Expression Of Fusion Transcript Research Articles

Modeling Extraordinary Response Through Targeting Secondary Alterations in Fusion-Associated Sarcoma.

Targeted therapy in translocation-associated sarcomas has been limited to oncogenic activation of tyrosine kinases or ligands while gene fusions resulting in aberrant expression of transcription factors have been notoriously difficult to target. Moreover, secondary genetic alterations in sarcomas driven by translocations are uncommon, comprising mostly alterations in tumor suppressor genes (TP53, CDKN2A/B). Our study was triggered by an index patient showing a dramatic clinical response by targeting the secondary BRAF V600E mutation in a metastatic angiomatoid fibrous histiocytoma (AFH) harboring the typical EWSR1::CREB1 fusion. The patient, a 28-year-old female, was diagnosed with an AFH of the thigh and followed a highly aggressive clinical course, with rapid multifocal local recurrence within a year and widespread distant metastases (adrenal, bone, liver, lung). The tumor showed characteristic morphologic features, with histiocytoid cells intermixed with hemorrhagic cystic spaces and lymphoid aggregates. In addition to the pathognomonic EWSR1::CREB1 fusion, targeted DNA sequencing revealed in both primary and adrenal metastatic sites a hot spot BRAF V600E mutation and a CDKN2A/B deletion. Accordingly, the patient was treated with a BRAF-MEK inhibitor combination (encorafenib/binimetinib) showing an excellent but short-lived response. Using a CRISPR-Cas9 approach, we introduced the BRAF c.1799 T>A point mutation in human embryonic stem (hES) cells harboring a conditional EWSR1 (exon7)::CREB1 (exon7) translocation and further differentiated to mesenchymal progenitors (hES-MP) before fusion expression. The cells maintained the fusion transcript expression and the AFH core gene signature while responding to treatment with encorafenib and binimetinib. These results highlight that additional targeted DNA NGS in chemotherapy-resistant translocation-associated sarcomas may reveal actionable oncogenic drivers occurring as secondary genetic events during disease progression.

STAT6-targeting antisense oligonucleotides against solitary fibrous tumor

Solitary fibrous tumor (SFT) is a rare, non-hereditary soft tissue sarcoma which is thought to originate from fibroblastic mesenchymal stem cells. The etiology of SFT is thought to be due to an environmental intrachromosomal gene fusion between NAB2 and STAT6 genes on chromosome 12. Wherein the activation domain of STAT6 is fused with the DNA-binding domain of NAB2 resulting in the oncogenesis of SFT. All NAB2-STAT6 fusion variations discovered in SFTs contain the C-terminal of STAT6 transcript, and thus can serve as target site for antisense oligonucleotides (ASOs)-based therapies. Indeed, our in vitro studies show the STAT6 3’-untranslated region (UTR)-targeting ASO (ASO 993523) was able to reduce expression of NAB2-STAT6 fusion transcripts in multiple SFT cell models with high efficiency (IC50: 116-300 nM). Encouragingly, In vivo treatment of SFT patient-derived xenograft (PDX) mouse models with ASO 993523 resulted in acceptable tolerability profiles, reduced expression of NAB2-STAT6 fusion transcripts in xenograft tissues (21.9%), and importantly, reduced tumor growth (32.4% decrease in tumor volume compared to the untreated control). Taken together, our study established ASO 993523 as a potential agent for the treatment of SFTs.

Novel MFSD7-ATP5I fusion promotes migration and invasion of human sarcoma.

Fusion genes have been implicated in the development and progression of several types of sarcomas, serving as valuable diagnostic and prognostic markers, as well as potential therapeutic targets. We discovered a novel major facilitator superfamily domain-containing 7 (MFSD7) and adenosine triphosphate 5I (ATP5I) gene fusion from sarcomas. In this study, the MFSD7-ATP5I fusion transcript was screened using RNA sequencing in 55sarcoma samples and sixteen normal samples. The MFSD7-ATP5I fusion transcript was detected in 58% of sarcoma samples. The correlation between the expression of MFSD7-ATP5I fusion transcript and clinicopathological information was analyzed, and MFSD7-ATP5I expression is associated with marked pleomorphism and lower tumor necrosis. Cell migration and invasion was significantly reduced by knock-down of MFSD7-ATP5I. Cell migration and invasion was increased by overexpression of MFSD7-ATP5I. A phosphokinase assay demonstrated that MFSD7-ATP5I is involved in the GSK-3 pathway. The current study found that MFSD7-ATP5I is associated with increasing pleomorphism and decreasing necrosis of tumors. And our gain and loss of function experiments prove that MFSD7-ATP5I promotes the invasiveness of tumor cells.

