High-Throughput Immunoglobulin Gene Sequencing Quantifies Minimal Residual Disease in CLL with 10e-6 Sensitivity and Strongly Predicts Relapse After Allogeneic Hematopoietic Cell Transplantation

Patients with high-risk chronic lymphocytic leukemia (CLL) uniformly relapse after conventional chemo-immunotherapy, but roughly half achieve long-term disease-free survival (DFS) following allogeneic hematopoietic cell transplantation (allo-HCT). Quantification of minimal residual disease (MRD) following allo-HCT predicts post-transplant relapse (PTR) when CLL disease burden remains greater than 10e-4 (ie, 1 leukemic cell in 10,000 peripheral blood mononuclear cells [PBMC]) when quantified by allele-specific oligonucleotide quantitative polymerase chain reaction (ASO-PCR) or flow cytometry. We previously demonstrated the feasibility of MRD quantification using consensus primers to amplify all immunoglobulin heavy chain (IGH) genes in a mixture of PBMC, followed by high-throughput sequencing (HTS) and clonotypic quantification. In our prior work, we used 454 pyrosequencing technology which enabled 10e-5 sensitivity. Here, we report 10e-6 MRD sensitivity using novel Illumina-based HTS that provides better prediction of disease recurrence than ASO-PCR.

We amplified IGH loci from genomic DNA extracted from PBMC (median input 2.4×10e6 cells; range 1.1–23.7×10e6) using V and J segment consensus primers. Amplified IGH molecules were then sequenced with one million or more dedicated reads using Illumina HiSeq and clones were quantified using Sequenta HTS bioinformatics. To verify 10e-6 sensitivity using this system, a clonal B cell population was diluted to 10e-6 in PBMC from a healthy donor with successful clonal detection. Disease-bearing samples (either pre-treatment or after PTR) were sequenced to verify applicability of consensus primers for each patient and to determine each patient's unique clonal IGH sequence. Thirty-seven PBMC samples from 14 patients which were either negative (n=30) by ASO-PCR or detectable below the linear limit of detection (n=7) were subjected to MRD quantification by HTS. The integrity of all samples was determined by preliminary IGH quantitative PCR.

CLL-specific IGH clonotypes from all 14 patients amplified successfully from samples with known disease burden, confirming the acceptability of consensus primers for all patients. Concordant MRD negativity by ASO-PCR and IGH-HTS was only observed in 14/37 samples (38%), while 16 samples (43%) were negative by ASO-PCR but detectable at the 10e-6 level using IGH-HTS (range 0.1–11 CLL IGH sequences per 10e6 PBMC genomes). Two of 37 samples (5%) exhibited concordant low-level positivity in the 10e-5 range. 4/37 samples (11%) were concordantly positive, but quantified more than or equal to 0.5log higher with ASO-PCR than HTS. One sample was positive below the linear limit of detection by ASO-PCR but negative by HTS. With median clinical follow-up of 1072 days (range 522–1986 days), one of 7 patients (14%) who exhibited MRD negativity by both ASO-PCR and IGH-HTS relapsed. All 5 patients found to have MRD negativity by ASO-PCR with concurrent MRD positivity using IGH-HTS relapsed. The association between IGH-HTS negativity and long-term DFS was highly significant (p=0.005), whereas ASO-PCR negativity was not significantly associated with DFS (p=0.47). In patients found to be MRD negative by ASO-PCR but positive by IGH-HTS, the HTS result predicted clinical relapse by a median 321 days (range 38–644 days).

Massively parallel immunoglobulin gene sequencing using Illumina HiSeq provides a heretofore unachievable level of MRD sensitivity in peripheral blood samples from patients with CLL. Samples found to be negative or below the linear limits of detection for CLL MRD using ASO-PCR were more accurately quantified using IGH-HTS. Quantification of CLL MRD using IGH-HTS has tremendous prognostic value since achievement of MRD negativity with 10e-6 sensitivity is highly associated with long-term DFS. To further validate the performance of Illumina-based HTS, we are currently sequencing 289 archived post-transplant PBMC from 42 CLL patients. This scalable and cost-effective platform for ultra-sensitive MRD quantification using consensus primers will broadly expand the availability and utility of post-transplant MRD assessment.

Faham:Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Zheng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.