Fusion Gene Transcripts Research Articles

Abstract P6-04-04: Functional characterization of ESR1 fusions in breast cancer

Abstract Background: We recently reported two in-frame estrogen receptor alpha gene (ESR1) fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, in endocrine therapy (ET)-refractory, metastatic estrogen receptor positive (ER+) breast cancer patients. Both fusions were causal in driving ET resistance and metastasis in experimental models. CDK4/6 inhibition suppressed tumor growth driven by ESR1 fusions, but did not induce tumor regression (Lei et al., Cell Rep., 2018). Additional ESR1 fusions with diverse partner genes have been recently reported (ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9) (Hartmaier et al., Ann Oncol., 2018), or communicated to us (ESR1-ARNT2, ESR1-PCMT1 and ESR1-ARID1B) (Dr. Dan R. Robinson, University of Michigan). All were identified in ET-refractory tumors. We therefore undertook detailed functional characterization of these new ESR1 fusions to examine their role as disease drivers. We also used mass spectrometry-based kinomics to identify therapeutic targets induced by individual ESR1 fusions. Methods: Unreported ESR1 fusions were identified by RNA sequencing followed by informatics designed to identify fusion gene reads. Stable ER+ breast cancer cell lines (T47D and MCF7) expressing in-frame ESR1 fusion proteins were generated by lentiviral transduction. ET-resistant cell proliferation was assessed in low estradiol medium and/or in the presence of anti-estrogen. Expression of estrogen responsive and epithelial-to-mesenchymal transition (EMT) genes were measured by mRNA-qPCR. Cell motility driven by these fusions was examined using scratch wound assay. Kinase expression patterns were examined using a Kinase Inhibitor Pulldown (KIP) mass spectrometry-based assay. Results: All the fusions we studied contain the first 6 exons (ESR1-e6) of ESR1 that provides the first 4 coding exons. In-frame C-terminal sequences were provided by different partner genes. In addition to previously described ESR1-YAP1 and ESR1-PCDH11X fusions, ESR1-SOX9 and ESR1-ARNT2 conferred estrogen-independent and fulvestrant-resistant growth in stable ER+ breast cancer cells (T47D and MCF7). In T47D cells, these ESR1 fusions upregulated not only classical ER targets, including GREB1, PGR, and TFF1, but also EMT-associated genes, SNAI1 (Snail) and VCAN in an estradiol-independent, fulvestrant-resistant manner. In addition, transcriptionally active ESR1 fusions promoted cell motility. KIP profiling of T47D cells expressing transcriptionally active ESR1 fusions and a patient-derived xenograft naturally harboring the ESR1-YAP1 fusion revealed an increase in abundance of multiple receptor tyrosine kinases including RET and insulin like growth factor 1 receptor (IGF1R). Responses to targeting these kinases have been observed in vitro. Other ESR1-e6 fusions produced stable proteins (ESR1-DAB2, ESR1-GYG1, ESR1-PCMT1, ESR1-ARID1B) but had much less clear transcriptional phenotypes and fewer upregulated kinases identified by KIP. Conclusion: A classification of ESR1 fusions in ET-refractory breast cancer is emerging from this study. About half of ESR1-e6 fusions studied are transcriptionally active, presumably because the 3’ partner in these examples is encoded by transcription factor gene (TF) (SOX9 or ARNT2) or co-activator gene (CoA) (YAP1). These can be classified as ESR1-e6 TF/CoA fusions. The phenotype of the remaining fusions remains under investigation because of the functionally diverse nature of the 3’ partners and the low activity in functional assays. While some of these may be passengers, the example of PCDH11X suggests a non-TF/CoA partner ESR1-e6 fusion can still be active and further studies on additional fusions will be necessary. Downstream targeting of fusion-dependent kinases may be effective. Citation Format: Xuxu Gou, Jonathan T Lei, Sinem Seker, Beom-Jun Kim, Saif Rehman, Adrian V Lee, Dan R Robinson, Matthew J Ellis. Functional characterization of ESR1 fusions in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-04.

Feasibility of Combined Detection of Gene Mutations and Fusion Transcripts in Bone Marrow Trephines from Leukemic Neoplasms

Chromosomal translocations resulting in fusion genes represent important oncogenic drivers and potential therapeutic targets in rare leukemia subtypes. Formalin-fixed, paraffin-embedded trephines are frequently used in hematologic diagnostic tests and provide relevant access to leukemic cells for further studies, for example, phenotyping in bone marrow fibrosis. However, high-throughput molecular analysis of nucleic acids obtained from this material is challenging, especially the reliable detection of RNA transcripts. Sixty-three formalin-fixed, paraffin-embedded bone marrow trephines of patients with chronic eosinophilic leukemia, chronic myeloid leukemia, acute myeloid leukemia, and myeloproliferative neoplasms were analyzed for gene mutations and the presence of fusion transcripts with a commercial amplicon-based next-generation sequencing approach. Fusion transcripts relevant for diagnosis and therapy could be detected and validated (by RT-PCR) in 25 patients (39.7%). Retrospectively selected material, up to 10 years old, was used for this purpose, and only one sample failed in the RNA analysis (1.6%). This study concludes that amplicon-based fusion transcript detection in bone marrow trephines is feasible and that bone marrow trephines taken for histologic assessment can also be applied for high-throughput molecular analysis.