New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.

Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.

Reduction of Tumor Growth with RNA-Targeting Treatment of the NAB2-STAT6 Fusion Transcript in Solitary Fibrous Tumor Models.

Solitary fibrous tumor (SFT) is a rare soft-tissue sarcoma. This nonhereditary cancer is the result of an environmental intrachromosomal gene fusion between NAB2 and STAT6 on chromosome 12, which fuses the activation domain of STAT6 with the repression domain of NAB2. Currently there is not an approved chemotherapy regimen for SFTs. The best response on available pharmaceuticals is a partial response or stable disease for several months. The purpose of this study is to investigate the potential of RNA-based therapies for the treatment of SFTs. Specifically, in vitro SFT cell models were engineered to harbor the characteristic NAB2-STAT6 fusion using the CRISPR/SpCas9 system. Cell migration as well as multiple cancer-related signaling pathways were increased in the engineered cells as compared to the fusion-absent parent cells. The SFT cell models were then used for evaluating the targeting efficacies of NAB2-STAT6 fusion-specific antisense oligonucleotides (ASOs) and CRISPR/CasRx systems. Our results showed that fusion specific ASO treatments caused a 58% reduction in expression of fusion transcripts and a 22% reduction in cell proliferation after 72 h in vitro. Similarly, the AAV2-mediated CRISPR/CasRx system led to a 59% reduction in fusion transcript expressions in vitro, and a 55% reduction in xenograft growth after 29 days ex vivo.

Impact of high-fat diet on mitochondrial dynamics in Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is an asymptomatic pathology, representing more than 75% of aortic aneurysms. It is characterized by a significant dilation of the aortic wall, which usually lead to severe complications as aneurysm rupture. Surgical repair (open or endovascular) is currently the only treatment available for AAA. As obesity is an established as a risk factor for cardiovascular disease, lipid metabolism involvement in AAA is not clearly understood. It would therefore be interesting to study its impact on mitochondrial dynamics, whose bioenergetic functions are necessary to maintain the function and integrity of the aortic wall. Our aim is to study the impact of lipid metabolism on mitochondrial dynamics in abdominal aortic aneurysm. We used a murine model of 6 months old of hypertensive Ldlr-/- male mice (angiotensin II, 1 mg/kg/day) fed with a standard diet (ND) or high-fat diet (HFD) for 28 days. Supra-renal abdominal aorta segment was removed to perform transcriptional and protein analysis (RT-qPCR and Western blot respectively) of the mitochondrial homeostasis actors. For HFD fed mice, we found an obvious obesity of Ldlr-/- hypertensive mice. Our results revealed a similar incidence of abdominal aortic aneurysm and mortality rate, between ND and HFD hypertensive Ldlr-/- mice. In these two groups, mice suffering from an abdominal aortic aneurysm showed a significant decrease, in mitochondrial fusion (Mfn1/2) and fission (Dnml1) transcript expression. Moreover, DRP1 protein expression and the mitophagy protein, PARKIN, are increased only in Ldlr-/- hypertensive ND mice. However, no significant difference was found with Ldlr-/- hypertensive HFD mice. These preliminary data suggest that the obesity observed in Ldlr-/- hypertensive mice by the HFD, has no impact on the development of the aortic aneurysm, as well as on mitochondrial homeostasis.

Integration and viral oncogene expression of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma and gastric cancer.

Persistent high-risk human papillomavirus (HR-HPV) infections cause cervical cancer and a fraction of head and neck cancer. To investigate whether HR-HPV infection might be also involved in the development of gastric cancer (GC), we developed a platform utilizing a rolling circle amplification (RCA)-based nested L1 polymerase chain reactionwith Sanger sequencing to genotype the HPV DNA in cancer tissues of 361 GC and 89 oropharyngeal squamous cell carcinomas (OPSCC). HPV transcriptional activity was determined by E6/E7 mRNA expression and a 3' rapid amplification of cDNA ends was performed to identify HPV integration and expression of virus-host fusion transcripts. Ten of 361 GC, 2of 89 OPSCC, and 1of 22 normal adjacent tissues were HPV L1 DNA-positive. Five of the 10 HPV-positive GC were genotyped as HPV16 by sequencing and 1of 2 GC with RCA/nested HPV16 E6/E7 DNA detection exhibited HPV16 E6/E7 mRNA. Two OPSCC displayed HPV16 L1 DNA and E6/E7 mRNA, of which 1OPSCC tissue showed virus-host RNA fusion transcripts from an intron region of KIAA0825 gene. Together, our data reveal viral oncogene expression and/or integration in GC and OPSCC and a possible etiology role of HPV infections in gastric carcinogenesis.

