Fusion Partner Genes Research Articles

Abstract 3109: Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite the identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathways resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we assessed the role of wild type TFE3 (wt-TFE3) in RCC cell bearing TFE3 fusion with genes associated with pre-mRNA splicing factor machinery (NONO, SFPQ and PRCC).Methods: Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft established in NSG mice. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. RNA interference (RNAi) mediated knockdown with differential exon targeting strategy was employed to study wt-TFE3 loss of function in RP-R07, UOK-109 and UOK-146, respectively. Real time monitoring of cell viability assay with multiplex readout were developed to investigate the effect of wt-TFE3 loss of function in 2D and 3D culture system. Two different 3D models were used;1) Tumor growth model, in which cancer cell interaction with extracellular matrix and stromal cells were represented in a multiculture-spheroid system; 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid, and the invasion trajectories were observed and quantified. Exogenous expression of wt-TFE3-EGFP was utilized to study the protein subcellular localization in RP-R07, UOK-109 and UOK-146 2D cultures and 3D culture system.Results: Consistent with the expression of chimeric TFE3, wt-TFE3-EGFP ectopic expression demonstrated strong nuclear localization in RP-R07, UOK-109 and UOK-146. Multiplex readout of cells viability assay showed that transient loss of wt-TFE3 in RP-R07, UOK-109 and UOK-146 curtails the proliferation rate of these cells in 2D and 3D models in fusion partner dependent manner (P<0.05). 3D invasion assay coupled with RNAi strategy in RP-R07, UOK-109 and UOK-146 demonstrated the role wt-TFE3 in invasion potential of these cell in fusion partner dependent fashion (P<0.05). Downstream analysis suggested that the wt-TFE3 transient silencing affected cell proliferation and invasion ability via inhibition of the IRS-PI3K-mTOR axis.Conclusion: Our results suggest the potential role of wt-TFE3 in tRCC oncogenesis irrespective of its different fusion partner. Citation Format: Nur P. Damayanti, Khunsha Ahmed, May Elbanna, Chinghai Kao, Roberto Pili. Investigating the role of wild-type TFE3 in renal cell carcinoma cells harboring TFE3 fusions with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3109.

Prevalence of ALK gene alterations among the spectrum of plexiform spitzoid lesions

ALK receptor tyrosine kinase gene (ALK) rearrangements have been described in spitzoid lesions with a plexiform growth pattern. To investigate the prevalence of ALK alterations in a large series of spitzoid lesions. ALK immunohistochemical and fluorescence in situ hybridization analyses of 78 spitzoid plexiform lesions including 41 Spitz nevi, 29 atypical Spitz tumors (ASTs), and 8 spitzoid melanomas. ALK immunohistochemical staining was observed in 14.6% of Spitz nevi (6 of 41) and 13.8% of ASTs (4 of 29); the spitzoid melanomas were ALK negative. Fluorescence in situ hybridization confirmed ALK translocation in 9 cases and amplification in 1 case. In 2 of the translocated cases it was possible to determine the fusion partner gene (ie, tropomyosin 3 gene [TPM3] or dynactin 1 gene [DCTN1]). Of the 4 cases of AST examined, 2 carried the B-Raf proto-oncogene, serine/threonine kinase gene (BRAF) V600E mutation. The 10 patients had a mean age of 18.7years (range, 1-39) and a female predominance (female-to-male ratio, 7:3). Seven lesions arose on the extremities; the 2 lesions occurring in infants were located on the face. The lesions' mean diameter was 6.2mm (range, 3-13), and their mean Breslow thickness was 1.83mm (range, 0.6-3.6). The results of sentinel node biopsy were negative in 2 ASTs. BRAF status was tested in only 4 of 10 samples because of the limited amount of material. ALK alterations characterize a significant subset of spitzoid lesions.

Characterization of Computed Tomography Imaging of Rearranged During Transfection-rearranged Lung Cancer