Diagnostic yield of NanoString nCounter FusionPlex profiling in soft tissue tumors.

Diagnostic histopathology of soft tissue tumors can be troublesome as many entities are quite rare and have overlapping morphologic features. Many soft tissue tumors harbor tumor‐defining gene translocations, which may provide an important ancillary tool for tumor diagnosis. The NanoString nCounter platform enables multiplex detection of pre‐defined gene fusion transcripts in formalin‐fixed and paraffin‐embedded tissue. A cohort of 104 soft tissue tumors representing 20 different histological types was analyzed for the expression of 174 unique gene fusion transcripts. A tumor‐defining gene fusion transcript was detected in 60 cases (58%). Sensitivity and specificity of the NanoString assay calculated against the result of an alternative molecular method were 85% and 100%, respectively. Highest diagnostic coverage was obtained for Ewing sarcoma, synovial sarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and desmoplastic small round cell tumor. For these tumor types, the NanoString assay is a rapid, cost‐effective, sensitive, and specific ancillary screening tool for molecular diagnosis. For other sarcomas, additional molecular testing may be required when a translocation transcript is not identified with the current 174 gene fusion panel.

Blinatumomab in Ph-like Acute Lymphoblastic Leukemia – an Institutional Experience

Background Blinatumomab is a landmark treatment for B-cell acute lymphoblastic leukemia (B-ALL) with approval in relapsed/refractory (R/R) and minimal residual disease (MRD) positive settings. Philadelphia chromosome-like (Ph-like) disease has been recognized as a high-risk subset (Jain et al, Blood 2017). However, efficacy of novel immunotherapies, such as blinatumomab, in these patients has not been described. Methods A retrospective chart review of all B-ALL patients diagnosed and/or treated since onset of standardized, Ph-like testing at our shared institutions was performed using clinical/pathological databases. Testing consisted of either 1) Ph-like FISH for CRLF2, ABL1, ABL2, JAK2, PDGFRb, or 2) ALL FISH panel with reflex gene fusion transcript analysis. Results 14 patients who received blinatumomab for R/R or MRD-positive disease were identified (8 R/R; 7 MRD-positive treatment settings – Table 1). 1 received blinatumomab for both indications at different time intervals. The majority of patients were Latino (79%) with CRLF2 rearrangement with age range (19-60, median 26). Most (71%) received induction therapy with pediatric-based regimen. Relapsed/Refractory Outcomes: Patients were treated for primary refractory to previously CR2 disease. 2 proceeded to allogeneic hematopoietic stem cell transplantation (allo-HSCT), with some (42%) refractory to treatment. No neurological (N) or cytokine release syndrome (CRS) toxicity of grade 3 or higher was noted. With allo-HSCT patients censored at time of transplant, mean event-free survival (EFS) was 3.1 months. MRD-positive Outcomes: Patients were treated for MRD-positive disease for CR1 (bridge to transplant) to after allo-HSCT. Remarkably, all 7 patients successfully achieved MRD-negative status. One patient, previously treated with blinatumomab in R/R setting with disease progression, had complete disease eradication post allo-HSCT in CR3. 2 N events of grade 3 or higher (aphasia, seizure) were noted, with both patients tolerating re-challenge. No CRS was seen. Conclusions Ph-like ALL is a challenging disease with clear high-risk implications; here we report our institutional experience with blinatumomab in Ph-like ALL. Blinatumomab was effective for select patients with R/R disease. However, it was more successful in MRD-positive ALL with all patients achieving MRD-negative status. Neurological toxicity was observed in 2 patients; both subsequently tolerated re-challenge. Ph-like ALL patients are more likely to remain MRD-positive after induction therapy (Jain et al, Blood 2017); utilizing blinatumomab as frontline or for MRD in first CR may improve outcomes. Further investigation with larger sample size and multi-center experience is required to fully determine the efficacy and role of blinatumomab, other immunotherapies, and allo-HSCT in the treatment of Ph-like ALL.

ИСПОЛЬЗОВАНИЕ ИММУНОФЕНОТИПИРОВАНИЯ ДЛЯ ПРОГНОЗИРОВАНИЯ ВЫЯВЛЕНИЯ ТРАНСЛОКАЦИИ t(12;21) (p13;q22)/ETV6-RUNX1 У ДЕТЕЙ С ОСТРЫМ ЛИМФОБЛАСТНЫМ ЛЕЙКОЗОМ ИЗ В-ЛИНЕЙНЫХ ПРЕДШЕСТВЕННИКОВ

