Advancing MRD Detection in Multiple Myeloma

Abstract

Multiple myeloma (MM) is a malignant hematologic tumor of plasma cells that presents significant challenges in treatment and management. Despite the advent of novel therapies in recent years, which have improved patient outcomes, complete eradication of the disease remains an elusive goal. This underscores the critical need for in-depth research and ongoing innovation to tackle MM. Minimal residual disease (MRD) detection has emerged as a vital tool for evaluating treatment efficacy and predicting prognosis in MM patients, garnering extensive attention and application in recent years. This paper provides a comprehensive review of recent advancements in major MRD detection methods for MM patients, including multiparametric flow cytometry (MFC), allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR), and next-generation sequencing (NGS). It delves into the clinical applications of MRD detection, anticipates future developments, and offers valuable insights for improving diagnostic and therapeutic strategies. Through persistent research and innovation, we hope to bring better therapeutic prospects to MM patients.

Multiple myeloma (MM) primarily originates from the abnormal proliferation of plasma cells in the bone marrow, leading to malignant hematologic changes. The hallmark of this disease is the clonal expansion of plasma cells in the bone marrow, often accompanied by the production of monoclonal immunoglobulins (M proteins).¹ Over the past decade, the introduction of novel therapies has significantly improved the life expectancy of MM patients. With modern multimodal therapeutic strategies, more than 70% of MM patients achieve complete remission (CR) during frontline treatment.² However, even among those achieving CR, some patients remain minimal residual disease (MRD) positive, which increases the risk of early relapse. Compared with patients with sustained MRD negativity, those who achieve CR but are MRD-positive experience significantly shorter progression-free survival (PFS) and overall survival (OS).^3,4 MRD detection enables the identification of residual tumor cells at a microscopic level, providing a scientific basis for implementing more precise treatment strategies, making it highly significant in the management of MM.⁵ In 2016, the International Myeloma Working Group introduced an innovative recommendation to incorporate MRD monitoring into the evaluation of treatment efficacy for MM. This initiative not only deepened the understanding of disease status but also established a consensus standard for reporting MRD status.⁶ Advanced detection technologies are essential to identify disease states beyond the traditionally defined CR. The primary techniques for detecting MRD in MM patients include multiparametric flow cytometry (MFC), allele-specific oligonucleotide real-time quantitative PCR (ASO-qPCR), and next-generation sequencing (NGS).^7,8 This paper explores these advanced detection technologies in detail, addressing the technical and clinical challenges of MRD detection while outlining future research directions. With the continued advancement and standardization of detection methods, MRD detection is poised to play an increasingly pivotal role in the treatment and management of MM.

MRD DETECTION METHODS

Multiparametric Flow Cytometry (MFC)

Flow cytometry is a highly sensitive detection technology widely used for monitoring MRD in MM patients. This technique leverages fluorescently labeled monoclonal antibodies that bind to specific surface and intracellular markers, enabling complex multiparametric analyses to precisely identify and quantify abnormal plasma cells in the bone marrow. In recent years, advancements in technology, particularly the use of 8 to 10-color flow cytometry, have significantly enhanced the accuracy of MRD detection. Traditional 4 to 6-color flow cytometry has certain limitations in distinguishing normal plasma cells from malignant plasma cells. In contrast, 8 to 10-color flow cytometry, by incorporating more markers, improves differentiation and achieves a sensitivity of 10⁻⁴ to 10⁻⁵. In 2016, clinical guidelines for MRD detection in MM patients recommended an 8-color approach. This method can accurately detect the core markers of abnormal plasma cells, including CD38, CD138, CD45, and CD19 negativity, as well as identify the expression of CD5 and cytoplasmic kappa (κ) or lambda (λ) immunoglobulin light chains.^9–11

In 2017, researchers compared MRD detection using next-generation flow (NGF) and conventional 8-color flow cytometry. NGF identified an MRD positivity rate of 47%, significantly higher than the 34% detected by traditional 8-color flow cytometry. Moreover, 25% of patients who tested MRD-negative using conventional MFC were found to be MRD-positive with NGF, highlighting NGF’s superior sensitivity in overcoming the limitations of traditional MFC methods.¹²

MFC provides real-time MRD detection results, enabling clinicians to rapidly adjust treatment plans based on these findings. Regular MRD testing during maintenance therapy facilitates the monitoring of disease progression, early identification of potential relapse, and timely interventions. Frequent MRD assessments allow for earlier relapse detection, optimizing treatment strategies accordingly.¹³

Allele-specific Oligonucleotide Real-time Quantitative PCR (ASO-qPCR)

ASO-qPCR is a highly sensitive molecular biology technique used to detect specific gene mutations or polymorphisms. This method distinguishes subtle differences between alleles by using allele-specific oligonucleotide primers (ASO primers) to target and amplify specific DNA sequences. ASO-qPCR can detect extremely low levels of residual disease, achieving a sensitivity of 10⁻⁵ to 10⁻⁶. This high sensitivity makes ASO-qPCR particularly advantageous for early detection and monitoring of residual disease.^4,14

