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KIT D816V dPCR in FFPE Sections
A
B
CD
E
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 vari- ant 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 neo- plasm (SM-AHN) [aggressive SM (ASM)-chronic myelomonocytic leukemia (CMML)] and response to cytoreductive therapy with cladribine (2-CDA). Later on, the patient pro- gressed 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 midostau- rin, 2-CDA and achieved complete remission after hematopoietic stem cell transplanta- tion (HSCT) (E).
whereas the mutation was detected in 98 patients (84%) by melting curve analysis. When analyzing SM subgroups, the mutation was detected with equal sensitivity in 17 patients with advanced SM (68%) by both methods. In contrast, 88 ISM patients (97%) were tested positive by dPCR compared to 81 (89%) by clamp PCR (Table 3). The observed difference in sensitivity was statistically signifi- cant (P<0.05) in favor of dPCR. To confirm the specificity of the results, we analyzed FFPE BM sections of control subjects (n=57). No KIT codon 816 mutation was detected by either method, indicating 100% specificity.
When we further characterized patients that were test- ed ‘false-negative’ by melting curve analysis after PNA- mediated PCR clamping (n=7), a relatively low VAF for KIT D816V was observed (median 0.7%, range 0.027- 2.1%). While some of these patients showed a mutant allele burden clearly below the limit of detection estab- lished for this method,15 others were found to have a low amount of total KIT copies (<1000) reflecting impaired quality or quantity of these specimens (Online
Supplementary Figure S3D). However, importantly, con- strained validity of the analysis was not recognizable by melting curve analysis after PNA-mediated PCR clamping. In this regard, quantitative dPCR results allow for an addi- tional quality control of the specimen within the same assay. Altogether, dPCR was superior over clamp PCR to detect the KIT D816V mutation in FFPE BM sections of patients with SM.
Discussion
Although it is generally appreciated that the quantifica- tion of the total burden of KIT-mutated neoplastic cells in SM is an important prognostic parameter, quantification of the KIT mutant allele burden has so far been limited to liquid specimens (PB or BM aspirate). This is a critical point, as neoplastic MC and their progenitors are not eas- ily aspirable from BM and are very rarely circulating in the PB; they are thus substantially underrepresented in liquid
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