Type Of Fusion Gene Research Articles

Investigation of -PRKACA/-PRKACB fusion genes in oncocytic tumors of the pancreatobiliary and other systems.

Intraductal oncocytic papillary neoplasms (IOPNs) of the pancreatobiliary system are tumors comprising oncocytic cells, in which three types of fusion genes involving -PRKACA/-PRKACB were recently identified. IOPNs infrequently combine with other histological subtypes of pancreatic intraductal papillary mucinous neoplasms (IPMNs) and intraductal papillary neoplasms of the bile duct (IPNBs). This study aimed to confirm the sensitivity/specificity of the fusion genes for IOPNs and to examine their significance in other oncocytic lesions. An RT-PCR, followed by DNA sequencing, was undertaken to examine the fusions in 18 histologically diagnosed IOPNs, including four combined IOPNs. Moreover, in two IOPN cases, invasive carcinomatous lesions were separately examined on their fusion status. Oncocytic thyroidal (n = 10), renal (n = 10), and salivary gland (n = 3) lesions and IPMNs (n = 9)/IPNBs (n = 4) with focal oncocytic changes were examined as controls. Fluorescence in situ hybridization using PRKACA break-apart probes was conducted for the combined IOPN cases. Target sequencing of KRAS exon2/3 and GNAS exon 8/9 was performed for IOPN cases. Fusions were detected in all IOPN cases including invasive lesions/none of the control cases. The fusion event was confirmed also in non-IOPN component in one of the four combined cases. Regarding mutation events, 5.6%/0% of IOPNs were KRAS-mt/GNAS-mt, respectively, and both components of combined IOPNs were all KRAS-wt/GNAS-wt. In conclusion, our study confirmed the sensitivity and specificity of these fusions for IOPNs. Here, we analyzed the roles of these fusion genes in combined IOPNs, proposing the possibility of IOPN development via IPMNs/IPNBs. Further studies with more combined cases are warranted.

Characteristics of fusion gene expression in acute lymphoblastic leukemia

Objective: To analyze the genetic landscape of 52 fusion genes in patients with de novo acute lymphoblastic leukemia (ALL) and to investigate the characteristics of other laboratory results. Methods: The fusion gene expression was retrospectively analyzed in the 1 994 patients with de novo ALL diagnosed from September 2016 to December 2020. In addition, their mutational, immunophenotypical and karyotypical profiles were investigated. Results: In the 1 994 patients with ALL, the median age was 12 years (from 15 days to 89 years). In the panel of targeted genes, 15 different types of fusion genes were detected in 884 patients (44.33%) and demonstrated a Power law distribution. The frequency of detectable fusion genes in B-cell ALL was significantly higher than that in T-cell ALL (48.48% vs 18.71%), and fusion genes were almost exclusively expressed in B-cell ALL or T-cell ALL. The number of fusion genes showed peaks at<1 year, 3-5 years and 35-44 years, respectively. More fusion genes were identified in children than in adults. MLL-FG was most frequently seen in infants and TEL-AML1 was most commonly seen in children, while BCR-ABL1 was dominant in adults. The majority of fusion gene mutations involved signaling pathway and the most frequent mutations were observed in NRAS and KRAS genes. The expression of early-stage B-cell antigens varied in B-cell ALL patients. The complex karyotypes were more common in BCR-ABL1 positive patients than others. Conclusion: The distribution of fusion genes in ALL patients differs by ages and cell lineages. It also corresponds to various gene mutations, immunophenotypes, and karyotypes.

A New Sensitive and Fast Molecular Analysis Method for the Identification of 30 Different Markers in Acute Myeloid and Lymphoid Leukemias at Diagnosis Time