BackgroundRearranged during transfection (RET)-rearranged non–small-cell lung cancer (NSCLC) is relatively rare and the clinical and computed tomography (CT) image characteristics of patients with an advanced disease stage have not been well documented. Patients and MethodsWe identified patients with advanced-stage RET-rearranged NSCLC treated in the Cancer Institute Hospital, Japanese Foundation for Cancer Research, and analyzed the clinical and CT imaging characteristics. ResultsIn 21 patients with advanced RET-rearranged NSCLC, RET rearrangements were identified using fluorescence in situ hybridization and/or reverse transcriptase-polymerase chain reaction. The fusion partner genes were identified as KIF5B (57%), CCDC6 (19%), and unknown (24%). CT imaging showed that 12 primary lesions (92%) were peripherally located and all were solid tumors without ground-glass, air bronchograms, or cavitation. The median size of the primary lesions was 30 mm (range, 12-63 mm). Of the 18 patients with CT images before initial chemotherapy, 12 (67%) showed an absence of lymphadenopathy. Distant metastasis included 13 with pleural dissemination (72%), 10 with lung metastasis (56%), 8 with bone metastasis (44%), and 2 with brain metastasis (11%). ConclusionAdvanced RET-rearranged NSCLC manifested as a relatively small and peripherally located solid primary lesion with or without small solitary lymphadenopathy. Pleural dissemination was frequently observed.

ALK-rearrangement in non-small-cell lung cancer (NSCLC).

The ALK gene encodes a transmembrane tyrosine kinase receptor. ALK is physiologically expressed in the nervous system during embryogenesis, but its expression decreases postnatally. ALK first emerged in the field of oncology in 1994 when it was identified to fuse to NPM1 in anaplastic large‐cell lymphoma. Since then, ALK has been associated with other types of cancers, including non‐small‐cell lung cancer (NSCLC). More than 19 different ALK fusion partners have been discovered in NSCLC, including EML4, KIF5B, KLC1, and TPR. Most of these ALK fusions in NSCLC patients respond well to the ALK inhibitor, crizotinib. In this paper, we reviewed fusion partner genes with ALK, detection methods for ALK‐rearrangement (ALK‐R), and the ALK‐tyrosine kinase inhibitor, crizotinib, used in NSCLC patients.

H2AFY is a novel fusion partner of MECOM in acute myeloid leukemia

The MECOM gene encoding a zinc finger protein that functions as a transcription factor, was located on chromosome 3q26, and rearrangements of MECOM often cause its overexpression in acute myeloid leukemia (AML). We identified H2AFY as a novel fusion gene partner of MECOM in an elderly male AML patient with cryptic 3q26 rearrangement using the whole transcriptome sequencing, who carried out abnormal karyotype of 46,XY,t(3;5)(q27;q31),add(14)(p11). We validated the existence of the unreported H2AFY-MECOM fusion gene by RT-PCR and Sanger DNA sequencing, and detected mutations of NRAS and BCOR in this patient. In addition, we found abnormally elevated expression of MECOM in this patient by quantitative-polymerase chain reaction (RQ-PCR). Further research is needed to investigate functional characterizations of this novel fusion in the development of AML.

ETV6 Gene Rearrangements Characterize a Morphologically Distinct Subset of Sinonasal Low-grade Non-intestinal-type Adenocarcinoma: A Novel Translocation-associated Carcinoma Restricted to the Sinonasal Tract.

Low-grade sinonasal adenocarcinomas (low-grade SNACs) of the sinonasal tract comprise a poorly characterized and histologically heterogeneous group of tumors. We describe three cases of a histologically distinct variant of low-grade SNAC characterized by ETV6 gene rearrangements. The patients included 2 women (aged 32 and 88 y) and a man (aged 75 y); all were initially treated with surgery alone. Follow-up ranged from 9 to 170 months with one patient having 2 local recurrences and none experiencing distant or regional metastases. Tumors were composed of cytologically bland columnar and cuboidal eosinophilic tumor cells with basally located nuclei arranged in tubular and tubulotrabecular patterns. Immunohistochemically, CK7, DOG1, GCDFP-15, and SOX10 were positive in all cases, and vimentin was positive in 2 cases. Scattered single cells or small groups of tumor cells were S-100 positive. Only one case had weak, focal expression of GATA3, and mammaglobin was consistently negative. Two cases had ETV6-NTRK3 gene fusions, whereas ETV6 had an unknown fusion partner gene in one case. The highly similar morphology, immunohistochemical profile, and genetics of the presented cases are suggestive of a specific disease. Although translocation-associated adenocarcinomas in the sinonasal tract have previously been described exclusively as salivary-type carcinomas, we present the first type of carcinoma characterized by recurrent genetic rearrangements and distinct phenotype occurring exclusively in the sinonasal tract with no known major salivary gland counterpart. We provisionally designate this tumor ETV6-rearranged low-grade SNAC. Identification of additional cases is necessary to fully appreciate the morphologic and biological spectrum of this disease.

