Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles

Walter W. Chen; Leonora Balaj; Linda M. Liau; Michael L. Samuels; Steve K. Kotsopoulos; Casey A. Maguire; Lori LoGuidice; Horacio Soto; Matthew Garrett; Lin Dan Zhu; Sarada Sivaraman; Clark Chen; Eric T. Wong; Bob S. Carter; Fred H. Hochberg; Xandra O. Breakefield; Johan Skog

doi:10.1038/mtna.2013.28

Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles

Walter W. Chen, Leonora Balaj, Linda M. Liau, Michael L. Samuels, Steve K. Kotsopoulos, Casey A. Maguire, Lori LoGuidice, Horacio Soto, Matthew Garrett, Lin Dan Zhu, Sarada Sivaraman, Clark Chen, Eric T. Wong, Bob S. Carter, Fred H. Hochberg, Xandra O. Breakefield, Johan Skog

Neurosurgery

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

286 Scopus citations

Abstract

Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.

Original language	English (US)
Article number	e109
Pages (from-to)	e109
Journal	Molecular Therapy Nucleic Acids
Volume	2
DOIs	https://doi.org/10.1038/mtna.2013.28
State	Published - 2013

Bibliographical note

Funding Information:
This work was supported by NIH/NCI grants CA069246 (F.H., X.O.B., B.C.); CA141226 (X.O.B.); CA156009 (X.O.B.); and CA141150 (X.O.B.); Brain Tumor Funders’ Collaborative (B.C., X.O.B.); American Brain Tumor Association (ABTA; J.S.); Harvard Catalyst (B.C.); A Reason To Ride research fund (E.T.W.); Hyugens Scholarship NL (L.B.), the Richard Floor Biorepository and the Accelerate Brain Cancer Cure (ABC2). All processing of samples by Exosome Diagnostics Inc and RainDance Technologies was carried out on de-identified samples. We thank Suzanne McDavitt for skilled editorial assistance. We thank N Agrawal for his advice and for providing us with BEAMing reagents and A Mandinova for providing access to the TissueLyser. We thank Karen Messer for help with statistical analysis. We also thank Winston Patrick Kuo and Fatemeh Momen-Heravi for help with TEM and Mikkel Noerholm for help with probe design. J.S. is an inventor on the microvesicle technology used in this study which has been licensed to Exosome Diagnostics, Inc. He holds equity in, and is an employee of that company. Breakefield and Carter are on the Scientific Advisory Board of the company for which they receive cash compensation. M.L.S. and S.K.K. are employees of RainDance Technologies Inc. The other authors declared no conflict of interest.

Keywords

BEAMing PCR
Biomarkers
Cancer
Droplet digital PCR
Extracellular vesicles

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access

10.1038/mtna.2013.28

OpenUrl availability

Full text

Cite this

Chen, W. W., Balaj, L., Liau, L. M., Samuels, M. L., Kotsopoulos, S. K., Maguire, C. A., LoGuidice, L., Soto, H., Garrett, M., Zhu, L. D., Sivaraman, S., Chen, C., Wong, E. T., Carter, B. S., Hochberg, F. H., Breakefield, X. O., & Skog, J. (2013). Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles. Molecular Therapy Nucleic Acids, 2, e109. Article e109. https://doi.org/10.1038/mtna.2013.28

Chen, WW, Balaj, L, Liau, LM, Samuels, ML, Kotsopoulos, SK, Maguire, CA, LoGuidice, L, Soto, H, Garrett, M, Zhu, LD, Sivaraman, S, Chen, C, Wong, ET, Carter, BS, Hochberg, FH, Breakefield, XO & Skog, J 2013, 'Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles', Molecular Therapy Nucleic Acids, vol. 2, e109, pp. e109. https://doi.org/10.1038/mtna.2013.28

@article{3666f845edae41c4890d359fe51cbb7e,

title = "Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles",

abstract = "Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.",

keywords = "BEAMing PCR, Biomarkers, Cancer, Droplet digital PCR, Extracellular vesicles",

author = "Chen, {Walter W.} and Leonora Balaj and Liau, {Linda M.} and Samuels, {Michael L.} and Kotsopoulos, {Steve K.} and Maguire, {Casey A.} and Lori LoGuidice and Horacio Soto and Matthew Garrett and Zhu, {Lin Dan} and Sarada Sivaraman and Clark Chen and Wong, {Eric T.} and Carter, {Bob S.} and Hochberg, {Fred H.} and Breakefield, {Xandra O.} and Johan Skog",