Background: Medical advances in the treatment of Acute and Chronic Leukemia in the last decade were exciting, thanks to the discovery of new drugs and transplantation of hematopoietic stem cells; but so that these weapons can be used in an optimal way, it is necessary that the leukemia is diagnosed as quickly as possible and accurately to limit the spread of leukemia cells in the patient. The ability to detect fusion genes characteristics of these diseases has become essential but laborious, time consuming and generally comprises karyotype analysis, followed by FISH (Fluorescent in situ hybridization) and molecular analysis (RT-PCR). New molecular diagnostic technologies are being developed that can identify disease markers with extreme accuracy in a few hours (2-3 hours). The Leukemia Fusion Genes (Q30) Screening Kit (QuanDx, San Jose, CA 95131 USA; Distr. Resnova S.r.l.) is a real-time multiplex RT-qPCR based assay for detection of leukemia associated fusion gene transcripts in total RNA from bone marrow or whole blood samples, it is a qualitative test for the simultaneous detection of 30 characteristic fusion genes of acute leukemias. For Acute Myeloid Leukemia (AML) transcripts we can expect tested 18 fusion genes with 72 different breakpoints: BCR-ABL1, MLL-AF9, CBFbeta-MYH11, PML-RARalpha, AML-MDS1, NPM-MLF1, AML-ETO, NPM-RARalpha, PLZF-RARalpha, DEK-CAN, MLL-ELL, AML1-EAP, MLL-AF10, ST-CAN, TEL-ABL1, TLS-ERG, FIP1L1-PDGFR alpha, TEL-PDGFRbeta. For Acute Lymphoid Leukemia (ALL) transcripts we will detected 15 fusion genes with 71 different breakpoints: MLL-AF4, TEL-AML1, MLL-ENL, E2APBX1, BCR/ABL1, SIL-TAL1, E2A-HLF, MLL-AF6, CALM-AF10, HOX11, HOX11L2, MLL-AF10, HOX11, HOX11L2, MLL-AF10, SET-CAN, TEL-ABL1, TLS-ERG.Aim: The aim of this study is to evaluate and to standardize a new commercial kit, the Leukemia Fusion Genes (Q30) Screening Kit, that is reliable, reproducible and able to detect simultaneously, in one real-time PCR reaction and in a short time different rearrangement involved in acute leukemia at diagnosis.Methods: 60 samples will be analyzed as follows: 51 AML and 9 ALL.All samples have been previously tested with classical PCR qualitative methods (Leukemia 1999). 55 RNAs were extracted using the Maxwell 16 Semi-automatic Extractor (Simply RNA Blood kit) from isolated cells from Ficoll or buffycoat of bone marrow or peripheral blood. 5 RNAs were extracted with manual phenol-chloroform method. Concentration was determined by spectrophotometric reading (from 20ng/µL to 200ng/µL). cDNA was added to 8 qPCR reaction tubes, which contain specific PCR primers and probes for detection of fusion genes and an internal control gene (GUSB). The qPCR is performed in a real-time thermal cycler, the kit is compatible with the ABI7500 system.Results: 21 out of 51 AML patients (pts), tested with routine approach, had no markers, the remaining 30 AML had rearrangements and mutations as follows:1 pt CBFβ-MYH11, 2 pts PML-RARalpha, 2 pts AML1-ETO, 1 pt DEK-CAN, 4 pts FLT3 ITD, 6 pts NPM, 8 pts FLT3/NPM, 1 pt DEK-CAN/FLT3, 3 pts PML-RARα/FLT3 ITD, 1 pt IDH mut, 1 pt MLL ITD. 5/9 ALL samples had no markers, the remaining presented with t (4;11) (2 pts), t (9;22) (2 pts).All the tested samples (60 samples) confirmed the molecular rearrangement where it was present (14 samples) using primer sets for molecular rearrangements by RT-PCR technique. 100% concordance, 14 samples out of 14.Significantly 5 out of 60 patients showed new (previously unidentified) rearrangements with the primer sets used by the laboratory routinely: 2 pts with MLL-ELL/t(11;19), 1 pt with MLL-AF10/t(10;11) and 2 pts with AML1-MTG16/t(16;21). The kit revealed a percentage of rearranged patients equal to 32% compared to 23% analyzed with the primer sets used routinely, using a time of two/three hours against 72 hours, considering the totality of the rearrangements.Conclusion: The Q-Fusion system provides a sensitive instrument specific, reliable and cost effective, easy to perform for routine screening in leukemias at diagnosis.The kit can detect 30 common fusion genes, it can only report the types of fusion genes, but cannot differentiate the splice variants. It is a Real-Time multiplex RT-qPCR and the Cq values cannot be used for quantification of the fusion transcript level. DisclosuresSaglio:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria.

Analysis of fusion gene expression in acute myeloid leukemia

目的分析初诊急性髓系白血病（AML）患者融合基因表达情况，进一步探讨不同融合基因家族患者的免疫表型及基因突变的特点。方法回顾性分析2016至2020年4192例初诊AML患者的多重融合基因筛查结果，并结合免疫表型分析以及高通量测序所获得的基因突变筛查结果，系统地进行差异性分析。结果①4192例AML患者中1948例(46.47％)检出融合基因，共检出29种不同的融合基因，其阳性率呈现为“幂律分布”。②随着AML患者年龄增长，融合基因的阳性率先上升而后逐步下降。儿童患者融合基因总阳性率及MLL相关融合基因（MLL-FG）阳性率均显著高于成人患者（69.18％对44.76％，15.35％对8.36％）。③MLL-FG及NUP98相关融合基因（NUP98-FG）阳性患者伴随的基因突变均以FLT3及RAS信号通路相关基因为主。④MLL-FG及NUP98-FG阳性患者未见特异性的免疫表型特点。结论采用多重PCR方法分析，近半数的AML患者伴有特异的融合基因表达，儿童和成人患者融合基因阳性类型构成比例存在差异。不同融合基因阳性AML患者常见的分子突变、免疫表型有一定规律。

