Detection of MRD Using IGH Assay By NGS after Second Cycle of Chemotherapy in Adult B-Lymphoblastic Leukemia Predicts Poor Clinical Outcomes: Results from a Multicenter Study
Authors: Polprasert Chantana , Chanswangphuwana Chantiya , Wudhikarn Kitsada , Limvorapitak Wasithep , Rattanathammethee Thanawat , Piyajaroenkij Thanakrit , Kobbuaklee Sirorat , Tantiworawit Adisak 
Published: December 2024

Introduction: B-lymphoblastic leukemia (B-ALL) is an aggressive hematologic malignancy. Chemotherapy has been the mainstay treatment for B-ALL, however, approximately 30% of adult B-ALL relapse after the treatment. Measurable residual disease (MRD) after intensive chemotherapy is one of predictors of disease relapse and inferior survival therefore being integrated into treatment scheme of B-ALL to individualize treatment for patients with B-ALL. Determining MRD status can be done by various methods. Multicolor flow cytometry has been widely integrated into clinical practice for MRD detection, however, there are certain technical limitations and variable sensitivity. Next-generation sequencing (NGS) detecting clonal rearrangement of immunoglobulin (Ig) gene has been an increasingly used technique with high sensitivity and unequivocal interpretation. However, an appropriate timepoint of Ig gene sequencing for MRD detection to correlate with clinical outcomes is not yet well validated. We aim to evaluate the role of MRD monitoring after intensive chemotherapy in adult B-ALL patients using Ig sequencing and its correlation with treatment outcomes.
Methods: Newly diagnosed adult (age&gt;15 years old) B-ALL patients from 4 university hospitals in Thailand between 2019 to 2023 were included (King Chulalongkorn Memorial Hospital, Thammasat university, Chiang Mai university and Ramathibodi hospital). DNA was extracted from bone marrow at the diagnosis, after the first and second cycle of chemotherapy (MRD1 and MRD2) and examined using NGS to analyze clonal rearrangement of Ig heavy chain (IGH) using LymphoTrack® IGHFR1 Assay (Invivoscribe) with IONS5 sequencer. Those that were negative for IGH clonality were further sequenced for clonal rearrangement of Immunoglobulin kappa light chain (IGK).
Results: A total of 25 B-ALL patients were sequenced for clonality by IGH assay. IGH clonality was demonstrated at diagnosis in 22 patients (88%) which were subsequently examined for IgH MRD analysis (9 male and 13 female). The median age at diagnosis was 30.5 years old (15-66), with 11/22 (50%) age older than 30 years. Unfavorable karyotypes were observed in 8 patients including 6 cases (27%) with Philadelphia chromosome-positive. Sixty-eight percent (15/22) of the patients were categorized as a high-risk group. Chemotherapy regimens for induction were selected by local hematologists upon institutional preference. There were 11 patients who received adult ALL protocol (adult ALL and HyperCVAD regimen), while 11 patients received pediatric-inspired regimens. All patients with Philadelphia chromosomes-positive ALL received imatinib concomitantly with chemotherapy. All patients achieved morphological complete remission after the first and second cycle of chemotherapy. Eighteen and 17 cases were evaluated for MRD1 and MRD2 by IGH sequencing, respectively. Five patients (22.7%) underwent allogeneic stem cell transplantation. After a median follow-up of 12 months (range, 2-61 months), 11/22 (50%) had disease relapse. Patients with positive MRD by IGH sequencing after second cycle of chemotherapy were significantly correlated with relapse disease (n=11) or not achieved complete remission (n=1) (HR 9.5, 95%CI 1.1-81.4; p=0.04), while those with positive MRD after first cycle of chemotherapy were not associated with relapse disease (p=0.43).
Conclusions: MRD detection using IGH NGS analysis can be applied for post-treatment response assessment of patients with B-ALL. Detection of IgH clonality after second cycle of chemotherapy correlates with inferior survival in B-ALL. Therefore, those patients should be considered for subsequent treatments, such as allogeneic stem cell transplantation or novel monoclonal antibody.