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Digital PCR
New techniques are on the horizon for the detection of small leukemic clones. A promising approach is based on digital PCR. Digital PCR is a breakthrough technology designed to provide absolute nucleic acid quantification. It is particularly useful in detecting low amounts of target; therefore, it is highly sensitive in detecting MRD. It is estimated that ddPCR can detect up to 0.001% mutated allele frequency.57
This technique can overcome some difficulties faced by conventional PCR. With ddPCR, a sample is partitioned in single nucleic acid molecules. As a result of the partitioning of the sample into some sort of “bubbles”, each bubble will contain zero or one molecule. After PCR amplification, nucleic acids may be quantified by counting the bubbles containing PCR end products. The main disadvantages for now are the cost of the analysis, the limited availability of the instruments that are not routinely introduced into diagnostic laboratories, and the lack of standardization.
In conventional PCR, the starting copy number is proportional to the number of PCR amplification cycles. Digital PCR, however, is not dependent on the number of amplification cycles to determine the initial sample amount, eliminating the reliance on uncertain exponential data to quantify target nucleic acids and providing absolute quantification.
Few studies have been published on MRD by ddPCR. We have already mentioned the study by Petrova et al35 based on IDH1/2 mutation. This technique has also been explored by Brambati et al34 in the setting of allogeneic bone marrow transplantation to identify the reappearance of small mutated clones of the recipient. In the same setting, Bill et al46 reported the prognostic significance of NPM1positivity by ddPCR. A new assay based on digital PCR technique composed of multiplex pools of insertion-specific primers that selectively detect mutated but not wild-type NPM1 has been described by Mencia-Trinchant et al.58
Next-generation sequencing
On the basis of the concept that MRD evaluation requires more than one technology and more than one marker, NGS is presently under investigation. Since there is a high degree of genotypic and phenotypic heterogeneity in AML, each patient should have a unique signature to be used to track MRD after therapy. Whole genome- or exome sequencing-based identification of clones and subclones in patients at diagnosis allows MRD to be followed by individualized monitoring. The usefulness of an NGS-based MRD assay is not only the prognostic stratification for relapse risk but also for the identification of different drugable targets for a personalized therapy.59 Prospective studies of the use of NGS in AML as a marker of MRD in the pre- and post-transplant setting are ongoing.
The main advantages of NGS technology are the reduced DNA sequencing time and cost, and the remarkably increased data-production capacity. NGS technologies rely on different methods for DNA template preparation, massively parallel reading of sequenced millions of short DNAs, real-time image capturing, alignment of sequences, sequence assembly, and variant detection. Each method has specific advantages: read length, accuracy, run time, and throughput. The main disadvantages of using NGS are shorter read length and the fact that its ability to detect MRD depends on the depth of sequencing and on the type of computational algorithms used. These aspects can complicate the process of standardization of the method for MRD detection.
Recently Onecha et al60 explored the possibility of using deep sequencing MRD approach. They analyzed 190 patients affected by AML. A total of 211 (80%) single nucleotide variations and 46 (20%) indels were detected using the NGS custom panel. They followed the aberrations during follow-up with the same approach and were able to demonstrate that MRD status (MRD levels >0.1%) at post-induction was associated with a significantly lower rate of OS (33% vs 78%), while MRD-positive status after induction chemotherapy (MRD levels >0.025%) was associated with shorter OS (33% vs 81%) and significantly shorter DFS.
Levis et al61 recently published a sensitive and specific MRD assay for FLT3-ITD mutations using NGS. They demonstrated a relationship between the mutation burden, as detected by their assay, and OS.
CONCLUSION
In acute leukemias, the detection of MRD is highly informative of the outcome of therapies, including HSCTs. The majority of the studies on MRD reported data based on MFC and real-time qPCR. Many attempts are ongoing to improve the sensitivity and to standardize the currently available techniques. This aspect is important to reach a common agreement on the threshold of MRD that triggers therapeutic decisions. New molecular targets are under investigation with encouraging results. Furthermore, the availability of new molecular targeted drugs that can potentially fully eradicate the residual small clones stimulates the interest in the detection of residual leukemic cells. New promising approaches are on the horizon to enlarge the spectrum of patients who can be monitored for the persistence of leukemic clones, including NGS.
Disclosure
The authors report no conflicts of interest in this work.
Giacomo Andreani, Daniela Cilloni
Department of Clinical and Biological Sciences, University of Turin, Turin, Italy
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