Genomic and Clinical Characterization of Adult Ph-Negative B-Cell Acute Lymphoblastic Leukemia

Although survival rate for children with Acute Lymphoblastic Leukemia (ALL) now exceeds about 90%, the outcome of adult patients with ALL is extremely poor. These differences might be attributed to the lack of insights into pathogenesis and clinical behavior of adult-ALL.Gross chromosomal alterations including chromosome translocations and aneuploidy are considered as early events in ALL and constitute disease subtypes. To identify chromosome translocations underlying adult with Ph-negative B-ALL, we performed RNA-seq analysis on RNA from individuals with B-ALL who had been treated on the Japan Adult Leukemia Study Group (JALSG) ALL202-O protocol (n = 149).We successfully identified chromosome translocations in 100 patients (67.1%). ZNF384 fusions were most frequently detected in 30 patients (20.1%) and they had wide range of fusion partners. DUX4- and MEF2D- fusions were also recurrently found in 7 (4.7%) and 9 (6.0%) patients, respectively. Chromosome translocations activating kinase and cytokine receptor were found in 25 patients (16.8%) with Ph-like gene expression profile. These alterations were almost completely mutually exclusive indicating these are likely to be primary genetic events.For simplicity, here we define (1) fusions involving ZNF384, DUX4, MEF2D, CEBP and PAX5 as well as TCF3-PBX1 and ETV6-RUNX1 as Transcription Factor fusions (TF fusions; 49% of patients), (2) fusions involving CRLF2, JAK2, PDGFRB, EPOR and ABL as Kinase/cytokine-receptor Activating fusions (KA fusions; 15%) and (3) non-recurrent fusions or the absence of fusions/aneuploidy as B-others (30%).First, we analyzed impact of the patient age on types of fusion genes, based of combined data of ALL202-O cohort, childhood B-ALL cohort (Lilljebjörn H, et al. 2016: n = 189) and ALL202-U cohort (Yasuda T, et al. 2016: n = 54). We found that incidence of ZNF384-, CEBP- fusions and B-others increases as patients age, whereas ETV6-RUNX1 and PAX5 fusions were more prevalent in younger patients, exhibiting negative association with age. DUX4 fusions and TF fusions were most prevalent in Adolescent and Young Adult (AYA) generation. JAK2-, PDGFRB-, EPOR- and KA- fusions were positively correlated with age.Next, we analyzed association between patient survival and types of fusions. In Japanese adult B-ALL cohort (ALL202-O and ALL202-U cohort), we observed ZNF384-, DUX4- fusions and TCF3-PBX1 were associated with better disease-free survival than B-others. Furthermore, when combined, MEF2D- (n = 14), CEBP- (n = 4), PAX5- fusions (n = 2) and ETV6-RUNX1 (n = 2) exhibited significantly better disease-free survival than B-others, indicating TF fusions were associated with an improved outcome. In contrast, KA fusions were associated with poorer disease-free survival than B-others. KMT2A fusions were comparable with B-others regarding to patient disease-free survival.These results allowed us to develop a prognostic schema to identify three distinct risk profile groups, based on types of fusion genes and cytogenetics (Table1); favorable-risk (5-year rate of disease-free survival 67.4%), intermediate-risk (5-year rate of disease-free survival 42.5%) and adverse-risk (5-year rate of disease-free survival 9.6%). This prognostic schema predicted the outcome independently of age, sex and methotrexate dose in multivariate analysis (p < 0.001).In conclusion, we promoted a better understanding of the genetic basis of adult B-ALL by focusing on fusion genes. Each chromosome translocations were closely associated with age. ZNF384-, KA fusions and B-others were characteristic for older-adult patients (40-65 years old) with B-ALL. We clearly demonstrated specific primary chromosome abnormalities are strong prognostic marker. Functional properties of primary genetic events (TF fusions vs. KA fusions) might be a key determinant of biological characteristics and clinical outcome. [Display omitted] DisclosuresKiyoi:Novartis Pharma K.K.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Phizer Japan Inc.: Research Funding; Eisai Co., Ltd.: Research Funding. Naoe:Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

Identification and characterization of novel fusion genes in prostate cancer by targeted RNA capture and next-generation sequencing.