Aim. The objective of the study was searching for surface antigen expression that could predict presence of translocation t(12;21)(p13;q22)/ ETV6-RUNX1 in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. Results. ETV6-RUNX1 fusion gene transcript was revealed in 72 (21.1%) out of 341 children with BCP-ALL. Leukemic blast cells in ETV6-RUNX1 — positive patients more frequently had high CD10 expression, myeloid markers co-expression, including CD13, CD33, CD117, and absence of CD20 than in ETV6-RUNX1 — negative ones. Nevertheless diagnostic test performance characteristics of each single parameter was not strong enough for predicting the presence of translocation t(12;21)(p13;q22)/ETV6-RUNX1. On the second step of analysis we used various combinations of the above mentioned markers. The highest diagnostic test performance parameters were obtained in case of combination of high CD117 and heterogeneous CD34 expressions. Specificity of this combination was 0.996 (95% CI 0.989-1.000), positive predictive value 0.947 (95% CI 0.847- 1.000), positive likelihood ratio 67.250 (95% CI 9.130-495.348), while negative likelihood ratio was 0.752 (95% CI 0.659-0.860). Conclusion. Thus, combination of CD34 heterogeneous expression and CD117 expression is highly specific for translocation t(12;21)(p13;q22)/ ETV6-RUNX1. However this combination had limited applicability, because it revealed in 25.0% percent of patients only.

Acute promyelocytic leukemia with t(2;17;15): report of one case and review of literature

Objective To explore the clinical and laboratory characteristics and therapeutic effect of acute promyelocytic leukemia (APL) with t(2;17;15). Methods The G-banding technique was used for karyotypic analysis in a female patient with APL who was admitted to the First Affiliated Hospital of Zhengzhou University in December 2018. PML-RARα fusion gene was quickly detected by fluorescence in situ hybridization (FISH). The real-time quantitative polymerase chain reaction (RT-PCR) was used to detection 43 kinds of fusion gene, and the gene mutations were detected by next generation sequencing (NGS). The induction therapy was given with oral retinoic acid+ intravenous infusion of arsenic trioxide, followed by 3 courses of retinoic acid+ arsenic trioxide consolidation therapy. Results The G-banding karyotypic analysis demonstrated 46, XX, t(2;17;15) (q31;q21;q22)[8]/46, XX[2]. FISH results indicated that 62.0% of analyzed cells were positive for the PML-RARα fusion gene. RT-PCR further revealed the positive PML-RARα fusion gene transcript. NGS detection of gene mutations showed no obvious abnormalities. After 39 days of induction therapy with retinoic acid and arsenic trioxide, the patient achieved complete remission (CR). The karyotype was 46XX[20], and PML-RARα/ABL was 0/100. Then, the patient was treated with 3 courses of consolidation therapy, and the results remained in CR. Conclusions APL with complex t(2;17;15) (q31;q21;q22) is rare, and the morphological characteristics are not typical, but it is still associated with the formation of PML-RARα fusion gene. Retinoic acid+ arsenic trioxide has a good therapeutic effect, and the long-term efficacy still needs follow-up. Key words: Leukemia, promyelocytic, acute; Translocation, genetic; Gene fusion; Tretinoin; Arsenites

Standardization of BCR-ABL1 quantification on the international scale in China using locally developed secondary reference panels

For patients with chronic myeloid leukemia, reverse transcription quantitative polymerase chain reaction is widely used in laboratories to quantify BCR-ABL1 fusion gene transcripts for disease management. Many efforts have been made to standardize the BCR-ABL1 testing assay, including the primary and secondary reference reagents, but the secondary standards have not been developed and used in the standardization program in China. With the use of armored RNA technology, armored RNA of BCR-ABL1 and control genes was manufactured to prepare the secondary reference material anchored to the World Health Organization primary reference calibrators for standardization of BCR-ABL1 testing assays. The secondary reference was sent to 30 laboratories in China for validation. Data from an external quality assessment after the standardization process were collected and analyzed as well. The assigned %BCR-ABL1/ABL1IS values of the four levels of the secondary material panels were 0.0118, 0.1345, 1.3808, and 19.4266, respectively. In validation trials, 70.0% (21/30) of laboratories obtained valid conversion factors for the BCR-ABL1 assay. All valid conversion factors from 11 international scale laboratories were equivalent to their respective previous values. External quality assessment data indicated that the accuracy and precision between laboratories were improved. Moreover, the quantity of the panels is abundant to be used as quality control samples for monitoring the shift of data. In this study, we established a secondary genetic reference panel for BCR-ABL1 quantification. This study will play a role in facilitating the worldwide dissemination of the international scale, especially in promoting the standardization of molecular monitoring in China.

NanoString nCounter Technology: High-Throughput RNA Validation.

NanoString nCounter is a multiplex nucleic acid hybridization technology that enables reliable and reproducible assessment of the expression of up to 800 genes or 228 gene fusions in 12 samples in a single assay. The technique works well with a variety of starting materials from fresh or formalin-fixed tissues, cell lysates or biological fluid samples. As low as 1ng RNA input per sample is needed. In addition to gene expression, copy number variation, single nucleotide variation and chimeric RNAs can be analyzed. In this chapter, we discuss the design, setup, and performance of a NanoString-based RNA assay for gene expression or fusion transcript assessment.

