Fig 1.
Performance of CALR qPCR assays for type 1 and type 2 mutations.
Logarithmic amplification curves, standard curves and measurements of assay reproducibility of (A) type 1 and (B) type 2 mutations. The qPCR amplification and standard curves were generated from 5-fold dilutions of mutated patient DNA in wildtype (wt) DNA. The amplification curves appearing at Cq values above 41 are unspecific amplification of wt DNA. Reproducibility of the type 1 and type 2 assays was evaluated by performing three independent runs on five patient samples with the CALR type 1 mutation and five patient samples with the CALR type 2 mutation with varying amounts of mutated alleles demonstrating robust performance suitable for clinical purposes.
Table 1.
Demographics and clinical baseline characteristics for molecular responders versus molecular non-responders and total cohort.
Fig 2.
Distributions of initial CALR mutant allele burdens according to diagnosis and mutation type.
Mutant allele burdens shown for type 1 ET (n = 2), type 1 pre-PMF (n = 2), type 1 PMF (n = 11), type 2 ET (n = 2), and type 2 pre-PMF (n = 2) patients. The mean is indicated by the horizontal line, and the type 1 PMF group is depicted with mean ±SD.
Fig 3.
Allele burden response to IFN in CALR mutated MPN patients.
Waterfall plot visualising percentage changes in the mutant allele burden of CALR from baseline (start of IFN treatment) to the latest CALR qPCR measurement in 21 patients with a median follow-up of 31 months (range 7–137 months).
Fig 4.
Dynamics of the CALR mutant allele burden during IFN treatment.
A) Dynamics of allele burden in patients from start of IFN treatment with black lines indicating molecular responder (MR) patients and grey lines indicating molecular non-responder (non-MR) patients. The dashed black line denotes a non-MR exhibiting MR dynamics, but with an insufficient reduction in mutant allele burden for obtaining MR status (45% reduction). B) Mutant allele burdens over time for MR patients with indications of reduction of IFN dose (maintenance phase) and IFN withdrawal (stop).
Fig 5.
CALR mutant allele burden dynamics are closely associated with those of platelets, leucocytes, LDH, and haemoglobin.
Representative graphs of the development of the CALR mutant allele burden and blood counts for a molecular responder (MR) patient (A-D) and a molecular non-responder (non-MR) patient (E-H) during and after IFN treatment. The values of the mutant allele burdens are shown on the left y-axis. (A and E) Mutant allele burden and platelet count. (B and F) Mutant allele burden and leucocyte count. (C and G) Mutant allele burden and LDH. (D and H) Mutant allele burden and concentration of haemoglobin. Take note of the close resemblance of the dynamics of the mutant allele burden with the blood counts and LDH both decreasing and increasing during relapse (A-D).