Gene fusions play critical roles in the development and progression of prostate cancer, and have been used as molecular biomarkers for diagnosis of the malignant disease. To further explore the novel fusions in prostate cancer, we performed targeted RNA capture and next-generation sequencing in a cohort of 52 prostate cancer patients, identified and validated 14 fusion events (7 types of fusion genes) in 12 cases, including three novel fusion genes. We characterized a chromosome rearrangement-induced trigenic KLK2-DGKB-ETV1 fusion, which may function as a non-coding RNA to upregulate the expression of the wild-type ETV1 protein in the tumor tissue. Additionally, we detected two novel fusion forms of HNRNPA2B1-ETV1 and SLC45A2-AMACR fusions, respectively. Interestingly, fusion events participated by kinase genes, which frequently occurred in other human cancers, were not present in these prostate cancer cases, suggesting discrepant gene fusion patterns in different cancers. These findings expand the genetic spectrum of prostate cancer and provide insight into diagnosis of this prevalent disease.

Additional cytogenetic aberrations in chronic myeloid leukemia: a single-center experience in the Middle East.

BackgroundAdditional cytogenetic aberrations are associated with disease progression in chronic myeloid leukemia (CML). This study was conducted to determine the type and frequency of these aberrations and their relationship with hematologic and molecular findings in the Middle East.MethodsIn this retrospective study, 134 well-established cases of CML were selected from 2010 to 2016. Their hematologic phase and type of fusion gene were determined. Finally, their karyotypes were analyzed and reported according to ISCN 2013.ResultsPatients had a mean age of 44 years. Twenty-two patients (16.4%) showed additional cytogenetic aberrations. Nine patients (6.7%) harbored a variant Philadelphia chromosome, and most were in the chronic phase. Seventeen patients (12.7%) had major and minor route abnormalities. There was a significant relationship between additional cytogenetic aberrations and major molecular response (P=0.032). Patient survival in the group with additional cytogenetic aberrations was significantly lower (49.7±11.1 mo) than that in the group without additional cytogenetic aberrations (77.3±3.1 mo) (P=0.031).ConclusionThis study revealed the same frequency of additional cytogenetic aberrations in CML as found in previous studies. Additional chromosomal aberrations led to shorter survival and lower rates of achievement of a major molecular response.

Cytogenetic characteristics of acute myeloid leukemia and effect of DA regimen with different doses of daunorubicin

Objective To analyze the genetic characteristics of chromosomes and related fusion genes in acute myeloid leukemia (AML) (non-M3), and to evaluate the prognosis of patients with chemotherapy of DA regimen with different doses of daunorubicin. Methods Fifty-six patients with newly diagnosed non-M3 AML from January 2013 to January 2015 were collected. Adopted short-term culture method was used to treat bone marrow, R-binding chromosome karyotyping was used to detect cytogenetic. Thirty-one types of fusion gene were identified by PCR and 10% agarose gel electrophoresis. All patients treated by DA regimen were divided into group A, group B and group C according to different dosage of daunorubicin. Then, complete remission (CR) rate and survival time in the 3 groups were observed. The effect of cytogenetic and molecular biology abnormality on the chemotherapy, CR rate and overall survival (OS) of the 3 groups were analyzed by the chi-square test. Results Among the 56 patients, 18 cases (32.1%) had abnormal chromosome karyotype, 6 cases (10.7%) had abnormal number of chromosome, 16 cases (28.6%) had abnormal structure of chromosome, and 4 cases (7.1%) had both abnormal number and structure of chromosome. Meanwhile, the most common abnormal structure was t(8;21), and the most common abnormal quantity were+8,-Y. Detective rate of genetic abnormality was raised to 62.00% through fusion gene and chromosome karyotype analysis. The total CR rate of DA-induced chemotherapeutic regimen was 73.2%, and the two-year OS rate was 42.9%. The remission rate of chemotherapy in the middle-risk group was significantly lower than that in the low-risk group (χ 2= 8.976, P= 0.002), but there was no significant difference between the low-dose chemotherapy group and the standard dose chemotherapy group (P > 0.05). The standard dose group showed a significant advantage in the OS rate (χ 2= 8.045, P= 0.005). Conclusions Adult acute leukemia has its unique cytogenetic characteristics, which can assist in guiding clinical diagnosis, classification and prognosis. The prognosis of middle-risk patients is significantly lower than the low-risk group. Low-risk patients could benefit from a reduced dose of DA regimen, but the standard dose DA regimen has a significant advantage in long-term survival. Key words: Leukemia, myeloid, acute; Chromosome karyotype; Fusion gene; Drug therapy, combination

Optical coding of fusion genes using multicolor quantum dots for prostate cancer diagnosis.

