Clinical impact of measurable residual disease monitoring by ultradeep next generation sequencing in NPM1 mutated acute myeloid leukemia

Abstract

Detection of measurable residual disease (MRD) by mutation specific techniques has prognostic relevance in NPM1 mutated AML (NPM1mut AML). However, the clinical utility of next generation sequencing (NGS) to detect MRD in AML remains unproven. We analysed the clinical significance of monitoring MRD using ultradeep NGS (NGS-MRD) and flow cytometry (FCM-MRD) in 137 samples obtained from 83 patients of NPM1mut AML at the end of induction (PI) and consolidation (PC). We could monitor 12 different types of NPM1 mutations at a sensitivity of 0.001% using NGS-MRD. We demonstrated a significant correlation between NGS-MRD and real time quantitative PCR (RQ-PCR). Based upon a one log reduction between PI and PC time points we could classify patients as NGS-MRD positive (<1log reduction) or negative (>1log reduction). NGS-MRD, FCM-MRD as well as DNMT3A mutations were predictive of inferior overall survival (OS) and relapse free survival (RFS). On a multivariate analysis NGS-MRD emerged as an independent, most important prognostic factor predictive of inferior OS (hazard ratio, 3.64; 95% confidence interval [CI] 1.58 to 8.37) and RFS (hazard ratio, 4.8; 95% CI:2.24 to 10.28). We establish that DNA based NPM1 NGS MRD is a highly useful test for prediction of relapse and survival in NPM1mut AML.

Next‐generation sequencing for measurable residual disease detection in acute myeloid leukaemia

“…Recent studies utilizing NGS for the detection of MRD in AML vary greatly in their design and technical aspects. Cohorts studied have included AML patients undergoing allogeneic haematopoietic cell transplantation (alloHCT) (Getta et al , ; Kim et al , ; Thol et al , ; Zhou et al , ; Press et al , ), receiving standard induction chemotherapy (Klco et al , ; Gaksch et al , ; Jongen‐Lavrencic et al , ; Morita et al , ; Onecha et al , ; Rothenberg‐Thurley et al , ; Thol et al , ; Wong et al , ), receiving novel therapies in clinical trials (Levis et al , ), or having only specific mutations (Thol et al , ; Kohlmann et al , ; Salipante et al , ; Levis et al , ; Patkar et al , ; Zhou et al , ; Patel et al , ). Also, since most studies to date have had access to diagnostic samples, de novo leukaemia‐associated mutation discovery using remission samples alone remains an important unmet challenge.…”

Minimal/Measurable Residual Disease Monitoring in NPM1-Mutated Acute Myeloid Leukemia: A Clinical Viewpoint and Perspectives

“…Based upon the above-mentioned biological and clinical characteristics, especially their homogeneous mutation pattern in AML patients, NPM1 mutations may be considered an ideal leukemia-specific target for MRD detection [ 2 ]. Since the first application by Gorello et al of sensitive and specific RQ-PCR assays as a reliable system to quantitatively assess NPM1 -mutated gene copies [ 30 ], several studies have investigated the clinical implications of MRD monitoring in NPM1 -mutated AML undergoing intensive therapeutic approaches ( Table 1 ) [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ].…”

“…Collectively, the results of the studies presented above confirmed that RQ-PCR is a reliable molecular tool to assess MRD in most patients with NPM1 -mutated AML [ 35 , 40 , 44 , 47 , 54 , 57 ]. The MRD monitoring, by either a kinetic measure [ 32 , 35 , 47 , 53 , 57 , 68 ] or the obtainment of an absolute threshold of NPM1 -mutated transcripts [ 31 , 33 , 35 , 38 , 40 , 44 , 47 , 48 , 49 , 50 , 51 , 52 , 54 , 55 , 56 , 58 , 61 , 63 , 65 , 66 , 67 , 69 ], was overall predictive of relapse and survival outcomes ( Table 2 ). However, due to differences in patients’ cohorts and the lack of standardization of NPM1 -mutated RQ-PCR MRD assays and analytical methods, any comparison between different studies remains extremely difficult, and evidence-based conclusions on the best MRD thresholds and timepoints cannot be definitely drawn [ 8 , 44 , 75 ].…”

“…However, after the first observation by Papadaki et al that two among 21 (9.5%) NPM1 -mutated AML patients experiencing relapse, lost NPM1 mutation at leukemia recurrence [ 32 ], many groups investigating NPM1 -mutated molecular MRD provided further information on stability of NPM1 mutation at relapse, as summarized in Table 2 . While in several cohorts, clonal AML evolution at disease recurrence was not observed [ 31 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 42 , 43 , 48 , 49 , 52 , 53 , 55 , 57 , 58 , 59 , 60 , 63 , 64 , 65 , 66 , 67 , 68 , 69 ], in the remaining studies the frequencies of patients experiencing AML relapse with undetectable NPM1 mutations were extremely variable, ranging from 1% in the prospective trial by Ivey et al [ 54 ] to 25% documented in smaller series [ 41 , 94 ]. Notably, cases with loss of NPM1 mutation at disease reoccurrence were initially considered as secondary therapy-related myeloid neoplasms rather than true relapses from the previously found NPM1 -mutated leukemic clone [ 14 , 32 , 40 ].…”

Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry‐based leukemic cell enrichment followed by mutational profiling

“…20,47 Recent studies using error-corrected sequencing approaches have shown increased sensitivities to detect leukemic cells 48,49 and were found to be highly predictive for relapse risk. 50 Application of such molecular barcoding in future studies might help to increase the sensitivity as well as specificity of our two-step method.…”