated and genuine RNA-Seq datasets. Paired-end Illumina RNA-Seq genuine data were obtained from 60 publicly available cancer cell line data sets. The results are compared against the state-of-the-art-methods such as STAR-Fusion, InFusion, and TopHat-Fusion. Our results show that FDJD exhibits superior accuracy compared to popular alternative fusion detection methods. We achieved 90% accuracy on simulated fusion transcript inputs, which is the highest among the compared methods while maintaining comparable run time.

Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma

Background Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma. Reverse transcription–polymerase chain reaction (RT–PCR) and fluorescence in situ hybridization are commonly used to detect the fusion gene, and assays using next-generation sequencing have recently been reported. However, at least 28 fusion transcript variants have been reported, making rapid and accurate detection difficult. Methods We constructed two sets of multiplex PCR assays and evaluated their utility using cell lines and clinical samples. Results EWSR1/FUS-ETS was detected in five of six tumors by the first set, and in all six tumors by the second set. The fusion gene detected only by the latter was EWSR1-ERG, which completely lacked exon 7 of EWSR1. The fusion had a short N-terminal region of EWSR1 and showed pathologically atypical features. Conclusions We developed multiplex RT–PCR assays to detect EWSR1-ETS and FUS-ETS simultaneously. These assays will aid the rapid and accurate diagnosis of Ewing sarcoma. In addition, variants of EWSR1/FUS-ETS with a short N-terminal region that may have been previously missed can be easily detected.

Cytogenetically cryptic TNIP1-PDGFRB and PCM1-FGFR1 fusion leading to myeloid/lymphoid neoplasms with eosinophilia (MLN-eo) in children

Introduction: MLN-eo associated with gene rearrangements of PDGFRA, PDGFRB, FGFR1, or PCM1-JAK2 are rare haematological neoplasms primarily affecting adults. Eosinophilia commonly occurs but may also be absent. The heterogeneous clinical picture and the rarity of the disease, especially in children, may delay an early diagnosis. MLN-eo are characterized by constitutive tyrosine kinase activity due to gene fusions. It is thus of prognostic importance to obtain a prompt genetic diagnosis to start a specific therapy. Here we report two female paediatric cases of MLN-eo (6 months and 13 years old at initial diagnosis). Methods: In both cases, bone marrow morphology, karyotyping, fluorescence in-situ hybridization analysis (FISH) via break apart probes (PDGFRB (5q32), FGFR1 (8p12), JAK2 (9p24), FIP1L1/CHIC2/PDGFRA (4q12)), targeted RNA sequencing and in one case array CGH were performed. Results: The 6 months old girl was admitted to hospital with a 3-month history of rash and leukocytosis with eosinophilia. The skin showed multiple purpuric lesions (Fig 1 A/B). Mild splenomegaly was noted. White blood count (WBC) was 48000/µl with 38% eosinophils. Bone marrow trephine showed hypercellular marrow with mild fibrosis and eosinophilia without increase in blasts. Biopsy of a skin nodule displayed a histological pattern of interface dermatitis with eosinophilic infiltrate. (Fig 1 C/D). Fluorescence R-banding showed a normal karyotype (46,XX) (Fig. 2 A). However, FISH and array CGH detected an interstitial deletion of 5` PDGFRB (5q32) in 61 % of interphase nuclei (Fig. 2 B-D). Targeted RNA sequencing (RNA-seq) confirmed, as the array CGH suggested, the suspected TNIP1/PDGFRB fusion. According to the WHO criteria, diagnosis of a myeloid neoplasia with PDGFRB rearrangement due to an interstitial deletion in 5q was made. Because of the PDGFRB rearrangement, imatinib (250 mg/m²/d) therapy was started. Leukocyte and eosinophil counts normalized within 4 days without signs of tumour lysis. Skin lesions disappeared within 2 weeks. After 4 weeks, the dose was reduced to 100 mg/m² 3 x/week. Now at 14 months of age, peripheral counts continue to be normal and the fusion transcript is not detectable in the peripheral blood. The 13 years old girl was admitted with severe tachypnoea due to pleural effusions, hepatosplenomegaly and lymphadenopathy. Echocardiography showed endocarditis, left ventricular fibrosis and mitral insufficiency. WBC was 112170 /µL with 39% eosinophils. Bone marrow aspirate and trephine showed a feature of myeloproliferative neoplasia (MPN) with eosinophilia. The karyotype was normal. A rearrangement involving the FGFR1 locus was detected by FISH (Fig. 3 B/C). Splitting of the probe signals indicated an inversion on chromosome 8. Targeted RNA sequencing revealed a PCM1-FGFR1 fusion transcript. Diagnosis of a MLN-eo with FGFR1 rearrangement and evidence of a PCM1-FGFR1 fusion, most likely caused by an inversion on chromosome 8, was made. The girl stabilized after therapy with prednisone, vincristine, hydroxycarbamide and anti-IL-5 antibody. Peripheral blood counts normalized within 2 weeks. Eight weeks after initial diagnosis she presented with signs of a transient ischemic attack, respiratory distress and arterial hypotension. At that time WBC was 139000/µl with 53% myeloid blasts and 5% eosinophils. Trisomy 8 was detected in all metaphases and 88% of cells in FISH (Fig.3 A-C). Diagnosis of a progression to a myeloid blast phase was made. Induction chemotherapy (cytarabine, idarubicin, etoposidphosphate) was administered. On day +22 bone marrow aspirates showed the persisting picture of MPN. Preparations for hematopoietic stem cell transplantation (HSCT) and ponatinib therapy were begun, but cardiac and respiratory insufficiency that developed during chemotherapy were fatal. Conclusion: As these two cases have shown, standard cytogenetic and molecular methods may not be sufficient to diagnose MLN-eo due to cytogenetically cryptic aberrations. Thus, genetic diagnosis must be precise and quick (e.g. break apart FISH, targeted RNA-seq) in order to initiate adequate therapies with tyrosine kinase inhibitors or HSCT. Patients with rearrangements of PDGFRA or PDGFRB usually respond well to imatinib, whereas patients with FGFR1 and JAK2 gene fusions exhibit more aggressive diseases with variable sensitivity to tyrosine kinase inhibitors and have an early indication for HSCT. Figure 1 Figure 1. Disclosures Reiter: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; AOP Orphan Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Deciphera: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support.