超高感度核酸解析を用いた研究に関する文献紹介

■BioMarkを用いた研究に関する文献のご紹介

Digital Arrayを用いたNSCLCにおける血漿からの変異型EGFRのコピー数解析

本研究では、新しい超高感度検出定量の技術であるdigital PCRを用いて肺がん患者の血液からEGFR遺伝子変異を検出しコピー数の定量を行いました。患者から切除した原発巣および採取した血漿から抽出したDNAを用いて検出・比較を行いました。その結果、原発巣がシークエンス法で変異陽性だった症例の92%において、digital PCRによって血漿から変異を検出することができました。また、従来のシークエンス法では変異が検出できなかった原発巣サンプルについても、digital PCRによって変異を検出することができました。さらに興味深いことに、EGFR-TKI(イレッサ、タルセバ)の投与前後の血漿中の変異型EGFRのコピー数をdigital PCRで測定した結果、EGFR-TKIによる治療効果に応じて変異型EGFRのコピー数が変動したことから、血漿中EGFR遺伝子変異のコピー数が治療効果の指標として利用できることが示唆されました。今後、非侵襲的なサンプルである血液サンプルからdigital PCRにより高感度にEGFR遺伝子変異を検出・コピー数定量することにより、より簡便にかつ高精度に治療の奏功性や予後の予測ができるようになることが期待されます。
（各文献の解釈につきましては、弊社は一切の責任を負いません）

Single-Molecule Detection of Epidermal Growth Factor Receptor Mutations in Plasma by Microfluidics Digital PCR in Non-Small Cell Lung Cancer Patients
Yung TK, Chan KC, Mok TS, Tong J, To KF, Lo YM.
Clinical Cancer Research.  2009;15:2076-2084.

【Abstract】

Pupose: We aim to develop a digital PCR-based method for the quantitative detection of the two common epidermal growth factor receptor (EGFR) mutations (in-frame deletion at exon 19 and L858R at exon 21) in the plasma and tumor tissues of patients suffering from non-small cell lung cancers. These two mutations account for >85% of clinically important EGFR mutations associated with responsiveness to tyrosine kinase inhibitors.
Experimental design: DNA samples were analyzed using a microfluidics system that simultaneously performed 9,180 PCRs at nanoliter scale. A single-mutant DNA molecule in a clinical specimen could be detected and the quantities of mutant and wild-type sequences were precisely determined.
Results: Exon 19 deletion and L858R mutation were detectable in 6 (17%) and 9 (26%) of 35 pretreatment plasma samples, respectively. When compared with the sequencing results of the tumor samples, the sensitivity and specificity of plasma EGFR mutation analysis were 92% and 100%, respectively. The plasma concentration of the mutant sequences correlated well with the clinical response. Decreased concentration was observed in all patients with partial or complete clinical remission, whereas persistence of mutation was observed in a patient with cancer progression. In one patient, tyrosine kinase inhibitor was stopped after an initial response and the tumor-associated EGFR mutation reemerged 4 weeks after stopping treatment.
Conclusion: The sensitive detection and accurate quantification of low abundance EGFR mutations in tumor tissues and plasma by microfluidics digital PCR would be useful for predicting treatment response, monitoring disease progression and early detection of treatment failure associated with acquired drug resistance.

PMID 19276259

■その他の文献リスト

Digital Array

Detection of Increased Amounts of Cell-Free Fetal DNA with Short PCR Amplicons
Sikora A, Zimmermann BG, Rusterholz C, Birri D, Kolla V, Lapaire O, Hoesli I, Kiefer V, Jackson L, Hahn S
Clin Chem.  2010;56:136-138.

Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy
Fan HC, Blumenfeld YJ, El-Sayed YY, Chueh J, Quake SR
American Journal of Obstetrics and Gynecology.  2009;200:543.e1-543.e7.

Noninvasive prenatal detection of fetal chromosomal aneuploidies by maternal plasma nucleic acid analysis: a review of the current state of the art
Lo Y. M. D.
BJOG: An International Journal of Obstetrics & Gynaecology.  2009;116:152-157.

