title: High-throughput VDJ sequencing for quantification of minimal residual disease in chronic lymphocytic leukemia and immune reconstitution assessment.
abstract: 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.
authors.affiliation: Divisions of Blood and Marrow Transplantation and Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Logan
authors.forename: Hong
authors.initials: H
authors.lastname: Gao
authors.forename: Chunlin
authors.initials: C
authors.lastname: Wang
authors.forename: Bita
authors.initials: B
authors.lastname: Sahaf
authors.forename: Carol D
authors.initials: CD
authors.lastname: Jones
authors.forename: Eleanor L
authors.initials: EL
authors.lastname: Marshall
authors.forename: Ismael
authors.initials: I
authors.lastname: Buño
authors.forename: Randall
authors.initials: R
authors.lastname: Armstrong
authors.forename: Andrew Z
authors.initials: AZ
authors.lastname: Fire
authors.forename: Kenneth I
authors.initials: KI
authors.lastname: Weinberg
authors.forename: Michael
authors.initials: M
authors.lastname: Mindrinos
authors.forename: James L
authors.initials: JL
authors.lastname: Zehnder
authors.forename: Scott D
authors.initials: SD
authors.lastname: Boyd
authors.forename: Wenzhong
authors.initials: W
authors.lastname: Xiao
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: David B
authors.initials: DB
authors.lastname: Miklos
mesh_terms: D019295:Computational Biology / Q000379:methods; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000517:prevention & control* / Q000628:therapy*; D018365:Neoplasm, Residual / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D055502:Secondary Prevention; D012680:Sensitivity and Specificity; D014184:Transplantation, Homologous
chemical_list: D007143:Immunoglobulin Heavy Chains; D009841:Oligonucleotides