Page 63 - 2021_12-Haematologica-web

A centralized NGS diagnostic platform for AML
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B
Figure 1. Variant allele frequency of samples included in the second cross validation round. (A) Variant allele frequency (VAF) >5% variants and (B) VAF ≤5% variants. Black dots indicate VAF reported for each center. Red dots mean not detected variant. Mean VAF is represented by a horizontal bar and whiskers represent the standard deviation.
diagnosis (IDH1 14.71% vs. 8.14%, P=0.040; IDH2 21.57% vs. 12.52%, P=0.020, and WT1: 7.84% vs. 3.13%, P=0.043). KRAS and PTPN11 variants were more frequent in refractory as compared to relapse stage (KRAS 9.76% vs. 1.96%, P=0.025, and PTPN11 8.54% vs. 1.10%, P=0.028) (Figure 4A and B).
Age-related mutations
At diagnosis, patients aged ≥65 years had more variants than younger (<65 years) patients (2.74 ± 0.81 vs. 2.18 ± 0.74 variants per patient, P<0.001). The following genes were more frequently mutated in patients aged ≥65 years vs. <65 years: ASXL1, EZH2, IDH2, JAK2, SF3B1, SRSF2, TET2, TP53, and U2AF1. FLT3-ITD and NPM1 mutations were more frequent in younger AML patients (Figure 5).
In relapsed AML, ASXL1 (20.00% vs. 3.57%, P=0.011) and IDH variants (46.70% vs. 25%, P=0.035) were associ- ated with patients aged ≥65 years (Online Supplementary Table S4).
Mutational stability in paired samples
Paired samples at DX-RP (n=14) and DX-RS (n=20) were obtained to assess clonal evolution. The following were stable variants: NPM1 (100%, as no patients acquired or lost variants), TP53, IDH2 (one acquisition at RS and one at RP in each gene), DNMT3A (100% stable at RS and one acquisition at RP), and RUNX1 (stable at RP and one loss at RS). The following variants were unstable: activating signaling pathways genes such as FLT3, NRAS, KRAS, BRAF, KIT and PTPN11 (Online Supplementary Figure S4). Interestingly for targeted therapy, 26.47% of patients changed the mutational status of FLT3 at RS or RP (FLT3- ITD: two gains and three losses; FLT3-PM: one gain and three losses). In all cases, the loss of function mutation of FLT3-PM was located on the Asp835 codon.
Clinically relevant mutations
Overall, 72.30% of patients harbored at least one clinical- ly relevant variant included in the AML clinical guidelines, clinical trials inclusion criteria or as a risk stratification bio- marker (ASXL1, CEBPA, FLT3, IDH1/2, NPM1, RUNX1 and TP53) (Online Supplementary Figure S5). Moreover, druggable mutations were present in a significant proportion of patients (FLT3 in 21.14% and IDH1/2 in 22.60%).
NPM1 mutations
NPM1 variants were found in 21.51% of samples being
78.53% type A (c.860_863dupTCTG), 6.21% type B (c.863_864insCATG), 6.21% type D (c.863_864insCCTG), and 9.04% had uncommon variants (Online Supplementary Figure S6; Online Supplementary Table S5).
FLT3 mutations
FLT3 was the most frequently mutated gene (24.06%).
FLT3 ITD was the most frequent FLT3 aberration (16.52%) followed by D835 and I836 variants (5.71%) and other vari- ants (3.16%) (Online Supplementary Figure S7A). Other vari- ants were mostly SNV (95.18%) located in the tyrosine kinase 1 domain (TKD1; 41.18%), juxtamembrane domain (JMD; 23.53%), tyrosine kinase 2 domain (TKD2; 20.59%), extracellular domain (ED; 11.76%) and kinase insert domain (KID 2.94%). No variants were detected in trans- membrane (TMD) and C-terminal domains (CTD). (Online Supplementary Figure S7B). 84.75% of all FLT3 variants were targetable with FLT3 inhibitors and had a direct clinical impact in 21.14% of patients through targeted therapy or clinical trials.
CEBPA mutations
CEBPA variants were found in 5.35% of samples, 3.52%
were monoallelic variants and 1.82% were biallelic variants
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