Through MRD detection, ASO-qPCR aids in predicting relapse risk and provides a more accurate prognosis for patients. It is applicable in various treatment settings, including induction chemotherapy, consolidation therapy, and autologous or allogeneic stem cell transplantation, thereby improving long-term survival rates.^4,15

In MM patients, common genetic alterations include rearrangements of immunoglobulin heavy and light chain genes. By designing ASO primers specific to these rearranged sequences, MRD can be detected with high sensitivity.^16,17 However, ASO-qPCR has certain limitations. Initially standardized for MRD detection in acute lymphoblastic leukemia (ALL) rather than MM, it faces challenges in MM due to high somatic hypermutation rates. These challenges limit the applicability of ASO-RQ-PCR for detecting myeloma cells.¹⁷ Achieving the high sensitivity and utility of ASO-qPCR for MRD detection requires repeated standard curve establishment, PCR cloning, and mismatch analysis.¹⁷

Next-generation Sequencing (NGS)

NGS is a high-throughput sequencing technology capable of rapidly sequencing millions of DNA fragments simultaneously. It provides comprehensive insights into genome structure, genetic variations, gene expression profiles, and epigenetic modifications, delivering unmatched capabilities for analyzing DNA and RNA molecules in a highly efficient and cost-effective manner.^18,19 In addition to IgH rearrangements, multiple driver gene mutations, such as KRAS, NRAS, and BRAF, are frequently observed in MM patients.²⁰ NGS can analyze a patient’s genome to accurately detect these mutations and rearrangements, thereby identifying resistant subclonal cancer populations that may lead to disease relapse. It can also help pinpoint potential targets for personalized therapy.²¹

NGS offers a sensitivity of up to 10⁻⁶, meaning it can detect one residual cancer cell among one million cells.²² The development of NGS has largely replaced ASO-qPCR. Although ASO-qPCR is highly sensitive, its applicability is limited; only 50% to 60% of patients have patient-specific probes capable of reliably identifying IgH clones after somatic hypermutation, whereas NGS methods (non-patient-specific) achieve applicability rates of 90% to 92%.^15,23 This underscores the broader applicability of NGS, eliminating the laborious need to develop patient-specific primers. Studies comparing the sensitivity of NGS and NGF show similar results at a sensitivity of 10⁻⁵, with both detecting MRD negativity in over 90% of CR patients. Differences in detection results become apparent only at a sensitivity of 10⁻⁶.^24,25 An MRD-negative status detected by NGS indicates a deeper remission, which is a critical prognostic indicator for patients who have already achieved CR.

Imaging Methods for MRD Detection

Whole-body magnetic resonance imaging (WB MRI), computed tomography with 18F-fluorodeoxyglucose (18F-FDG-PET/CT), and positron-emission tomography are sensitive methods for identifying widespread tumor infiltration in the BM, extramedullary disease, and localized lesions.²⁶ In assessing treatment responses, multiple cohorts have been rigorously analyzed using these methods, demonstrating an association between negative results and favorable responses along with prolonged survival rates.^27–29 The recent guidelines from the IMWG established PET/CT as the reference tool for defining imaging and MRD negativity when combined with NGS or MFC.⁶ Imaging, by capturing the expansion of tumor cells at a whole-body level and distinguishing between active and inactive lesions, may be considered as an additional layer of minimal residual disease (MRD) interpretation.³⁰

A study by Zamangi and colleagues confirms that FDG-PET/CT after therapy serves as a reliable predictor of long-term outcomes in patients with NDTEMM. The findings of this study suggest that hepatic uptake should be adopted as a straightforward and reproducible reference for determining PET negativity post-therapy, applicable in both clinical trials and routine clinical practice. The agreement between CMR and MFC negativity was 0.76, specifically 0.64 for FLs and 0.92 for BM. Patients who attained both PET/CT complete metabolic response and minimal residual disease negativity at post-treatment monitoring exhibited significantly prolonged progression-free survival, with survival probabilities at 24 and 48 months being 95% and 81%, respectively, compared with 70% and 59% in other patients (HR 0.45, CI 0.23-0.88, P 0.020).³¹

APPLICATIONS OF MRD DETECTION IN MM TREATMENT

Prognostic Assessment

Extensive studies have demonstrated that MRD-negative status is significantly associated with longer PFS and OS in MM patients. Joaquin Martinez-Lopez and colleagues found that MM patients with MRD negativity detected by NGS had significantly higher 5-year progression-free survival rates compared with MRD-positive patients.³² Thoma and colleagues reported that achieving MRD negativity within 12 months reduces the risk of disease progression, and the impact of treatment on MRD correlates with its influence on PFS.³³ In addition, a large meta-analysis confirmed the positive role of MRD negativity in long-term survival outcomes for MM patients.³⁴

PET-positive lesions after therapy are significantly associated with poor prognosis. Several studies have indicated that patients achieving complete remission (CR) who exhibit FDG-PET/CT negativity after ASCT experience a reduced risk of progression or mortality compared with those with metabolically active disease sites.^35–41

Treatment Adjustment

MRD detection results serve as a critical basis for treatment adjustments. For MRD-positive patients, intensifying treatment through extended cycles or modified drug combinations may be necessary to eliminate residual cancer cells. Conversely, for MRD-negative patients, reducing treatment intensity could minimize side effects and improve quality of life.^32–34,42

MRD detection not only evaluates the effectiveness of current treatments but also serves as a tool for relapse monitoring. Regular MRD monitoring improves the success rate of therapy by enabling timely intervention at the early stages of disease recurrence.

