Whole Exome Sequencing Reveals Clonal Evolution of Myeloproliferative Neoplasms to Acute Myeloid Leukemia

Disease progression to acute myeloid leukemia (AML) is observed in 7% of the cases with the three classical BCR-ABL1 negative myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). According to the WHO, the presence of ≥ 20% blasts in bone marrow or peripheral blood is the diagnostic criterion for establishing leukemic transformation. PMF patients are at the highest risk, while PV and ET patients often develop secondary myelofibrosis prior to the leukemic transformation. Post-MPN AML patients have poor prognosis and clonal evolution of MPN chronic phase to AML is not well understood. Here we aimed to study the clonal evolution from MPN to AML in 7 cases, by performing whole exome sequencing (WES) on samples taken at various disease stages from individual patients.

From the 7 post-MPN AML patients included in the study, 4 were diagnosed with PV and 3 with ET during chronic phase of the disease. For all 7 patients WES was performed on DNA samples from the control tissue, chronic phase and/or accelerated phase, and the leukemic phase of the disease. All variants identified by WES were validated using Sanger sequencing. In addition, tumor samples were analyzed for genomic deletions, gains and uniparental disomies (UPD) using SNP microarrays.

We identified on average 16 somatic mutations (range 12-27) and 4 chromosomal aberrations (range 0-9) per patient in the leukemic stage of the disease. All patients were JAK2-V617F positive. 115 validated somatic mutations affected a total of 100 different genes. Most mutations were found in genes that were previously not linked to myeloid cancers, however, they were not recurrent. Besides JAK2-V617F, recurrent mutations were found in TP53 (N=3/7), RUNX1 (N=2/7), TET2 (N=2/7) and MPL (N=2/7). Biallelic TP53, RUNX1 and TET2 mutations were present in single patients. Known MPN and AML-related genes such as DNMT3A, SRSF2, U2AF1, IDH2, KIT, and PHF6 were mutated in single patients. We identified 25 chromosomal aberrations in 7 patients. Del5q, del6p, del7q and 9pUPD were recurrent. UPDs and trisomies of chromosomes 9, 12q, 17p and 21 were coupled with mutations in JAK2, SH2B3, TP53 and RUNX1. One patient harbored focal deletions of <1Mb on chromosomes 10 and 12, targeting TET1 and ETV6, respectively.          

We used variant frequencies detected by WES and copy number ratios and allelic difference values detected by microarrays at various stages of the disease to reconstruct the clonal evolution from chronic phase to AML in the 7 studied cases. Mutations with similar variant frequencies showing changes of allelic frequency in the same direction were assumed to be part of the same clone. Figure 1 illustrates an example of the proposed model for clonal evolution in Patient 6. As in this patient we had WES data from chronic, accelerated and leukemic stage of the disease, we first analyzed the clonal evolution from chronic to the accelerated phase. In the chronic phase the ~60-80% of granulocytes were derived from a single clone carrying 5 somatic mutations (JAK2, CAD, PPFIA2, SCNG, USH2A) and 2 chromosomal aberrations (1q gain and trisomy 9). At the accelerated phase of the disease we could observe that the main clone acquired somatic mutations in ADIPOQ, EYA3 and FAM123C and that there is at least one subclone (~40% of cells) appearing with mutations in OGDH, PHF6, USH2A, del7q and 9qUPD. At the leukemic stage, the clone with 9qUPD was suppressed by the outgrowing clone carrying OGDH and other mutations. We could also show that at the final leukemic stage the dominant clone acquired RUNX1 S400X mutation, amplified with a trisomy of chromosome 21, while the other RUNX1 allele mutated to Q262X.

In each of the 7 studied patients the clonal evolution was unique and complex process with a few common features. Loss of TP53 is the most common genetic lesion. TET2 mutations are early events in clonal evolution of MPN and often precede the acquisition of JAK2-V617F, while RUNX1 mutations seem to be late events, leading to differentiation arrest and appearance of blasts. We demonstrated that in 6/7 studied cases the clonal evolution was a linear process, led by sequential acquisition of somatic mutations on the basis of the same clone causing the chronic disease. This finding is in line with the results of our previous study where we showed that the genetic basis of secondary AML is significantly different from de novo AML.