Endogenous megakaryocytic colonies underline association between megakaryocytes and calreticulin mutations in essential thrombocythemia.

Calreticulin (CALR) mutations occur in 20%-25% of myeloproliferative neoplasms (MPN). At least 40 CALR mutations have been reported to date, all located in exon 9. The most frequent CALR mutations are a 52-bp deletion (type 1) and a 5-bp insertion (type 2). Expression of type 1 CALR mutation was shown to induce constitutive activation of JAK-STAT signaling pathway in a Ba/F3-cell line with STAT5 phosphorylation leading to spontaneous growth in the absence of interleukin-3. Furthermore, megakaryocyte lineage has been reported to play a major role in MPN pathophysiology. In particular, hematopoietic colony formation independent of exogenous cytokines, including endogenous megakaryocytic (EMC) and endogenous erythroid colonies (EEC) was shown to be a functional sign of clonal hematopoiesis due to deregulated signaling pathways in MPN. Altogether, these data suggest a particular link between megakaryocytic (MK) proliferation and deregulation of signaling due to driver mutations. However, to our knowledge, spontaneous growth of hematopoietic progenitors (EMC and EEC) has not been characterized in type 1 or type 2 CALR -mutated patients. In the current study, we analyzed patterns of EMC and EEC according to molecular status (JAK2V617F, CALR, MPL, triple negative) in a cohort of 302 essential thrombocythemia (ET) patients from 3 French University Hospitals [Grenoble (n=121), Dijon (n=121), and Nantes (n=60)]. The inclusion criteria were ET patients who had benefited from in vitro cultures from bone marrow at diagnosis and for whom DNA was available. The JAK2V617F mutation was assessed using purified granulocytes by tetra-primer ARMS-PCR or by allele-specific quantitative PCR.MPL mutations were screened by high resolution melt assay and confirmed by sequencing. The mutational status of CALR was determined using previously described high-resolution sizing of fluorescent dyelabeled PCR amplificons of exon 9, with Sanger sequencing controls. To standardize EMC and EEC, cultures were performed from bone marrow samples using the same standardized collagen medium in all three centers. Megakaryocytic colonies composed of at least 4 MKs were counted by microscopy after MGG staining of dry collagen dishes. We analyzed EMC and EEC results both qualitatively [presence or not of EMC and/or EEC (so-called “positive or negative EMC/EEC”)] and quantitatively (number of EMC and/or EEC per 10 cells plated). For statistical analysis, non-parametric tests were applied: Mann-Whitney test (for comparison of two groups) and McNemar test (paired data). c or Fisher tests were used to compare nominal variables, and Spearman rank correlation to compare two continuous variables. P<0.05 was considered statistically significant. The mutational distribution was 50.8% (153 of 302) for JAK2V617F, 23% (69 of 302) for CALR, 5.3% (17 of 302) for MPL and 19% (57 of 302) for “triple negative”. Six patients lacked MPL and/or CALR data (1.9%). Among the 69 patients with CALR mutations, 35 (50.7%) patients harbored type 1 and 24 (34.8%) patients had type 2. Irrespective of mutational status, overall endogenous hematopoietic growth (defined by presence of EMC and/or EEC) was 58.9% (178 of 302). We observed significantly more EMC (56.3%; 170 of 302) than EEC (13.6%; 41 of 302) whatever the mutational status (P<0.001). In order to determine the relationship between growth profile and genotype, we compared global spontaneous growth defined by EMC and/or EEC positivity according to mutational status. Patients harboring mutations such as JAK2V617F or CALR showed a significantly higher proportion of overall spontaneous growth (respectively 66% and 73.9%) compared to “triple negative” ones (25%; P<0.001). No difference in frequency of EMC and/or EEC between JAK2V617F and CALR was noted (P=0.24). On the contrary, MPL mutations were less frequently associated with EMC and/or EEC compared to CALR (47% and 73.9%, respectively; P=0.03). As higher platelets counts were reported in patients with CALR mutations in comparison with JAK2V617F patients, we attempted to compare the proportion of EMC in these two subgroups (Figure 1). EMC were more frequently observed in CALR than in JAK2V617F (73.9% and 61.4%, respectively) without reaching statistical significance (P=0.07). To strengthen the link between genotypes and megakaryocytic proliferation, we also compared the mean number of EMC according to the molecular status. CALR mutations were associated with a significantly higher mean number of EMC compared to “triple negative” and JAK2V617F patients: 9.36 (0-81.5), 1.75 (0-23.3), and 5.5 (0-53.3), respectively; P<0.01 and P=0.02. A tendency towards a higher number of EMC was observed between CALR and MPL but without statistical significance (P=0.07). Interestingly, concerning EEC, CALR-mutated patients displayed a significantly weaker proportion of EEC positivity compared to JAK2V617F-mutated patients (1.4% vs. 22.8%; P<0.001). This percentage of EEC in JAK2V617F ET is consistent with previous data obtained with a serum-free assay. EEC were rare in the CALR-mutated population; only one type 2-mutated patient among 69 showed 2 EEC per 10 cells. A significantly higher proportion of EEC were associated with JAK2V617F compared to triple negative (P<0.001) and MPL (P<0.05). Next, the endogenous megakaryocytic profile between types 1 and 2 CALR mutants and “variants” was compared. “Variants” have neither type 1 nor type 2 CALR mutations (n=10 patients). EMC frequencies were similar in type 1 and type 2 patients (82.8% and 70.8%, respectively; P=0.34). The proportion of EMC in “variants” was