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

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.