Comparison of Multiple Clinical Testing Modalities for Assessment of NPM1-Mutant AML

Abstract

BackgroundNPM1 mutation status can influence prognosis and management in AML. Accordingly, clinical testing (i.e., RT-PCR, NGS and IHC) for mutant NPM1 is increasing in order to detect residual disease in AML, alongside flow cytometry (FC). However, the relationship of the results from RT-PCR to traditional NGS, IHC and FC is not widely known among many practitioners. Herein, we aim to: i) describe the performance of RT-PCR compared to traditional NGS and IHC for the detection of mutant NPM1 in clinical practice, and also compare it to FC, and ii) provide our observations regarding the advantages and disadvantages of each approach in order to inform future clinical testing algorithms.MethodsPeripheral blood and bone marrow samples collected for clinical testing at variable time points during patient management were tested by quantitative, real-time, RT-PCR and results were compared to findings from a Myeloid NGS panel, mutant NPM1 IHC and FC.ResultsRT-PCR showed superior sensitivity compared to NGS, IHC and FC with the main challenge of NGS, IHC and FC being the ability to identify a low disease burden (<0.5% NCN by RT-PCR). Nevertheless, the positive predictive value of NGS, IHC and FC were each ≥ 80% indicating that positive results by those assays are typically associated with RT-PCR positivity. IHC, unlike bulk methods (RT-PCR, NGS and FC), is able provide information regarding cellular/architectural context of disease in biopsies. FC did not identify any NPM1-mutated residual disease not already detected by RT-PCR, NGS or IHC.ConclusionOverall, our findings demonstrate that RT-PCR shows superior sensitivity compared to a traditional Myeloid NGS, suggesting the need for “deep-sequencing” NGS panels for NGS-based monitoring of residual disease in NPM1-mutant AML. IHC provides complementary cytomorphologic information to RT-PCR. Lastly, FC may not be necessary in the setting of post-therapy follow up for NPM1-mutated AML. Together, these findings can help inform future clinical testing algorithms.

Detection of Hybrid Fusion Transcripts, Aberrant Transcript Expression, and Specific Single Nucleotide Variants in Acute Leukemia and Myeloid Disorders with Recurrent Gene Rearrangements

“…NPM1 mutations correspond to various types of frameshift insertion/deletion mutations in the c-terminus [54]. This NGS assay could detect NPM1 mutations (regardless of type) at the same time as assessing gene fusion status; indeed, since NPM1-mutated AML cases often have normal karyotype, the NPM1 variant data can inform AML classification and prompt the appropriate mutationspecific qRT-PCR required to monitor NPM1 MRD [55]. The limitations of this RNA NGS assay include the analytical sensitivity (i.e., limit of detection), which necessitates separate assays (e.g., BCR::ABL qRT-PCR, CBFB::MYH11 qRT-PCR, NPM1 qRT-PCR) for MRD testing; this is similar to the conclusion of another group using a similar platform [26].…”

“…NPM1 mutations correspond to various types of frameshift insertion/deletion mutations in the c-terminus [54]. This NGS assay could detect NPM1 mutations (regardless of type) at the same time as assessing gene fusion status; indeed, since NPM1 -mutated AML cases often have normal karyotype, the NPM1 variant data can inform AML classification and prompt the appropriate mutation-specific qRT-PCR required to monitor NPM1 MRD [55].…”

Detecting the Neuraminidase R294K Mutation in Avian Influenza A (H7N9) Virus Using Reverse Transcription Droplet Digital PCR Method

“…Meanwhile, there are still some limitations to the RT-dd PCR method developed in this study. Compared with the advantages of NGS in detecting various types of mutations (including new or unknown drug-resistant mutations) [23,24], RT-dd PCR can only detect specific known mutation sites and cannot discover new or rare mutations [25,26]. In addition, RT-dd PCR also requires larger sample sizes and higher mutation frequency to achieve more reliable results [27].…”

Managing leukemia patients via liquid biopsy and super rolling circle amplification (superRCA)

“…The assays can be applied in subsets of patients carrying NPM1 mutations or fusion genes, including PML/RARA, RUNX1-RUNX1T1, and CBFB-MYH11 [18,27,28]. The technique can detect single nucleotide variants at frequencies down to around 1/100, whereas prominent sequence changes such as insertion and deletions may be detected at proportions of 1/10,000 [29], sometimes allowing peripheral blood to be used as a source for MRD testing. Although very well established as an MRD method, RT-qPCR is mostly done on cDNA and therefore requires the isolation of RNA and the use of external standard curves.…”