Authors
Alicia Serrano-Alcalá, Laura Ventura-López, Azahara Fuentres-Trillo, Blanca Ferrer-Lores, Felipe Javier Chaves, María José Terol-Casterá.
Introduction
Measurable residual disease (MRD) is a key biomarker for assessing treatment response and prognosis in chronic lymphocytic leukemia (CLL). Flow cytometry (FC), the standard MRD method, offers rapid immunophenotyping with sensitivity around 10⁻⁴ and established prognostic value, especially for undetectable MRD (uMRD). However, its limited sensitivity and inability to track clonal heterogeneity restrict its use. Next-generation sequencing (NGS) of the immunoglobulin heavy chain variable region (IGHV) advances MRD detection by providing higher sensitivity (~10⁻⁶) without patient-specific primers. It also allows simultaneous determination of IGHV mutational status and detailed longitudinal clonal tracking within a polyclonal background. This study evaluates IGHV analysis by NGS for mutational status and dynamic MRD monitoring to improve prognostic accuracy and treatment guidance in CLL.
Patients and methods
A retrospective observational study included 58 untreated patients diagnosed with CLL/CLL-like monoclonal B-cell lymphocytosis (MBL) from 1999 to 2024. IGHV mutational status was determined by Sanger sequencing and compared to an automated method using shorter reads (Illumina 2 × 150 bp), and the subsequent analysis with in-house pipeline (https://github.com/afuentri/B347 MyRepCLL). Clonotype monitoring by NGS was performed on 127 sequential samples from 23 patients treated with chemoimmunotherapy (CIT; 40%) and 35 patients treated with ibrutinib (60%). Flow cytometry was conducted on peripheral blood after erythrocyte lysis using the ERIC 8-color panel following EuroFlow guidelines. Data acquisition was performed on a FACSCanto II (BD) and analyzed with Infinicyt™ (Cytognos). MRD was expressed as the fraction of CLL cells among nucleated cells, with negativity defined as < 1×10⁻⁴.
Results
The mean number of sequences analyzed per patient was comparable between groups—112,885 for CIT (range: 1,471–1,427,745) and 118,308 for ibrutinib (range: 1,000–585,659). The average dominant clonotype count was higher in CIT-treated patients (104,823; range: 0–1,427,019) than in those receiving ibrutinib (48,288; range: 0–488,194). V-gene mapping efficiency was lower in CIT (mean: 25.85%) compared to ibrutinib (mean: 80.46%).
In the CIT subgroup, the primary IGVH rearrangement was undetectable in 34% of samples (19/56). Among these, 100% (19/19) of patients achieved complete remission (CR). In contrast, 81.3% (13/16) of patients with an NGS IGVH ratio >10% relapsed (P < 0.001). In 5% of cases (3/56), the clonotype read ratio was < 1%, while in 29% (16/56) it remained >18%.
Among ibrutinib-treated patients, the initial clonotype was undetectable in only 3% of samples (2/80), both from the same individual. After excluding 28 samples (35%) with persistent lymphocytosis, 62% (50/80) of the remaining samples showed an NGS IGVH ratio >14%, with a mean ALC of 2.95 × 10⁹/L (range: 0.90–4.98).
A separate comparative analysis of MRD detection by NGS and flow cytometry (FC) was conducted in 42 CIT-treated patients (67 follow-up samples). At baseline, 81% had a single dominant clonotype, while 19% had dual IGVH rearrangements. The median number of sequences per sample was 148,381 (range: 1,897–1,845,191), with a mean dominant clonotype count of 111,219 and median V-gene mapping of 48.68%. NGS demonstrated >90% concordance with FC at the < 0.01% ( < 10⁻⁴) threshold, with two discordant cases in which NGS detected low-level MRD (0.4% and 0.9%) not identified by FC. Both cases converted to MRD-negative in subsequent samples. In samples with MRD between < 10⁻⁴ and 10⁻⁴, polyclonal patterns were observed (mean maximum inter-rearrangement difference: –1.25×).
In dual-rearranged cases, MRD was undetectable by FC in 11 of 16 samples. NGS showed full concordance (11/11) when tracking only the productive clonotype; non-productive rearrangements yielded detectable MRD down to 0.020%.
Conclusions
NGS-based MRD monitoring offers high sensitivity and strong clinical correlation in CLL. Undetectable clonotypes post-chemoimmunotherapy were associated with complete remission, while persistent clonotypes predicted relapse. In ibrutinib-treated patients, clonotypes often remained detectable, reflecting distinct clonal dynamics. NGS showed high concordance with flow cytometry, particularly when tracking productive rearrangements. These results support NGS as a reliable tool for MRD assessment and risk stratification in CLL.
Keywords : Next-Generation Sequencing (NGS), Immunoglobulin Heavy Chain (IGHV), Minimal Residual Disease
Please indicate how this research was funded. :
Please indicate the name of the funding organization.: Lymphoproliferative Syndrome Group, INCLIVA Biomedical Research Institute, Hematology Service, Valencia University Clinical Hospital.