Monitoring of Minimal Residual Disease in NPM1 Mutated Acute Myeloid Leukemia (AML) – a Single Center Experience in 27 Patients.

Mutations within NPM1 gene occurs in about 60% of adult cytogenetic normal AML (CN-AML) and represent the single most frequent molecular aberration in this subgroups of patients. These mutations usually occur at exon 12 and induce most frequently a net insertion of four base pairs.

To examine the applicability and sensitivity of DNA-based real-time quantitative polymerase chain reaction (RQ-PCR) with mutation-specific reverse primers and common minor groove binding (MGB) probe and to evaluate whether minimal residual disease levels are of prognostic relevance in CN-AML patients with NPM1 mutations.

Patients were treated within different AML trials and follow-up samples of peripheral blood or bone marrow were referred to perform an RQ-PCR. Samples were analysed at diagnosis, during, and after therapy. The NPM1 mutations were A (17 pts), B (1 pt), D (2 pts) and 7 patients with individual rare types. For all cases, levels of minimal residual disease were determined by DNA-based RQ-PCR with mutation-specific reverse primer, one common forward primer and one common MGB probe. The NPM1 mutation value was normalized on the number of albumin gene copies and expressed as the number of NPM1 mutations every 10⁶ genomic equivalents.

This assay is highly specific as no wildtype NPM1 could be detected. Maximal reproducible sensitivity was 10 plasmide molecules per reaction.

A total of 950 samples of bone marrow and/or peripheral blood from 27 patients have been analyzed. Twenty of 27 patients (74%) achieved molecular remission (MR), twenty-six of 27 patients (96%) achieved hematological remission (HR). 6 of 27 (22%) patients achieved HR without MR and one patient failed therapy. 8 of 20 patients (40%) with MR after treatment relapsed at molecular level and except one in all these patients hematological relaps occured (one patient is still in HR with bone marrow blast present, but < 5%). Considering relapsed patients, time from molecular to hematological relapse was 1 to 5 months (median: 3 months).

Considering all 14 patients with HR without MR (6 pts) or with molecular relapse (8 pts), in 11 of them hematological relaps occured (79%) and molecular positivity anticipating hematological relaps with median of 3,5 month (1-7 months). 3 of these 14 patients are still in HR.

Mutations within NPM1 gene are a sensitive marker for monitoring minimal residual disease in CN-AML patients. RQ-PCR using a MGB probe is an efficient approach to long-term follow-up of residual leukemia cells and frequent quantitative monitoring is useful for reliably predicting hematological relapse. Achievement of negativity appears to predict favorable clinical outcome.

This work was partially supported by research grant No. MSM0021622430

No relevant conflicts of interest to declare.