Next-generation sequencing (NGS) has been applied to monitor minimal residual disease (MRD) in multiple myeloma (MM). Standardized DNA input and sequencing depth is essential for achieving a uniform sensitivity in NGS-based MRD study. Herein, the sensitivity of 10 −5 was verified by a standardized experimental design based on triplicate measurements of 1 μg DNA input and 1 million sequencing reads using the LymphoTrack-MiSeq platform. MRD level was defined as the mean MRD burden of the triplicates. Two spike-in controls at concentrations of 0.001% tumor plasma cells (PC) for verifying the sensitivity of 10 −5 and 0.01% (or 0.005%) for MRD normalization were systematically analyzed. The spike-in control of 0.001% MRD was consistently detected in all samples, confirming a sensitivity of 10 −5 . Moreover, this standardized NGS approach yielded MRD measurements concordant with serological response and comparable to allele-specific oligonucleotide (ASO) real-time quantitative (RQ)-PCR. Moreover, NGS showed an improved sensitivity and provided quantification of MRD for cases assigned “positive but not quantifiable” (PNQ) by ASO RQ-PCR, even without the use of patient-specific probes/primers. Issues regarding the specificity of myeloma-specific sequences as MRD target, optimal input for spike-in normalization, and interpretation of MRD from triplicates are discussed. Herein, the standardized LymphoTrack-MiSeq-based method is verified to carry a sensitivity of 10 −5 , hence an effective tool for MRD monitoring in MM. As only a small number of samples are tested here, further study with a larger number of patients is warranted.
Despite the significantly higher complete remission rates and improved survival achieved in the last decade, multiple myeloma (MM) patients continue to relapse due to persistence of minimal residual disease (MRD). Generally, MRD refers to persistence of low levels of disease in the order of one tumour cell in ≥10 normal cells. Currently, molecular and immunophenotypic techniques are employed for MRD detection. This review focuses on MRD detection by molecular techniques, including allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), next-generation sequencing (NGS) and digital PCR (dPCR), in addition to a brief description of, and comparison with, multiparameter flow cytometry. The basic principles, technical advantages and limitations, and the clinical impact of all three molecular techniques are reviewed and compared. They all have a sensitivity of at least 10 , among which ASO real-time quantitative PCR is the most well-standardized, and NGS carries the highest sensitivity and applicability, while dPCR is still under investigation. Furthermore, molecular MRD negativity is a favourable prognostic factor for survival of patients with MM. However, several challenges inherent to molecular detection of MRD still remain to be overcome, particularly false negativity and failure to detect extramedullary disease. Finally, detection of MRD from peripheral blood remains challenging.
Allele-specific oligonucleotide real-time quantitative PCR (ASO-RQPCR) is a standardized technique for detection and monitoring of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) but not multiple myeloma (MM) due to a low applicability inherent with presence of somatic hypermutation. Herein, by a staged PCR approach and sequencing, clonality and tumor-specific complementarity-determining region 3 (CDR3) sequence were identified in 13/13 MM by sequential PCR of IgH VDJ (n = 10), IgH DJ (n = 2), or IgK VJ (n = 1). Using consensus primers/probes conventionally employed in ALL, ASO-RQPCR worked in three (23.1 %) cases only. Conversely, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in eight (61.5 %) cases with a sensitivity of 5 × 10−4–10−5. Moreover, using standard curves constructed by serial dilution of plasmids cloned with patient-specific CDR3, ASO-RQPCR was successful in 12 (92.3 %) cases with a sensitivity of 10−4–10−5, but not in a case lacking an N region, in which design of a tumor-specific ASO primer was precluded. Finally, in a patient in complete response (CR), further reduction of MRD after autologous stem cell transplantation (ASCT) was demonstrated. In summary, using entirely patient-specific primers/probes, ASO-RQPCR was applicable in >90 % MM patients and enabled detection of dynamic changes of MRD before and after ASCT despite conventional CR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0336-4) contains supplementary material, which is available to authorized users.
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