USP6 activation in nodular fasciitis by promoter-swapping gene fusions

Nodular fasciitis is a self-limited myofibroblastic lesion that can be misdiagnosed as a sarcoma as a result of its rapid growth, cellularity, and sometimes prominent mitotic activity. A recurrent translocation t(17;22) has been identified in nodular fasciitis, fusing the coding region of USP6 to the promoter region of MYH9, and resulting in increased USP6 expression. A subset of cases show USP6 rearrangement without the typical fusion variants by RT-PCR, or any MYH9 rearrangement by FISH. We sought to further characterize such tumors using molecular diagnostic assays. A novel RT-PCR assay was designed to detect the two known MYH9–USP6 fusion types in formalin-fixed paraffin-embedded and frozen tissue, and a break-apart FISH assay was designed to detect USP6 rearrangement. Twenty-six cases of nodular fasciitis diagnosed between 2002 and 2013 were retrieved from the pathology files of our institutions and were confirmed to be positive by FISH and/or RT-PCR. Seven samples showed USP6 rearrangement by FISH but were negative for MYH9–USP6 fusion by RT-PCR; these cases were subjected to a next-generation sequencing assay utilizing anchored multiplex PCR technology. This assay targets a single partner gene associated with fusions in bone and soft tissue tumors for agnostic detection of gene fusion partners. Novel fusion partners were identified in all seven cases and confirmed by RT-PCR. Structurally, all fusions consisted of the juxtaposition of the entire coding region of USP6 with the promoter of the partner gene, driving increased USP6 expression. This study confirms the neoplastic nature of nodular fasciitis, defines additional pathogenic fusion partners, and adds to the growing body of literature on USP6-associated neoplasia. Given the diagnostic challenges of these tumors, molecular assays can be useful ancillary tools; however, the prevalence of promoter swapping must be recognized when interpreting results.

Discovery of Human-Similar Gene Fusions in Canine Cancers.

Canine cancers represent a tremendous natural resource due to their incidence and striking similarities to human cancers, sharing similar clinical and pathologic features as well as oncogenic events, including identical somatic mutations. Considering the importance of gene fusions as driver alterations, we explored their relevance in canine cancers. We focused on three distinct human-comparable canine cancers representing different tissues and embryonic origins. Through RNA-Seq, we discovered similar gene fusions as those found in their human counterparts: IGK-CCND3 in B-cell lymphoma, MPB-BRAF in glioma, and COL3A1-PDGFB in dermatofibrosarcoma protuberans-like. We showed not only similar partner genes but also identical breakpoints leading to oncogene overexpression. This study demonstrates similar gene fusion partners and mechanisms in human-dog corresponding tumors and allows for selection of targeted therapies in preclinical and clinical trials with pet dogs prior to human trials, within the framework of personalized medicine. Cancer Res; 77(21); 5721-7. ©2017 AACR.

Involvement of DPP9 in gene fusions in serous ovarian carcinoma

BackgroundA fusion gene is a hybrid gene consisting of parts from two previously independent genes. Chromosomal rearrangements leading to gene breakage are frequent in high-grade serous ovarian carcinomas and have been reported as a common mechanism for inactivating tumor suppressor genes. However, no fusion genes have been repeatedly reported to be recurrent driver events in ovarian carcinogenesis. We combined genomic and transcriptomic information to identify novel fusion gene candidates and aberrantly expressed genes in ovarian carcinomas.MethodsExamined were 19 previously karyotyped ovarian carcinomas (18 of the serous histotype and one undifferentiated). First, karyotypic aberrations were compared to fusion gene candidates identified by RNA sequencing (RNA-seq). In addition, we used exon-level gene expression microarrays as a screening tool to identify aberrantly expressed genes possibly involved in gene fusion events, and compared the findings to the RNA-seq data.ResultsWe found a DPP9-PPP6R3 fusion transcript in one tumor showing a matching genomic 11;19-translocation. Another tumor had a rearrangement of DPP9 with PLIN3. Both rearrangements were associated with diminished expression of the 3′ end of DPP9 corresponding to the breakpoints identified by RNA-seq. For the exon-level expression analysis, candidate fusion partner genes were ranked according to deviating expression compared to the median of the sample set. The results were collated with data obtained from the RNA-seq analysis. Several fusion candidates were identified, among them TMEM123-MMP27, ZBTB46-WFDC13, and PLXNB1-PRKAR2A, all of which led to stronger expression of the 3′ genes. In view of our previous findings of nonrandom rearrangements of chromosome 19 in this cancer type, particular emphasis was given to changes of this chromosome and a DDA1-FAM129C fusion event was identified.ConclusionsWe have identified novel fusion gene candidates in high-grade serous ovarian carcinoma. DPP9 was involved in two different fusion transcripts that both resulted in deregulated expression of the 3′ end of the transcript and thus possible loss of the active domains in the DPP9 protein. The identified rearrangements might play a role in tumorigenesis or tumor progression.