Detecting the Unusual without Compromising Diagnostic Accuracy - a Prospective WGS/Wts Pilot Study in Acute Leukemias Provided Additional Information for Diagnosis, Prognosis and Treatment

Background: Following WHO and ELN guidelines, standard diagnostics and risk stratification for acute leukemias (AML and ALL) are based on genetic information gathered by a combination of cytogenetics and targeted molecular profiling. Today, comprehensive techniques, i.e WGS and WTS (whole genome and transcriptome sequencing) and the accompanying clinical bioinformatics workflows may even refine and individualize diagnostics. Aim: To evaluate the clinical utility and added value of WGTS in a real-world setting in comparison to standard of care genetic diagnostics. Patients and Methods: The study enrolled 113 patients, 97 with AML and 16 with ALL, diagnosed according to WHO criteria based on cytomorphology, immunophenotyping, cyto- and molecular genetics. WGS (100x) and WTS (50 Mio reads) were performed on NovaSeq instruments. For selected AML cases CD3+ MAC sorted cells were used as normal controls (30x). Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta. Results: All patients were analyzed for structural variants (SV), copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) based on WGS data and the presence of fusion transcripts and expression patterns based on WTS data. Samples without a normal control (n = 74) were analyzed for 121 genes recurrently mutated in myeloid/lymphoid neoplasms, whereas for 39 AML cases with CD3+ normal controls the whole exome was assessed. Overall, 308/333 (92%) gene mutations were consistently identified by targeted sequencing and WGS. Due to the higher read depth, 25/333 (8%) sub-clonal mutations were only detected by targeted sequencing, with 23/25 showing a variant allele fraction <10%. However, 40 gene mutations were identified exclusively by WGS due to the unrestricted analytical scope (e.g. PTPN11, DDX54). Every patient carried on average 3.3 gene mutations within the 121 genes of interest. The number increased to 63 variants (mean; range: 46-116) when analyzing the exome. 52/63 variants are most likely germline variants based on occurrence in CD3+ control sample. Hence, the CD3+ control samples reduced the number of somatic variants in the exome to 11 (mean; range: 1-23) mostly patient-specific mutations that can be explored for personalized treatment options. Addressing cytogenetics with relevance to risk stratification and assignment to AML/ALL genetic subgroups, WTS identified all entity defining fusions and WGS and chromosome banding analysis (CBA) showed a very high concordance. In 10 patients WGS missed subclonal aberrations due to small clone size (median clone size detected by IP-FISH analysis: 6%). However, WGS provided additional information in 40 patients, mostly SV, CNV and cnLOH of unknown significance, but also alterations of biological interest and prognostic and/or therapeutic importance: in one AML case WGTS revealed a KMT2A::MLLT10 rearrangement that was cytogenetically cryptic and not detectable by FISH (KMT2A break apart probe), assigning the patient to the adverse risk group. WGS also identified a cytogenetically cryptic SV leading to an ETV6::ABL1 rearrangement in an ALL patient with complex karyotype. Retrospective analysis showed that the aberration had already been present when the patient presented with an MPN and a normal karyotype (CBA) two years earlier. Hence, WGS led to the diagnosis of an ETV6::ABL1-positive neoplasm in blast phase (poor prognosis), which might be treated with tyrosine kinase inhibitors. Of note, in 3 AML patients a 7q deletion or complex SV leading to a 7q deletion resulted in a CDK6::NOM1 fusion transcript and high MNX1 expression. In addition, WGS revealed in one patient with ETP-ALL a cytogenetically cryptic translocation t(7;14)(q36;q32) affecting the IGH locus and the NOM1 gene, accompanied by high MNX1 expression. Elevated MNX1 expression as a result of t(7;12)(q36;p13) is a very rare event in pediatric AML, but our findings might suggest a more important role of MNX1 expression in adult acute leukemia than previously known. Conclusions: This prospective study in a real-world setting 1) demonstrated the diagnostic power of WGTS with an accuracy comparable to gold standard techniques, 2) added important prognostic information, 3) identified potential new therapeutic targets and 4) gave new insights into disease biology. WGTS significantly will improve and refine diagnostics in the future.

