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KIT D816V dPCR in FFPE Sections
other genes can be monitored. In particular, mutations of SRSF2, ASXL1 and RUNX1 are found in aggressive forms of mastocytosis and TET2 mutations have been described to precede the acquisition of KIT D816V in some patients.37,44 KIT D816V-negative clonal cells in advanced SM might be overlooked by dPCR while sequencing of a gene panel would identify them. Thus, additional next generation sequencing (NGS)-based monitoring of the dis- ease might be warranted, particularly when there is clini- cal suspicion of disease progression despite a low KIT D816V allele burden. Here we show a higher clinical sen- sitivity of dPCR to detect KIT D816V in BM sections of SM patients compared to the clamp PCR that is widely used for tissue analysis. This is partly due to the higher analytical sensitivity of dPCR, which allows low mutant alleles to be detected.16 The analytical sensitivity of clamp PCR can be increased by analysis of micro-dissected neo- plastic MC.14 Micro-dissection of MC has been used to detect KIT D816V in SM patients but requires time-con- suming sample processing and is not widely available for routine diagnostics. In this regard, dPCR seems sufficient- ly sensitive, as it allows the detection of KIT D816V with- out micro-dissection in >95% of all ISM patients. However, micro-dissection of MC is still valuable to detect non-D816V mutations in KIT by clamp PCR or sequencing. Thus, both molecular techniques for KIT D816V detection are necessary and helpful, but the dPCR technique may have some advantages and should be con- sidered in the future. One advantage of dPCR is the quan- tification of KIT wild-type alleles in the sample as quality control. Samples with borderline quality/quantity of DNA isolated from FFPE material are easily identified. In a num- ber of these samples, clamp PCR could have shown false negative results if no additional (quantitative) quality con- trol measurements for amplifiable DNA had been applied. Beside these assays, a number of other molecular tests, such as qPCR or ultra-sensitive NGS, have been described for quantification of KIT D816V in PB or BM aspirate.13,17,45
We have previously shown an excellent correlation of dPCR and qPCR in these liquid specimens with easier inter-laboratory standardization and lack of amplification bias in highly fragmented DNA as potential advantages of dPCR.16 For diagnostic assessment, the high analytical sen- sitivity of digital PCR is sufficient to use not only trephines but also BM aspirates.16 In addition, qPCR and dPCR detect the mutation in the PB of the majority of patients,16,46,47 arguing for an early molecular assessment during the diagnostic workup of SM.9
In summary, dPCR for KIT D816V is a valuable diagnos- tic test to detect and quantify the mutation in FFPE BM sections of SM patients. The clinical sensitivity to detect the mutation is superior to clamp PCR without micro-dis- section of MC. The KIT D816V mutant allele burden in FFPE BM sections correlates with BM MC infiltration and serum tryptase levels and represents a novel molecular parameter which differs from the mutant allele burden in PB or BM aspirate. Importantly, dPCR-based measure- ment of KIT D816V mutation burden in the tissue repre- sents a novel biomarker with independent prognostic sig- nificance that can also be employed for follow-up analyses in SM. We therefore propose to include the measurement of tissue mutation burden in future studies for prognosis, SSM definition, and monitoring of disease progression and treatment response in SM patients.
Acknowledgments
The authors thank Jana Strasakova (Department of Laboratory Medicine, Medical University of Vienna) for technical support.
Funding
This study was supported by the Austria Science Fund (FWF) project P26079-B13, the SFB projects F4701-B20 and F4704- B20, the Medical-Scientific Fund of the Mayor of Vienna and a research grant of the Austrian Society of Laboratory Medicine and Clinical Chemistry (ÖGLMKC).
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