Transcriptome sequencing in pediatric acute lymphoblastic leukemia identifies fusion genes associated with distinct DNA methylation profiles

BackgroundStructural chromosomal rearrangements that lead to expressed fusion genes are a hallmark of acute lymphoblastic leukemia (ALL). In this study, we performed transcriptome sequencing of 134 primary ALL patient samples to comprehensively detect fusion transcripts.MethodsWe combined fusion gene detection with genome-wide DNA methylation analysis, gene expression profiling, and targeted sequencing to determine molecular signatures of emerging ALL subtypes.ResultsWe identified 64 unique fusion events distributed among 80 individual patients, of which over 50% have not previously been reported in ALL. Although the majority of the fusion genes were found only in a single patient, we identified several recurrent fusion gene families defined by promiscuous fusion gene partners, such as ETV6, RUNX1, PAX5, and ZNF384, or recurrent fusion genes, such as DUX4-IGH. Our data show that patients harboring these fusion genes displayed characteristic genome-wide DNA methylation and gene expression signatures in addition to distinct patterns in single nucleotide variants and recurrent copy number alterations.ConclusionOur study delineates the fusion gene landscape in pediatric ALL, including both known and novel fusion genes, and highlights fusion gene families with shared molecular etiologies, which may provide additional information for prognosis and therapeutic options in the future.

Detection of ROS1 rearrangement in non-small cell lung cancer: current and future perspectives.

ROS1 rearrangement characterizes a small subset (1%–2%) of non-small cell lung cancer and is associated with slight/never smoking patients and adenocarcinoma histology. Identification of ROS1 rearrangement is mandatory to permit targeted therapy with specific inhibitors, demonstrating a significantly better survival when compared with conventional chemotherapy. Detection of ROS1 rearrangement is based on in situ (immunohistochemistry, fluorescence in situ hybridization) and extractive non-in situ assays. While fluorescence in situ hybridization still represents the gold standard in clinical trials, this technique may fail to recognize rearrangements of ROS1 with some gene fusion partner. On the other hand, immunohistochemistry is the most cost-effective screening technique, but it seems to be characterized by low specificity. Extractive molecular assays are expensive and laborious methods, but they specifically recognize almost all ROS1 fusions using a limited amount of mRNA even from formalin-fixed, paraffin-embedded tumor tissues. This review is a discussion on the present and futuristic diagnostic scenario of ROS1 identification in lung cancer.

Abstract 2416: Transcriptome and cluster analysis of infant acute lymphoblastic leukemia cases with and without MLL (KMT2A) rearrangement

Abstract Background: Infant acute lymphoblastic leukemia (ALL) is a high-risk cancer with poor survival due to treatment failure and relapse. The majority of infant ALL cases possess an MLL (KMT2A) translocation (MLL-R), where MLL joins with AF4 (AFF1), ENL (MLLT1), or another partner gene to create a novel fusion gene. A subset of infant ALL cases do not involve an MLL rearrangement (non-MLL-R). Gene expression profiling delineates MLL-R from non-MLL-R cases. Here, we describe transcriptome profiling and cluster analysis of MLL-R and non-MLL-R cases and compare between partner genes. Methods: We performed RNA sequencing (RNA-seq) on diagnostic blood or bone marrow samples from 40 infants with ALL. Cohort A included 16 patients with MLL-R ALL known to have relapsed; cohort B, 10 patients with MLL-R ALL not known to have relapsed; and cohort C, 14 patients with non-MLL-R ALL whom to date have also not relapsed. RNA-seq was performed to an average of 8.8Gb and analyzed using Bowtie2, RSEM, and limma-voom. Multiple different groups of cases (e.g. cohort A versus B) were compared for differential gene expression; the resulting genes were ranked by adjusted P value with a significance cutoff of 0.01 and further evaluated for pathway analysis with Ingenuity software. Hierarchical clustering of samples was conducted using distance measures computed from normalized gene expression. Results: Transcriptome analysis of MLL-R versus non-MLL-R cases found 2,334 differentially expressed genes. Ingenuity pathway analysis of these genes showed enrichment in specific gene sets, such as those involved in PTEN, EGF, and NF-κB signaling. Ingenuity predicted increased PTEN signaling and diminished EGF and NF-κB signaling in MLL-R cases relative to non-MLL-R cases. Transcriptome analysis of relapsed versus non-relapsed MLL-R cases found no differentially expressed genes meeting significance criteria, while analysis of MLL fusion partners identified 46 differentially expressed genes including BMI1 and MYC. Cluster analysis demonstrated a distinct grouping of non-MLL-R cases relative to MLL-R cases. Additionally, MLL-R samples sub-clustered according to their MLL fusion gene partners. However, no difference in clustering was appreciated between MLL-R cases that later went on to relapse versus those that did not. Conclusions: Our findings show that MLL-R infant ALL has a distinct gene expression signature at diagnosis that correlates with the type of MLL translocation present. This is important because it suggests treatment strategies could be tailored to the MLL partner gene. Risk of relapse in MLL-R cases could not be predicted by this signature due to a paucity of differentially expressed genes between relapsed and non-relapsed groups at diagnosis. Similar to prior profiling studies, we find that non-MLL-R cases independently cluster together and have a gene expression signature distinct from MLL-R cases.[B.Y. and M.F. contributed equally to this work.] Citation Format: Byunggil Yoo, Midhat S. Farooqi, Rumen Kostadinov, Neil Miller, Jeff Johnston, Emily Farrow, Shannon Kelley, Margaret Gibson, Patrick Brown, Erin Guest. Transcriptome and cluster analysis of infant acute lymphoblastic leukemia cases with and without MLL (KMT2A) rearrangement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2017-2416

