PMC10200979_01
PMC10200979_02
(A) Normocellular bone marrow not diagnostic for myeloproliferative neoplasia, only a few megakaryocytes with myeloproliferative features in sample from 2017. (B) Hypercellular bone marrow with clusters of atypical megakaryocytes with myeloproliferative features without fibrosis in sample from 2022. (C) Reticulin staining in sample from 2017 reflecting a normal bone marrow. (D) Loose networks of reticulin staining grade 0-1 in bone marrow sample from 2022. (A, B) Hematoxylin and eosin staining; (C, D) Reticulin staining; magnification x400).
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The blood cell counts and bone marrow biopsy were normal although, when reviewed, early MPN features could be discerned ( Figures 1A, C ). This included forms with hyper-lobulated, as well as bulbous and hyperchromatic, nuclei ( Figure 1B ). In addition, silver staining revealed focally lightly accentuated reticulin fibrosis grade 0-1 reminiscent of an early pre-fibrotic myelofibrosis ( Figure 1D ).
Exponential growth of CALR mutant allele burden from CH to MPN (A) Graph illustrating the mutant allele burden of CALR type 1 from September 2010 to November 2022 demonstrating exponential growth of the mutant allele burden and the platelet count. Note the close correlation between the CALR mutant allele burden and the platelet count once it exceeds 5%, including the transient drop even from 19% mutant alleles to 15% indicating a biological phenomenon rather than a technical discrepancy. The left Y-axis indicates the mutant allele burden, where black dots indicate CALR data points. The right Y-axis describes the number of platelets/microl and the red circles measurements of platelets. The dashed lines indicate the upper and lower boundary for the reference range of platelet count. (B) Backward extrapolation used to estimate when the mutation occurred. A heterozygous mutation in 1 out of 100.000 (dashed blue line) or 200.000 (dashed red line) stem cells.
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Table 1 ), but at this point, the exponential growth of the mutated clone became evident ( Figure 2A ). Although within the reference range, the platelet count clearly increased alongside the mutant allele burden demonstrating significant correlation (Pearson: R2 = 0.9259, P<0.0001) ( Figures 2A ,  3A ). Figure 2A ). Fitting the exponential expression , where  is the time measured in years after the first measurement, to the CALR allele burden (using the fit function in MATLAB's curve fitting toolbox, version R2021b) results in  (95% CI ) and,  (95% CI , with , corresponding to a doubling time of  (95% CI ) and a yearly growth rate of  (95% CI ). Different sources have different estimates for the number of hematopoietic stem cells in a human, and therefore we have calculated the estimated time of mutation for a wide range of the assumed number of stem cells ( Figure 2B ). A heterozygous mutation in one out of N =  stem cells (corresponding to an allele burden of ) is estimated to have occurred  (95% CI  based on the 95% CI of ) before the first measurement, and a heterozygous mutation in one out of N =  stem cells (corresponding to an allele burden of ) is estimated to have occurred  (95% CI  based on the 95% CI of ) before the first measurement, i.e. somewhere between the age of 45 and 53 for the patient based on the assumed number of stem cells ( Figure 2B ).
Significant correlation of longitudinal CALR mutant allele burden with (A) platelet counts, (B) the neutrophil-to-lymphocyte ratio, (C) hemoglobin and (D) erythrocyte count. R squared and p values from Person correlation are shown in graphs. NLR, Neutrophil-to-lymphocyte ratio; Hb, Hemoglobin; eryt, Erythrocyte count.
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Although within the reference range, the platelet count clearly increased alongside the mutant allele burden demonstrating significant correlation (Pearson: R2 = 0.9259, P<0.0001) ( Figures 2A ,  3A ). The mutant allele burden demonstrated a significant correlation with the neutrophil-to-lymphocyte ratio (NLR) (Pearson: R2 = 0.6422, P=0.0006) ( Figure 3B ). An inverse correlation was observed between the mutant allele burden and both hemoglobin (Hb) (Pearson: R2 = 0.7819, P<0.0001) and erythrocytes (Pearson: R2 = 0.6843, P=0.0003) ( Figures 3C, D ).