Mast cells and fibrosis in myeloproliferative neoplasms.

The spectrum of BCR-ABL negative myeloproliferative neoplasms (MPNs) consists
of 3 diseases that are characterized by aberrant proliferation of the myeloid
lineage.1 MPNs are commonly associated with activating somatic mutations in the
JAK2, CALR or MPL pathway of hematopoietic stem cells (HSC).2
The subsequent proliferation of myeloid cells leads to an overproduction of
erythrocytes or thrombocytes and granulocytes resulting in polycythemia vera
(PV), essential thrombocythemia (ET) or primary myelofibrosis, respectively.
Remodelling of the hematopoietic niche by the MPN-clone and its progeny can
lead to secondary myelofibrosis.
Evidence suggests an elevation of proinflammatory cytokines as one of the main
contributors to the fibrotic remodeling of the hematopoietic bone marrow.3,4

Over the past years the crucial role of the hematopoietic niche in MPNs have
been increasingly elucidated.5 Gli1+ mesenchymal stromal cells have recently
been identified by investigators of this proposal to be the major
fibrosis-driving cells. Inhibition of these cells leads to a strong reduction
of fibrosis as well as clinical burden in vivo.6
The phenotypical and transcriptional changes of these cells during
pharmacotherapy with tyrosine kinase inhibitors such as Ruxolitinib are however
not yet studied. Treatment with Ruxolitinib has been described to not only
ameliorate clinical symptoms in MPN-patients but to also improve the degree of
fibrosis.7 Hence, it is clear that the remodeling of the niche responsible for
synthesis and deposition of extracellular matrix in myelofibrosis is directly
or indirectly influenced by pharmacotherapy.
Unraveling the transcriptional changes in the hematopietic niche will allow us
to map the changes induced by pharmacotherapy. Comparing patients with
different response rates will enable us to identify mechanisms of resistance
and patients at risk that might need to undergo more intense treatment.

Several studies provide evidence pointing to mast cells as an important player
in MPN and especially progression to myelofibrosis. An increased number of mast
cells as well as the presence of aberrant mast cells is frequently found in the
bone marrow of MPN patients.8 There seems to be a correlation between mast cell
proliferation and the degree of bone marrow fibrosis in MPN patients.9 As part
of the myeloid lineage, mast cells can also harbor MPN specific mutations such
as the JAK2V617F substitution leading to an atypical phenotype and function.10
Similar results could also be replicated in JAK2 V617F transgenic mice.11
Furthermore, these mast cells appear *more active* compared to healthy
controls. Mast cells in the bone marrow of MPN patients produce more TGF-* and
IL-13 in vitro12, and skin mast cells of MPN patients are more easily activated
compared with healthy controls13.
Clinically, the numerical and functional aberrancy of mast cells can contribute
to symptoms commonly afflicting MPN patients such as fatigue and pruritus.14
Especially severe pruritus has been described to have a significant impact on
the quality of life of MPN Patients.
Ruxolitinib has been shown to inhibit mast cell degranulation in vitro. 16 It
has been shown to effectively reduce the symptom burden of MPN patients in
clinical trials.17,18 It even has been described to ameliorate symptoms in
systemic mastocytosis, thereby indicating a relevant effect on atypical mast
cells.19,20
In spite of the evidence suggesting a role of mast cells in fibrogenesis,
information on the effect of Ruxolitinib on mast cells is scarce. We
hypothesize that mast cells are a source of proinflammatory and profibrotic
cytokines in the bone marrow, and that Ruxolitinib works at least partly via
inhibition of mast cell activation.

Taken together, this study will allow us to map the fibrotic alterations in the
hematopoietic niche over the course of therapy in detail and it will enable us
to investigate mast cells as therapeutic target and potential driver of
myelofibrosis.

The primary objective is to investigate the role of the hematopoeitic niche and
mast cells in the bone marrow of MPN, and the effect of pharmacotherapy on
these cells.

This is an observational, case-control study performed at the hematology
department of the Erasmus MC, IJsselland hospital and Albert Schweitzer
hospital. The duration of follow-up will be 3 months, with 2 contact moments
for the collection of data (at inclusion and at 3 months).

We will include 62 patients with suspected MPN, or previously diagnosed MPN who
did not receive treatment for the MPN until the moment of inclusion.
Data will be collected at two time points: at inclusion and after three months.
For the most main research objectives, we will compare subgroups of patients
(e.g. with/without pruritus, or with/without JAK2 mutation). Furthermore, the
data obtained at the moment of inclusion will be compared with the data
obtained after 3 months. Based on a retrospective search of the MPN cohort of
the Erasmus MC in the last three years, we expect that approximately 50% of
patients will receive ruxolitinib.

Most of the investigations described above (visits to the outpatient clinic,
venapunctions, and the first bone marrow punction) are part of the normal
work-up of MPN and therefore do not form an extra burden to the participants.
The second bone marrow punction and the two questionnaires do form a small
study-related burden. The risks of participation are negligible.