Recent studies have found that prostate cancer expresses abnormal genetic markers including multiple types of TMPRSS2–ERG fusion genes. The expression level of different TMPRSS2–ERG fusion genes is correlated to pathologic variables of aggressive prostate cancer and disease progression. State-of-the-art methods for detection of TMPRSS2–ERG fusion genes include reverse transcription polymerase chain reaction (RT-PCR) with a detection limit of 1 fmol at urinary condition. RT-PCR is time consuming, costly, and inapplicable for multiplexing. Ability to identify multiple fusion genes in a single sample has become important for diagnostic and clinical purposes. There is a need for a sensitive diagnostic test to detect multiple TMPRSS2–ERG fusion genes for an early diagnosis and prognosis of prostate cancer. Here, we propose to develop an assay for prostate cancer diagnosis using oligonucleotide-functionalized quantum dot and magnetic microparticle for optical detection of rearranged TMPRSS2–ERG fusion genes at a low concentration in urine. We found that our assay was able to identify three different types of fusion gene with a wide detection range and detection limit of 1 fmol (almost the same level of the RT-PCR result reported). Here, we show detection of multiple TMPRSS2–ERG fusion genes using color-coded oligonucleotides in cell lysate and urine.

High throughput differential identification of TMPRSS2-ERG fusion genes in prostate cancer patient urine.

Identifying genetic diversity is important for studies in cancer as it can provide insights on disease progression and treatment. Although clinical outcome and major symptom of cancer might be same in all patients, the type of overexpressed gene could be different. Even though prostate-specific antigen assay is a good tool widely used for prostate cancer diagnosis, it is not capable of providing information on genetic differences. Therefore, screening method that can differentiate genetic differences is necessary. Here we detected different types of TMPRSS2-ERG, prostate cancer specific fusion genes, to verify the genetic diversity between the patients using high throughput screening method, bio-barcode assay. Prostate cancer patients with different types of fusion gene were successfully differentiated directly from untreated patients' urine, while traditional PSA assay could not. This non-invasive assay, when used with PSA assay, can be a strong secondary screening method which can offer new insights on disease progression and clinical outcome.

FUSI GEN BREAKPOINT CLUSTER REGION ABELSON KINASE (BCRABL) DAN UJI HEMATOLOGIS RUTIN

Chronic myeloid leukemia (CML) is a type of Chronic myeloproliferative disorders in pluripotencial stem cell haematopoiesis cell disease caused by somatic mutation chromosomal translocation of the Abelson (ABL) and Breakpoint Cluster Region (BCR) genes on chromosomes 9 and 22. The Breakpoint Cluster Region Abelson Kinase (BCR-ABL) gene encodes different fusion transcripts of messenger Ribo Nucleic acid (m RNA)/type of fusion gene that vary in size depending on the breakpoint in the BCR gene. The majority of CMLcases have been shown to have either b3a2 or b2a2 fusion gene. This research is a preliminary study designed to know how to identify a quantification BCR-ABL gene and expression fusion of gene and its relation to routine haematological parameters. The researchers analyzed 12 adults who were positive using a quantification ratio BCR-ABL and Glucose-6-Phosphate Dehydrogenase (G6PDH) as a house keeping gene by real-time quantitative polymerase chain reaction (RQ PCR) of chronic phase of CML patients, qualitative of translocation of BCR-ABL gene by gel agarose and routine haematological tests by a haematologic analyzer. The average quantification ratio of BCRABL gene and G6PDH was 0.0881, 50% patients had b3a2 fusion gene, 41.6% had b2a2 dan 0.4% had e1a1. Fusion gene b3a2 showed a quantification ratio, haemoglobin level and leukocyte count higher compared to b2a2 fusion gene.

ZNF384 -related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384 Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

Detection of Rearrangements of Mixed Lineage Leukemia Gene and Its clinical relevance

