Clinical molecular testing for ASXL1 c.1934dupG p.Gly646fs mutation in hematologic neoplasms in the NGS era

Abstract

ASXL1 (additional sex combs like 1) is a gene that is mutated in a number of hematological neoplasms. The most common genetic alteration is c.1934dupG p.Gly646fs. Previous publications have shown that ASXL1 mutations have a negative prognostic impact in patients with MDS and AML, however, controversy exists regarding the molecular testing of ASXL1 c.1934dupG as polymerase splippage over the adjacent homopolymer could lead to a false-positive result. Here, we report the first study to systematically test different targeted next generation sequencing (NGS) approaches for this mutation in patients with hematologic neoplasms. In addition, we investigated the impact of proofreading capabilities of different DNA polymerases on ASXL1 c.1934dupG somatic mutation using conventional Sanger sequencing, another common method for ASXL1 genotyping. Our results confirm that ASXL1 c.1934dupG can be detected as a technical artifact, which can be overcome by the use of appropriate enzymes and library preparation methods. A systematic study of serial samples from 30 patients show that ASXL1 c.1934dupG is a somatic mutation in haematological neoplasms including MDS, AML, MPN and MDS/MPN and often is associated with somatic mutations of TET2, EZH2, IDH2, RUNX1, NRAS and DNMT3A. The pattern of clonal evolution suggests that this ASXL1 mutation might be an early mutational event that occurs in the principal clonal population and can serve as a clonal marker for persistent/relapsing disease.

Fig 1

A. VAF scatterplot plotting all the clinical samples studied by NGS with direct PCR amplification (PA) based library preparation chemistry found to harbor ASXL1 c.1934dupG (n = 2870). Majority of the cases (2636, 92%) have a variant frequency below 10%. Y axis: VAF. B. 234 samples (8%) were identified with ASXL1 c.1934dupG with VAF≥10%. The maximum VAF observed in the series was 51.4. X axis: VAF, Y axis: number of samples. C. VAF scatterplot showing all the clinical samples studied by HCA-based NGS library preparation chemistry found to harbor ASXL1 c.1934dupG (n = 250).Y axis: VAF D. Left panel: VAF scatterplot showing all the clinical samples studied by NGS with amplicon based library preparation chemistry found to harbor ASXL1 c.1934del p.G645fs (n = 2864). As depicted, most of the cases (99%) have a variant frequency below 10%. The maximum VAF observed in the series was 40.36. Y axis: VAF. Right panel: VAF scatterplot showing all the clinical samples studied by HCA-based NGS found to harbor ASXL1 c.1934del p.G645fs (n = 74). Y axis: VAF. E. Three electropherograms of cases with ASXL1 c.1934dupG quantified as 3.59, 7.78 and 30.31 respectively by NGS after amplicon based library preparation. As shown, no minor clonal sequence is found in the case with a VAF<5%.

Fig 2

A. Variant allele frequency (X axis) was plotted against variant coverage (log10 scale, Y axis) in patient n2. ASXL1 c.1934dupG was present at a VAF different than those found for well recognized SNPs (TP53 c215 C>G, EGFR c1562 G>A and ASXL1 c2444 T>C). B, C, D and E. VAF (Y axis) is plotted in two different time points (X axis) in four different patient samples.B. Patient n2 (male, 65 year old) samples are shown. First sample (MDS RAEB-2, 15% blasts in BM aspirate count) was obtained prior to therapy. A second 28-gene NGS analysis was done 20 months after alo-SCT (trilineage haematopoiesis, 1% blast count in BM aspirate). A dramatic reduction in VAF for all the mutations found at diagnosis (ASXL1 c.1934dupG, DNMT3A c.2408G>A, DNMT3A c.2026 C>T and IDH2 c515G>A) is evident after SCT. C. Patient n6 (female, 62 year old) was diagnosis as AML with MDS related changes (52% blasts in BM aspirate count)). The patient underwent haploidentical SCT. On day #76 after transplant the patient relapsed (29% blasts in BM aspirate count). As shown, both samples share the same mutational profile, characterized by ASXL1 c.1934dupG, DNMT3A c.1948del, DNMT3A c.1685G>A, EGFR c.2988C>A and IDH2 c.419G>A. D. Patient n30 (male, 64 year old) samples are shown. First and second samples were diagnosed as MPN (JAK2 negative post ET-myelofibrosis, fibrosis grade 3 and osteosclerosis, blast count in BM aspirate was 1% and 8%). The third sample was obtained 30 days after allogeneic SCT. Pathological diagnosis was hypocellular BM (5% blasts) with osteosclerosis. The fourth sample was obtained 7 months after SCT and the pathological diagnosis was cellular bone marrow with fibrosis grade 2. A decline in the donor chimerism both in the myeloid and T-lineage cell types was observed at that time point. As shown in the figure, a dramatic reduction of both ASXL1 c.1934dupG and TET2 c.2108T>A p.1703* is seen after allogeneic SCT, in correlation with BM cellularity. Both ASXL1, TET2 and a new clonal population characterized by TP53 c.818G>A p.R273H mutation arises after therapy. E. Patient n1 (male, 79 year old) samples are shown. First sample was diagnosed as MDS/MPN (7% blast count). After 16 cycles of Vidaza/Revlimid the patient showed persistent MDS/MPN with 17% blast count. As shown in the figure, there is a shift in the clonal composition of the disease with a decrease in the three IDH1 mutations first identified (IDH1 c603_606del p.S202fs*7, IDH1 c624T>G p.Y208* and IDH1 c611G>A p.G204D) after therapy, but the emergence of new clones characterized by FLT3 c.1471G>C p.V491L, NPM1 c393_395dupGGA p. E131dup, KRAS c35G>C p.G12A and NRAS c.37G>C p.G13R. Both ASXL1 c.1934dupG p.G646fs*11 and MLL/KMT2A c4737_4739dupTGA p.D1580dup remain relatively constant along time.