DNMT3A,   FLT3,  General AML,  TP53

MRD detection by NGS before allogeneic HCT in patients with acute myeloid leukemia

Current next-generation sequencing (NGS) based measurable residual disease (MRD) detection is characterized by a sequencing error of up to 1% at each nucleotide, which is a major problem as there is a challenge in differentiating true mutations with low variant allele frequency (VAF) from sequencing errors.

A group of German researchers adapted an error-corrected sequencing concept in a NGS-based approach for MRD assessment and evaluated its ability to predict relapse and survival post-allogeneic hematopoietic cell transplantation (HCT) in patients with acute myeloid leukemia (AML). This error-corrected NGS entails the use of NGS-grade polymerase chain reaction (PCR) primers with random barcodes for error correction, a proofreading polymerase, avoidance of barcode contamination in consecutive sequencing run, a standardized bioinformatics approach for error correction that can be automated. The results of the study were published in a September 2018 issue of Blood.

One-hundred and sixteen patients with AML who underwent allogeneic HCT in complete morphologic remission (CR) between 1996 and 2016 at the Hannover Medical School and have DNA samples available at diagnosis and in CR just before allogeneic HCT were included in this study. Patients underwent myeloid panel sequencing in order to identify a suitable MRD marker. Using this analysis, it was observed that at diagnosis, 7% (8/116) of patients did not have any applicable MRD markers and were thus excluded from the study. Another 12 patients were excluded from the analysis due to the presence of an ancestral clone of VAF > 5% in CR samples before allogeneic HCT. Thus, the analysis cohort consisted of 96 patients who had an evaluable MRD marker with a VAF of 5% or less at the time of CR before allogeneic HCT.

Genomic DNA obtained from bone marrow (n = 40) or peripheral blood (n = 56) was used to analyze for 1 (n = 66), 2 (n = 28), or 3 (n = 2) mutations per patient for MRD in the pre-transplant CR sample using NGS. MRD positivity (median VAF = 0.33, range 0.016–4.91%) was seen in 45% (43/95) of patients, while 55% (53/96) of patients were MRD negative.

Key findings:
  • Median follow-up in 96 evaluable patients: 6.2 years
  • Twenty-seven (63%) MRD positive patients relapsed after allogeneic HCT
  • Eight MRD negative patients relapsed after allogeneic HCT
  • Cumulative incidence of relapse (CIR) was significantly higher in MRD positive patients than MRD negative patients: 5-year CIR; 66% vs 17%, HR = 5.58 (95% CI, 0.51–2.37), P < 0.001
  • Non-relapse mortality (NRM) rate was not significantly different between MRD positive and negative patients: 5-year NRM: 9% vs 11%, HR = 0.61 (95% CI, 0.15–2.37), P = 0.47
  • Relapse-free survival (RFS) and overall survival (OS) were significantly better in MRD negative patients than MRD positive patients
    • 5-year RFS in MRD negative and positive patients: 74% vs 31%, HR = 3.56 (95% CI, 1.86–6.81)
    • 5-year OS in MRD negative and positive patients: 78% vs 41%, HR = 3.06 (95% CI, 1.53–6.12)
  • MRD positivity was found to be an independent predictor of CIR (HR = 5.67, P < 0.001) besides DNTM3A, NPM1 and FLT3-ITD mutation status
  • MRD positivity was found to be an independent predictor of OS (HR = 3.01, P = 0.004) besides conditioning regimen, KRAS, and TP53 mutation status
  • MRD positivity was found to be an independent predictor of RFS (HR = 3.41, P = 0.001) besides patient age, KRAS, and TP53 mutation status

In summary, NGS-MRD positivity is associated with a poor prognosis independently of other prognostic risk factors. The authors noted that their study is limited by the use of 1–3 MRD markers per patient rather than using the whole gene panel to monitor clonal evolution.

The researchers concluded that “sensitive NGS-based MRD is widely applicable to AML patients, is highly predictive of relapse and survival when measured in CR” before allogeneic HCT. In addition, it may help in “refining transplant and post-transplant management” in patients with AML.

References
  1. Thol F. et al. Measurable residual disease (MRD) monitoring by NGS before allogeneic hematopoietic cell transplantation in AML. Blood. 2018 Sep 6. DOI: 10.1182/blood-2018-02-829911. [Epub ahead of print].
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