Background: The mixed lineage leukemia (MLL) gene located on chromosome 11 band q23 normally functions as a transcription regulator of the HOX genes and is essential for normal mammalian development and hematopoiesis. Chromosomal translocations involving MLL gene represent frequent cytogenetic abnormalities found in aggressive acute leukemia, both lymphoblastic and myeloid. AL MLL rearrangements have the unique clinical, hematological and prognostic features. We aimed to study the incidence and the types of fusion genes and the clinical relevance.Results: Study samples were from 60 acute leukemia (AL) patients from Sep. 2003 to Dec. 2005. There were 28 males and 32 females. The ages were from 3 months to 54 years old. 17 patients were less than 15 years old and 43 patients were more than 15 years old. All patients were diagnosed based on FAB diagnostic criteria. The patients included 30 with ALL (10 of ALL-L1 patients, 15 of ALL-L2, 5 of ALL-L3), 28 with AML (2 of AML-M1 patients, 5 of AML-M2, 5 of AML-M4, 13 of AML-M5, 1 of AML-M6, 2 of AML-M7), 1 with mixed cell leukemia (AMLL), and 1 with NK cell leukemia. 59 patients were newly diagnosed and 1 patient was refractory. The rearrangements of MLL gene were detected by fluorescence in situ hybridization (FISH) and 6 types of common fusion genes (MLL / AF4, MLL / ENL, MLL / AF9, MLL / ELL MLL / AF6, MLL / AF10) resulting from the rearrangements of MLL gene were detected by nested RT-PCR. There arrangements of MLL gene was found in 7 out of 60 AL patients, (11.67%). Among these 7 patients, 2 were diagnosed with AML- M5 and 5 patients were diagnosed with B-ALL. The fusion genes of the 2 AML-M5 patients who had the rearrangements of MLL gene were MLL/AF9. Among 5 B-ALL patients, 2 patients were confirmed to express MLL/ENL, 1 patient was confirmed to express MLL/AF4, and the other 2 patients did not express the fusion genes. 1 of 2 AML-M5 MLL fusion gene positive patients had invasion of leukemia cell in the left leg and CR was achieved after the first course of chemotherapy. The central nervous system leukemia was got after 1 year and died. The other achieved CR with the third of chemotherapy. 1 of 5 patients died from DIC and the cerebral hemorrhage on the second day after diagnosis B-ALL of MLL fusion gene positive. 2 of 5 patients were not in remission and died from multi-organ failures and infection 3 weeks after diagnosis with positive MLL fusion gene. 1 of 5 patients B-ALL of MLL fusion gene positive got invasion of leukemia cell in thoracic and achieved CR after the second course of chemotherapy. 1 of 5 patients of B-ALL of MLL fusion gene positive achieved CR after the first course of chemotherapy and was relapse 1 year after the bone marrow transplant and then died.Conclusions: We conclude that nested RT-PCR is convenient and feasible method to detect the types of fusion genes resulting from the rearrangements of MLL gene. The clinical features of AL with MLL fusion gene include high white blood cell, invasion of multiple organs, resistant to conventional chemotherapy, easy to relapse after remission, and poor prognosis. The detection of MLL gene rearrangement is of great importance in predicting prognosis and guiding therapy in AL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5167: Identification of inactivating mutations in stepwise hepatocarcinogenesis using next generation sequencer

Abstract Early hepatocellular carcinoma (HCC) is histologically diagnosed as the existence of intratumoral portal tracts with stromal invasion. It consists of small hepatocytes with little cellular atypia but with structural atypia. This type of very well-differentiated cancerous tissue is usually characterized by indistinct margins and many portal tracts within the tumor, and develops into classical HCC. To identify sequential genomic changes in stepwise hepatocarcinogenesis, we analyzed fusion genes and mutations using next generation sequencer about 43 early and 105 advanced HCCs. First, exome sequence showed that base sequence aberrations (non-silent) were more frequent in advanced HCC than early HCC (70.9 ± 29.0 vs 53.7 ± 23.8 genes per tumor, P = 0.0002). Mutations in p53/RB pathway including TP53, ATM, RB1, CDKN2A, and CDKN1A were observed in 27.9% and 38.1% in early and advanced HCCs, respectively (P = 0.161) and those of WNT/b-catenin pathways consisting of CTNNB1, AXIN1, and APC were recurrently observed in both early and advanced HCCs (27.9% vs 41.0%, P = 0.095). On the other hand, mutations in SWI/SNF complex genes were more frequently observed in advanced HCC (35.2%) than early HCC (16.3%, P = 0.016). Especially, inactivating mutation of ARID was found in 16 advanced HCCs (15.2%) while only 1 case in early HCC (2.3%, P = 0.018). Consistent with our previous report homozygous deletions near CSMD1 and CDKN2A were also found in 8 and 5 cases, respectively, only in advanced HCC. Next, we detected 4 types of fusion gene, including interchromosomal (translocation), intrachromosomal (deletion), intrachromosomal (translocation), and inversion by RNA sequencing. We also found that every HCC harbored 2 to 5 fusion genes, which were more frequent in advanced than early HCC, although there were no recurrent rearrangements in neither advanced nor early HCCs. Especially chromathripsis, in which 10 to 100 rearrangements were localized in the specific genomic regions and genomic features imply chromosome breaks occur in one-off crisis, was the event observed only in advanced HCC. Taken together, genomic changes were more frequent in advanced HCC, and inactivating mutations of p53/RB and WNT/b-catenin signaling pathways are the driver mutation in the initiation of hepatocarcinogenesis and that of ARID2 may play pivotal roles in the progression from early to advanced HCC. Citation Format: Yutaka Midorikawa, Shogo Yamamoto, Hiroo Ueda, Kotaro Sonoda, Shingo Tsuji, Kenji Tatsuno, Tatsuhiro Shibata, Kyle Covington, David Wheeler, Tadatoshi Takayama, Hiroyuki Aburatani. Identification of inactivating mutations in stepwise hepatocarcinogenesis using next generation sequencer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5167. doi:10.1158/1538-7445.AM2014-5167

Abstract 4020: Genome-wide analysis of stepwise hepatocarcinogenesis using next generation sequencer.