The location of the t(4;14) translocation breakpoint within the NSD2 gene identifies a subset of patients with high-risk NDMM

AbstractAlthough translocation events between chromosome 4 (NSD2 gene) and chromosome 14 (immunoglobulin heavy chain [IgH] locus) (t(4;14)) is considered high risk in newly diagnosed multiple myeloma (NDMM), only ∼30% to 40% of t(4;14) patients are clinically high risk. We generated and compared a large whole genome sequencing (WGS) and transcriptome (RNA sequencing) from 258 t(4;14) (n = 153 discovery, n = 105 replication) and 183 non-t(4;14) NDMM patients with associated clinical data. A landmark survival analysis indicated only ∼25% of t(4;14) patients had an overall survival (OS) <24 months, and a comparative analysis of the patient subgroups identified biomarkers associated with this poor outcome, including translocation breakpoints located in the NSD2 gene and expression of IgH-NSD2 fusion transcripts. Three breakpoints were identified and are designated as: “no-disruption” (upstream of NSD2), “early-disruption” (in the 5' UTR), and “late-disruption” (within the NSD2 gene). Our results show a significant difference in OS based on the location of DNA breakpoints (median OS 28.6 “late-disruption” vs 59.2 “early disruption” vs 75.1 months “no disruption”). These findings have been replicated in an independent replication dataset. Also, univariate and multivariate analysis suggest high-risk markers such as del17p, 1p independently contribute to poor outcome in t(4;14) MM patients.

Fusion transcript analysis reveals slower response kinetics than multiparameter flow cytometry in childhood acute myeloid leukaemia.

Analysis of measurable residual disease (MRD) is increasingly being implemented in the clinical care of children and adults with acute myeloid leukaemia (AML). However, MRD methodologies differ and discordances in results lead to difficulties in interpretation and clinical decision-making. The aim of this study was to compare results from reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiparameter flow cytometry (MFC) in childhood AML and describe the kinetics of residual leukaemic burden during induction treatment. In 15 children who were treated in the NOPHO-AML 2004 trial and had fusion transcripts quantified by RT-qPCR, we compared MFC with RT-qPCR for analysis of MRD during (day 15) and after induction therapy. Eight children had RUNX1::RUNX1T1, one CBFB::MYH11 and six KMT2A::MLLT3. When ≥0.1% was used as cut-off for positivity, 10 of 22 samples were discordant. The majority (9/10) were MRD positive with RT-qPCR but MRD negative with MFC, and several such cases showed the presence of mature myeloid cells. Fusion transcript expression was verified in mature cells as well as in CD34 expressing cells sorted from diagnostic samples. Measurement with RT-qPCR suggests slower response kinetics than indicated from MFC, presumably due to the presence of mature cells expressing fusion transcript. The prognostic impact of early measurements with RT-qPCR remains to be determined.

Population-based targeted RNA sequencing reveals novel disease-related gene fusions in pediatric and adult T-ALL

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a genetically diverse disease characterized by a heterogeneous profile of genetic lesions. Recent transcriptome studies identified a number of new T-ALL-related gene fusions. Novel gene fusions could be employed as disease-specific molecular markers and provide a better understanding of T-ALL biology, proving the need for further omics sequencing studies. MethodsWe performed a population-based analysis of 65 (26 pediatric and 39 adults) Lithuanian T-ALL patients. Targeted RNA sequencing (RNA-seq) was used to detect recurrent and novel T-ALL-related fusion transcripts and gene sequence variants. RT-qPCR was used to calculate the relative gene expression of fusion transcripts. ResultsWe identified disease-related gene alterations in 57/65 (87.7%) T-ALL cases, of which four patients harbored gene fusions that affect ABL-class or JAK-STAT signaling pathways. Five novel gene fusions were detected in 4/65 (6.2%) T-ALL cases: CD99::ABL2 and ZEB1::GNAS in the same case, CTCF::ENKD1, DIAPH1::JAK2, and CDK6::NEK1. Novel fusion transcripts encode chimeric proteins that can potentially affect T-cell proliferation, apoptosis, and help to maintain leukemic cells. Importantly, novel fusion transcripts were not detected in the clinical remission samples attributing these fusions to the leukemic compartment. ConclusionsWe report a similar incidence rate of recurrent gene alterations affecting T-ALL-related molecular signaling pathways in both Lithuanian T-ALL patients and other T-ALL studies. Our data suggest that newly identified gene fusions are specific to leukemic T-cells and provide new molecular insights on T-ALL pathogenesis. Further research is needed to confirm these findings.

