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. 2020 Jan 31;105(2):366-374.
doi: 10.3324/haematol.2019.217950. Print 2020.

Molecular quantification of tissue disease burden is a new biomarker and independent predictor of survival in mastocytosis

Georg Greiner  1   2 , Michael Gurbisz  1 , Franz Ratzinger  1 , Nadine Witzeneder  1   3 , Svenja Verena Class  4 , Gregor Eisenwort  2   3 , Ingrid Simonitsch-Klupp  4 , Harald Esterbauer  1   2 , Matthias Mayerhofer  5 , Leonhard Müllauer  4 , Wolfgang R Sperr  2   3 , Peter Valent  2   3 , Gregor Hoermann  6   2   7
Affiliations

Molecular quantification of tissue disease burden is a new biomarker and independent predictor of survival in mastocytosis

Georg Greiner et al. Haematologica. .

Abstract

A high allele burden of the KIT D816V mutation in peripheral blood or bone marrow aspirates indicates multi-lineage hematopoietic involvement and has been associated with an aggressive clinical course of systemic mastocytosis. Since mast cells are substantially underrepresented in these liquid specimens, their mutation burden likely underestimates the tumor burden of the disease. We used a novel previously validated digital polymerase chain reaction (PCR) method for KIT D816V analysis to systematically analyze the mutation burden in formalin-fixed, paraffin-embedded bone marrow tissue sections of 116 mastocytosis patients (91 with indolent and 25 with advanced systemic mastocytosis), and to evaluate for the first time the clinical value of the tissue mutation burden as a novel biomarker. The KIT D816V mutation burden in the tissue was significantly higher and correlated better with bone marrow mast cell infiltration (r=0.68 vs 0.48) and serum tryptase levels (r=0.68 vs 0.58) compared to that in liquid specimens. Furthermore, the KIT D816V tissue mutation burden was: (i) significantly higher in advanced than in indolent systemic mastocytosis (P=0.001); (ii) predicted survival of patients in multivariate analyses independently; and (iii) was significantly reduced after response to cytoreductive therapy. Finally, digital PCR was more sensitive in detecting KIT D816V in bone marrow sections of indolent systemic mastocytosis patients than melting curve analysis after peptide nucleic acid-mediated PCR clamping (97% vs 89%; P<0.05). In summary, digital PCR-based measurement of KIT D816V mutation burden in the tissue represents a novel biomarker with independent prognostic significance that can also be employed for monitoring disease progression and treatment response in systemic mastocytosis.

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Figures

Figure 1.
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 proportional 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 showing a skewing towards higher results in the tissue for samples with low average VAF.
Figure 2.
Figure 2.
Association of KIT D816V allele burden with biomarkers of disease burden in systemic mastocytosis (SM). Correlation of KIT D816V mutation burden in 185 formalin-fixed paraffin-embedded (FFPE) bone marrow (BM) sections [variant allele frequency (VAF) tissue: A and C] and 108 BM aspirate/peripheral blood (PB) samples (VAF liquid: B and D) with immunohistologically determined BM mast cell (MC) infiltration (A and B) and serum tryptase (C and D). r: Spearman’s correlation coefficient.
Figure 3.
Figure 3.
Biomarkers of disease burden in indolent and advanced systemic mastocytosis (SM). Bone marrow (BM) mast cell (MC) infiltration (A), serum tryptase levels (B), and KIT D816V mutant allele burden in BM aspirate/peripheral blood samples [variant allele frequency (VAF) liquid: C] and formalin-fixed paraffin-embedded (FFPE) BM sections (VAF tissue; D) were assessed for indolent (ISM) (blue, n=91) and advanced (green, n=25) SM patients. Samples from smoldering SM patients within the ISM cohort are shown in orange. **P<0.01; ***P<0.001.
Figure 4.
Figure 4.
KIT D816V tissue mutation burden for prognostication and therapy response monitoring in systemic mastocytosis (SM). (A and B) Kaplan-Meier plot for progression-free survival (PFS) (A) and overall survival (OS) (B) of SM patients with a KIT D816V variant allele frequency (VAF) <9% or ≥9% in the bone marrow (BM) tissue. (C-E) Follow up of KIT D816V tissue mutation burden (blue) and serum tryptase (black) in three patients with SM who received cytoreductive treatment is shown: a patient initially diagnosed as indolent SM (ISM) with disease progression to SM with an associated hematologic neoplasm (SM-AHN) [aggressive SM (ASM)-chronic myelomonocytic leukemia (CMML)] and response to cytoreductive therapy with cladribine (2-CDA). Later on, the patient progressed to acute myeloid leukemia (AML) (C). A patient diagnosed with mast cell leukemia who responded to alternating treatment with the tyrosine kinase inhibitor midostaurin and 2-CDA (D). A patient diagnosed with ASM who responded to treatment with midostaurin, 2-CDA and achieved complete remission after hematopoietic stem cell transplantation (HSCT) (E).
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