Networking for advanced molecular diagnosis in acute myeloid leukemia patients is possible: the PETHEMA NGS-AML project

Abstract

Next-Generation Sequencing has recently been introduced to efficiently and simultaneously detect genetic variations in acute myeloid leukemia. However, its implementation in the clinical routine raises new challenges focused on the diversity of assays and variant reporting criteria. To overcome this challenge, the PETHEMA group established a nationwide network of reference laboratories aimed to deliver molecular results in the clinics. We report the technical cross-validation results for next-generation sequencing panel genes during the standardization process and the clinical validation in 823 samples of 751 patients with newly diagnosed or refractory/relapse acute myeloid leukemia. Two cross-validation rounds were performed in seven nationwide reference laboratories in order to reach a consensus regarding quality metrics criteria and variant reporting. In the pre-standardization cross-validation round, an overall concordance of 60.98% was obtained with a great variability in selected genes and conditions across laboratories. After consensus of relevant genes and optimization of quality parameters the overall concordance rose to 85.57% in the second cross-validation round. We show that a diagnostic network with harmonized next-generation sequencing analysis and reporting in seven experienced laboratories is feasible in the context of a scientific group. This cooperative nationwide strategy provides advanced molecular diagnostic for acute myeloid leukemia patients of the PETHEMA group.

Prognostic significance of FLT3-ITD length in AML patients treated with intensive regimens

“…Samples from 118 of the 362 AML patients with FLT3-ITD mutations were analyzed with an NGS panel of 39 genes (see Supplementary Fig. S2) in PETHEMA centralized diagnostic laboratories as previously described 33 . Information regarding the ITD insertion site and mutational status of another 38 genes recurrently mutated in myeloid neoplasms was available in 106 and 118 patients, respectively.…”

NGS-defined measurable residual disease (MRD) after initial chemotherapy as a prognostic biomarker for acute myeloid leukemia

“…However, as we have shown, these less-sensitive methods may yield false-negative MRD results in a significant fraction of patients. Given the current lack of standardization of AML NGS assays in different labs [ 19 ], the specific reagent and bioinformatic technical details of these other comparable MRD studies were expectedly quite heterogeneous. In contrast, our assay used a standardized NGS protocol that was thoroughly validated, by rigid government-mandated regulatory guidelines, for routine diagnostic use in a licensed clinical laboratory.…”

NGS-defined measurable residual disease (MRD) after initial chemotherapy as a prognostic biomarker for acute myeloid leukemia

“…However, as we have shown, these less-sensitive methods may yield false-negative MRD results in a signi cant fraction of patients. Given the current lack of standardization of AML NGS assays in different labs [19], the speci c reagent and bioinformatic technical details of these other comparable MRD studies were expectedly quite heterogeneous. In contrast, our assay used a standardized NGS protocol that was thoroughly validated, by rigid government-mandated regulatory guidelines, for routine diagnostic use in a licensed clinical laboratory.…”