Current Challenges and Future Prospects

Despite significant advancements in MRD detection technology, several challenges remain in its practical application. Advanced MRD detection methods such as NGS and NGF involve high costs for instruments, reagents, and consumables. Although NGS offers high specificity and enables the detection of multiple gene mutations and rearrangements simultaneously and provides comprehensive genomic information; its data analysis is complex, and the associated costs remain prohibitive for widespread use. NGF, although relatively more cost-effective, requires high technical expertise, complex downstream data analysis, and significant cellular input.^30,43

Another major challenge in MRD detection is the lack of standardized protocols across laboratories. Differences in methodologies, sample handling, and data interpretation can lead to inconsistent results, making it difficult to compare outcomes across studies and clinical settings.^30,44

Future advancements in MRD detection technologies, such as improved NGS sensitivity and the development of new methods like single-cell sequencing, hold the promise of overcoming existing limitations. These advances will enable more precise and comprehensive MRD evaluations. Large-scale, multicenter clinical studies will help establish unified standards and guidelines for MRD detection, promoting its global application. As technology matures and becomes more widely adopted, associated costs are expected to decrease. Economies of scale and innovations will make these methods more affordable, facilitating their integration into routine clinical practice.

Liquid biopsy is a noninvasive strategy for disease monitoring through the analysis of circulating tumor DNA (ctDNA) or circulating tumor plasma cells (CTCs). This approach is becoming a promising noninvasive tool, notably for monitoring response to treatment in lymphomas.^45–47 Moreover, the development of these blood-based MRD strategies is crucial to overcome pitfalls related to BM samples. Detecting ctDNA or CTCs is of particular relevance in MM, as both sources better represent the multifocal “patchy” nature of the disease rather than relying on aspiration at a single marrow site to reflect the complete cancer milieu, which is not realistic. In addition, recurrent and frequent sampling of peripheral blood is feasible and painless as opposed to BM. Although its clinical utility is still under investigation, new emergent data is becoming available in the context of plasma cell disorders (PCDs).⁴⁸ Overall, liquid biopsies may provide a dynamic and comprehensive picture of the genomic landscape in MM and, even more, a noninvasive approach to monitor tumor burden. However, these methods are still novel and demand further research, especially when comparing results with matched BM assessments.^49,50 Therefore, the implementation of liquid biopsies for MM requires validation and harmonization of the assays.

All mass spectrometry (MS) methods have a similar basis for detecting M-proteins: the unique sequence of the 3 CDRs of the Ig. Two main MS approaches have been described so far by the IMWG,⁵¹ both taking the enrichment of each patient’s Igs as the starting point but with different downstream detection and analysis of the target molecule. One of these methods divides the Ig into peptides specific to the CDR by enzymatic digestion (clonotypic peptide approach), whereas the other one chemically reduces and denatures Igs into heavy and light chains to measure the distribution of the LC mass (intact LC approach). Besides these 2 approaches, the first results of a third MS version termed ‘quantitative immunoprecipitation mass spectrometry’ (QIP-MS) have been recently presented.⁵² This assay enables the identification, quantification, and typing of complete and LC-only M-proteins at once. Preliminary results from the Spanish GEM group showed a strong association of MRD and a shorter survival detected by either QIP-MS or NGF, with a high correlation between techniques.⁵³ However, more studies are needed to discern the best MS method and its clinical implications.

CONCLUSION

MRD detection plays a pivotal role in the diagnosis and treatment of MM. Utilizing advanced detection technologies such as multiparametric flow cytometry, ASO-qPCR, and NGS significantly enhances the sensitivity and specificity of MRD detection, providing strong support for personalized treatment plans and early identification of relapse risks. However, challenges remain in the standardization of techniques, cost management, and clinical integration. Consistency and comparability of MRD detection results are undoubtedly affected by procedural and standard differences across laboratories and institutions. Furthermore, the high costs of equipment and reagents limit the widespread adoption of these advanced technologies, particularly in resource-limited settings. The role of MRD detection in the precision treatment of MM will continue to grow. Future research directions include optimizing existing technologies, exploring new biomarkers and algorithms, and promoting standardization and cost reduction. These efforts will further enhance the accuracy and accessibility of MRD detection, providing patients with more effective treatment options and personalized care strategies.

In summary, MRD detection has become an essential component of MM diagnosis and treatment. It not only provides scientific evidence for treatment planning but also helps adjust strategies in a timely manner to improve patients’ quality of life and extend survival. With advancements in medical technology and accumulation of clinical experience, the critical role of MRD detection in cancer therapy will become increasingly evident, offering better outcomes and long-term prognoses for every patient.