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G. Greiner et al.
specimens compared to BM biopsy material. We had pre- viously investigated the analytical validity of dPCR for KIT D816V in detail according to laboratory standards before applying the technology here.16 In the current study, we used dPCR for mutant allele burden measure- ment in FFPE BM sections of SM patients. To the best of our knowledge, this is the first study that comprehensive- ly assesses the tissue mutation burden as a novel molecu- lar biomarker in SM.
Our results suggest that the KIT D816V allele burden in BM tissue was not interchangeable with that in liquid specimens (PB or BM aspirate). In particular, a number of ISM patients showed substantially higher levels in FFPE tissue. In SM patients with multi-lineage involvement, the KIT D816V mutation is also present in CD34+ hematopoi- etic stem/precursor cells, eosinophils, basophils, mono- cytes or neutrophils.26-28 These cells are abundant in the liq- uid specimens and represent the main source of KIT D816V in PB of ISM patients with high mutant allele bur- den.19 In contrast, a MC infiltration of >10% is commonly found in the BM tissue of ISM patients.22,23 The mutation burden in FFPE BM sections, therefore, reflects both the ‘infiltration burden’ of KIT D816V+ MC in the tissue and the multi-lineage involvement of the mutation in non- MC. Accordingly, the mutation burden in BM tissue cor- related better with the established markers of disease bur- den in SM (serum tryptase levels and BM MC infiltration) than molecular parameters performed on liquid speci- mens.
A high MC burden in the BM biopsy (>30% infiltration of cellularity) and high serum total tryptase (>200 ng/mL) also represent B-findings (‘burden of disease’) for defini- tion of SSM.4 Although the clinical course in SSM is often stable for many years, progression to advanced SM can occur. Therefore, SSM represents a rare high-risk subcate- gory compared to ISM.29 Quantification of KIT D816V allele burden in FFPE might be useful as an additional molecular marker of disease burden in SSM since it includes all KIT D816V positive cells in the tissue. In our study, a particularly high tissue allele burden was found in SSM patients. However, the numbers of samples tested were too small to allow us to draw a final conclusion as to the value of KIT D816V allele burden measurement in FFPE BM sections as an additional criterion of SSM. Multi- center studies with larger patient cohorts are currently being prepared to investigate the definitive value of meas- urement of tissue mutation burden and its applicability as a B-finding in SSM.
The current definition of advanced SM relies largely on the presence of C-findings (‘cytoreduction-requiring’) as markers of organopathy in ASM.1,4 For these patients, cytoreductive treatment is required.4,30 With the develop- ment of tyrosine kinase inhibitor (TKI) treatment in SM as a more specific therapeutic option,31,32 additional sub- groups of patients might benefit from treatment and are the subject of ongoing clinical trials.33-35 Thus, prognostic biomarkers are warranted to define patients at risk that do not meet the current ASM criteria. We and others have shown that multi-lineage involvement of KIT D816V indi- cated by a high mutant allele burden in PB was associated with an aggressive clinical course.17-20 In liquid specimens, we have previously used a 2% VAF cut-off to stratify OS in SM patients.18 Jara-Acevedo et al. used a 6% VAF cut-off in PB to discriminate between MC-restricted versus multi- lineage SM.19 In this study, we used a higher cut-off of 9%
VAF in BM sections to take into account the higher ISM mutational burden in the tissue. Using this cut-off, highly significant differences in both PFS and OS were observed and the tissue mutation burden remained an independent poor risk marker in multivariate analysis when B- and C- findings were considered. This is an important and novel finding as all previous studies assessing the mutation bur- den in liquid samples (PB and BM aspirate samples) found a significant effect on survival only in univariate but not in multivariate analyses.17,18,20,36,37 This difference might be explained by the close association of multi-lineage KIT D816V involvement (indicated by a high liquid mutation burden) with advanced SM (indicated by the presence of C-findings). Our observations have a clear clinical impact and strongly argue for inclusion of assessment of the KIT D816V mutation burden in prognostic scoring systems for mastocytosis. A potential limitation of our study is that different cytoreductive treatment modalities were applied in this retrospective analysis with a relatively high propor- tion of interferon-α or cladribine. In fact, more effective treatment regimens may improve OS in the future.31 However, the vast majority of patients with a low tissue mutation burden experienced no events despite the lack of any cytoreductive treatment, suggesting mutation burden analysis is important irrespective of treatment.
Therapy response criteria in advanced SM mainly rely on resolution of C-findings as markers of SM-mediated organopathy to define major response.38 In addition, reduction of MC infiltration and/or of serum tryptase lev- els is used to define complete remission, incomplete remission, and pure clinical response.38 The TKI midostau- rin showed high efficacy in advanced SM with 45% major response and marked decreases in BM MC burden and serum tryptase.31 Both measurements are valuable surro- gate parameters of disease burden in SM, but they do have some limitations.39 While the basal level of total tryptase is well established as being quite a stable parameter in SM, single time-point measurements might be substantially influenced by MC activation or allergic reactions.40-42 On the other hand, quantification of MC burden is a rather robust parameter of disease burden, but relies on experi- enced hematopathologists.43 In this regard, measurement of KIT D816V allele burden might be useful as an addi- tional objective response parameter. Recently, a ≥25% reduction in expressed KIT D816V allele burden in PB was described as an independent 'on treatment' marker for improved OS in midostaurin-treated patients with advanced SM.24 Based on the results of individual patients before and after cytoreductive treatment, FFPE-based allele burden measurement as a more direct marker of the number of all KIT D816V positive cells in the tissue might be an interesting additional follow-up parameter for treat- ment response. This might be of particular relevance for ISM patients undergoing TKI treatment in the future, since the mutation burden in liquid specimens substantially underestimates the disease burden in ISM. Furthermore, the high sensitivity of the assay makes it applicable for KIT D816V-based MRD measurement in patients that achieve complete remission in the histopathological assessment. However, further multi-centric studies are needed to definitively establish the tissue mutation bur- den as a parameter for therapy response in SM.
A potential limitation of dPCR and any other molecular test detecting specifically KIT D816V is that neither rare non-D816V mutations of KIT nor somatic mutations in
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haematologica | 2020; 105(2)