Principles of standard and next-generation technologies in MRD monitoring
| Technology . | References . | Potential advantages and open issues . |
|---|---|---|
| RQ-PCR | 1,35 | The most validated and standardized quantitative method for MRD detection in FL. Despite remarkable sensitivity and specificity, RQ-PCR has notable limitations due to the lack of BCL2/IGH target in about 40% of advanced FL and 65% to 70% of localized FL, currently not eligible for MRD assessment, and to the challenge of very low MRD levels, where it is difficult to dissect if the signal observed by PCR (not quantifiable) is due to few residual leukemic cells or to a nonspecific amplification of normal DNA. |
| NGS | 34,36 | Potential deeper sensitivity compared with the classic methods. A sensitivity of 10−6 is achievable only when high amounts of DNA are used. Broad spectrum of identifiable molecular targets: a “capture-based” protocol covering the coding V, D, J genes of the IGH loci was capable of detecting clonal rearrangements in 87% (21/24) of lymphoproliferative disorders. |
| TLA | 37,38 | Alternative NGS tool capable of sequencing structural variants, usually not detected by conventional PCR approaches, thanks to the selective amplification and sequencing of entire genes on the basis of the cross-linking of physically proximal DNA loci. |
| ddPCR | 39,40 | A third-generation quantitative method based on the partition of the template DNA into water-in-oil droplets in which PCR amplification occurs, allowing the quantification of nucleic acid targets without the need of the calibration curve. Sensitivity, accuracy, and reproducibility at least comparable to that of RQ-PCR. A good performance in the MRD quantification in at least 20% to 30% of samples resulting positive but not quantifiable by RQ-PCR. |
| cfDNA | 41-43 | Plasma is a potentially important source of DNA (ie, cfDNA), potentially useful to identify distinct biological subtypes of lymphomas and to provide insights into the patterns of genomic evolution/resistance throughout treatment in all compartments, not only PB and BM. A noninvasive tool to monitor MRD in non-Hodgkin lymphoma through patient-specific IGH rearrangements, to identify individuals at an increased risk of relapse and to detect relapse before clinical evidence of disease. Most cfDNA originates from leukocytes, and only a small fraction (<10%) is tumor derived, known as ctDNA. ctDNA concentration varies among patients and differs according to the type, location, and stage of cancer, with some producing extremely low concentrations. Thus, it is unlikely that cfDNA will reach the sensitivity of MRD analysis on circulating cells in FL. Before broad clinical implementation, issues on preanalytical factors must be addressed in order to achieve consistent and reproducible results. |
| Technology . | References . | Potential advantages and open issues . |
|---|---|---|
| RQ-PCR | 1,35 | The most validated and standardized quantitative method for MRD detection in FL. Despite remarkable sensitivity and specificity, RQ-PCR has notable limitations due to the lack of BCL2/IGH target in about 40% of advanced FL and 65% to 70% of localized FL, currently not eligible for MRD assessment, and to the challenge of very low MRD levels, where it is difficult to dissect if the signal observed by PCR (not quantifiable) is due to few residual leukemic cells or to a nonspecific amplification of normal DNA. |
| NGS | 34,36 | Potential deeper sensitivity compared with the classic methods. A sensitivity of 10−6 is achievable only when high amounts of DNA are used. Broad spectrum of identifiable molecular targets: a “capture-based” protocol covering the coding V, D, J genes of the IGH loci was capable of detecting clonal rearrangements in 87% (21/24) of lymphoproliferative disorders. |
| TLA | 37,38 | Alternative NGS tool capable of sequencing structural variants, usually not detected by conventional PCR approaches, thanks to the selective amplification and sequencing of entire genes on the basis of the cross-linking of physically proximal DNA loci. |
| ddPCR | 39,40 | A third-generation quantitative method based on the partition of the template DNA into water-in-oil droplets in which PCR amplification occurs, allowing the quantification of nucleic acid targets without the need of the calibration curve. Sensitivity, accuracy, and reproducibility at least comparable to that of RQ-PCR. A good performance in the MRD quantification in at least 20% to 30% of samples resulting positive but not quantifiable by RQ-PCR. |
| cfDNA | 41-43 | Plasma is a potentially important source of DNA (ie, cfDNA), potentially useful to identify distinct biological subtypes of lymphomas and to provide insights into the patterns of genomic evolution/resistance throughout treatment in all compartments, not only PB and BM. A noninvasive tool to monitor MRD in non-Hodgkin lymphoma through patient-specific IGH rearrangements, to identify individuals at an increased risk of relapse and to detect relapse before clinical evidence of disease. Most cfDNA originates from leukocytes, and only a small fraction (<10%) is tumor derived, known as ctDNA. ctDNA concentration varies among patients and differs according to the type, location, and stage of cancer, with some producing extremely low concentrations. Thus, it is unlikely that cfDNA will reach the sensitivity of MRD analysis on circulating cells in FL. Before broad clinical implementation, issues on preanalytical factors must be addressed in order to achieve consistent and reproducible results. |
A major standardization effort is under way within the EuroClonality (https://www.euroclonalityngs.org/usr/pub/pub.php) and EuroMRD Consortium (www.euromrd.org), to establish guidelines for NGS and ddPCR MRD analysis and their future application in standard clinical practice.
cfDNA, cell-free DNA; NGS, next-generation sequencing; TLA, target locus amplification.