Lymphoid blast transformation in an MPN with BCR-JAK2 treated with ruxolitinib: putative mechanisms of resistance

AbstractThe basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)–JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)–like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.

Challenging conventional karyotyping by next-generation karyotyping in 281 intensively treated patients with AML

AbstractAlthough copy number alterations (CNAs) and translocations constitute the backbone of the diagnosis and prognostication of acute myeloid leukemia (AML), techniques used for their assessment in routine diagnostics have not been reconsidered for decades. We used a combination of 2 next-generation sequencing–based techniques to challenge the currently recommended conventional cytogenetic analysis (CCA), comparing the approaches in a series of 281 intensively treated patients with AML. Shallow whole-genome sequencing (sWGS) outperformed CCA in detecting European Leukemia Net (ELN)–defining CNAs and showed that CCA overestimated monosomies and suboptimally reported karyotype complexity. Still, the concordance between CCA and sWGS for all ELN CNA–related criteria was 94%. Moreover, using in silico dilution, we showed that 1 million reads per patient would be enough to accurately assess ELN-defining CNAs. Total genomic loss, defined as a total loss ≥200 Mb by sWGS, was found to be a better marker for genetic complexity and poor prognosis compared with the CCA-based definition of complex karyotype. For fusion detection, the concordance between CCA and whole-transcriptome sequencing (WTS) was 99%. WTS had better sensitivity in identifying inv(16) and KMT2A rearrangements while showing limitations in detecting lowly expressed PML-RARA fusions. Ligation-dependent reverse transcription polymerase chain reaction was used for validation and was shown to be a fast and reliable method for fusion detection. We conclude that a next-generation sequencing–based approach can replace conventional CCA for karyotyping, provided that efforts are made to cover lowly expressed fusion transcripts.

Clinical Significance of MLL-AF4 Fusion Transcript Expression in the Absence of a Cytogenetically Detectable t(4;11)(q21;q23) Chromosomal Translocation

AbstractLeukemic cells from bone marrow (BM) of 17 infants and 127 children with newly diagnosed ALL, as well as fetal liver and BM and normal infant BM samples, were analyzed for presence of a t(4;11) translocation using standard cytogenetic techniques and expression of an MLL-AF4 fusion transcript using standard reverse transcriptase-polymerase chain reaction (RT-PCR) assays as well as nested RT-PCR that is 100-fold more sensitive than standard RT-PCR. Overall, 9 of 17 infants and 17 of 127 noninfant pediatric ALL patients were positive for expression of MLL-AF4 fusion transcripts, as determined by standard and/or nested RT-PCR assays. None of theMLL-AF4+ cases were positive for E2A-PBX1or BCR-ABL fusion transcript expression. Although 8 of 9MLL-AF4+ infants had cytogenetically detectable t(4;11)(q21;q23), 15 of the 17 MLL-AF4+noninfants were t(4;11)−. Infants withMLL-AF4+ ALL had poor outcomes, whereas non-infant MLL-AF4+/t(4;11)−patients had favorable outcomes similar toMLL-AF4− patients. Notably, MLL-AF4transcripts also were detected by nested RT-PCR in 4 of 16 fetal BMs, 5 of 13 fetal livers, and 1 of 6 normal infant BMs, but not in any of the 44 remission BM specimens from pediatric ALL patients. Our results provide unprecedented evidence that MLL-AF4 fusion transcripts can be present in normal hematopoietic cells, indicating that their expression is insufficient for leukemic transformation of normal lymphocyte precursors.© 1998 by The American Society of Hematology.

