Multiplexed automated digital quantification of fusion transcripts

Comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients

Ariz Akhter, Muhammad Kashif Mughal, Ghaleb Elyamany, Gary Sinclair, Raja Zahratul Azma Raja Sabudin, Noraidah Masir, Salwati Shuib, Fariborz Rashid-Kolvear, Meer Taher Shabani-Rad, Douglas Allan Stewart, Adnan Mansoor

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Article number89
JournalDiagnostic Pathology
Volume11
Issue number1
DOIs
Publication statusPublished - 15 Sep 2016

Fingerprint

Fluorescence In Situ Hybridization
Leukemia
Karyotype
Genetic Translocation
Sensitivity and Specificity
Bone Marrow
Technology
Gene Expression
Validation Studies
Cytogenetics
Paraffin
Formaldehyde
RNA
Pathology
Biopsy
Costs and Cost Analysis

Keywords

  • Acute leukemia
  • Chromosomal translocation
  • Fluorescent in-situ hybridization (FISH)
  • Fusion transcript
  • Prognosis

ASJC Scopus subject areas

  • Histology
  • Pathology and Forensic Medicine

Cite this

Multiplexed automated digital quantification of fusion transcripts : Comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients. / Akhter, Ariz; Mughal, Muhammad Kashif; Elyamany, Ghaleb; Sinclair, Gary; Raja Sabudin, Raja Zahratul Azma; Masir, Noraidah; Shuib, Salwati; Rashid-Kolvear, Fariborz; Shabani-Rad, Meer Taher; Stewart, Douglas Allan; Mansoor, Adnan.

In: Diagnostic Pathology, Vol. 11, No. 1, 89, 15.09.2016.

Research output: Contribution to journalArticle

Akhter, Ariz ; Mughal, Muhammad Kashif ; Elyamany, Ghaleb ; Sinclair, Gary ; Raja Sabudin, Raja Zahratul Azma ; Masir, Noraidah ; Shuib, Salwati ; Rashid-Kolvear, Fariborz ; Shabani-Rad, Meer Taher ; Stewart, Douglas Allan ; Mansoor, Adnan. / Multiplexed automated digital quantification of fusion transcripts : Comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients. In: Diagnostic Pathology. 2016 ; Vol. 11, No. 1.
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AU - Mughal, Muhammad Kashif

AU - Elyamany, Ghaleb

AU - Sinclair, Gary

AU - Raja Sabudin, Raja Zahratul Azma

AU - Masir, Noraidah

AU - Shuib, Salwati

AU - Rashid-Kolvear, Fariborz

AU - Shabani-Rad, Meer Taher

AU - Stewart, Douglas Allan

AU - Mansoor, Adnan

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N2 - 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.

AB - 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.

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KW - Prognosis

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