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Initial diagnosis of chronic myelogenous leukemia based on quantification of M-BCR status using droplet digital PCR

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Abstract

Formed from a reciprocal translocation t(9:22)(q34;q11) of genetic material between the long arms of human chromosomes 9 and 22, the constitutively active breakpoint cluster region (BCR) Abelson 1 (ABL) tyrosine kinase BCR-ABL is known to be causative of chronic myelogenous leukemia (CML). In 98 % of CML patients harboring the t(9:22)(q34;q11) translocation, known as the Philadelphia chromosome, the chimeric BCR-ABL oncogene is created through cleavage of the BCR gene within its major breakpoint region (M-BCR) and breakage of the ABL gene within a 100-kbp region downstream of exon 2a. Clinical detection of the fused BCR-ABL oncogene currently relies on direct visualization by fluorescence in situ hybridization (FISH), a relatively tedious assay that typically offers a detection limit of ca. 2 %. Here, we describe a novel assay that uses droplet digital PCR (ddPCR) technology to reliably measure M-BCR status and the presence of BCR-ABL. When applied to cell-line models of CML, the assay accurately quantifies BCR-ABL frequency to a detection limit of 0.25 %. It therefore offers improved specificity relative to FISH, and may allow identification of variant translocation patterns, including derivative chromosome 9 deletions.

A new assay based on droplet digital PCR is described for highly sensitive detection and quantification of the BCR-ABL1 t(9:22)(q34;q11) reciprocal translation that is causative of chronic myelogenous leukemia

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Notes

  1. KU812 (60,XYY,−2,−3,add(4)(p11),−5,+6,−7,+8,−9,t(9;22)(q34;q11.2)x2,−10,i(11)(q10),−12,−16, del(17)(q11.2q24),i(17)(q10),−18,+19,−20,−22.ish del(17)(TP53+),add(4) (p11)(wcp4)(18)/60, sl,+19)

  2. K562 (67∼70,XX,−X,add(2)(q3?3),−3,+5,add(5)(q11.2),ins(6;?)(p21;?),+7,der(7)t(7;7) (p1?1.1;q22), −9,del(9)(p13), der(9)t(9;9)(p1?3;q22), der(10)t(3;10)(p21;q2?3),−13, add(13)(p11.2),−14,+17,der(17) t(10;17)(q11.2;q11.2)der(10)t(3;10),der(17)t(17;20) (p11.2;p11.2),+19,−20,?der(21)t(1;21) (q21;p11.1),−22,+4mar.ish add(2) (BCR+,ABL1+,BCR con ABL1x1), add(5)(D5S23+,EGR1−), der(9)t(9;9) (ABL1++),der(17)t(17;20) (TP53−), der(17)t(10;17)der(10)t(3;10)(p53+),mar1(BCR+,ABL1+,BCR con ABL1++++), mar2(BCR+,ABL1+,BCR con ABL1++++)[cp7])

  3. MEG01 (97∼100<4n>,XX,−Y,−Y,−1,der(1)t(1;2)(p21;q21), der(2;14)(q10;q10)x2,+6,+6,−7,+8, +8,add(8)(p13),−9,t(9;22)(q34;q11.2)x3,−10,−10,−10,add(10)(p12),+11,del(11)(q13q23)x2,−12,−13,−13, add(13)(q34),+17,+19,+19,+19,+19,+19, add(19)(p13.1)x2,add(19)(q13.1),+21,+21,−22,+der(22)t(9;22), +9mar[cp8])

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Acknowledgments

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institutes of Health Research (CIHR), and Genome BC. C. Haynes receives salary support as a Canada Research Chair.

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Authors and Affiliations

  1. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada

    Helen Louise Lund, Curtis B. Hughesman, Leonard J. Foster & Charles Haynes

  2. Genome Science and Technology Program, University of British Columbia, 100-570 West 7th Avenue, Vancouver, BC, V5Z 4S6, Canada

    Helen Louise Lund

  3. RES’EAU NSERC Research Network, Department of Chemical and Biological Engineering, University of British Columbia, 2360 East Mall, Vancouver, BC, V6T 1Z3, Canada

    Charles Haynes

  4. Cancer Genetics Laboratory, Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, BC, V6T 2B5, Canada

    Kelly McNeil, Shahira Clemens, Kimberly Hocken, Ryan Pettersson & Aly Karsan

  5. Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada

    Leonard J. Foster

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  1. Helen Louise Lund
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Lund, H.L., Hughesman, C.B., McNeil, K. et al. Initial diagnosis of chronic myelogenous leukemia based on quantification of M-BCR status using droplet digital PCR. Anal Bioanal Chem 408, 1079–1094 (2016). https://doi.org/10.1007/s00216-015-9204-2

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