High-throughput amplicon-based copy number detection of 11 genes in formalin-fixed paraffin-embedded ovarian tumour samples by MLPA-seq

Australian Ovarian Cancer Study (AOCS) Group

doi:10.1371/journal.pone.0143006

High-throughput amplicon-based copy number detection of 11 genes in formalin-fixed paraffin-embedded ovarian tumour samples by MLPA-seq

Australian Ovarian Cancer Study (AOCS) Group

Bar-Ilan University - Azrieli Faculty of Medicine

Research output: Contribution to journal › Article › peer-review

10 Scopus citations

Abstract

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

Original language	English
Article number	e0143006
Journal	PLoS ONE
Volume	10
Issue number	11
DOIs	https://doi.org/10.1371/journal.pone.0143006
State	Published - 1 Nov 2015

Bibliographical note

Publisher Copyright:
© 2015 Kondrashovaet al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding

The AOCS was supported by the U.S. Army Medical Research and Materiel Command under DAMD17-01-1-0729, The Cancer Council Tasmania and The Cancer Foundation of Western Australia and the National Health and Medical Research Council of Australia (NHMRC, ID400413), the Peter MacCallum Cancer Foundation and Ovarian Cancer Australia (OCA). This work was supported by research grants from the Victorian Comprehensive Cancer Centre, the Victorian Cancer Agency (TRP13088), and the National Health and Medical Research Council(APP1034301) with infrastructure support for sequencing from the University of Melbourne and Therapeutic Innovation Australia. The Victorian Cancer Biobank is supported by the Victorian Government. GT is part supported by the Herman Trust. The authors thank MRC-Holland for making the probe sequences used in MLPA kits publically available. The authors thank Kathryn Alsop (AOCS), Benhur Amanuel (PathWest), Michael Christie (Royal Melbourne Hospital), Diar Aziz and Irma Gresshoff (University of Melbourne) and Audrey Partenen (Victorian Cancer Biobank) for providing DNA and tumour samples with known genotypes. The authors thank Kym Pham (University of Melbourne) for sequencing infrastructure management and support. The authors also thank Kandavel Shanmugan, Andrea Muranyi and Leanne Hendrickson (Ventana) for providing the chr19q12 ISH probe, protocol and reagents. The management group of the AOCS group included D Bowtell, G Chenevix-Trench, A Green, P Webb, A DeFazio and D Gertig. The project and data managers included N Traficante, S Fereday, S Moore, J Hung, K Harrap, T Sadkowsky, N Pandeya. The Reseach Nurses and Assistants of the AOCS group included: MMalt, A Mellon, R Robertson, T Vanden Bergh, MJones, P Mackenzie, J Maidens, K Nattress, YE Chiew, A Stenlake, H Sullivan, B Alexander, P Ashover, S Brown, T Corrish, L Green, L Jackman, K Ferguson, K Martin, A Martyn, B Ranieri, J White, V Jayde, P Mamers, L Bowes, L Galletta, D Giles, J Hendley, K Alsop, T Schmidt, H Shirley, C Ball, C Young, S Viduka, Hoa Tran, Sanela Bilic, Lydia Glavinas, Julia Brooks. The Clinical and Scientific Collaborators of the AOCS group included: R Stuart-Harris, F Kirsten, J Rutovitz, P Clingan, A Glasgow, A Proietto, S Braye, G Otton, J Shannon, T Bonaventura, J Stewart, S Begbie MFriedlander D Bell, S Baron-Hay, A Ferrier (dec.), G Gard, D Nevell, N Pavlakis, S Valmadre, B Young, C Camaris, R Crouch, L Edwards, N Hacker, D Marsden, G Robertson, P Beale, J Beith, J Carter, C Dalrymple, R Houghton, P Russell, M Links, J Grygiel, J Hill, A Brand, K Byth, R Jaworski, P Harnett, R Sharma, G Wain, B Ward, D Papadimos, A Crandon, MCummings, K Horwood, A Obermair, L Perrin, D Wyld, J Nicklin, MDavy, MK Oehler, C Hall, T Dodd, T Healy, K Pittman, D Henderson, J Miller, J Pierdes, P Blomfield, D Challis, R McIntosh, A Parker, B Brown, R Rome, D Allen, P Grant, S Hyde, R Laurie, MRobbie, D Healy, T Jobling, T Manolitsas, J McNealage, P Rogers, B Susil, E Sumithran, I Simpson, K Phillips, D Rischin, S Fox, D Johnson, S Lade, MLoughrey, N O’Callaghan, WMurray, P Waring, V Billson, J Pyman, D Neesham, MQuinn, C Underhill, R Bell, LF Ng, R Blum, V Ganju, I Hammond, Y Leung, A McCartney (dec.), MBuck, I Haviv, D Purdie, D Whiteman, N Zeps. We also acknowledge the contribution of the study nurses and research assistants and would like to thank all of the women who participated in the study.