Dazl Functions in Maintenance of Pluripotency and Genetic and Epigenetic Programs of Differentiation in Mouse Primordial Germ Cells In Vivo and In Vitro
Haston Kelly M., Tung Joyce Y., Reijo Pera Renee A.
PLoS ONE.  2009;4:e5654.
 
Digital PCR provides sensitive and absolute calibration for high throughput sequencing
White Richard, Blainey Paul, Fan H. Christina, Quake Stephen
BMC Genomics.  2009;10:116.

Single-molecule sequencing of an individual human genome
Pushkarev Dmitry, Neff Norma F., Quake Stephen R.
Nature Biotechnology.  2009;27:847-850.

Population-genetic nature of copy number variations in the human genome
Kato M, Kawaguchi T, Ishikawa S, Umeda T, Nakamichi R, Shapero MH, Jones KW, Nakamura Y, Aburatani H, Tsunoda T
Hum. Mol. Genet.  2009:ddp541.

Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number
Bhat S, Herrmann J, Armishaw P, Corbisier P, Emslie KR
Analytical and Bioanalytical Chemistry.  2009;394:457-467.

Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte
Sindelka R, Jonak J, Hands R, Bustin SA, Kubista M.
Nucl. Acids Res.  2008;36:387-392.

Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR
Oehler VG, Qin J, Ramakrishnan R, Facer G, Ananthnarayan S, Cummings C, Deininger M, Shah N, McCormick F, Willis S, Daridon A, Unger M, Radich JP
Leukemia  2008;23:396-399.

DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight
Weisenberger DJ, Trinh BN, Campan M, Sharma S, Long TI, Ananthnarayan S, Liang G, Esteva FJ, Hortobagyi GN, McCormick F, Jones PA, Laird PW
Nucl. Acids Res. 2008;36:4689-4698.

Digital PCR: a powerful new tool for noninvasive prenatal diagnosis?
Zimmermann BG, Grill S, Holzgreve W, Zhong XY, Jackson LG, Hahn S
Prenatal Diagnosis. 2008;28:1087-1093

Studying copy number variations using a nanofluidic platform
Qin Jian, Jones Robert C., Ramakrishnan Ramesh
Nucl. Acids Res..  2008;36:e116.

Microfluidics Digital PCR Reveals a Higher than Expected Fraction of Fetal DNA in Maternal Plasma
Lun FM, Chiu RW, Allen Chan KC, Yeung Leung T, Kin Lau T, Dennis Lo YM
Clin Chem.  2008;54:1664-1672.

Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device
Dube Simant, Qin Jian, Ramakrishnan Ramesh
PLoS ONE.  2008;3:e2876.

Detection of Aneuploidy with Digital Polymerase Chain Reaction
Fan H. Christina, Quake Stephen R.
Analytical Chemistry.  2007;79:7576-7579.

Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR
Warren Luigi, Bryder David, Weissman Irving L., Quake Stephen R.
Proceedings of the National Academy of Sciences.  2006;103:17807-17812.

Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria
Ottesen EA, Hong JW, Quake SR, Leadbetter JR
Science.  2006;314:1464-1467.

Dynamic Array

Nuclear Receptor DHR96 Acts as a Sentinel for Low Cholesterol Concentrations in Drosophila melanogaster
Bujold Mattea, Gopalakrishnan Akila, Nally Emma, King-Jones Kirst
Mol. Cell. Biol..  2010;30:793-805.

Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-I± Monoclonal Antibody Trials in Systemic Lupus Erythematosus
Yihong Y., Brandon W. H., Chris M.
Human Genomics and Proteomics.  2009.

Circulating Tumor Cells Are Transcriptionally Similar to the Primary Tumor in a Murine Prostate Model
Helzer KT, Barnes HE, Day L, Harvey J, Billings PR, Forsyth A
Cancer Res.  2009;69:7860-7866.

Enhanced Generation of Induced Pluripotent Stem Cells from a Subpopulation of Human Fibroblasts
Byrne James A., Nguyen Ha N., Reijo Pera Renee A.
PLoS ONE.  2009;4:e7118.

Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility
Nicholas CR, Haston KM, Grewall AK, Longacre TA, Reijo Pera RA
Hum. Mol. Genet..  2009;18:4376-4389.
 
Pharmacogenetic Pathway Analysis for Determination of Sunitinib-Induced Toxicity
van Erp NP, Eechoute K, van der Veldt AA, Haanen JB, Reyners AK, Mathijssen RH, Boven E, van der Straaten T, Baak-Pablo RF, Wessels JA, Guchelaar HJ, Gelderblom H
J Clin Oncol.  2009;27:4406-4412.

Evaluation of current methods used to analyze the expression profiles of ATP-binding cassette transporters yields an improved drug-discovery database
Orina JN, Calcagno AM, Wu CP, Varma S, Shih J, Lin M, Eichler G, Weinstein JN, Pommier Y, Ambudkar SV, Gottesman MM, Gillet JP
Molecular Cancer Therapeutics.  2009;8:2057-2066.

Association of reactive oxygen species levels and radioresistance in cancer stem cells
Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, Qian D, Lam JS, Ailles LE, Wong M, Joshua B, Kaplan MJ, Wapnir I, Dirbas FM, Somlo G, Garberoglio C, Paz B, Shen J, Lau SK, Quake SR, Brown JM, Weissman IL, Clarke MF
Nature.  2009;458:780-783.

Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion
Sato T, Onai N, Yoshihara H, Arai F, Suda T, Ohteki T
Nature Medicine.  2009;15:696-700.

Bioinformatics construction of the human cell surfaceome
da Cunha JP, Galante PA, de Souza JE, de Souza RF, Carvalho PM, Ohara DT, Moura RP, Oba-Shinja SM, Marie SK, Silva WA Jr, Perez RO, Stransky B, Pieprzyk M, Moore J, Caballero O, Gama-Rodrigues J, Habr-Gama A, Kuo WP, Simpson AJ, Camargo AA, Old LJ, de Souza SJ
Proceedings of the National Academy of Sciences of the United States of America.  2009;106:16752-16757.

Inhibition of prolyl hydroxylase protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity: model for the potential involvement of the hypoxia-inducible factor pathway in Parkinson disease
Lee DW, Rajagopalan S, Siddiq A, Gwiazda R, Yang L, Beal MF, Ratan RR, Andersen JK
Journal of Biological Chemistry.  2009;284:29065-29076.

High-throughput single nucleotide polymorphism genotyping using nanofluidic Dynamic Arrays
Wang J, Lin M, Crenshaw A, Hutchinson A, Hicks B, Yeager M, Berndt S, Huang WY, Hayes RB, Chanock SJ, Jones RC, Ramakrishnan R
BMC Genomics.  2009;10:561.

Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells
Yuan P, Han J, Guo G, Orlov YL, Huss M, Loh YH, Yaw LP, Robson P, Lim B, Ng HH
Genes & Development.  2009;23:2507-2520.

Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo
Jedrusik A, Parfitt DE, Guo G, Skamagki M, Grabarek JB, Johnson MH, Robson P, Zernicka-Goetz M
Genes & Development.  2008;22:2692-2706.

In-depth characterization of the microRNA transcriptome in a leukemia progression model
Kuchenbauer F, Morin RD, Argiropoulos B, Petriv OI, Griffith M, Heuser M, Yung E, Piper J, Delaney A, Prabhu AL, Zhao Y, McDonald H, Zeng T, Hirst M, Hansen CL, Marra MA, Humphries RK
Genome research.  2008;18:1787-1797.

High Throughput Gene Expression Measurement with Real Time PCR in a Microfluidic Dynamic Array
Spurgeon Sandra L., Jones Robert C., Ramakrishnan Ramesh
PLoS ONE.  2008;3:e1662.

Lola regulates Drosophila olfactory projection neuron identity and targeting specificity
Spletter ML, Liu J, Liu J, Su H, Giniger E, Komiyama T, Quake S, Luo L
Neural Development.  2007;2:14.

なお、本件に関するお問い合わせは、お問い合わせフォームよりお願いいたします。