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.

Fig. 1.

454 pyrosequencing MRD quantification and artifact characteristics. (A) The final MRD count for each patient sample with the composition of identical reads, homopolymeric indels, random single-nucleotide indels, and single-nucleotide substitutions is shown. (B) The number of unique reads with each error type is indicated for each patient sample. (C–E) The correlation between IGH-HTS MRD quantification and the number of unique reads requiring correction for homopolymeric indels (C), random single-nucleotide indels (D), and single-nucleotide substitutions or gaps (E) are demonstrated.

Fig. 2.

Phylogenetic analysis of CLL subclones. Neighbor-joining phylogenetic trees are depicted for patients SPN3975 (A) and SPN3860 (B). The number of nucleotide differences are represented by horizontal proximity to the dominant CLL clone (Dc). We required subclones (Sc) contributing to these phylogenetic trees to be seen at least twice in two or more time points. The specific time points in which each clonotype was observed is indicated at each node.

Fig. 3.

Sensitivity of CLL MRD detection by 454 pyrosequencing determined by limiting dilution. (A) Consensus primer groups in the FR1 or FR2 regions of V_H were used in conjunction with a consensus J_H primer to create IGH amplicon libraries by multiplexed PCR, which were then sequenced by synthesis using a 454 pyrosequencer. (B and C) CLL cells purified by FACS were diluted into PBMC from a healthy donor leukapheresis sample to a level of 1:1,000 (10⁻³), 1:10,000 (10⁻⁴), and 1:100,000 (10⁻⁵). Clonotypic IGH quantifications at each dilution are demonstrated for FR1 (B) and FR2 (C) primer sets with reference values for expected quantifications at ≥95% Poisson probability.

Fig. 4.

Correlation of CLL MRD quantification by IGH-HTS, ASO-PCR, and FC. CLL MRD quantification for samples with 15,000 or fewer CLL copies per microgram of DNA using 454 IGH-HTS are graphed with paired analyses using ASO-PCR (A) and FC (B). The Pearson r correlation coefficient between the methods was 0.85 for 454 IGH-HTS vs. ASO-PCR and 0.49 for 454 IGH-HTS vs. FC. For this comparison, FC data were converted as described in SI Materials and Methods.

Fig. 5.

IGH-HTS reveals kinetics of IGH repertoire reconstitution following allo-HCT for CLL. All V-J recombinations detected in peripheral blood from patient SPN4077 at diagnosis and days +56, +180, +365, and +550 following allo-HCT are demonstrated. The x axis at the bottom of each section represents IGH V segments 1–49, which combined with IGH J segments 1–6 as defined along the x axis at the top of each section. The y axis represents the number of total reads for that recombination pair. The CLL clonotype is demarcated by an asterisk. The repertoire of this patient's donor is demonstrated for comparison.