Abstract 4475: Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes

Abstract Background: Translocation Renal Cell Carcinoma (tRCC) represents an aggressive subtype of kidney cancer associated with various gene fusions involving translocation of one of two members of micropthalmia transcription factor (MiT) family, TFE3 or TFEB. Despite identification of multiple TFE3 gene fusions in tRCC, heterogeneous phenotype and various dysregulated signaling pathway resulting from variety of gene fusion partners pose a challenge to establish effective treatments for these patients. In this work, we sought to study the biology underlying oncogenesis in tRCC involving TFE3 fusion with genes associated with pre-mRNA splicing factor machinery; NONO, SFPQ and PRCC. Methods: To investigate the biology of different TFE3 fusion proteins, oncogenic phenotypes and oncogenic signaling were compared among cells with distinct endogenous expression of TFE3 fusions such as SFPQ-TFE3, NONO-TFE3, PRCC-TFE3. Endogenous SFPQ-TFE3 expressing cells, RP-RO7, were generated from patient derived xenograft. UOK-109 and UOK-146 (kindly provided by Dr. Marston Lenehan, NCI) were used as endogenous NONO-TFE3 and PRCC-TFE3 expressing cells, respectively. TFE3 fusion transcripts were identified with RT-PCR. TFE3 fusion preferential DNA binding was assessed with ChIP-sequencing. Oncogenic phenotype assessment was done in 2D culture and two different 3D models; 1) Solid tumor model, in which cancer cell interaction with extracellular matrix and stromal cells, were represented in multiculture-spheroid system, 2) Invasion model, in which cell ability to invade basement membrane barrier was modeled with matrix restricted spheroid. Results: Dissimilar oncogenic phenotypes were seen among RP-R07, UOK-109 and UOK-146 in 2D and 3D culture. The presence of nuclear E-Cadherin was observed in all three cells model, with increasing level of nuclear expression in fusion partner dependent manner (P<0.05). Distinctive profiles of global phosphotyrosine and global phosphoserine were observed in cells model harboring different TFE3 fusions, despite common nuclear signal of TFE3. Nuclear expression of selected kinases and phosphokinases such as AKT, FAK, mTOR, VGEFR-2 were identified among TFE3 fusion cells with different level of expression (P<0.05). These results suggest distinct alteration of kinases nucleocytoplasmic shuttling regulation in tRCC cells (P<0.05), as compared to non TFE3 fusion cells. TFE3 ChIP-seq data in RP-R07 indicate novel DNA target involving metabolism and TGF-β signaling related genes. Conclusions: Our results indicate a distinct biology underlying tRCC oncogenesis in different TFE3 fusion models involving kinase reprograming and nucleocytoplasmic shuttling regulation. These differences also suggest the possibility of generating personalized treatments targeting specific TFE3 fusion genes associated with the spliceosome machinery. Citation Format: Nur P. Damayanti, Sreenivasulu Chintala, Ashley Orillion, Remi Adelaiye-Ogala, May F. Elbanna, Pete Hollenhorst, Roberto Pili. Delineating translocation renal cell carcinoma oncogenesis in cells harboring TFE3 fusion with spliceosome machinery associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4475. doi:10.1158/1538-7445.AM2017-4475

