JAK2V617F and CALR double mutations are more frequently encountered in patients with low JAK2V617F allelic burdens

We have read with great interest the comment by Dr He and coworkers clarifying the significance of various kinds of CALR mutations. [1] The authors stress that, among the CALR mutations recently reported in patients with essential thrombocythemia or primary myelofibrosis,[2,3] some do not follow the specific pattern observed in myeloproliferative neoplasms patients. Indeed, while pathogenic mutations generate a one base pair frameshift, deletions of ‘‘multiples of 3’’ base pairs are sometimes found, generally thought to be constitutional, and they should be distinguished from ‘‘true’’ pathogenic, acquired CALR mutations. The exclusion of these in frame, non pathogenic mutations decreases the frequency of true ‘‘doublemutants’’ carrying both a JAK2 and a CALR mutation, as clearly demonstrated by the analysis of published series provided by Dr He. Therefore, it is probably not interesting to look for a CALR mutation in all JAK2-mutated patients for such rare events. Moreover, this finding generally does not modify patients’ management since no series of patients with double mutations has reported specific clinical characteristics yet. However, we believe some circumstances could justify testing for CALR mutations in a subgroup of JAK2 mutated patients. Indeed, in a collaborative study of patients presenting with low allele burdens of JAK2V617F, we found a relatively high proportion (2/11) of CALR mutations in patients with thrombocytosis. [4] The clinical significance of a low JAK2V617F allelic burden may be difficult to interpret since it can be observed in patients with no evidence of myeloproliferative neoplasm. Finding an additional CALR mutation in this context thus provides a more definitive proof of clonality. We then wondered whether CALR mutations would be more frequently found in patients with low JAK2V617F allelic burden. To test this hypothesis, we searched the database of the hematology laboratory at CHU de Bordeaux for patients with positive JAK2V617F from 2006 to 2015. 1895 patients had between 5% and 99% JAK2V617F as determined by two different allele specific quantitative PCR. [5,6] In the same period, 227 patients (10.7% of the total) had a JAK2V617F allelic burden between 0.01% and 4%. CALR mutations were investigated using a PCR fragment length analysis, as described in [7], in 412 patients with45% JAK2V617F and 133 patients with55% JAK2V617F who had given written informed consent for the use of remaining DNA for research purposes. Only two patients of the first cohort (0.5%) had a CALR mutation: one had 50% JAK2V617F and a type 2 CALR mutation (c.1154_1155insTTGTC, p.K385fs*47); the second also had a type 2 CALR mutation but only 5% JAK2V617F. Among the ‘‘low JAK2V617F allelic burden’’ group, 19 patients (14.29%) had a CALR mutation: 9 type 1 (c.1099_1150del, p.L367fs*46), 7 type 2, 4 other types and one carrying both type 1 and type 2 CALR mutants (Table 1). The repartition of these mutation types is similar to results reported in the literature. [2] Follow-up samples were available for 7 patients, between 1 and 101 months after the initial assessment. All patients remained double positive for JAK2 and CALR mutations. JAK2V617F allelic burdens were very stable in all 7 patients, except patient P14who had the longest followup (101 months). This patient’s JAK2V617F burden decreased from 2.5 to 0.3% while her CALR burden increased from 43 to 50%, suggesting that mutations occurred in different clones. Similarly, CALR mutational burden increased modestly in two additional patients (P15: +5% and P21:+8%), but decreased in two other patients: -5% both (P17 treated by hydroxyurea and P16 untreated). The presence of two mutations made us