A Multicenter Nonrandom Clinical Study Using Induction Regimen of FLAG-IDA or Dae in Treatment of 101 Children with Newly Diagnosed CBF-AML in South of China: Clinical Responses and the Midterm Report of Survival

OBJECTIVE: Patients with core binding factor (CBF) AML generally have a favorable prognosis, but the appropriate intensity of chemotherapy and number of courses are still key points for further exploration by the different leukemic group. Our prospective multicenter clinical study was designed to use high intensity, 4-courses of chemotherapy (backbone of MRC AML 15 protocol) to treat childhood CBF-AML and to observe the feasibility and effectiveness in China. METHODS: Total of 101 children with CBF-AML from 9 centers of south of China were enrolled on the C-HUANAN Children AML 2015 protocol from 2015.1 to 2018.11, accounting for 30.6% of total AML (non-APL) cases. There were 59 boys and 42 girls, aged between 2- 14 years old. The diagnosis of CBF-AML was established by PCR-based detection of RUNX1-RUNX1T1 and CBFβ-MYH11 fusion gene transcripts or by FISH based detection of t(8;21) and inv(16) aberration, respectively. Nine cases were combined with FTL3-ITD. Eleven cases were combined with ASXL1 mutation, and 23 cases were combined with c-KIT mutation. The induction regimen were non-randomized to DAE group and FLAG-IDA group. The induction regimen of DAE group including coures 1 (DAE 3+5+10), course 2 (DAE 3+5+8) . The induction regimen of FLAG-IDA including two courses of FLAG-IDA. Consolidation treatment was same and consisted of 2 courses: 1.HAE or MACE; 2.MidAC. Follow-up observation was then started after 4 courses of treatment. All of patients were followed-up to 06/30/2019, median follow-up time was 25 months. RESULTS: The overall complete remission rate was 93.1% after two courses of induction, 93.8% in the DAE group and 92.8% in the FLAG-IDA group. There was no statistical difference between the two groups. The MRD negative rate (<0.1%) detected by the flow cytometry after first course was 57/87 (65.5%) and 67/77 (87.0%) after second course respectively. The RUNX1-RUNX1T1 or CBFβ-MYH11 gene negative rate was 28/60 (46.7%) and 25/43 (58.1%) after first course and second course of treatment respectively .The overall survival rate (OS) was 83.2%, and the leukemia-free survival rate was 81.1%.Among them, the OS and LFS of the DAE group were 76.4% and 73.2%, respectively, and the OS and LFS of the FLAG-IDA group were 86.3% and 84.6%, respectively( P= 0.331 and 0.241).The OS and LFS of the RUNX1-RUNX1T1 group were 81.8% and 79.2%, respectively.The OS and LFS of the CBFβ/MYH11 group were both 88.9%( P= 0.542 and 0.403). The OS and LFS in CBF-AML with FLT3-ITD positive were both 62.2%. The OS and LFS in CBF-AML without FLT3-ITD were 85.3% and 82.9%, respectively (P=0.045 and 0.081),there was significantly inferior OS in CBF-AML with FLT3-ITD subgroup. Among them, the OS was 60.0% and 85.9% in RUNX1-RUNX1T1 with or without FLT3-ITD group respectively( P=0.041), there was significantly inferior OS in RUNX1-RUNX1T1 with FLT3-ITD subgroup. There were no significant differences in OS and LFS between the CBF with or without C-kit and ASXL1 genes. The overall relapse was 10.9%, and treatment related mortality was 6.9%. Conclusion: The childhood CBF-AML accounted for 30.6% of children's newly diagnosed AML in the South China Children's AML Group. The complete remission rate reached to 93.1% by using 2 courses of intensive induction regimen of DAE or FLAG-IDA. Only 2 courses of consolidation chemotherapy were administered. Two years OS and LFS were higher than 80%.There were significant difference in OS of CBF-AML with FLT3-ITD, and RUNX1-RUNX1T1 AML with FLT3-ITD. There were no significant differences in that of LFS. The overall LFS was close to the outcome of the MRC AML 15 protocol, but there was a gap in OS. The main reason was that the most of relapsed patients abandoned treatment. On other hand the treatment related mortality need to be further reduced. Figure Disclosures No relevant conflicts of interest to declare.

Recreation of t(7;12)(q36;p13) in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Generates a Useful Model for Studying Acute Myeloid Leukemia

Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.

Gene Fusion Detection By RNA-Seq in Acute Myeloid Leukemia (AML)