Abstract B17: Functional characterization of HPV-human fusion transcripts in oropharyngeal cancer

Abstract Human papillomavirus (HPV) genomic integration is frequently seen in oropharyngeal squamous cell carcinoma (OPSCC). Integration into cellular genes can cause the generation of HPV-host fusion transcripts, which have been associated with worse survival in OPSCC patients, but their functional consequences are unknown, as HPV alone is capable of transformation. The forced expression of fusion transcripts versus HPV transcripts in spontaneously immortalized human oral keratinocytes (NOKSI) could demonstrate their effects on cell behavior. Our research focuses on characterizing HPV integration sites and fusion transcripts in cell lines and tumors, followed by in vitro analysis of fusion transcript function. By Detection of Integrated Papillomavirus Sequences (DIPS-PCR) and RT-PCR, we have identified integration events and fusion transcripts in a subset of samples in both intergenic and genic regions of the genome. We began our analysis with two fusion transcripts, one reading from HPV16 E6*-E7-E1 into the tumor suppressor gene TP63 from UM-SCC-47 and the other reading from HPV16 E6*-E4 into the solute carrier gene SLC47A2 from UM-SCC-104. The full fusion transcripts and associated controls were cloned into lentiviral vectors, and stable keratinocyte populations expressing these transcripts were selected. These cultures were subjected to proliferation, migration, and invasion assays. Subsequent in-depth molecular characterization and in vivo studies will help clarify their effects on cancer formation and metastasis. These studies will advance our understanding of how fusion transcripts lead to worse patient survival and help develop new therapies for these patients. Citation Format: Lisa M. Pinatti, Heather M. Walline, Thomas E. Carey. Functional characterization of HPV-human fusion transcripts in oropharyngeal cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B17.

Intergenic Breakpoints Identified by DNA Sequencing Confound Targetable Kinase Fusion Detection in NSCLC.

Next-generation sequencing (NGS) based on genomic DNA has been widely applied for gene rearrangement detection in patients with NSCLC. However, intergenic-breakpoint fusions, in which one or both genomic breakpoints localize to intergenic regions, confound kinase fusion detection. We evaluated the function of intergenic-breakpoint fusions with multiplex molecular testing approaches. NSCLCs with intergenic-breakpoint fusion identified by DNA-based NGS were analyzed by RNA-based NGS, immunohistochemistry (IHC), and fluorescence in situ hybridization. A total of 26 cases with single intergenic-breakpoint fusion were identified from a large cohort of NSCLCs using DNA-based NGS. Of the 26 cases, RNA-based NGS detected expressed fusion transcripts in 11 cases, but the genomic breakpoint position did not logically predict breakpoint of the fusion transcript in these cases, possibly owing to complex rearrangements (n= 5), alternative splicing (n= 2), and reciprocal rearrangement (n= 4). Nonetheless, no expressed fusion transcript was detected in five cases. Moreover, positive anaplastic lymphoma receptor tyrosine (ALK) finding was observed in three of the remaining 10 cases with IHC but not with RNA-based NGS. Three patients with intergenic-breakpoint ALK fusion with or without RNA-based NGS or IHC confirmation who received crizotinib treatment were found to have partial responses. However, one patient with intergenic-breakpoint ROS1, given the positive fluorescence in situ hybridization result, received crizotinib but developed progressive disease within 1 month, possibly owing to no functional fusion transcript detected by RNA-based NGS. Intergenic-breakpoint fusions detected by DNA sequencing confound kinase fusion detection in NSCLC, as functional fusion transcripts may be generated or not. Additional validation testing using RNA/protein assay should be performed in intergenic-breakpoint fusion cases to guide optimal treatment.

CBFA2T3-GLIS2 induces Malignant Transformation of Primitive CD34+ Cord Blood Cells and Recapitulates Infant Megakaryocytic Leukemia

