Clonal heterogeneity in polycythemia vera patients with JAK2 exon12 and JAK2-V617F mutations

Abstract

We studied the lineage distribution of JAK2 mutations in peripheral blood of 8 polycythemia vera (PV) patients with exon 12 mutations and in 21 PV patients with JAK2-V617F. Using a quantitative allele discrimination assay, we detected exon 12 mutations in purified granulocytes, monocytes, and platelets of 8 patients studied, but lymphoid cells showed variable involvement and the mutation was absent in T cells. Endogenous erythroid colonies grew in all patients analyzed. One patient displayed erythroid colonies homozygous for the exon 12 mutation with evidence for mitotic recombination on chromosome 9p. In some patients with exon 12 mutations or JAK2-V617F, a proportion of endogenous erythroid colonies were negative for both JAK2 mutations. One patient carried 2 independent clones: one with an exon 12 mutation and a second with JAK2-V617F. IntroductionMutations in exon 12 of JAK2 are detected selectively in patients with polycythemia vera (PV) that are negative for JAK2-V617F and in some patients with idiopathic erythrocytosis. 1 The JAK2-V617F and exon 12 mutations represent clonal markers useful to track the hematopoietic lineages involved in myeloproliferative disorder (MPD). [2][3][4][5][6] In patients with MPD, JAK2-V617F is present in purified hematopoietic stem cells, in myeloid lineages of the peripheral blood, and in variable proportions of lymphoid cells. [7][8][9][10][11] Using a novel sensitive assay, we quantitated the involvement of exon 12 mutations in purified peripheral blood lineages and in erythroid progenitor assays. In addition, we addressed the question of whether JAK2-V617F is present in T cells by clonal analysis. Methods PatientsThe screening for JAK2 exon 12 mutation in MPD patients was performed by DNA sequencing. 12 All patients except p024 fulfilled the diagnostic criteria of PV according to the World Health Organization (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). 13,14 Patient p024 was initially diagnosed with essential thrombocythemia, and several months later phlebotomies were started because of rising hemoglobin (175 g/L). Two patients with JAK2 exon 12 mutations (Vi064, Vi327) were from Vienna, Austria. All other patients were from Basel, Switzerland. The collection of patient samples was approved by the "Ethik Kommission Beider Basel" and the "Ethik Kommission der Universität Wien und des Allgemeinen Krankenhauses der Stadt Wien-AK." Written consent was obtained from all patients in accordance with the Declaration of Helsinki. Cells, DNA, and RNA analysesIsolation of granulocytes, platelets, and peripheral blood mononuclear cells was performed as described. 5,15 Sorting of peripheral blood mononuclear cells, colony assays in methylcellulose, T-cell cloning, 16 and the SNaPshot assay for RNA samples are described in Document S1. The allele discrimination assay for detection and quantification of JAK2 exon 12 mutations is described in Figure S1. Allele-specific polymerase chain reaction (PCR) for the detection ...

Aquagenic pruritus in polycythemia vera: Characteristics and influence on quality of life in 441 patients

“…The excellent response to JAK2-inhibitors and the observations that bone marrow derived basophils and mast cells harbor the JAK2-mutation suggests an explanation: depending upon at which stage of hematopoietic development the mutation is introduced into the differentiation pathway, cells differentiate into differently affected cells such as granulocytes, monocytes, basophils, or platelets [26]. The occurrence of AP may depend upon whether or not there is a sufficient amount of JAK2-positive mast cells present in the patient.…”

Genetic complexity of myeloproliferative neoplasms

“…A number of patients with a simultaneous presence of JAK2-V617F and MPL-W515L/K, and JAK2-V617F and JAK2-ex12 mutations have been reported. 33,55 However, only one study proved that two independent clones of progenitors co-existed, one positive for JAK2-V617F and the other for JAK2-ex12 mutations. 96 Presence of del20q has been reported either as a mutation establishing clonal hematopoiesis before the acquisition of JAK2-V617F 44 or, in a different recent study, del20q-positive progenitors have been observed as a minor clone arising from JAK2-V617F-positive cells.…”

“…97 (f) A patient with polycythemia vera with both JAK2-V617F and JAK2-ex12 mutations was reported. 55 The two mutations were representing two clones of cells. (g, h) Simple clonal structure is likely in patients carrying a single MPL-W515L mutation.…”

“…This broad inhibitory effect of drugs may increase their [20][21][22][23][24] (d) Some patients with JAK2-ex12 acquire 9pUPD. 55 (e) Deletions on chromosome 20q (del20q) were reported to occur on the background of JAK2-V617F-positive cells in some patients. 97 (f) A patient with polycythemia vera with both JAK2-V617F and JAK2-ex12 mutations was reported.…”

“…The presence of low mutational burden detected in some patients carrying MPL and JAK-ex12 mutations predicts that these mutations may also occur on a clonal background. 33,55 The presence of clonal hematopoiesis has been a fundamental part of MPN definition and played an important role in distinguishing MPN from reactive conditions such as secondary polycythemia or reactive thrombocythemia. The discovery of polyclonal ET 56,57 initially raised questions on the diagnostic criteria applied for ET and suggested the genetic heterogeneity of this MPN entity.…”