High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment

被引:142
作者
Logan, Aaron C. [2 ]
Gao, Hong [1 ]
Wang, Chunlin [1 ]
Sahaf, Bita [2 ]
Jones, Carol D. [4 ]
Marshall, Eleanor L. [4 ]
Buno, Ismael [5 ]
Armstrong, Randall [7 ]
Fire, Andrew Z. [4 ]
Weinberg, Kenneth I.
Mindrinos, Michael [1 ]
Zehnder, James L. [3 ]
Boyd, Scott D. [4 ]
Xiao, Wenzhong [1 ,6 ]
Davis, Ronald W. [1 ]
Miklos, David B. [2 ]
机构
[1] Stanford Genome Technol Ctr, Stanford, CA 94304 USA
[2] Stanford Univ, Div Blood & Marrow Transplantat, Sch Med, Stanford, CA 94305 USA
[3] Stanford Univ, Dept Med, Div Hematol, Sch Med, Stanford, CA 94305 USA
[4] Stanford Univ, Dept Pathol, Sch Med, Stanford, CA 94305 USA
[5] Hosp Gen Univ Gregorio Maranon, Dept Hematol, Madrid 28007, Spain
[6] Harvard Univ, Sch Med, Massachusetts Gen Hosp, Boston, MA 02114 USA
[7] Stanford Hosp & Clin, Stanford Cellular Therapeut & Transplantat Lab, Stanford, CA 94305 USA
基金
美国国家卫生研究院;
关键词
next-generation sequencing; consensus-primed polymerase chain reaction; immune reconstitution; STEM-CELL TRANSPLANTATION; POLYMERASE-CHAIN-REACTION; VERSUS-HOST-DISEASE; ALLOGENEIC TRANSPLANTATION; FLOW-CYTOMETRY; RQ-PCR; CLL; IMMUNOGLOBULIN; ERADICATION; RITUXIMAB;
D O I
10.1073/pnas.1118357109
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention to prevent clinical relapse; however, MRD quantification remains an uncommon diagnostic test because of logistical and financial barriers to widespread use. Here we describe a method for quantifying CLL MRD using widely available consensus primers for amplification of all Ig heavy chain (IGH) genes in a mixture of peripheral blood mononuclear cells, followed by high-throughput sequencing (HTS) for disease-specific IGH sequence quantification. To achieve accurate MRD quantification, we developed a systematic bioinformatic methodology to aggregate cancer clone sequence variants arising from systematic and random artifacts occurring during IGH-HTS. We then compared the sensitivity of IGH-HTS, flow cytometry, and allele-specific oligonucleotide PCR for MRD quantification in 28 samples collected from 6 CLL patients following allogeneic HCT. Using amplimer libraries generated with consensus primers from patient blood samples, we demonstrate the sensitivity of IGH-HTS with 454 pyrosequencing to be 10(-5), with a high correlation between quantification by allele-specific oligonucleotide PCR and IGH-HTS (r = 0.85). From the same dataset used to quantify MRD, IGH-HTS also allowed us to profile IGH repertoire reconstitution after HCT-information not provided by the other MRD methods. IGH-HTS using consensus primers will broaden the availability of MRD quantification in CLL and other B cell malignancies, and this approach has potential for quantitative evaluation of immune diversification following transplant and nontransplant therapies.
引用
收藏
页码:21194 / 21199
页数:6
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