Accurate and complete genetic classification of AML is crucial for the prediction of clinical outcome and treatment stratification. Deciphering the spectrum of genetic abnormalities by polymerase chain reaction (PCR), karyotyping and fluorescence in situ hybridization (FISH) in routine diagnostics is the current gold standard, however, fusion genes might potentially be missed by these assays. Recently, several methods have been developed to improve the detection of gene fusion transcripts based on RNA sequencing data, providing robust results. To test the detection power and assess the applicability of RNA-Seq based methods in clinical diagnostics we applied two different algorithms, namely FusionCatcher (Nicorici D et al., bioRxiv, 2014) and Arriba (Uhrig S et al., DKFZ, https://github.com/suhrig/arriba), to the transcriptomes of 895 well-characterized AML samples from three independently sequenced cohorts: AMLCG (Herold T et al., Haematologica, 2018, n=261), DKTK (Greif PA et al., Clin Cancer Res, 2018 and unpublished data, n=166), BeatAML (Tyner JW et al., Nature 2018, n=468) and publicly available healthy control samples (SRA studies: SRP018028, SRP047126, SRP050146, SRP105369, SRP115911, SRP133442, n=38). According to karyotyping, 31% (277/895) of samples harbored chromosomal aberrations putatively causing gene fusions (i.e. translocations, interstitial deletions, duplications, inversions, insertions). Analyses by FISH and/or PCR confirmed these rearrangements in 51.3% (142/277) of samples, whereas fusion detection by the means of RNA-Seq showed evidence for fusion genes corresponding to these rearrangements in 60.3% (167/277) of samples. Chromosomal aberrations, identified by karyotyping, which are known to result in clinically relevant fusions (e.g. RUNX1-RUNX1T1, KMT2A fusions) were confirmed by FISH/PCR (AMLCG: n=27/27, DKTK: n=21/21, BeatAML: n=54/57) and RNA-Seq based methods (AMLCG: n=17/27, DKTK: n=21/21, BeatAML: n=56/57) in most of the cases. Of note, the AMLCG cohort was sequenced using the SENSE mRNA Library Prep Kit from Lexogen which seems to be not optimal for fusion detection. Furthermore, 19 samples (AMLCG: n=12, DKTK: n=4, BeatAML: n=3) were found to harbor known pathogenic fusions, described in previous studies, which were not reported by routine diagnostics: NUP98-NSD1 (n=11); CBFB-MYH11, RUNX1-RUNX1T1 and DEK-NUP214 (n=2 each); RUNX1-CBFA2T2 and RUNX1-CBFA2T3 (n=1 each). Reanalysis of six of these samples by PCR confirmed three fusions which were initially missed by routine diagnostics. In general, the amount of reported fusion events by RNA-Seq is high (on average 69 and 39 per sample as detected by FusionCatcher and Arriba respectively), even after applying the built-in filters, indicating a high false positive rate. To robustly identify putative novel fusions, we developed a filtering pipeline and incorporated two new filtering steps. The promiscuity score (PS) of a fusion measures the amount of further distinct fusion partners which were detected in the respective cohort for the 5' and 3' gene. The fusion transcript score (FTS) measures the relative abundance of a fusion transcript to its 5' and 3' partner gene. PS and FTS of known, clinically relevant fusions confirmed by FISH/PCR were used to define cut-offs. To further maximize specificity while maintaining sensitivity, we excluded fusion events which we detected in publicly available healthy samples and subsequently filtered for overlapping calls from FusionCatcher and Arriba (Fig. 1A). Additionally, we obtained further evidence for a fusion event by an elevated transcription of the 3' fusion partner. In case of a fusion event, the transcription of the 3' partner gene likely gets under the control of the promoter of the 5' partner gene. This results in an elevated transcription of genes which are otherwise transcribed at low levels (Fig. 1B-C). Thus, we identified five putatively novel recurrent fusion genes which were detected in two cohorts independently: NRIP1-MIR99AHG, LATS2-ZMYM2, ATP11A-ING1, MBP-SLC66A2, PRDM16-SKI (Fig. 1D-F). Although these events were called with high evidence, we aim at independent validation by complementary methods. In our study, we have not only demonstrated that the application of RNA-Seq to the detection of fusion genes is a valuable complement to diagnostic routine but also has the potential to discover novel putatively pathogenic fusions. Disclosures No relevant conflicts of interest to declare.