Clinical and radiological features of advanced RET-rearranged lung cancer.

e23104 Background: RET fusion gene was found in 1–2% of non-small cell lung cancer (NSCLC). Most of the reports published so far investigated only surgical specimens, and details of advanced tumors were unknown. This study explored the clinical and radiological characteristics of RET-rearranged lung cancers in advanced stages. Methods: Among 1,074 advanced NSCLC patients who were treated at The Cancer Institute Hospital, Tokyo, from 2012 to 2016, 14 cases (14/1,074 = 1.3%) were treated as RET- rearranged lung cancer. The cases were identified by FISH and / or by RT-PCR (FISH 3, RT-PCR 2, FISH and RT-PCR 8, unknown 1). The fusion partner genes included KIF5B (n = 10), CCDC6 (n = 1). Three were unknown. The tumor size, location, and shape/margins of the primary tumor as well as lymphadenopathy and site of metastasis were recorded based on computed tomography (CT) images taken right before the initial chemotherapy. Results: The median age of the 14 patients was 64 years (range, 34–79), with 10 patients (71%) over 60 years old. Nine patients (64%) were women, whereas 10 patients (71%) were never smokers. Eight patients were classified as stage IV and 6 underwent recurrence after surgery. We successfully evaluated CT images at the initial chemotherapy of 12 patients. Of the 7 patients whose primary lesions were detectable, all were located peripherally and were of a solid tumor type without ground-glass, air bronchograms, or cavitation. The median size of the primary lesion was 3.0 cm (range, 1.2–6.8), and 3 lesions were less than 3.0 cm. Only 5 patients had lymphadenopathy (4 were of Stage IV, 1 was a recurrence), most of which were isolated with a median size of 1.5 cm (range, 1.2–3.5). The sites of distant metastases included 8 pleural disseminations, 5 lungs, 5 bones, 3 livers, 2 brains, and 0 adrenals. Conclusions: Advanced RET-rearranged lung cancer manifested as a relatively small and peripherally located solid primary lesion with isolated lymphadenopathy. Pleural dissemination was frequently observed, whereas brain metastasis was less frequent. These features differ from EGFR-mutated or ALK-fused lung cancers.

A new GTF2I-BRAF fusion mediating MAPK pathway activation in pilocytic astrocytoma.

Pilocytic astrocytoma (PA) is the most common pediatric brain tumor. A recurrent feature of PA is deregulation of the mitogen activated protein kinase (MAPK) pathway most often through KIAA1549-BRAF fusion, but also by other BRAF- or RAF1-gene fusions and point mutations (e.g. BRAFV600E). These features may serve as diagnostic and prognostic markers, and also facilitate development of targeted therapy. The aims of this study were to characterize the genetic alterations underlying the development of PA in six tumor cases, and evaluate methods for fusion oncogene detection. Using a combined analysis of RNA sequencing and copy number variation data we identified a new BRAF fusion involving the 5’ gene fusion partner GTF2I (7q11.23), not previously described in PA. The new GTF2I-BRAF 19–10 fusion was found in one case, while the other five cases harbored the frequent KIAA1549-BRAF 16–9 fusion gene. Similar to other BRAF fusions, the GTF2I-BRAF fusion retains an intact BRAF kinase domain while the inhibitory N-terminal domain is lost. Functional studies on GTF2I-BRAF showed elevated MAPK pathway activation compared to BRAFWT. Comparing fusion detection methods, we found Fluorescence in situ hybridization with BRAF break apart probe as the most sensitive method for detection of different BRAF rearrangements (GTF2I-BRAF and KIAA1549-BRAF). Our finding of a new BRAF fusion in PA further emphasis the important role of B-Raf in tumorigenesis of these tumor types. Moreover, the consistency and growing list of BRAF/RAF gene fusions suggests these rearrangements to be informative tumor markers in molecular diagnostics, which could guide future treatment strategies.