note = "Funding Information: This work was supported by NIH/NCI grants CA069246 (F.H., X.O.B., B.C.); CA141226 (X.O.B.); CA156009 (X.O.B.); and CA141150 (X.O.B.); Brain Tumor Funders{\textquoteright} Collaborative (B.C., X.O.B.); American Brain Tumor Association (ABTA; J.S.); Harvard Catalyst (B.C.); A Reason To Ride research fund (E.T.W.); Hyugens Scholarship NL (L.B.), the Richard Floor Biorepository and the Accelerate Brain Cancer Cure (ABC2). All processing of samples by Exosome Diagnostics Inc and RainDance Technologies was carried out on de-identified samples. We thank Suzanne McDavitt for skilled editorial assistance. We thank N Agrawal for his advice and for providing us with BEAMing reagents and A Mandinova for providing access to the TissueLyser. We thank Karen Messer for help with statistical analysis. We also thank Winston Patrick Kuo and Fatemeh Momen-Heravi for help with TEM and Mikkel Noerholm for help with probe design. J.S. is an inventor on the microvesicle technology used in this study which has been licensed to Exosome Diagnostics, Inc. He holds equity in, and is an employee of that company. Breakefield and Carter are on the Scientific Advisory Board of the company for which they receive cash compensation. M.L.S. and S.K.K. are employees of RainDance Technologies Inc. The other authors declared no conflict of interest.",

year = "2013",

doi = "10.1038/mtna.2013.28",

language = "English (US)",

volume = "2",

pages = "e109",

journal = "Molecular Therapy Nucleic Acids",

issn = "2162-2531",

publisher = "Nature Publishing Group",

}

TY - JOUR

T1 - Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles

AU - Chen, Walter W.

AU - Balaj, Leonora

AU - Liau, Linda M.

AU - Samuels, Michael L.

AU - Kotsopoulos, Steve K.

AU - Maguire, Casey A.

AU - LoGuidice, Lori

AU - Soto, Horacio

AU - Garrett, Matthew

AU - Zhu, Lin Dan

AU - Sivaraman, Sarada

AU - Chen, Clark

AU - Wong, Eric T.

AU - Carter, Bob S.

AU - Hochberg, Fred H.

AU - Breakefield, Xandra O.

AU - Skog, Johan

N1 - Funding Information: This work was supported by NIH/NCI grants CA069246 (F.H., X.O.B., B.C.); CA141226 (X.O.B.); CA156009 (X.O.B.); and CA141150 (X.O.B.); Brain Tumor Funders’ Collaborative (B.C., X.O.B.); American Brain Tumor Association (ABTA; J.S.); Harvard Catalyst (B.C.); A Reason To Ride research fund (E.T.W.); Hyugens Scholarship NL (L.B.), the Richard Floor Biorepository and the Accelerate Brain Cancer Cure (ABC2). All processing of samples by Exosome Diagnostics Inc and RainDance Technologies was carried out on de-identified samples. We thank Suzanne McDavitt for skilled editorial assistance. We thank N Agrawal for his advice and for providing us with BEAMing reagents and A Mandinova for providing access to the TissueLyser. We thank Karen Messer for help with statistical analysis. We also thank Winston Patrick Kuo and Fatemeh Momen-Heravi for help with TEM and Mikkel Noerholm for help with probe design. J.S. is an inventor on the microvesicle technology used in this study which has been licensed to Exosome Diagnostics, Inc. He holds equity in, and is an employee of that company. Breakefield and Carter are on the Scientific Advisory Board of the company for which they receive cash compensation. M.L.S. and S.K.K. are employees of RainDance Technologies Inc. The other authors declared no conflict of interest.

PY - 2013

Y1 - 2013

N2 - Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.

AB - Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.

KW - BEAMing PCR

KW - Biomarkers

KW - Cancer

KW - Droplet digital PCR

KW - Extracellular vesicles

UR - http://www.scopus.com/inward/record.url?scp=84880339763&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84880339763&partnerID=8YFLogxK

U2 - 10.1038/mtna.2013.28

DO - 10.1038/mtna.2013.28

M3 - Article

C2 - 23881452

AN - SCOPUS:84880339763

SN - 2162-2531

VL - 2

SP - e109

JO - Molecular Therapy Nucleic Acids

JF - Molecular Therapy Nucleic Acids

M1 - e109

ER -

Beaming and droplet digital pcr analysis of mutant idh1 mrna in glioma patient serum and cerebrospinal fluid extracellular vesicles

Abstract

Bibliographical note

Keywords

UN SDGs

Access

OpenUrl availability

Other files and links

Fingerprint

Cite this