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KIT D816V dPCR in FFPE Sections
These last three entities are collectively referred to as advanced mastocytosis, based on their increased risk of progression and death.4,7
Molecular diagnostics has become increasingly important in SM. Molecular techniques with a high analytical sensitiv- ity, such as allele-specific quantitative polymerase chain reaction (qPCR) or digital PCR (dPCR), are required to reli- ably detect the KIT mutation in liquid specimens (PB or BM aspirate).8-13 Melting curve analysis after peptide nucleic acid (PNA)-mediated PCR clamping (clamp-PCR) is widely used for qualitative detection of the KIT mutation in formalin- fixed paraffin-embedded (FFPE) tissue biopsies despite its limited analytical sensitivity that requires micro-dissection of BM MC in a number of cases.14,15 We have recently shown that dPCR is suitable as a new sensitive method for KIT D816V testing in SM that also reliably quantifies the variant allele frequency (VAF).16 A high mutant allele burden in liquid specimens was indicative of multi-lineage involve- ment with KIT D816V and was associated with an aggres- sive clinical course and advanced forms of SM.16-20 However, in the majority of patients, only a small fraction of KIT D816V+ MC and/or MC precursors, if any, is found in liquid specimens.21 Therefore, quantification of KIT D816V VAF in liquid specimens typically only evaluates multi-lineage involvement in the non-MC compartment. In line with this observation, only a moderate correlation of KIT D816V VAF in liquid specimens with MC infiltration or serum tryptase as surrogate markers for disease burden in SM has been described.17-20 In contrast, the percentage of KIT D816V+ MC is typically much higher in BM tissue biopsies compared to BM aspirates.22,23 Therefore, molecular meas- urements in liquid specimens substantially underestimate the disease burden in SM, in particular in ISM. Moreover, while a reduction of the mutation burden in liquid speci- mens has been described in response to cytoreductive treat- ment in patients with advanced SM and multi-lineage involvement,18,24 its value as a follow-up parameter in ISM and advanced SM without multi-lineage involvement remains uncertain. Molecular quantification of KIT D816V disease burden in the tissue has the potential to overcome these limitations as a biomarker of disease burden in SM.
The quantification of KIT D816V VAF has not been assessed systematically in BM tissue sections of SM patients. Here we investigated the clinical value of dPCR- based KIT D816V tissue mutation burden quantification as a novel biomarker in SM.
Methods
Patients
We examined 390 samples (211 FFPE BM sections, 106 BM aspi- rates, 73 PB) from 116 SM patients (58 females, 58 males), diag- nosed between April 1988 and February 2016 and included in a
local registry. PB and BM samples at diagnosis and during follow up were obtained after informed consent and the study was approved by the institutional review board (EK:1750/2017). According to WHO criteria,1,6 83 patients were diagnosed with ISM, 8 with SSM, 7 with ASM, 3 with MCL, and 15 with SM- AHN. Patients’ characteristics are shown in Table 1. During the course of disease, 36 patients (31%) received a cytoreductive treat- ment with a median of two different regiments (range 1-5) (Online Supplementary Table S1). Fifty-seven FFPE BM sections from lym- phoma patients undergoing a staging biopsy, and in whom no BM infiltration was detected, were used as control material (Online Supplementary Table S2). Quantification of BM infiltration by MC, flow cytometry of MC, and measurement of serum tryptase is described in the Online Supplementary Methods.
Molecular analysis of KIT D816V
Genomic DNA was extracted from (FFPE) BM sections as well
as PB and/or aspirated BM cells as previously described and dPCR for KIT D816V was performed with the PrimePCR ddPCR muta- tion assay for KIT wild-type and the KIT D816V point mutation (Bio-Rad Laboratories, Munich, Germany) and analyzed on the QX-200 droplet-reader (Bio-Rad Laboratories), as described in detail in the Online Supplementary Methods.16 In addition, qualita- tive detection of KIT codon 816 mutations was performed using melting curve analysis after PNA-mediated PCR clamping as described.14-16
Statistical analysis
Statistical analysis was performed using R (version 3.4.2, Vienna, Austria)25 and GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Applied tests are described in detail in the Online Supplementary Methods and P<0.05 was considered to be signifi- cant.
Results
The KIT D816V tissue allele burden is a novel biomarker in systemic mastocytosis
The KIT D816V tissue allele burden was studied by dPCR in 211 FFPE BM sections from 116 SM patients (Table 1). Median VAF was 1.9%, with a range between 0.027% and 60% indicating a substantial difference in the tissue mutation burden over various orders of magnitude between patients. When we compared the KIT D816V VAF in FFPE BM section with that in matched PB or BM aspirates samples, higher tissue allele burden levels were observed (Figure 1A), whereas a strong correlation was found between PB and BM aspirates (r=0.99) (Online Supplementary Figure S1). A comparison between the log transformed KIT D816V VAF in the tissue and liquid specimens (BM aspirate n=96, PB n=12 in cases for which no BM aspirate was available for molecular analysis) in 108 matched samples from 79 patients, revealed a direct correlation in non-parametric analysis (r=0.87) but a con- stant and proportional deviation was found in Passing Bablok regression analysis (intercept: 1.72, 95%CI: 1.53- 1.91; slope: 0.59, 95%CI: 0.52-0.65) (Figure 1B). In addi- tion, Bland-Altman plot displayed a deviation tendency to higher KIT D816V allele burden in FFPE BM sections, par- ticularly in samples with low VAF (Figure 1C). In summa- ry, these findings indicate that the KIT D816V VAF in FFPE BM sections was not interchangeable with allele burden measurements in liquid specimens and represents a new biological variable of disease burden in SM patients.
Table 1. Patients’ characteristics.
Disease subtype
Patients‘ characteristics
Age (median, range)
ISM (n=91)
50 (23–82)
Advanced SM (n=25)
64 (21–91)
Total cohort (n=116)
53 (21–91)
Sex(female|male) 50|41 8|17 58|58
KIT D816V positive * 88/91 (97%) 17/25 (68%)# 105/116 (91%)
ISM: indolent systemic mastocytosis; n: number. *As assessed by digital polymerase chain reac- tion. #One additional patient was tested positive for KIT D816H.
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