title: Multiplexed automated digital quantification of fusion transcripts: comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients.
abstract: BACKGROUND
  The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL.
  
  
  METHODS
  "nCounter® Leukemia fusion gene expression assay" by NanoString was employed to detect various fusion transcripts in a large set samples (n = 94) utilizing RNA from formalin fixed paraffin embedded (FFPE) diagnostic bone marrow biopsy specimens. This series included AL patients with various recurrent translocations (n = 49), normal karyotype (n = 19), or complex karyotype (n = 21), as well as normal bone marrow samples (n = 5). Fusion gene expression data were compared with results obtained by conventional karyotype and FISH technology to determine sensitivity/specificity, as well as positive /negative predictive values.
  
  
  RESULTS
  Junction probes for PML/RARA; RUNX1-RUNX1T1; BCR/ABL1 showed 100 % sensitivity/specificity. A high degree of correlation was noted for MLL/AF4 (85 sensitivity/100 specificity) and TCF3-PBX1 (75 % sensitivity/100 % specificity) probes. CBFB-MYH11 fusion probes showed moderate sensitivity (57 %) but high specificity (100 %). ETV6/RUNX1 displayed discordance between fusion transcript assay and FISH results as well as rare non-specific binding in AL samples with normal or complex cytogenetics.
  
  
  CONCLUSIONS
  Our study presents preliminary data with high correlation between fusion transcript detection by a throughput automated multiplexed platform, compared to conventional karyotype/FISH technique for detection of chromosomal translocations in AL patients. Our preliminary observations, mandates further vast validation studies to explore automated molecular platforms in diagnostic pathology.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.
authors.forename: Ariz
authors.initials: A
authors.lastname: Akhter
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.
authors.forename: Muhammad Kashif
authors.initials: MK
authors.lastname: Mughal
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.
authors.forename: Ghaleb
authors.initials: G
authors.lastname: Elyamany
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.
authors.forename: Gary
authors.initials: G
authors.lastname: Sinclair
authors.affiliation: Department of Pathology, University Kebangsaan Malaysia, Kuala Lumpur, Cheras, Malaysia.
authors.forename: Raja Zahratul
authors.initials: RZ
authors.lastname: Azma
authors.affiliation: Department of Pathology, University Kebangsaan Malaysia, Kuala Lumpur, Cheras, Malaysia.
authors.forename: Noraidah
authors.initials: N
authors.lastname: Masir
authors.affiliation: Department of Pathology, University Kebangsaan Malaysia, Kuala Lumpur, Cheras, Malaysia.
authors.forename: Salwati
authors.initials: S
authors.lastname: Shuib
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.
authors.forename: Fariborz
authors.initials: F
authors.lastname: Rashid-Kolvear
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada.
authors.forename: Meer-Taher
authors.initials: MT
authors.lastname: Shabani-Rad
authors.affiliation: Departments of Oncology and Medicine, University of Calgary, Calgary, AB, Canada.
authors.forename: Douglas Allan
authors.initials: DA
authors.lastname: Stewart
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Calgary and Calgary Laboratory Services, Calgary, AB, Canada. Adnan.Mansoor@cls.ab.ca.|Division of Haematology and Transfusion Medicine, University of Calgary/Calgary Laboratory Services, Room 7522, 7th floor, McCaig tower building; 3134 Hospital drive NW, Calgary, AB, T2N 5A1, Canada. Adnan.Mansoor@cls.ab.ca.
authors.forename: Adnan
authors.initials: A
authors.lastname: Mansoor
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000235:genetics*; D001706:Biopsy; D001856:Bone Marrow Examination; D002648:Child; D002675:Child, Preschool; D005260:Female; D050939:Gene Fusion; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000235:genetics*; D011237:Predictive Value of Tests; D012333:RNA, Messenger / Q000235:genetics*; D015203:Reproducibility of Results; D014178:Translocation, Genetic; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger
keywords: Acute leukemia; Chromosomal translocation; Fluorescent in-situ hybridization (FISH); Fusion transcript; Prognosis