P652: MEASURING MINIMAL RESIDUAL DISEASE BEYOND 10-4 THROUGH IGHV LEADER-BASED NEXT GENERATION SEQUENCING IMPROVES PROGNOSTIC STRATIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract

Background: The sensitivity of conventional techniques for reliable quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between CLL patients with durable remission and those at risk of early relapse. Aims: To develop an academically sourced IGHV leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. Methods: The IGHV leader primer set developed by the EuroClonality-NGS working group was used to amplify all IGH rearrangements present in an end of treatment (EOT) DNA pool. The IGH clonal target was amplified in a single-step PCR and subsequent sequencing was performed on the Illumina MiSeq platform. To calculate MRD depth, NGS output was annotated through in the ARResT/Interrogate immunoprofiler. Technical validation of the IGHV leader-based assay was performed on contrived MRD samples, created by serial dilution of pretreatment CLL DNA pooled PBMC DNA from healthy donors. Clinical validation of the IGHV leader-based assay was performed on EOT samples from the CLL11 trial (NCT01010061). Results: In 176/183 (96%) measurements on contrived samples, the CLL-specific rearrangement was detected. The limit of detection and limit of quantitation were estimated at 3.4 [95% CI 1.9-16.0] and 3.8 [95% CI 1.7-8.2] malignant cells per assay, respectivelly. The limit of blank was found to be 0. Linearity was established in the MRD 10-2-10-5 range (r=0.94 [95%CI 0.91-0.96]). Of note, when provided with sufficient DNA input, MRD could even be detected down to MRD 10-6. There was high inter-assay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative PCR (r=0.92, [95%CI 0.86-0.96]) and droplet digital PCR (r=0.93, [95%CI 0.88-0.96]). In a cohort of 67 patients from the CLL11 trial, using 5μg DNA input and MRD 10-5 as a cut-off, undetectable MRD (uMRD) was associated with superior progression-free survival (PFS) and time to next treatment. Importantly, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10-5. Patients with MRD in this range had a significantly shorter PFS, compared to patients with uMRD <10-5, but significantly longer, compared to patients with MRD ≥10-4 (median PFS: ≥10-4, 10.4 months; <10-4 but ≥10-5, 27.5 months; uMRD <10-5, NR, 4-year PFS rate: ≥10-4, 66.7%; <10-4 but ≥10-5, 25.0%; uMRD <10-5, 7.2%, P<0.001) (Figure 1). Image:Summary/Conclusion: We here present an academically-developed, IGHV leader-based NGS assay for the detection and quantification of MRD in CLL beyond MRD 10-4. The assay has high sensitivity and is quantitative and linear up to MRD 10-5 when using 5μg DNA input. Measurement to MRD 10-6 is feasible, conditional on sufficient DNA input. The deeper MRD measurements enabled by the IGHV leader-based NGS assay resulted in improved stratification of CLL patients following treatment.