Leukemic transformation driven by an ASXL1 mutation after a JAK2V617F-positive primary myelofibrosis: clonal evolution and hierarchy revealed by next-generation sequencing

Abstract

We have characterized the molecular changes underlying the transformation of a JAK2V617F+-myelofibrosis with trisomy 8, into a JAK2V617F-negative leukemia. Leukemic clone did not carry JAK2V617F mutation, but showed ASXL1 mutation (R693X). This mutation was identified in a low percentage at diagnosis by next-generation sequencing. Using this technology in serial specimens during the follow-up, we observed a progressive expansion of the ASXL1-mutated minor clone, whereas the JAK2V617F+-clone carrying trisomy 8 decreased. Hematologic progression occurred simultaneously with an ASXL1-R693X-negative lung-cancer. This is the first report showing a clear association between the expansion of an ASXL1-mutated clone and the leukemic transformation of myelofibrosis.

Figure 1

Histologic and radiologic studies at the time of leukemic transformation. (A) BM biopsy showing immature myeloid proliferation without segmentation (blasts >20%) and dysplastic megakaryocytes. With silver staining (right panel), marked reticulin fibrosis associated with osteoesclerosis was also shown. (B) Chest CT scan showing an increase in bone density of the vertebral bodies and a right lung nodule of 2 × 2 cm in size with speculated edges. (C) Positron-emission tomography with CT showing a markedly increased of ¹⁸ F-FDG uptake in the BM of the vertebral bodies; sacrum; extremity bones (specially left humerus); and focal in the spleen.

Figure 2

Clonal progression from diagnosis of PMF to leukemic transformation and death. (A) Dynamic changes in the size of the two mutated clones, JAK2V617F and ASXL1-R693X, in three time points during follow up: at the time of presentation of PMF, during the evolution and in blast-phase. (B) Microsatellite analysis on PB granulocytes at diagnosis (left panel) and blast-phase of MF (right panel) for 4 markers on chromosome 9p flanking JAK2. The positions of microsatellite markers used to identify the common 9pLOH region are shown as vertical lines (C) ASXL1 sequencing in paired samples of cDNA from PB granulocytes showing one nonsense mutation (R693X) in the ASXL1 gene at blast-phase, but not at diagnosis (left panel). This mutation was not detected in the hepatic metastatic tissue of lung cancer (right panel).