Novel t(5;11)(q32;q13.4) with NUMA1-PDGFRB fusion in a myeloid neoplasm with eosinophilia with response to imatinib mesylate

We report a NUMA1-PDGFRB fusion in a myeloproliferative neoplasm with eosinophilia in a 61-year old man, with response to imatinib mesylate therapy. A t(5;11) chromosome translocation involving bands 5q32 and 11q13.4 was identified by metaphase chromosome analysis, and rearrangement of the platelet-derived growth factor receptor beta (PDGFRB) gene on 5q32 was demonstrated by FISH using a PDGFRB break-apart probe set. Bacterial artificial chromosome (BAC) FISH mapping of the PDGFRB fusion partner gene narrowed the breakpoint at 11q13.4 to a 150 kb genomic region containing three genes, including NUMA1. Mate pair sequencing analysis demonstrated NUMA1-PDGFRB fusion. The fusion protein includes coiled-coil domains of nuclear mitotic apparatus protein 1 (NuMA1, involved in protein homodimerization and heteroassociation) and tyrosine kinase domains of PDGFRB. Diverse rearrangements involving the PDGFRB gene have been identified in myeloid and lymphoid neoplasms with eosinophilia, but rearrangement of the nuclear mitotic apparatus protein 1 (NUMA1) gene has previously been reported in a human malignancy in only one instance, a NUMA1-RARA fusion caused by a t(11;17) translocation in a patient with acute promyelocytic leukemia. The NUMA1-PDGFRB fusion is the second instance of rearrangement of NUMA1, encoding an element of the mitotic apparatus, in human cancer.

Abstract P1-05-14: Copy number aberration-induced gene breakage analysis identifies recurrent FOXP1 fusions in breast cancer

Abstract Background: Genomic instability is a critical feature of breast cancers, which manifests in genome-wide copy number aberrations (CNA), often causing “gene breakage” and the generation of fusion genes. We aimed to identify aborted transcripts with underlying CNAs and to investigate the molecular landscape of breast cancers harbouring such events. Methods: A walking student's t-test algorithm was applied to Affymetrix Exon 1.0ST array data of 123 breast cancers to identify regions of aborted transcription and overlaid with DNA breakpoints derived from matched Affymetrix SNP6 ASCAT-segmented copy number. Aborted transcripts were investigated as potential fusion gene partners through RNA-seq analysis of 151 breast cancer samples (TCGA) and 51 breast cancer cell lines (BCCL) using ChimeraScan. Clinical correlates were established for clinicopathological features, genomic instability measures, and gene expression-based molecular classifiers including PAM50, TNBCtype, IntClust subtypes and immune signatures. Results: One hundred and six genes with recurrent CNA-induced aborted transcription were identified. Aborted transcription showed hormone receptor subtype-specificity for 7 genes (nTNBC=1, nNon-TNBC=6) and was less prevalent in samples of IntClust 2 and IntClust 4 subtypes (p: 0.0043, 0.0011). Aborted transcripts were more frequently observed in samples with greater copy-neutral loss of heterozygosity (p=0.012), while aborted transcription of 54/106 genes significantly affected enrichment of 27 tumor-infiltrating lymphocyte subpopulations.14 aborted transcripts were found as a fusion gene with one partner in RNA-seq of TCGA and BCCL, while 19 were involved in multiple fusion events (range=1-6, median=2). Nine of 106 genes displayed gene breakage and fusion events exclusively in samples with an enriched tandem duplication phenotype. Notably, FOXP1, localised to a tumour suppressor locus at 3p14.1, reported the highest number of fusion configurations (n=6) with concurrent aborted transcription across all RNA-seq datasets (nPRADA=9, nTCGA=38, nBCCL=6). Conclusion: CNA-induced gene breakage affects the molecular landscape of breast cancers and is linked with many genomic configurations of interest including copy-neutral loss of heterozygosity and tandem duplications. In particular, the role of recurrent gene fusions of the tumour suppressor, FOXP1, in tumourigenesis warrants further investigation. Citation Format: Mohd Noor A, Maguire S, Watkins J, Quist J, Mirza H, Tutt A, Gillett C, Natrajan R, Grigoriadis A. Copy number aberration-induced gene breakage analysis identifies recurrent FOXP1 fusions in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-14.

AMKL chimeric transcription factors are potent inducers of leukemia.

Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts that are predicted to be pathological including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2, which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain-of-function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathological drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.

Therapeutic strategies and mechanisms of drug resistance in anaplastic lymphoma kinase (ALK)-rearranged lung cancer.

Anaplastic lymphoma kinase (ALK) gene encoding the receptor tyrosine kinase ALK is expressed as a fusion gene in a variety of carcinomas. The expression of ALK is nearly undetectable in adults, and its activation is normally regulated by its ligands, FAM150A/B. However, ALK gene rearrangements result in constitutive ALK fusion proteins expression via the active promoter of fusion partner genes. ALK fusion proteins dimerize in a ligand-independent manner and lead to the dysregulation of cell proliferation via abnormal constitutive activation of ALK tyrosine kinase. Many ALK tyrosine kinase inhibitors (TKIs) have been developed to date, three of which are currently in clinical use for the treatment of ALK-rearranged non-small cell lung cancer (NSCLC). ALK TKIs often achieve marked tumor regression in NSCLC patients with ALK rearrangements; however, ALK TKI-resistant tumors inevitably emerge within a few years in most cases. In this review, we summarize diverse ALK TKI resistance mechanisms identified in NSCLC with ALK rearrangements, and review potential therapeutic strategies to overcome ALK TKI resistance in these patients.

