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G. Greiner et al.
Table 2. Parameters for progression-free survival (PFS) in systemic mastocytosis.
Progression-free survival (PFS)
Molecular quantification of KIT D816V allele burden VAF in liquid specimen ≥2%
VAF in tissue ≥9%
Clinical characteristics
Age > 65 years Sex B-findings
MC infiltration in BM biopsy >30% Serum tryptase level >200 μg/L Organomegaly without dysfunction *
C-findings
Hemoglobin <10 g/dL
Platelets <100x109/L Hepatomegaly with dysfunction# Alkaline phosphatase >150 U/L Weight loss
Albumin levels <35 g/L
Univariate
HR [95%CI] P
5.99 [2.41-14.88] <0.0001 15.82 [5.31- 47.16] <0.0001
1.06 [1.04-1.10] <0.0001 3.25 [1.26-8.38] 0.015
3.12 [1.32-7.37] 0.010 2.43 [0.90-6.58] n.s. 3.93 (1.65-9.35) 0.001
16.59 [5.43-50.67] <0.0001 17.44 [5.93-51.29] <0.0001 6.97 [1.52-31.93] 0.012 3.32 [1.36-8.19] 0.008 3.41 [1.40-8.27] 0.007
3.38 [0.98-11.57] n.s.
Multivariate
HR [95%CI] P
50.71 [4.23-607.90]
21.26 [2.64-171.30]
n.s. 0.002
n.s. n.s.
n.s. n.s. n.s.
n.s. 0.004 n.s. n.s. n.s. n.s.
Multivariate analyses regarding the prognostic impact of KIT D816V tissue variant allele frequency (VAF) and clinical characteristics (including B- and C-findings) on PFS of 103 patients with SM. HR: hazard ratio; CI: confidence interval; MC: mast cell; BM: bone marrow; n.s.: not significant. *Organomegaly including hepatomegaly, splenomegaly, or lym- phadenopathy. #Hepatomegaly with ascites and/or portal hypertension.
and progression-free survival (PFS) in KIT D816V+ SM patients (n=103; no survival data available for 2 patients) in univariate and multivariate analyses including KIT D816V VAF in liquid specimens, age, sex, B-findings (BM MC infiltration, serum tryptase, organomegaly), and C- findings (anemia, thrombocytopenia, hypalbuminemia, weight loss, hepatomegaly with liver dysfunction, and increased alkaline phosphatase) as additional variables. In univariate analyses, PFS was adversely influenced by the majority of established risk factors including a high KIT D816V tissue allele burden with a hazard ratio (HR) of 15.82 (95%CI: 5.31-47.16) and a high KIT D816V liquid allele burden with a HR of 5.99 (95%CI: 2.41-14.88) (Table 2). In multivariate analysis including all molecular and clinical variables, only thrombocytopenia (HR: 21.26, 95%CI: 2.64-171.30; P=0.004) and a high KIT D816V allele burden in the tissue (HR: 50.71, 95%CI: 4.23-607.90; P=0.002) remained independent risk markers for PFS (Table 2). Similar results were obtained for OS with a HR of 12.79 (95%CI: 4.22-38.76) for a high KIT D816V tissue allele burden and 4.69 (95%CI: 1.84-11.98) for a high KIT D816V liquid allele burden in univariate analysis and a sig- nificant independent influence in multivariate analysis only for the tissue mutation burden (HR: 18.12, 95%CI: 1.98-165.57; P=0.01) (Online Supplementary Table S3). Using the maximum selected rank statistics method, 9% VAF in the tissue represents an optimal cut-off differenti- ating between surviving and non-surviving patients. In the group with <9% tissue mutant allele burden (n=79), the median PFS and OS was not reached, whereas in patients with a KIT D816V allele burden of ≥9% (n=24) the medi- an PFS was 4.1 years (Figure 4A) and the median OS 4.6 years (Figure 4B). The observed differences in survival were highly significant both for OS and PFS (Log-rank test; P<0.0001 each).
Moreover, the KIT D816V tissue allele burden could be followed in 26 patients over a median observation time of 42 months (range 2-172 months). In patients with stable disease and no cytoreductive therapy, no substantial increase or decrease in the KIT D816V allele burden was
Table 3. Sensitivity of molecular techniques to detect the KIT D816V in the tissue.
P<0.05
n. (%)
negative
positive
total
Clamp PCR
negative
3 (3%)
7 (8%)
10 (11%)
positive
0 (0%)
81 (89%)
81 (89%)
Total
3 (3%)
88 (97%)
91 (100%)
Results of digital polymerase chain reaction (dPCR) and melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp PCR) to detect KIT D816V in formalin-fixed paraffin-embedded bone marrow sections of indolent systemic mas- tocytosis patients (n=91).
observed. In contrast, marked changes in KIT D816V tis- sue VAF were found in patients with advanced SM receiv- ing cytoreductive therapy. In particular, six patients who showed a response to cytoreductive treatment (cladribine n=3, midostaurin and cladribine n=3) showed a significant decrease in the KIT D816V tissue burden (92% median reduction comparing post- with pre-therapeutic samples; P<0.01). Representative examples of two patients are shown in Figure 4C and D. In addition, Figure 4E shows a patient with complete remission after allogeneic stem cell transplantation, in whom low minimal residual disease (MRD) was detected by dPCR at an early time point (day 93) after transplantation. In total, our data show that dPCR-based tissue mutation burden measurement is fea- sible for monitoring treatment responses and to assess MRD levels in patients with SM.
Digital polymerase chain reaction is a sensitive diagnostic test to detect KIT D816V in formalin-fixed paraffin-embedded bone marrow sections
Finally, we assessed the performance of dPCR as a diag- nostic test to detect the KIT D816V mutation in FFPE BM section compared to melting curve analysis after PNA- mediated PCR clamping on diagnostic BM section from 116 SM patients (Table 1). In the total cohort, 105 patients (91%) were tested positive for KIT D816V by dPCR
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dPCR