Universal Minimal Residual Disease Quantification Using Consensus Primers and High-Throughput Igh Sequencing Predicts Post-Transplant Cll Relapse Better Than Patient-Specific PCR

Methods for broadening the availability of minimal residual disease (MRD) quantification in chronic lymphocytic leukemia (CLL) are needed to improve patient management. Here, we report the performance of consensus primed PCR to amplify immunoglobulin heavy chain (IGH) loci from polyclonal B cells in peripheral blood, followed by high-throughput sequencing (IGH-HTS) to ascertain CLL burden in patients treated with reduced intensity allogeneic hematopoietic cell transplant (HCT). We achieved 10e-6 MRD sensitivity and found this approach provides better prediction of relapse than allele-specific quantitative PCR (ASO-PCR). We amplified IGH loci from genomic DNA extracted from PBMC (median input 1.4x10e6 cells; range 0.2-23.7x10e6) using consensus V and J segment primers. IGH molecules were sequenced with one million or more dedicated reads using Illumina HiSeq and clonotypes were quantified with Sequenta HTS bioinformatics. 289 samples collected prospectively from 42 patients at 10 post-HCT time points (range 0-1090 days) were analyzed. CLL-specific IGH clonotypes were identified for 37 patients either by Sanger sequencing performed for pre-transplant prognostication, or by IGH-HTS using disease bearing samples. Five patients did not have samples with sufficient disease burden to identify the CLL clone and have been excluded from analysis. 24 patients were assessed by ASO-PCR and IGH-HTS, 13 by IGH-HTS alone. We observed no failures of the IGH-HTS technique to recover CLL-specific IGH clones, even in patients with somatically mutated CLL. With median follow-up of 927 days (range 297-2265 days), 18 patients (49%) relapsed and 7 (19%) died. Of 17 patients positive for MRD by IGH-HTS between 270 to 365 days post-HCT, 100% relapsed, whereas 1 of 12 MRD negative patients (8.3%) relapsed (p = 0.0001). In contrast, ASO-PCR negativity during this time frame was not associated with sustained remission (p = 0.21). Owing to its higher sensitivity, the median time to MRD negativity was longer with IGH-HTS compared with ASO-PCR (365 versus 270 days). 80% of patients who achieved ASO-PCR negativity at any time, but who never subsequently achieved negativity by IGH-HTS, relapsed. IGH-HTS provides heretofore unachievable MRD sensitivity for patients with CLL. The highly prognostic value of achieving MRD negativity with 10e-6 sensitivity within one year post-HCT may provide a benchmark for treatment success and a basis for further therapy in patients failing to achieve this status.