Abstract Early hepatocellular carcinoma (HCC) originates from the hepatocytes of chronic liver disease and develops into classical HCC. To identify sequential genomic changes in multistep hepatocarcinogenesis, we analyzed fusion genes and mutations using next generation sequencer about 40 early and 80 advanced HCCs. First, exome sequence showed that base sequence aberrations were more frequent in advanced HCC than early HCC, and mutations including TP53 (24%), CTNNB1 (24%), and ARID1A (16%) were recurrently observed in both early and advanced HCCs. Consistent with our previous report homozygous deletions near CSMD1 and CDKN2A were also found in 8 and 5 cases, respectively, only in advanced HCC. Next, we detected 4 types of fusion gene, including interchromosomal (translocation), intrachromosomal (deletion), intrachromosomal (translocation), and inversion by RNA sequencing. We also found that every HCC harbored 2 to 5 fusion genes, which were more frequent in advanced than early HCC, although there were no recurrent rearrangements in neither advanced nor early HCCs. Especially chromathripsis, in which 10 to 100 rearrangements were localized in the specific genomic regions and genomic features imply chromosome breaks occur in one-off crisis, was the event observed only in advanced HCC. Taken together, genomic changes were more frequent in advanced HCC, suggesting early HCC originates in the chronic liver disease and progresses into advanced HCC with genomic aberrations. Citation Format: Yutaka Midorikawa, Tadatoshi Takayama, Shogo Yamamoto, Hiroo Ueda, Kotaro Sonoda, Kiyoko Takane, Shingo Tsuji, Genta Nagae, Kenji Tatsuno, Margaret Morgan, David Wheeler, Hiroyuki Aburatani. Genome-wide analysis of stepwise hepatocarcinogenesis using next generation sequencer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2013-4020

Poorly differentiated synovial sarcoma is associated with high expression of enhancer of zeste homologue 2 (EZH2)

BackgroundEnhancer of zeste homologue 2 (EZH2) is a polycomb group (PcG) family protein. Acting as a histone methyltransferase it plays crucial roles in maintaining epigenetic stem cell signature, while its deregulation leads to tumor development. EZH2 overexpression is commonly associated with poor prognosis in a variety of tumor types including carcinomas, lymphomas and soft tissue sarcomas. However, although the synovial sarcoma fusion proteins SYT-SSX1/2/4 are known to interact with PcG members, the diagnostic and prognostic significance of EZH2 expression in synovial sarcoma has not yet been investigated. Also, literature data are equivocal on the correlation between EZH2 expression and the abundance of trimethylated histone 3 lysine 27 (H3K27me3) motifs in tumors.MethodsImmunohistochemical stains of EZH2, H3K27me3, and Ki-67 were performed on tissue microarrays containing cores from 6 poorly differentiated, 39 monophasic and 10 biphasic synovial sarcomas, and evaluated by pre-established scoring criteria. Results of the three immunostainings were compared, and differences were sought between the histological subtypes as well as patient groups defined by gender, age, tumor location, the presence of distant metastasis, and the type of fusion gene. The relationship between EZH2 expression and survival was plotted on a Kaplan-Meier curve.ResultsHigh expression of EZH2 mRNA and protein was specifically detected in the poorly differentiated subtype. EZH2 scores were found to correlate with those of Ki-67 and H3K27me3. Cases with high EZH2 score were characterized by larger tumor size (≥ 5cm), distant metastasis, and poor prognosis. Even in the monophasic and biphasic subtypes, higher expression of EZH2 was associated with higher proliferation rate, larger tumor size, and the risk of developing distant metastasis. In these histological groups, EZH2 was superior to Ki-67 in predicting metastatic disease.ConclusionsHigh expression of EZH2 helps to distinguish poorly differentiated synovial sarcoma from the monophasic and biphasic subtypes, and it is associated with unfavorable clinical outcome. Importantly, high EZH2 expression is predictive of developing distant metastasis even in the better-differentiated subtypes. EZH2 overexpression in synovial sarcoma is correlated with high H3K27 trimethylation. Thus, along with other epigenetic regulators, EZH2 may be a future therapeutic target.