Identification of Two Distinct Poor Prognostic Subgroups Related to High Expression of BMP2 or PRDM16 in Pediatric AML

[Background]Pediatric acute myeloid leukemia (AML) remains to be a disease with a poor prognosis, with a long-term survival rate of approximately 60%. We have recently reported that high PRDM16 expression (high-PRDM16) was common in t(5;11)(q35;p15.5)/NUP98-NSD1-positive patients, and that high-PRDM16 was found to be a novel poor prognostic factor in this disease. However, this finding was not observed in FAB-M7. The presence of inv(16)(p13.3q24.3)/CBFA2T3-GLIS2 was postulated to be a reason. This fusion was reported as the most frequent fusion gene associated with a poor prognosis in FAB-M7, and we found that all CBFA2T3-GLIS2-positive patients had a low PRDM16 expression (low-PRDM16). Furthermore, some studies reported that bone morphogenetic protein 2 (BMP2) was frequently and highly expressed in CBFA2T3-GLIS2-positive patients, indicating the importance of BMP2 in pathogenesis in a subset of AML. In this study, we analyzed the expression status of BMP2 and PRDM16 and their relationship to prognosis in pediatric AML.[Patients and Methods]A total of 369 AML patient samples, who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial, were analyzed. Expression level of BMP2 and PRDM16 is analyzed by real-time quantitative PCR (Taqman Gene Expression Assay), using ABL1 as a control gene. The cutoff value for high-BMP2 was defined as the lowest expression level among 11 CBFA2T3-GLIS2-positive patients, and that for high-PRDM16 was defined as the lowest expression level among 10 NUP98-NSD1-positive patients. We also screened fusion genes and gene mutations of CBFA2T3-GLIS2, NUP98-JARID1A, RBM15-MKL1, MLL rearrangements, NUP98-NSD1, FUS-ERG, DEK-NUP214, RUNX1-RUNX1T1, CBFB-MYH11, and gene mutations of KIT, NRAS, KRAS, WT1, NPM1, MLL-PTD, and FLT3-ITD.[Results]Out of 369 patients, high-BMP2 and high-PRDM16 were identified in 47 (12.7%) and 44 (11.9%) patients, respectively. Only three (0.8%) of 369 patients had both high-BMP2 and high-PRDM16 (high-BMP2/PRDM16). In 44 high-BMP2 patients and 41 high-PRDM16 patients (3 high-BMP2/PRDM16 patients were excluded), MLL rearrangements were only found in high-BMP2 patients (25.5% vs 0%, p < 0.001), whereas FLT3-ITD was significantly much more frequent in high-PRDM16 patients (4.5% vs 56.1%, p < 0.001). Core-Binding-Factor (CBF)-AML was rare in both groups (4.5% and 0%, p = 0.495). Kaplan|Meier analyses revealed that high-BMP2 patients and high-PRDM16 patients had a low 5-year overall survival (OS) (46.3% and 29.3%, respectively). In molecular subgroup analyses, the prognosis of MLL-rearranged patients, including 12 high-BMP2 patients (21.8%), was not influenced by BMP2 expression (5-year OS: high-BMP2 66.7% vs low-BMP2 64.9%, p = 0.488), suggesting that the prognosis of MLL-rearranged patients might primarily be defined by their fusion partner genes and other gene aberrations as previously reported (e.g., EVI1 expression). Considering this result, further survival analyses of the patients lacking CBF-AML or MLL-rearrangements were performed (n = 174, 5-year OS: 50.5%). In this subgroup, 5-year OS of high-BMP2 patients (n = 30) and high-PRDM16 patients (n = 41) were significantly lower than that of low-BMP2/low-PRDM16 patients (36.7% vs 67.2%, p < 0.001, and 29.3% vs 67.2%, p < 0.001, respectively).[Conclusions]High-BMP2 and high-PRDM16 could identify distinct subgroups with poor prognosis in pediatric AML. Further investigation of molecular-targeted therapy against these genes may contribute to the improvement of clinical outcome in these subgroups. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.