Funders	Funder number
Benhur Amanuel
Cancer Foundation of Western Australia
Diar Aziz and Irma Gresshoff
G Chenevix-Trench
Herman Trust
MRC-Holland
University of Melbourne and Therapeutic Innovation Australia
Ventana
Victorian Cancer Biobank
Victorian Comprehensive Cancer Centre
Medical Research and Materiel Command	DAMD17-01-1-0729
Victorian Cancer Agency	TRP13088
Ovarian Cancer Australia
Royal Melbourne Hospital
National Health and Medical Research Council	APP1034301, ID400413
Cancer Council Tasmania
University of Melbourne
State Government of Victoria
Peter MacCallum Foundation

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title = "High-throughput amplicon-based copy number detection of 11 genes in formalin-fixed paraffin-embedded ovarian tumour samples by MLPA-seq",

abstract = "Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.",

author = "{Australian Ovarian Cancer Study (AOCS) Group} and Olga Kondrashova and Love, {Clare J.} and Sebastian Lunke and Hsu, {Arthur L.} and D. Bowtell and G. Chenevix-Trench and A. Green and P. Webb and A. DeFazio and D. Gertig and N. Traficante and S. Fereday and S. Moore and J. Hung and K. Harrap and T. Sadkowsky and N. Pandeya and M. Malt and A. Mellon and R. Robertson and {Vanden Bergh}, T. and M. Jones and P. Mackenzie and J. Maidens and K. Nattress and Chiew, {Y. E.} and A. Stenlake and H. Sullivan and B. Alexander and P. Ashover and S. Brown and T. Corrish and L. Green and L. Jackman and K. Ferguson and K. Martin and A. Martyn and B. Ranieri and J. White and V. Jayde and P. Mamers and L. Bowes and L. Galletta and D. Giles and J. Hendley and K. Alsop and T. Schmidt and H. Shirley and C. Ball and I. Haviv",

note = "Publisher Copyright: {\textcopyright} 2015 Kondrashovaet al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

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AU - Kondrashova, Olga

AU - Love, Clare J.

AU - Lunke, Sebastian

AU - Hsu, Arthur L.

AU - Bowtell, D.

AU - Chenevix-Trench, G.

AU - Green, A.

AU - Webb, P.

AU - DeFazio, A.

AU - Gertig, D.

AU - Traficante, N.

AU - Fereday, S.

AU - Moore, S.

AU - Hung, J.

AU - Harrap, K.

AU - Sadkowsky, T.

AU - Pandeya, N.

AU - Malt, M.

AU - Mellon, A.

AU - Robertson, R.

AU - Vanden Bergh, T.

AU - Jones, M.

AU - Mackenzie, P.

AU - Maidens, J.

AU - Nattress, K.

AU - Chiew, Y. E.

AU - Stenlake, A.

AU - Sullivan, H.

AU - Alexander, B.

AU - Ashover, P.

AU - Brown, S.

AU - Corrish, T.

AU - Green, L.

AU - Jackman, L.

AU - Ferguson, K.

AU - Martin, K.

AU - Martyn, A.

AU - Ranieri, B.

AU - White, J.

AU - Jayde, V.

AU - Mamers, P.

AU - Bowes, L.

AU - Galletta, L.

AU - Giles, D.

AU - Hendley, J.

AU - Alsop, K.

AU - Schmidt, T.

AU - Shirley, H.

AU - Ball, C.

AU - Haviv, I.

N1 - Publisher Copyright: © 2015 Kondrashovaet al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Y1 - 2015/11/1

N2 - Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

AB - Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

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High-throughput amplicon-based copy number detection of 11 genes in formalin-fixed paraffin-embedded ovarian tumour samples by MLPA-seq

Abstract

Bibliographical note

Funding

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