A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity

Teoman Deger; Pauline A.J. Mendelaar; Jaco Kraan; Wendy J.C. Prager-van der Smissen; Michelle van der Vlugt-Daane; Eric M.J. Bindels; Anieta M. Sieuwerts; Stefan Sleijfer; Saskia M. Wilting; Antoinette Hollestelle; John W.M. Martens

doi:10.1002/1878-0261.13174

A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity

Teoman Deger, Pauline A.J. Mendelaar, Jaco Kraan, Wendy J.C. Prager-van der Smissen, Michelle van der Vlugt-Daane, Eric M.J. Bindels, Anieta M. Sieuwerts, Stefan Sleijfer, Saskia M. Wilting, Antoinette Hollestelle, John W.M. Martens^*

^*Corresponding author for this work

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

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Abstract

Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

Original language	English
Pages (from-to)	2981-3000
Number of pages	20
Journal	Molecular Oncology
Volume	16
Issue number	16
Early online date	29 Dec 2021
DOIs	https://doi.org/10.1002/1878-0261.13174
Publication status	Published - Aug 2022

Bibliographical note

Funding Information:
We would like to thank Stanley van Herk, Paulette M.H. van Strien and Rui Neves for their technical support. We also thank Raimo Tanzi (Menarini) and his team for technical support as well as Lisa Oomens and Michiel Stevens (VyCAP) for providing WGA samples and technical support. We thank Mark van de Wiel for his support in implementing the R packages he developed. TD, this study and the VyCAP single‐cell puncher were funded by the Daniel den Hoed Foundation. AMS was supported by the Cancer Genomics Center, the Netherlands (CGC.nl).

Publisher Copyright:
© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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A pipeline for copy number profiling of single circulatingtumour cells to assess intrapatient tumour heterogeneityFinal published version, 1.73 MB

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Deger, T., Mendelaar, P. A. J., Kraan, J., Prager-van der Smissen, W. J. C., van der Vlugt-Daane, M., Bindels, E. M. J., Sieuwerts, A. M., Sleijfer, S., Wilting, S. M., Hollestelle, A., & Martens, J. W. M. (2022). A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity. Molecular Oncology, 16(16), 2981-3000. https://doi.org/10.1002/1878-0261.13174

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title = "A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity",

abstract = "Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1{\texttrademark} methods, as well as the results from the Illumina Truseq and the Ampli1{\texttrademark} LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1{\texttrademark} WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.",

author = "Teoman Deger and Mendelaar, {Pauline A.J.} and Jaco Kraan and {Prager-van der Smissen}, {Wendy J.C.} and {van der Vlugt-Daane}, Michelle and Bindels, {Eric M.J.} and Sieuwerts, {Anieta M.} and Stefan Sleijfer and Wilting, {Saskia M.} and Antoinette Hollestelle and Martens, {John W.M.}",

note = "Funding Information: We would like to thank Stanley van Herk, Paulette M.H. van Strien and Rui Neves for their technical support. We also thank Raimo Tanzi (Menarini) and his team for technical support as well as Lisa Oomens and Michiel Stevens (VyCAP) for providing WGA samples and technical support. We thank Mark van de Wiel for his support in implementing the R packages he developed. TD, this study and the VyCAP single‐cell puncher were funded by the Daniel den Hoed Foundation. AMS was supported by the Cancer Genomics Center, the Netherlands (CGC.nl). Publisher Copyright: {\textcopyright} 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.",

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month = aug,

doi = "10.1002/1878-0261.13174",

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Deger, T, Mendelaar, PAJ, Kraan, J, Prager-van der Smissen, WJC, van der Vlugt-Daane, M, Bindels, EMJ, Sieuwerts, AM, Sleijfer, S , Wilting, SM, Hollestelle, A & Martens, JWM 2022, 'A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity', Molecular Oncology, vol. 16, no. 16, pp. 2981-3000. https://doi.org/10.1002/1878-0261.13174

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T1 - A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity

AU - Deger, Teoman

AU - Mendelaar, Pauline A.J.

AU - Kraan, Jaco

AU - Prager-van der Smissen, Wendy J.C.

AU - van der Vlugt-Daane, Michelle

AU - Bindels, Eric M.J.

AU - Sieuwerts, Anieta M.

AU - Sleijfer, Stefan

AU - Wilting, Saskia M.

AU - Hollestelle, Antoinette

AU - Martens, John W.M.

N1 - Funding Information: We would like to thank Stanley van Herk, Paulette M.H. van Strien and Rui Neves for their technical support. We also thank Raimo Tanzi (Menarini) and his team for technical support as well as Lisa Oomens and Michiel Stevens (VyCAP) for providing WGA samples and technical support. We thank Mark van de Wiel for his support in implementing the R packages he developed. TD, this study and the VyCAP single‐cell puncher were funded by the Daniel den Hoed Foundation. AMS was supported by the Cancer Genomics Center, the Netherlands (CGC.nl). Publisher Copyright: © 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

PY - 2022/8

Y1 - 2022/8

N2 - Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

AB - Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

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DO - 10.1002/1878-0261.13174

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SN - 1574-7891

VL - 16

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EP - 3000

JO - Molecular Oncology

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ER -

A pipeline for copy number profiling of single circulating tumour cells to assess intrapatient tumour heterogeneity

Abstract

Bibliographical note

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