CBFA2T3-GLIS2 fusions are uniquely expressed in infants with AML and its presence is associated with refractory disease with high induction failure and nearly uniform fatality. Recent studies into varying features of this refractory leukemia have defined a unique immunophenotype that is referred to as the RAM phenotype with exceptionally high CD56 expression. Further, deep sequencing of patients with this fusion has failed to identify additional recurrent sequence or structural variants, suggesting that the presence of this fusion might be sufficient for malignant transformation. Given its presence uniquely in infants and lack of cooperating events, the premise of malignant transformation by a single, highly penetrant event might be justified. Despite discovery efforts to define genes, pathways, and networks that are dysregulated in this AML subset, lack of appropriate model systems has limited pre-clinical evaluation of potential therapies. Prior efforts in generating cell lines and xenograft models have met with limited success. We inquired whether primitive CD34+ cord blood cells might provide the appropriate developmental milieu to study fusion-induced transformation. To investigate this possibility, we transduced human CD34+ cord blood cells with a lentivirus encoding CBFA2T3-GLIS2 fusion transcript and GFP. Transduced and mock-transduced control cells were cultured in bulk in a myeloid promoting condition (IMDM with 15% FBS and 10 ng/mL of each growth factor SCF, TPO, FLT3L, IL3, and IL6). The transduction efficiency was approximately 9% at MOI of 50 and expression of fusion transcript was confirmed by RT-PCR. Growth kinetics and surface expressions were assessed by flow cytometry. Evaluation of the growth properties showed that CBFA2T3-GLIS2 transduced cells exhibited enhanced proliferation, with a doubling time (DT) of 93 hours after 5 weeks, whereas DT for mock-transduced control cells was 124 hours (Figure 1A). Further, CBFA2T3-GLIS2 transduced cells had significant expansion of the GFP+ cells, an increase from 9% at the initiation of cultures to 95% by week 8 (Figure 1B). After 12 weeks in culture, fusion-positive cells continue their rapid expansion. Surface CD34 expression was lost after the first week of culture for both CBFA2T3-GLIS2 and mock-transduced cells (Figure 1C), indicative of rapid differentiation in myeloid promoting condition. Morphological evaluation of cells expressing CBFA2T3-GLIS2 reveal a homogenous population with open chromatin, occasional multinucleated cells, prominent nucleoli, and abundant focally basophilic and vacuolated cytoplasm with cytoplasmic blebs (Figure 1D) which are morphologic features suggestive of megakaryocytic differentiation. Megakaryocytic lineage of the fusion transduced cells was confirmed by demonstration of high expression of CD41 and CD61 on the surface of transduced cells (Figure 1E). We further evaluated immunophenotypic alterations as a result of CBFA2T3-GLIS2 fusions, by comparing the surface expression of 16 different markers including CD11b, CD33, CD34, CD36, CD38, CD56, CD64, CD117, CD123, and HLA-DR between CBFA2T3-GLIS2 and mock transduced cells. Mock-transduced cells showed expression of antigens associated with monocytic differentiation (CD36, CD64, and CD11b) (Figure 1F) whereas the CBFA2T3-GLIS2 transduced cells were consistent with more immature cells expressing CD117 and lacking CD11b, CD36 and CD64 (Figure 1G). Of significance, fusion-transduced cells had remarkably high CD56 expression (mean linear MFI of 43 and 3556 for the mock-transduced and transduced cells respectively), similar to that observed in patients with fusion-positive RAM phenotype (average MFI=2040) (Figure 1H), suggesting that CD56 expression is causally linked to the expression of the fusion transcript. This study demonstrates that the CBFA2T3-GLIS2 fusion is sufficient for malignant transformation and recapitulates megakaryocytic AML that is seen in infants. This further demonstrates that engineered primitive cord blood cells can be utilized for generation of appropriate cell lines that can be used for therapeutic development. Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Loken:Hematologics, Inc: Employment, Equity Ownership.

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.

Rapid detection of chromosomal translocation and precise breakpoint characterization in acute myeloid leukemia by nanopore long-read sequencing

Detection of chromosomal translocation is a key component in diagnosis and management of acute myeloid leukemia (AML). Targeted RNA next-generation sequencing (NGS) is emerging as a powerful and clinically practical tool, but it depends on expression of RNA transcript from the underlying DNA translocation. Here, we show the clinical utility of nanopore long-read sequencing in rapidly detecting DNA translocation with exact breakpoints. In a newly diagnosed patient with AML, conventional karyotyping showed translocation t(10;12)(q22;p13) but RNA NGS detected NUP98-NSD1 fusion transcripts from a known cryptic translocation t(5;11)(q35;p15). Rapid PCR-free nanopore whole-genome sequencing yielded a 26,194 bp sequencing read and revealed the t(10;12) breakpoint to be DUSP13 and GRIN2B in head-to-head configuration. This translocation was then classified as a passenger structural variant. The sequencing also yielded a 20,709 bp sequencing read and revealed the t(5;11) breakpoint of the driver NUP98-NSD1 fusion. The identified DNA breakpoints also served as markers for molecular monitoring, in addition to fusion transcript expression by digital PCR and sequence mutations by NGS. We illustrate that third-generation nanopore sequencing is a simple and low-cost workflow for DNA translocation detection.