Order of Mutation Acquisition Predicts Clinical Progression in MPNs

Alexander M. Castellino, PhD

February 11, 2015

In myeloproliferative neoplasms (MPNs), the order in which mutations in JAK2 and TET2 are acquired in hematopoietic stem cells and progenitor cells determines the clinical presentation and blood cell characteristics of patients with polycythemia vera (PV), essential thrombocytopenia (ET), or myelofibrosis (MF), according to researchers from the University of Cambridge in the United Kingdom.

In addition, the researchers showed that the order in which the mutations are acquired predicts whether cells are susceptible to treatment with the JAK kinase inhibitor  ruxolitinib (Jakafi/Jakavi, Incyte/Novartis).

The findings were published in the February 12 issue of the New England Journal of Medicine.

The order in which the mutations are acquired may have implications for the clinical management of these patients down the road, lead investigator David G. Kent, PhD, research associate from the Department of Haematology at the University of Cambridge, told Medscape Medical News.

This surprising finding could improve the accuracy of prognoses for MPN patients, and mutation order could be used to tailor potential therapies. For example, our results predict that targeted JAK2 inhibitors will be more effective in patients with one mutation order than the other, Dr Kent said in a university press release.

Charles Swanton, MD, PhD, from the Translational Cancer Therapeutics Laboratory at the CR-UK London Research Institute in the United Kingdom, concurs.

In their investigation of the combination and the order of driver mutations JAK2 and TET2 in myeloproliferative disorders, the researchers "provide compelling evidence that mutation order influences the biology and outcome of the disease," Dr Swanton writes in an accompanying editorial.

This is the first study to show the order of mutational events in MPNs and the significance of that order, he told Medscape Medical News.

Determining Mutational Evolution in MPNs

The researchers screened 246 patients (92 with ET, 107 with PV, and 47 with MF) with JAK2 V617F mutations for mutations in TET2. Mutational order was determined in 24 patients who carried TET2 mutations (7 with ET, 11 with PV, and 6 with MF).

To study mutation order in cancer, it is necessary to have a clonal assay and to look at an early stage of disease; both criteria are satisfied in MPNs, Dr Kent told Medscape Medical News. These are neoplasms that develop in early progenitor cells and are easy to grow clonally in semisolid media.

To determine mutation order, peripheral blood mononuclear cells were used to grow hematopoietic cell (specifically erythroid) colonies, each of which was derived from a single cell or clone. The researchers sequenced approximately 8000 colonies for JAK2 and TET2.

Colonies from a single patient will show either no mutation, a single mutation (JAK2 or TET2), or both mutations (JAK2 and TET2). The presence of clones with a single mutation allowed the researchers to assign order in individual patients, Dr Kent explained.

In colonies from a patient with PV, for example, 40% had no mutation, 2% had a JAK2 mutation, and 9% had JAK2 and TET2 mutations. In addition, 49% of colonies had TET2 mutations but were homozygous for JAK2; these were considered JAK2-first patients.

In colonies from a patient with ET, 9% had no mutation, 8% had a TET2 mutation, and 81% had JAK2 and TET2 mutations. In addition, 2% of colonies had TET2 mutations and were homozygous for JAK2; these were considered TET2-first patients.

The researchers demonstrated that in any patient, clonal architecture is typically quite stable. When they followed-up at 12 to 67 months, clonal distribution — no mutation, one mutation, or both — was similar.

Mutation Order Predicting Disease Progression

By examining patient-derived cell populations enriched for hematopoietic stem and progenitor cells, the researchers showed that when a JAK2 mutation is acquired first, progenitor cells expand into megakaryocytes and erythroid cells. However, when a TET2 mutation is first acquired, although the hematopoietic stem and progenitor cells proliferate, there is minimal expansion into cells of the erythroid and megakaryocyte lineage

This suggests that the transcriptional machinery has to be appropriately regulated. To confirm this, the researchers did transcriptional profiling on individual nonmutant, single-mutant, and double-mutant cells.

They pooled colonies (nonmutant, single mutant, and double mutant) that were only JAK2-first or only TET2-first from their patient samples to control for confounding factors, such as age, sex, and treatment background. They identified an upregulation of the transcriptional machinery and an increased  expression of cell-cycle genes in JAK2-first patients. This corresponded to a significant expansion of the double mutants to erythroid and megakaryocytes in JAK2-first patients.

In essence, cells with the exact same genetic mutations behave completely differently, depending on the order in which the two were acquired, Dr Kent told Medscape Medical News.

Mutational Order Predicting Disease Phenotype

How can the observations on mutational order predict disease phenotype? In their initial cohort of 24 patients, 12 were TET2-first and 12 were JAK2-first. Of the JAK2-first patients, seven had PV, two had ET, and three had MF. Although this suggests that patients with PV are more likely to be JAK2-first, the researchers confirmed these observations in 24 additional patients who came from an expansion cohort of 918 other patients, 90 of whom harbored both mutations.

This suggests that JAK2-first patients present with PV at a younger age than TET2-first patients, are more likely to present with PV, and are more likely to experience thrombotic events, Dr Kent explained to Medscape Medical News.

Implications for Clinical Practice

When asked about the clinical significance of the observation that JAK2-first cells are more susceptible in vitro to ruxolitinib, a JAK inhibitor, Dr Kent indicated that a clinical trial needs to be conducted in patients with MPNs. The acquisition order of the mutations will need to be determined to test the efficacy of ruxolitinib in clinical trials.

In fact, ruxolitinib was recently recommended by the European Medicines Agency for the second-line treatment of PV on the basis of data from a phase 3 study, as reported by Medscape Medical News. Although the clinical benefit was considered to be significant, as mandated by the trial's primary end point, only 21% of patients showed a response to ruxolitinib.

Although these observations suggest that the likelihood of acquiring a JAK2 mutation is high in patients with PV, there are patients with PV who present with a TET2-first mutation. According to the data collected so far, these patients are much less likely to experience significant thrombotic events, Dr Kent explained.

This is the first time that mutation order has been shown to affect any cancer, and it is likely that this phenomenon occurs in many types of malignancy," said lead investigator Anthony R. Green, FRCPath, FMedSci, from the Department of Haematology at the University of Cambridge.

These results show how the study of MPNs provides unparalleled access to the earliest stages of tumor development (inaccessible in other cancers, which usually cannot be detected until many mutations have accumulated). This should give us powerful insights into the origins of cancer, he added.

This work was supported in part  by Leukaemia & Lymphoma Research and Cancer Research UK. The authors have disclosed no relevant financial relationships.

N Engl J Med. 2015;372:601-612, 661-663. Abstract, Editorial

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