Sequential RT-qPCR monitoring in prediction of relapse among Patients with NPM1 mutations

At present there is growing interest in whether prognostication can be improved through more extensive molecular profiling that capitalizes on advances in sequencing technology. Ivey et al., conducted molecular profiling and sequential monitoring of Minimal Residual Disease (MRD) in a large series of patients (n=346) with NPM1-mutated Acute Myeloid Leukemia (AML) who had undergone intensive treatment in the National Cancer Research Institute (NCRI) AML17 trial. Their results were published in the New England Journal of Medicine in February 2016.

Here are the key findings with reference to sequential monitoring

• Among 53 patients in morphologic remission, a rising NPM1-mutated transcript level in preceding samples on RT-qPCR reliably predicted the occurrence of a hematologic relapse (median increment, 0.7 log10 per month; range, 0.3 to 2.0)
• The analysis of the time from the first molecular positivity to relapse showed a longer time to relapse if MRD was first detected in bone marrow than if it was first detected in peripheral blood (median, 133 days vs. 87 days; P=0.65)
• Targeted sequencing at the time of molecular or hematologic relapse showed that the variant allele frequencies in some genes exceeded those in NPM1 in 34 of 49 patients (69%) who were tested
• Mutations in DNMT3A, TET2, and IDH2 were detected at low levels in remission samples that were used as controls for exome sequencing and that were negative for NPM1 mutations at a sensitivity of at least 1.0×10−4
• Regardless of mutations associated with preleukemic clones, RT-qPCR analysis of available relapse samples showed that NPM1 mutations were a stable marker of AML disease status and were detectable in 69 of 70 patients (99%) at the time of relapse

In conclusion, this study demonstrated that the presence of MRD in the bone marrow rather than in peripheral blood was a predictor of relapse in patients with NPM1 mutations. These findings also suggest that focusing more on MRD may be of benefit for informing decisions relating to stem cell transplantation.

The question of whether outcomes might be improved by more rapid deployment of transplantation is being studied in the ongoing NCRI AML 19 trial (Current Controlled Trials number, ISRCTN78449203).

Abstract

Background

Despite the molecular heterogeneity of standard-risk acute myeloid leukemia (AML), treatment decisions are based on a limited number of molecular genetic markers and morphology-based assessment of remission. Sensitive detection of a leukemia-specific marker (e.g., a mutation in the gene encoding nucleophosmin [NPM1]) could improve prognostication by identifying submicroscopic disease during remission.

Methods

We used a reverse-transcriptase quantitative polymerase-chain-reaction assay to detect minimal residual disease in 2569 samples obtained from 346 patients with NPM1-mutated AML who had undergone intensive treatment in the National Cancer Research Institute AML17 trial. We used a custom 51-gene panel to perform targeted sequencing of 223 samples obtained at the time of diagnosis and 49 samples obtained at the time of relapse. Mutations associated with preleukemic clones were tracked by means of digital polymerase chain reaction.

Results

Molecular profiling highlighted the complexity of NPM1-mutated AML, with segregation of patients into more than 150 subgroups, thus precluding reliable outcome prediction. The determination of minimal-residual-disease status was more informative. Persistence of NPM1-mutated transcripts in blood was present in 15% of the patients after the second chemotherapy cycle and was associated with a greater risk of relapse after 3 years of follow-up than was an absence of such transcripts (82% vs. 30%; hazard ratio, 4.80; 95% confidence interval [CI], 2.95 to 7.80; P<0.001) and a lower rate of survival (24% vs. 75%; hazard ratio for death, 4.38; 95% CI, 2.57 to 7.47; P<0.001). The presence of minimal residual disease was the only independent prognostic factor for death in multivariate analysis (hazard ratio, 4.84; 95% CI, 2.57 to 9.15; P<0.001). These results were validated in an independent cohort. On sequential monitoring of minimal residual disease, relapse was reliably predicted by a rising level of NPM1-mutated transcripts. Although mutations associated with preleukemic clones remained detectable during ongoing remission after chemotherapy, NPM1 mutations were detected in 69 of 70 patients at the time of relapse and provided a better marker of disease status.

Conclusions

The presence of minimal residual disease, as determined by quantitation of NPM1-mutated transcripts, provided powerful prognostic information independent of other risk factors. (Funded by Bloodwise and the National Institute for Health Research; Current Controlled Trials number, ISRCTN55675535.)