Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of plasma cells in the bone marrow. Advances in treatment, including proteasome inhibitors, immunomodulatory drugs, and autologous stem cell transplantation (ASCT), have improved progression-free survival (PFS) and overall survival (OS). However, relapse occurs due to minimal residual disease (MRD), undetectable by conventional methods. Highly sensitive techniques are needed to detect MRD and predict patient outcomes. Two main approaches exist: immunophenotyping (multiparametric flow cytometry, MFC) and molecular techniques (next-generation sequencing – NGS, digital PCR). MFC, particularly the EuroFlow NGF methodology, offers high sensitivity (2 × 10⁻⁶) but requires expertise and high-quality samples. Molecular methods, such as NGS, offer advantages in terms of sensitivity and quantification but have limitations in terms of cost and availability. This study aimed to validate a commercial NGS panel (LymphoTrac®) for MRD detection in MM and compare its performance with NGF.

The literature review section extensively discusses the limitations of conventional methods for detecting minimal residual disease (MRD) in multiple myeloma and the emergence of more sensitive methods like multiparametric flow cytometry (MFC) and next-generation sequencing (NGS). It highlights the development of standardized procedures for MRD assessment using MFC (EuroFlow) and NGS, emphasizing the high sensitivity achieved by these methods in detecting residual tumor cells. The review also covers the challenges associated with existing molecular methods like quantitative PCR, noting its limitations in terms of labor intensity, cost, and applicability to only a subset of myeloma patients. The need for further validation of newer NGS methods, like the LymphoTrac® panel used in this study, and the importance of comparing the performance of different MRD assessment strategies are underscored.

This study included 106 newly diagnosed, transplant-candidate MM patients from the Spanish GEM2012 clinical trial. Bone marrow samples were collected 100 days post-transplantation, and two independent pulls were processed for NGF and NGS analysis. NGS used the LymphoTrac® IGH panel to detect previously characterized clonotypic immunoglobulin heavy chain rearrangements. The process included PCR amplification, sequencing on a MiSeq platform, and analysis using the MRD Data Analysis Tool, Vidjil, and Arrest/Interrogate. NGF followed the EuroFlow guidelines using an eight-color workflow. MRD positivity was defined differently for each method (NGS: at least two identical clonotypic reads; NGF: at least 20 aberrant plasma cells). Statistical analysis included Fisher's exact test, Mann-Whitney test, Bland-Altman plots, Kaplan-Meier method, and Cox regression to compare PFS and OS between MRD-positive and -negative patients, considering various clinical parameters like cytogenetics and conventional response.

The study found a high correlation between NGS and NGF results (r = 0.951; R² = 0.905) despite some discordant cases. NGS applicability was affected by sample quality and concentration, with successful analysis in 89.6% of samples. MRD negativity (by both NGS and NGF) was associated with significantly better 3-year PFS and OS rates compared to MRD positivity (p<0.001 for both comparisons). Five patients with undetectable MRD by both methods experienced progression, all of whom achieved complete response (CR) at the time of MRD assessment, suggesting the possibility of occult MRD or other factors influencing progression. Multivariate Cox regression analysis confirmed that MRD negativity by NGF (HR: 0.20, p<0.001) and R-ISS I or II (HR: 0.37, p=0.01) were independently associated with better PFS, while MRD negativity by NGS (HR: 0.21, p=0.02) and R-ISS (HR: 0.13, p<0.001) were associated with better OS. No significant association was found between MRD status and other variables like conventional response or cytogenetics alone, except for CR/sCR which showed a clear association with MRD negativity.

The findings of this study reinforce the clinical significance of MRD assessment in multiple myeloma, demonstrating the prognostic value of MRD negativity regardless of the method used (NGS or NGF). The high correlation between NGS and NGF results validates the LymphoTrac® panel as a suitable alternative to NGF, despite limitations related to sample processing. The study confirms that MRD status is a powerful predictor of patient outcomes, surpassing other factors such as conventional response and cytogenetics in prognostic ability. While some patients with undetectable MRD progressed, this highlights the limitations of current MRD detection methods and the possibility of other factors influencing relapse. The consistent prognostic value of MRD across different methodologies underscores the importance of standardizing MRD assessment protocols and incorporating MRD status as a primary endpoint in clinical trials.

This study demonstrates the strong prognostic value of MRD assessment in multiple myeloma using both NGS and NGF, highlighting the importance of MRD negativity in predicting better PFS and OS. The high correlation between NGS and NGF validates the LymphoTrac® IGH panel as a potential alternative to NGF, though challenges related to sample quality remain. Future research should focus on optimizing sample processing for NGS and exploring the underlying mechanisms that drive progression in patients with undetectable MRD. Standardization of MRD assessment across different methodologies is crucial for improving patient care and guiding treatment decisions.

The study's limitations include the impact of sample processing on NGS applicability, leading to a smaller number of evaluable samples for NGS compared to NGF. The discordant cases between NGS and NGF require further investigation to understand the reasons for discrepancies. The study is limited to patients from a single clinical trial and might not be fully generalizable to other populations. Further studies with larger cohorts and direct comparison of MRD quantification under identical conditions are needed to comprehensively assess the relative strengths and weaknesses of NGS and NGF.