Table 2 Comparison among techniques used in clonality assessment by IG rearrangements
From: Next-generation sequencing for MRD monitoring in B-lineage malignancies: from bench to bedside
Techniques | ASO-RQ-PCR | BIOMED-2 Strategy | NGS |
|---|---|---|---|
Samples | DNA, including low-quality DNA from small biopsies and FFPE tissues (low amplification efficacy) | DNA, high-quality DNA is required at diagnosis | |
Mechanism | Consensus primers are used to sequence and design precise primers for specific amplification of rearranged fragments | Multiplex PCR and capillary electrophoresis (GeneScan), 97 new primers | Obtain all information of the IG rearrangements then compare the results with germline sequences |
Sensitivity | 1 × 10–4 ~ 10–5 | 1 × 10–3 | 1 × 10–6 |
Clonal evolution | Cannot be detected | Present or absent | Sequences can be detected |
Standardisation | Poor-standardised | Well-standardised | Well-standardised |
Specific request | Design patient-specific primers by sequencing the junction region of first PCR products | Analysis of PCR products: monoclonal (1–2 peak) polyclonal (Gaussian distribution) | Higher DNA input is needed for higher sensitivity |
Advantages | Relatively high sensitivity | Wider application range | Higher sensitivity and resolving power |
Fewer technical requirements | Good reproducibility | More convenient and rapid operation: synchronized detection, serial monitoring during follow-up | |
Short turn-around time | High accuracy | Relatively objective interpretation | |
Economical and affordable | Low DNA requirements | EuroClonality-NGS working group | |
Suitable for MRD monitoring | Already commercialised and instituted in most laboratories | Better identification of bi-allelic rearrangements and oligoclonality | |
Multiple target genes increase the accuracy | Recommended as standard method for clonality assessment in lymphoproliferative diseases | Bioinformatic identification and correction | |
Established guidelines for the analysis of RQ-PCR data | Monitor MRD status using peripheral blood | ||
Disadvantages | Pseudo-clonality (false-positive) and oligoclonality (weak clonal products) due to non-specific amplification and insufficient discernibility | Â | On-going optimisation of techniques |
Mismatches (false-negative) due to SHM | Â | Criterions for statistics analysis are not consistent | |
Time-consuming and labour-intensive | Unsuitable for MRD monitoring | Applied in limited laboratories | |
High standards of experimental condition | Separate PCR products by the lengths but not the sequences | High requirements for DNA input quality at diagnosis | |
Lack of sufficient diagnostic materials which may influencing the standard curve | Separate PCR products by the lengths but not the sequences | Well-functioning networks and collaboration between centres are needed | |
Not suitable when clonal evolution or a secondary malignancy occurs, or tumours originate from immature cells | Cumbersome operations due to the multi-step approach | A large-scale validation study is needed | |
Do not harbour correction mechanisms | |||