In Search of Missed Tumors: Next-Generation Sequencing for Minimal Residual Disease Detection in Multiple Myeloma Comes of Age

Multiple myeloma (MM) is an incurable plasma cell malignancy that resides in the bone marrow. Despite significant advances in treatment and prognosis, most patients eventually relapse, presumably because of undetected residual disease that in due course gives rise to a new clonal wave.¹  Achieving complete response (CR), as assessed clinically merely by means of blood tests and a bone marrow biopsy, does not preclude relapse or death.²  Instead, a molecular or immunophenotypic CR that identifies residual tumor at increased depth, compared to standard clinical assessment, is a better predictor of disease outcome.³  Naturally, the accuracy of those predictions is a function of technological limitations and assay design, and to some degree, spatial heterogeneity and stochastic sampling.⁴  The principle, however, is quite simple: The more sensitive the method, the more reliable the predictions.

Minimal residual disease (MRD) in MM is currently assessed by multicolor flow cytometry, allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), and next-generation sequencing (NGS) of bone marrow samples.⁵  Eight-color flow cytometry and ASO-PCR both have a sensitivity of 10^–5 (allowing for detection of one tumor cell in a background of 100,000 normal cells), and have been shown to be significant predictors of progression-free survival (PFS) and overall survival (OS).^6-8  On the other hand, clonotype analysis by VDJ sequencing has an increased sensitivity of 10^–6, allowing for more accurate measurement of residual disease.⁹  Of note, following significant optimization, so-called next-generation eight-color flow cytometry (NGF) achieved comparable sensitivity.¹⁰ 

NGS has significantly improved our understanding of cancer biology and informed cancer taxonomy, prognosis, and treatment. Moreover, as sequencing costs have dropped dramatically, NGS is now also increasingly used as a clinical detection tool.¹¹  Examples of this include the Dana-Farber Cancer Institute's (DFCI) targeted NGS assay, OncoPanel, and the U.S. Food and Drug Administration (FDA)–approved Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-Impact).^12,13  Although capture-based deep targeted sequencing assays like those can be useful for the characterization of the mutational landscape of certain cancers, MM exhibits significant genetic heterogeneity that effectively prohibits one-size-fits-all approaches for residual disease detection, presumably due to a high false-negative rate. In fact, the most common mutations only occur in approximately 20 to 25 percent of MM patients and at variable cancer cell fractions at that.¹⁴  Yet every single cell of all MM patients carries a particular VDJ rearrangement that can serve as the tumor’s molecular signature. Although lymphoid cancers of more immature lineage, such as acute lymphoblastic leukemia, can undergo clonotypic evolution, resulting in multiple clonal VDJ rearrangements in a single tumor, MM plasma cells are mature and almost always exhibit clonotypic monotony.¹⁵ 

The recently FDA-approved clonoSEQ assay takes advantage of the uniform presence of single VDJ rearrangements across all cells of an individual tumor to detect MRD in patients with MM.¹⁶  More specifically, the IgH locus of diagnostic samples is amplified in a multiplex fashion using consensus primers followed by targeted sequencing and clonotype calling; the clonotype can then be tracked by NGS.⁹  Although the method is simple in its perception, IgH locus amplification through multiplex PCR represents a practical challenge due to significant sequence homology in the region and variable primer specificity and kinetics. To address that issue, clonoSEQ uses proprietary chemistry based on a synthetic IgH receptor library which allowed for experimental and computational optimization of the multiplex PCR assay.¹⁷  These technological advances led to a sensitivity lower than 10^–6, which made clonoSEQ an important tool for MRD detection in B-cell malignancies including MM.^9,18  Sensitive MRD detection is likely to not only provide better survival estimates for patients, but also redefine the risk factor landscape in MM and help distinguish variables that truly reflect aggressive biology.

A recent study by Dr. Aurore Perrot and colleagues in patients from the Intergroupe Francophone Du Myelome (IFM)/DFCI cohort confirmed that MRD negativity, as assessed by clonoSEQ, is a major prognostic factor in MM.¹⁹  Notably, despite a significantly higher rate of MRD negativity in patients who underwent transplantation, compared to those who were treated with RVD (lenalidomide, bortezomib, dexamethasone) alone, MRD-negative patients had similar outcomes (PFS/OS) irrespective of treatment arm.¹⁹  Additionally, patient outcome was independent of cytogenetics and International Staging System (ISS) stage.¹⁹  These results seem to suggest that in patients who achieve MRD-negative status by clonoSEQ, the effect of transplantation as well as the prognostic significance of cytogenetics and ISS stage, which have long been considered important outcome predictors, should be investigated anew. It should be mentioned though that statistical significance is largely a function of sample and effect size and there is a clear trend in favor of transplantation, standard-risk cytogenetics, and ISS Stage I, even in patients who achieved MRD negative status (Figure).¹⁹  In fact, only three of 28 patients with del(17p) (11%) achieved MRD negativity.¹⁹  As always, larger cohorts with longer follow up are needed for definitive conclusions.

Despite its increased sensitivity, clonoSEQ can still miss residual tumor in patients.^18,19  Those who achieve MRD negative status by means of VDJ targeted sequencing can still relapse, leaving significant room for improvement of MRD detection methods. Nevertheless, MRD assessment by NGS in MM is now a confirmed, strong biomarker of PFS and OS. Its gradual incorporation into clinical trial design and clinical practice is poised to change MM patient management as we know it.