Genotyping of Chimerical BCR-ABL1 RNA in Chronic Myeloid Leukemia by Integrated DNA Chip

Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) are associated with fusion of the BCR and ABL1 genes by chromosome translocation. The chimerical BCR-ABL1 gene encodes different fusion proteins that vary in size, depending on the breakpoint in the BCR region. Different types of fusion genes in CML and Ph(+) ALL are thought to be related to the clinical course and outcome of each patient. Currently, the genotypes are determined by PCR, followed by gel electrophoresis or DNA sequencing (among other methodologies). Our major aim was to develop a diagnostic method for CML genotyping by means of an integrated process of DNA microarray. Here, we describe a method of integrated multiplex reverse transcription, asymmetric PCR, and hybridization, all in the same reaction mixture in a chamber assembled on the surface of capture oligonucleotide probes immobilized on a glass slide. The integrated system successfully identified the four predominant types of chimerical BCR-ABL1 RNA (b3a2, b2a2, e1a2, and c3a2), which together account for 98% of CML cases. The integrated multiplex system also had a high sensitivity of detection (as little as 200 molecules of target RNA molecules). The integrated process saves time and effort, and it also the advantage of minimizing the steps needed for automated detection of different types of chimerical CML RNA.

Fusion genes in angiomatoid fibrous histiocytoma

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue tumor of low malignant potential and uncertain differentiation. Only three genetically investigated cases of AFH have been reported. Two of them displayed a FUS-ATF1 fusion gene and one showed an EWSR1-ATF1 chimera. Using RT-PCR analysis, we have identified the EWSR1-ATF1 fusion transcript, and delineated the genomic breakpoints, in two new cases of AFH. Previously, the EWSR1-ATF1 fusion protein has been suggested to activate expression of the MITF-M transcript, and therefore the expression pattern of the MITF gene was studied. The MITF-M transcript was not detected in either AFH, in line with the finding that the co-activator SOX10 was not expressed. Thus, of the five AFH that have been molecularly analyzed to date, two have displayed a FUS-ATF1 fusion gene and three have shown an EWSR1-ATF1 chimera. There is no apparent correlation between the type of fusion gene and clinicopathologic features. Nonetheless, RT-PCR for these fusion transcripts remains a valuable diagnostic adjunct in the distinction between AFH and other soft tissue tumors or metastases that may simulate it.

Participation of nuclear localization signal 2 in the 3'-ETS domain of FLI1 in nuclear translocation of various chimeric EWS-FLI1 oncoproteins in Ewing tumor

A t(11;22)(q24;q12) translocation is present in 90% of Ewing's sarcoma, and results in the formation of the EWS-FLI1 fusion gene encoding an oncogenic transcription factor. To clarify the function of chimeric EWS-FLI1 proteins, an identification of a nuclear localization signal (NLS) in the EWS, FLI1 and EWS-FLI1 proteins is important because the chimeric oncoprotein may lose or gain NLS function different from native proteins resulting in different subcellular localization, and in deregulated gene expression. Furthermore, some studies reported that patients with one type of fusion gene ('type 1') had better overall survival than those with other types, suggesting that functional differences may be present among various fusion proteins. There has been only one study reporting a NLS in EWS, but none reporting those in FLI1 and EWS-FLI1. To clarify the molecular mechanisms of Ewing tumor development, we first identified the NLSs of EWS and FLI1. We allocated the NLS to amino acid residues 632-656 near the C-terminal region of EWS that is different from the previous study, and identified two NLSs of FLI1, NLS1 (63-90) in the N-terminal domain and NLS2 (319-360) in the 3'-ETS domain. In addition, the present study showed that all of the EWS-FLI1 fusion proteins completely reside in the nucleus without affecting the frequency of nuclear localization among variants, suggesting that NLS2 of FLI1 was used for nuclear translocation of various EWS-FLI1 fusion proteins.

A novel FISH assay for SS18–SSX fusion type in synovial sarcoma

Synovial sarcoma is a morphologically, clinically and genetically distinct entity that accounts for 5–10% of all soft tissue sarcomas. The t(X;18)(p11.2;q11.2) is the cytogenetic hallmark of synovial sarcoma and is present in more than 90% of the cases. It produces three types of fusion gene formed in part by SS18 from chromosome 18 and by SSX1, SSX2 or, rarely, SSX4 from the X chromosome. The SS18–SSX fusions do not seem to occur in other tumor types, and it has been shown that in synovial sarcoma a clear correlation exists between the type of fusion gene and histologic subtype and, more importantly, clinical outcome. Previous analyses regarding the type of fusion genes have been based on PCR amplification of the fusion transcript, requiring access to good-quality RNA. In order to obtain an alternative tool to diagnose and follow this malignancy, we developed a fluorescence in situ hybridization (FISH) assay that could distinguish between the two most common fusion genes, that is, SS18–SSX1 and SS18–SSX2. The specificity of the selected bacterial artificial chromosome clones used in the detection of these fusion genes, as well as the sensitivity of the analysis in metaphase and interphase cells, was examined in a series of 28 synovial sarcoma samples with known fusion gene status. In all samples, the type of fusion was correctly identified by FISH. Thus, the assay described here should be useful for clarifying unresolved chromosome markers and for identifying fusion gene status in samples from which RNA of sufficient quality for PCR could not be extracted.