Page 134 - 2020_02-Haematologica-web

G. Greiner et al.
The KIT D816V tissue allele burden correlates with mast cell infiltration and serum tryptase levels
In a next step, we correlated KIT D816V VAF with BM MC infiltration and serum tryptase levels as established surrogate parameters of disease burden in KIT D816V pos- itive SM patients (n=105). The correlation of mutant allele burden with BM MC infiltration as determined by immunohistochemistry was higher for DNA isolated from BM FFPE sections (r=0.68) (Figure 2A) than from liquid specimens (r=0.48) (Figure 2B). Likewise, a higher correla- tion with serum tryptase was observed for tissue mutation burden (r=0.68) (Figure 2C) than for liquid mutation bur- den (r=0.58) (Figure 2D). When we stratified the samples into ISM and advanced SM, a higher correlation of BM MC infiltration with the mutation burden in the tissue (compared to liquid samples) was observed for both sub- groups, with a generally higher correlation in ISM patients (Online Supplementary Figure S2A and B). In contrast, we found only a modest correlation of the mutation burden with serum tryptase for both specimens in advanced SM, while the correlation with tryptase substantially increased from liquid (r=0.55) to tissue VAF (r=0.70) in ISM (Online Supplementary Figure S2C and D). Together, the KIT D816V allele burden in FFPE BM sections reflects the burden of neoplastic cells in SM better than established mutant allele burden measurements in PB or BM aspirate.
The KIT D816V tissue allele burden is higher in advanced than in indolent systemic mastocytosis
Next, we compared surrogate markers of disease burden
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in SM patients between ISM (n=91) and advanced SM (n=25). The median BM MC infiltration was higher in advanced SM (20%) compared to ISM (7%) (P<0.01) (Figure 3A). Likewise, a tendency towards higher levels of serum tryptase levels were observed in advanced SM (median 140 ng/mL vs. 37 ng/mL; P=0.07) (Figure 3B). Within the KIT D816V positive patients, a significantly higher mutant allele burden was observed in advanced SM both in liquid specimens (21.31% vs. 0.34%; P<0.01) (Figure 3C) as well as in BM tissue samples (23.40% vs. 1.65%; P<0.001) (Figure 3D).
However, the observed difference was bigger in liquid specimens due to the higher KIT D816V allele burden in BM tissue (compared to liquid specimens) in our ISM patients. When we analyzed SSM patients within the ISM cohort (n=8), particularly high levels of MC infiltration (median: 35%, range: 20-80%), serum tryptase (median: 234 ng/mL, range: 188-545 ng/mL), liquid mutant allele burden (median: 35.40%, range: 10.60-39.10%) as well as tissue mutant allele burden (median: 10.94%, range: 3.90- 36.90%) were found (Figure 3). In summary, our data indi- cate that tissue mutation burden is a promising novel bio- marker of disease burden in SM.
The KIT D816V tissue allele burden predicts survival in systemic mastocytosis and reflects response to cytoreductive treatment
To define the prognostic value of tissue mutation bur- den quantification, we associated KIT D816V tissue VAF results with the clinical end points overall survival (OS)
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Figure 1. Relation of KIT D816V allele burden in formalin-fixed paraffin- embedded (FFPE) bone marrow (BM) sections of systemic mastocytosis (SM) patients with that in liquid specimens. (A) Ternary plot of KIT D816V variant allele frequency (VAF) from 54 paired FFPE BM section (red axis), BM aspirate (green axis) and peripheral blood (PB) (blue axis) samples. (B) Comparison of KIT D816V VAF in 108 paired FFPE BM (VAF) tissue] and BM aspirate/PB samples (VAF liquid) showing a systematic constant and propor- tional deviation to higher VAF in the tissue (r=0.87; slope: 0.59 (95%CI: 0.52- 0.65), intercept: 1.72 (95%CI: 1.53-1.91) for log transformed data). (C) Bland-Altman plot of KIT D816V VAF in the tissue and liquid specimens show- ing a skewing towards higher results in the tissue for samples with low aver- age VAF.
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haematologica | 2020; 105(2)