Role of Vav1 Genomic Alterations in T-Cell Differentiation and Transformation in Peripheral T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature post-thymic T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphomas not otherwise specified (PTCL, NOS) account for >45% diagnoses, show limited response to intensified chemotherapy treatment with dismal overall survival rate. Our genomic studies have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS, supporting a role for the VAV1 oncogene in the pathogenesis of PTCL. Genetic alterations in VAV1 found in PTCL included recurrent deletions resulting in the in-frame deletion of 9 codons (DV778_T786) in the linker region connecting the C-terminal SH2 domain to the downstream SH3 domain on VAV1 as well as recurrent different gene-fusion transcripts encoding proteins in which the C-terminal SH3 domain of VAV1 is replaced by different domains of the fusion partner. Consistent with the auto-inhibitory role of the C-SH3 domain, the PTCL-associated VAV1 alterations resulted in increased VAV1 activity and strong activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) pathways. Interestingly, mutation co-occurrence analysis shows significant mutual exclusivity of the highly prevalent AITL RHOA G17V mutation and VAV1 genomic alterations (p-value 0.0142) in AITL and PTCL, NOS. This observation supports that VAV1 genetic alterations and RHOA G17V could share a common mechanism of action. To study the role and mechanisms of VAV1 alterations in PTCL in vivo we have engineered a C57/BL6 mouse lines with conditional expression of the recurrent Vav1-Myo1f fusion. By crossing Vav1-myo1f conditional knockin with CD4-CreERT2 mouse deletor line, we have generated animals with CD4+ T cell-specific and tamoxifen-inducible expression of Vav1-myo1f. Preliminary studies indicate that expression of Vav1-myo1f in CD4+ cells induces expansion of TFH cells. This phenotype partially resembles that of animals with CD4 specific expression of Rhoa G17V, indicating a potential convergent effect and mechanisms for VAV1 alterations and RHOA G17V in T-cell development and transformation. However, and more interestingly, Vav1-myo1f expressing cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features that had not been observed in the Rhoa G17V model. Analysis of signaling pathways activated in control and Vav1-myo1f expressing CD4+ cells showed increased phosphorylation of Vav1 and increased activation of the MAPK pathway upon expression of Vav1-myo1f, supporting the role of Vav1 alterations as regulators of T-cell activation. Notably, single cell transcriptome analysis of bulk splenic cells isolated from CD4-CreERT2 control and CD4-CreERT2Vav1co-Vav1-myo1f/WT mice indicate that expression of Vav1-myo1f in CD4+ cells leads to a significant expansion of monocytes and TFH cells and a decrease in B-cell, both naïve and memory. These results support that expression of Vav1-myo1f in CD4+ cells can deeply modify the lymphoid microenvironment. Finally, preliminary analysis of a limited number of mice with targeted expression of the Vav1-Myo1f fusion in CD4+ cells (CD4-Cre Vav1co-Vav1-Myo1f /WT) showed development of a fatal CD4+ malignant lymphoproliferation with a latency of 6-14 months. Histological examination showed disrupted splenic architecture accompanied with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS characteristics and verified expression of both wild type Vav1 and Vav1-myo1f alleles. Interestingly, Vav1-myo1f lymphoma cells showed Th2 associated features including expression of Gata3, c-Maf and IL4 and Il10. These results support an important role for VAV1 alterations in T-cell differentiation, remodeling of the tumor microenvironment and T-cell lymphoma development and highlight the importance of our model as a new experimental system to study the evolution of PTCL and develop novel targeted therapies for this disease. Disclosures Palomero: Kura Oncology: Research Funding.

A tailored approach to fusion transcript identification increases diagnosis of rare inherited disease

Background RNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5–35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data. Methods We describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization. Results We demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance. Conclusions The approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.

JAK2 (V617F) positively regulates PD-L1 mRNA expression via STAT3/5 activation in MNP (PV and ET) patients

Abstract Background Escalated PD-L1 expression is reported in many cancer types initiating an immune escape mechanism. This led to the development of checkpoint inhibitors against PD-1/PD-L1. However, the mechanisms underlying escalated production of PD-L1 in many cancers is not clear yet. Methods In this study, we studied PD-L1 mRNA expression levels in concert to JAK2 (V617F) mutation in a group of 72 MPN patients (38 PV and 33 of ET). Clinical and demographical characters were noted carefully. Results Confirmed MPN patients were screened for JAK2 (V617F) mutation by tetra-primer ARMS-PCR. We next quantified JAK2 (V617F) with ASO-PCR. The patients were also screened for BCR/ABL1 fusion gene transcripts to clarify Ph negative MPN status. The mRNA expression levels of PD-L1, STAT3/5 in all MPN patients were evaluated and it was identified that PD-L1, STAT3 but not STAT5 mRNA levels were significantly high in JAK2 (V617F) patients compared to JAK2 (WT) ones. It was also identified that PD-L1, STAT3/5 mRNA expression was upregulated in MPN patients with more JAK2 (V617F) percentage/allele burden than those MPN patients with less JAK2 (V617F) percentage/allele burden. Finally, we evaluated correlation of JAK2 (V617F) percentage with PD-L1, STAT3/5 mRNA expression levels and discovered that PD-L1 and STAT3 are in strong and direct correlation with JAK2 (V617F) burden while STAT5 was found to be moderately correlated. In addition, we identified strong coexpression of PD-L1 and STAT3 and as expected STAT5 moderately coexisted with PD-L1. Conclusion In summary,this study shows that increased PD-L1 expression accompanies JAK2 (V617F) mutation. The increased expression of PD-L1 may be caused by excessive activation of STAT3/5 which are regulators of PD-L1. The study finds that PD-L1 expression is mainly mediated by JAK2 (V617F) via STAT3 and thatSTAT5 only plays a minor role.The study supports the concept of using PD-L1/STAT3/5 axes as targets for developing checkpoint inhibitors.

Escalated activation ofSTAT3 due to JAK2 V617F activatingmutation causesdownregulation ofPTEN via miRNA 21overexpression

Abstract Background Mutations in Janus kinase 2 (JAK2) genes are the genetic characteristic of BCR-ABL1-negative myeloproliferative neoplasms (MPN). JAK2 V617F mutation is frequently found in Polycythemia Vera (PV) and Essential Thrombocythemia (ET). We aimed to determine the effect of JAK2 V617F on expression of phosphatase and tensin homolog (PTEN) and its regulator miR-21. Materials and Methods Seventy two (72) (MPN) patients were screened for JAK2 V617F burden using ASO-PCR. Out of 72 MPN patients, 58 were JAK2 V617F positive and 14 were JAK2 V617F negative. BCR/ABL1 fusion gene transcript screening by multiplex PCR was done to rule out Philadelphia positive MPN. We performed SYBR green based qRT-PCR assay to characterize relative STAT3, PTEN mRNA expression and miR21 expression in 72 MPN patients. Results The mRNA expression levels of STAT3 and PTEN and mi-21 expression in all MPN patients were evaluated and compared withJAK2 (V617F) and JAK2 (WT) MPN patients. We identified that STAT3 mRNA expression was significantly upregulated in patients with JAK2 (V617F) genotype (mean fold±SD=2.14 ±1.2) compared to patients with JAK2 (WT) genotype (mean fold±SD=1.23±0.95). Further, we observed a significantly increased miR 21 expression (mean fold±SD=2.18±0.44) in JAK2 (V617F) patients in comparison to JAK2 (WT) patients (mean fold±SD=1.70±0.49) (p = 0.0003). In addition, we observed that PTEN expression was substantially and significantly downregulated in JAK2 (V617F) patients (mean fold±SD= 1.73 ± 0.36) compared JAK2 (WT) patients (mean fold±SD=2.55 ± 0.25) (p Conclusion In summary, STAT3 activation caused by JAK (V617F) mutation in MPN patients causes overexpression of miR-21 which leads to downregulation of tumour supressor PTEN MPN patients. Thus, our suggests that JAK2 activated STAT3/miRNA-21 may play a valuable prognostic biomarker for MPN.

Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR

To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.

PS1137 COMPLEX STRUCTURAL VARIANTS LEAD TO GENE INACTIVATION RATHER THAN EXPRESSION OF DE NOVO FUSION GENES IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:Complex structural variants (cSVs) represented by chromothripsis have recently been identified in various tumor types, including chronic lymphocytic leukemia (CLL). They lead to extensive chromosomal rearrangements due to chromosome shattering and consequent random re‐joining of scattered fragments. Substantial parts of DNA are typically lost, and other chromosomal parts potentially containing oncogenes are amplified. Importantly, albeit not common in CLL, de novo fusion gene variants with novel functions can be formed. Overall, chromothripsis is associated with unfavorable prognosis, which can be in part attributed to the frequent co‐occurrence with TP53 gene defects.Aims:To perform a detailed study of cSVs in CLL and to identify putative de novo fusion genes and their possible functions.Methods:cSVs were screened among TP53‐aberrant CLL cases using CytoScanHD arrays (ThermoFisher Scientific). In parallel, conventional cytogenetics, mFISH, iFISH and chromosome painting methods were used to supplement array findings. Transcriptome analysis was performed using TruSeq Stranded Total RNA Library Prep Kit on HiSeq 2000–1T (Illumina). Obtained data were analyzed with in‐house bioinformatic pipelines developed for specific detection of fusion gene variants in transcriptomic data.Results:cSVs suggestive of chromothripsis were screened among 93 CLL patients (111 samples in total) with various types of wild‐type TP53 gene inactivation using CytoScan assay. Out of them, cSVs were identified in 20 patients (21.5%; 26 samples) and were associated with defect of both TP53 alleles (p = 0.0006). In 13/20 patients we predominantly observed clustered deletions; in the remaining 7/20 patients, deletions and also amplifications were present in localized regions. The number of breakpoints ranged from 30 to 154 per case (median 54); 10 to 65 coding genes (median 24) were present in the breakpoints. cSVs were clustered in one chromosome in 9/20 cases, whereas in the rest they were detected in ≥ 2 chromosomes. Comparing the array results to cytogenetic findings, typically a higher number of chromosomes (up to 8) was involved in cSVs within the genome. It can be explained by balanced abnormalities undetectable by the array. Several chromosomes were affected recurrently with the most frequent being chr6 and chr13 in 8 cases, and chr8 and chr9 in 6 cases, respectively. Ten cases with cSVs were subjected to the transcriptome analysis. Although a high number of genomic breakpoints was detected in these cases (555 breakpoints in total affecting 236 genes), they mostly did not lead to formation of expressed fusion genes. Altogether 15 fusion gene transcripts were noted in 8/10 cases (range 1 to 4 per case); 10 fusions were validated by PCR. They mostly consisted of two fusion gene partners, however, in 2 instances, fusions of 3 and 5 genes were detected, respectively. Using online tools, we tested whether the 10 validated fusion transcripts could be translated in proteins and found out that 4 of them encoded abnormal proteins containing domains with predicted functions.Summary/Conclusion:Using our comprehensive approach we showed that chromothripsis affects over a fifth of CLL cases with aberrant TP53 and mostly leads to inactivation of genes present in genomic breakpoints. De novo fusion transcripts supposedly encoding abnormal proteins are expressed only to a limited extent.Supported by CEITEC2020 LQ1601, MZCR‐RVO 65269705, MUNI/A/1105/2018, MZCR‐AZV 16–34272A, GACR 19–11299S, H2020 116026 and 825872.

PS1138 TUMOR MUTATIONAL BURDEN ASSESSED BY TARGETED HIGH THROUGHPUT SEQUENCING PREDICTS TREATMENT FREE SURVIVAL IN CLL: RESULTS FROM A BICENTER STUDY

Background: Complex structural variants (cSVs) represented by chromothripsis have recently been identified in various tumor types, including chronic lymphocytic leukemia (CLL). They lead to extensive chromosomal rearrangements due to chromosome shattering and consequent random re-joining of scattered fragments. Substantial parts of DNA are typically lost, and other chromosomal parts potentially containing oncogenes are amplified. Importantly, albeit not common in CLL, de novo fusion gene variants with novel functions can be formed. Overall, chromothripsis is associated with unfavorable prognosis, which can be in part attributed to the frequent co-occurrence with TP53 gene defects. Aims: To perform a detailed study of cSVs in CLL and to identify putative de novo fusion genes and their possible functions. Methods: cSVs were screened among TP53-aberrant CLL cases using CytoScanHD arrays (ThermoFisher Scientific). In parallel, conventional cytogenetics, mFISH, iFISH and chromosome painting methods were used to supplement array findings. Transcriptome analysis was performed using TruSeq Stranded Total RNA Library Prep Kit on HiSeq 2000–1T (Illumina). Obtained data were analyzed with in-house bioinformatic pipelines developed for specific detection of fusion gene variants in transcriptomic data. Results: cSVs suggestive of chromothripsis were screened among 93 CLL patients (111 samples in total) with various types of wild-type TP53 gene inactivation using CytoScan assay. Out of them, cSVs were identified in 20 patients (21.5%; 26 samples) and were associated with defect of both TP53 alleles (p = 0.0006). In 13/20 patients we predominantly observed clustered deletions; in the remaining 7/20 patients, deletions and also amplifications were present in localized regions. The number of breakpoints ranged from 30 to 154 per case (median 54); 10 to 65 coding genes (median 24) were present in the breakpoints. cSVs were clustered in one chromosome in 9/20 cases, whereas in the rest they were detected in ≥ 2 chromosomes. Comparing the array results to cytogenetic findings, typically a higher number of chromosomes (up to 8) was involved in cSVs within the genome. It can be explained by balanced abnormalities undetectable by the array. Several chromosomes were affected recurrently with the most frequent being chr6 and chr13 in 8 cases, and chr8 and chr9 in 6 cases, respectively. Ten cases with cSVs were subjected to the transcriptome analysis. Although a high number of genomic breakpoints was detected in these cases (555 breakpoints in total affecting 236 genes), they mostly did not lead to formation of expressed fusion genes. Altogether 15 fusion gene transcripts were noted in 8/10 cases (range 1 to 4 per case); 10 fusions were validated by PCR. They mostly consisted of two fusion gene partners, however, in 2 instances, fusions of 3 and 5 genes were detected, respectively. Using online tools, we tested whether the 10 validated fusion transcripts could be translated in proteins and found out that 4 of them encoded abnormal proteins containing domains with predicted functions. Summary/Conclusion: Using our comprehensive approach we showed that chromothripsis affects over a fifth of CLL cases with aberrant TP53 and mostly leads to inactivation of genes present in genomic breakpoints. De novo fusion transcripts supposedly encoding abnormal proteins are expressed only to a limited extent. Supported by CEITEC2020 LQ1601, MZCR-RVO 65269705, MUNI/A/1105/2018, MZCR-AZV 16–34272A, GACR 19–11299S, H2020 116026 and 825872.

ABL1 tyrosine kinase domain mutations in chronic myeloid leukemia treatment resistance.

The development of mutations in the BCR-ABL1 fusion gene transcript causes resistance to tyrosine kinase inhibitors (TKIs) based therapy in chronic myeloid leukemia (CML). Thereby, screening for BCR-ABL1 mutations is advised especially in patients undergoing poor response to treatment. In the current study the authors investigated 43 patients with CML that failed or had suboptimal response to TKIs treatment. Blood samples were collected from patients that were treated with TKIs. The analysis of genetic mutations was performed using a semi-nested PCR assay, followed by Sanger sequencing. The analysis revealed 15 mutations (32.55%): 14 point mutations and an exon 7 deletion. In roughly 30% of cases, mutations in the BCR-ABL1 fusion gene are common causes for treatment resistance.

A Deep Learning Approach to the Screening of Oncogenic Gene Fusions in Humans.

Gene fusions have a very important role in the study of cancer development. In this regard, predicting the probability of protein fusion transcripts of developing into a cancer is a very challenging and yet not fully explored research problem. To this date, all the available approaches in literature try to explain the oncogenic potential of gene fusions based on protein domain analysis, that is cancer-specific and not easy to adapt to newly developed information. In our work, we choose the raw protein sequences as the input baseline, and propose the use of deep learning, and more specifically Convolutional Neural Networks, to infer the oncogenity probability score of gene fusion transcripts and to group them into a number of categories (e.g., oncogenic/not oncogenic). This is an inherently flexible methodology that, unlike previous approaches, can be re-trained with very less efforts on newly available data (for example, from a different cancer). Based on experimental results on a large dataset of pre-annotated gene fusions, our method is able to predict the oncogenity potential of gene fusion transcripts with accuracy of about 72%, which increases to 86% if we consider the only instances that are classified with a high confidence level.