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Macrophage-derived cytokines are crucially involved in myelofibrosis by supporting the differentiation of mesenchymal stromal cells to profibrotic myofibroblasts, a key step in bone marrow (BM) fibrosis. Investigating the function of such tissue-resident macrophages in subtypes of MPN may help to better understand the development of BM fibrosis and could reveal potential cellular targets for therapy.
Classical MPN includes Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML), characterized by BCR-ABL1 translocation, and essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) as the three main subtypes of Philadelphia chromosome-negative (Ph−) MPN, which are driven by JAK-STAT signaling. A study recently published in Annals of Haematology by Molitor et al.1 explored the abundance of macrophages in BM from patients with these subtypes of MPN using the surrogate macrophage markers CD68 and CD163.
The study analyzed BM biopsies from 61 patients, comprising:
BM sections were examined using confocal multiplex microscopy for immunohistochemistry analysis. The frequency of CD68 single-, CD163 single-, and CD68/CD163 double-positive cells was quantified as a percentage of the total number of nucleated cells.
Table 1. Patient characteristics1
BM, bone marrow; CML, chronic myeloid leukemia; ET, essential thrombocythemia; MF, myelofibrosis; PMF, primary myelofibrosis; PV, polycythemia vera; WBC, white blood cells. |
||||||
Characteristic |
PMF |
PV |
ET |
CML |
Control BM |
|
---|---|---|---|---|---|---|
Median age, years (range) |
63 (40–82) |
59.5 (17–77) |
64.5 (21–81) |
58 (31–78) |
59 (22–81) |
|
Male, n (%) |
11 (61) |
6 (50) |
6 (43) |
5 (46) |
5 (63) |
|
MF grade, n (%) |
|
|
|
|
|
|
0 |
1 (6) |
5 (42) |
13 (93) |
1 (11) |
7 (88) |
|
1 |
10 (56) |
5 (42) |
1 (7) |
8 (89) |
1 (13) |
|
2 |
2 (11) |
2 (17) |
0 (0) |
0 (0) |
0 (0) |
|
3 |
5 (28) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|
Mutation, n (%) |
|
|
|
|
|
|
BCR-ABL1 |
0 (0) |
0 (0) |
0 (0) |
9 (100) |
0 (0) |
|
JAK2 V617F |
15 (83) |
11 (92) |
12 (86) |
0 (0) |
0 (0) |
|
CALR |
2 (11) |
1 (8) |
2 (14) |
0 (0) |
0 (0) |
|
MPL W515 |
1 (6) |
0 (0) |
0 (0) |
0 (0) |
0 (0) |
|
Mean hemoglobin, g/dL (range) |
12.2 |
16.2 |
14.0 |
10.8 |
11.6 |
|
Mean platelets, ×109/L (range) |
692.9 |
477.3 |
862.2 |
232.0 |
108.0 |
|
Mean WBC, ×109/L (range) |
14.3 |
15.6 |
10.0 |
146.5 |
9.8 |
|
As summarized in Table 2, staining with anti-CD68 and anti-CD163 revealed the following:
Table 2. Mean percentage of CD68- and CD163-positive cells per all nucleated cells within BM spaces1
BM, bone marrow; CML, chronic myeloid leukemia; ET, essential thrombocythemia; PMF, primary myelofibrosis; PV, polycythemia vera. |
||||
Subtype |
CD68 |
CD163 |
||
---|---|---|---|---|
Mean frequency, % |
p value (when compared to PMF) |
Mean frequency, % |
p value (when compared to PMF) |
|
PMF (n = 18) |
27 |
— |
28 |
— |
PV (n = 12) |
13 |
< 0.001 |
8 |
< 0.001 |
ET (n = 14) |
6 |
< 0.001 |
7 |
< 0.001 |
CML (n = 9) |
2 |
0.017 |
1 |
0.017 |
Control BM (n = 8) |
5 |
< 0.001 |
2 |
< 0.001 |
Morphologically, in Ph− MPN, CD68- and CD163-positive macrophages had an irregular stellate shape with slender cytoplasmic processes, whereas in CML, macrophages were not only sparser but more ovaloid in appearance.
CD68 and CD163 were frequently co-expressed in macrophages across all MPN types, although some cells only expressed CD68 or CD163 (Table 3).
Table 3. Mean percentage of CD68 and CD163 single- or double-positive cells, per all nucleated cells1
CML, chronic myeloid leukemia; ET, essential thrombocythemia; PMF, primary myelofibrosis; PV, polycythemia vera. |
|||
|
CD68 single-positive, % |
CD163 single-positive, % |
CD68/CD163 double- positive, % |
---|---|---|---|
PMF |
60 |
62 |
53 |
PV |
42 |
80 |
40 |
ET |
56 |
70 |
52 |
CML |
18 |
26 |
16 |
Frequencies of CD68- and CD163-positive cells did not correlate with severity of myelofibrosis when looking at the entire study population. Although data indicated some correlation when analyzing PMF and ET separately, only limited conclusions could be drawn as the majority of these PMF cases had MF Grade 1 (10/11), whereas ET cases mostly had MF Grade 0 (13/14).
The comparison of macrophage frequencies in different MPN subtypes found CD68- and CD163-positive cells to be more abundant in PMF than in all other MPN subtypes and control BM, showing a gradual decline in levels from PMF to PV to ET. Furthermore, in Ph− MPN, CD68 and CD163 were often co-expressed in macrophages with stellate morphology, in a sponge-like reticular network.
Macrophages play a role in the differentiation of mesenchymal stromal cells into myofibroblasts and the development of myelofibrosis, which is associated with poor prognosis in MPN. This study showed that the abundance of macrophages differs between Ph+ MPN and Ph− MPN, and between subtypes of Ph− MPN, with the highest frequencies observed in PMF, followed by PV and ET. The stellate morphology of macrophages seen in Ph− MPN, and the resulting sponge-like reticular network, may explain the unbalanced hematopoiesis in the BM space, suggesting that the monocyte/macrophage system contributes to the disease process and may be a potential target for novel therapeutics.
Furthermore, it may be possible to use CD68 or CD163 expression to distinguish between these subtypes at an early stage. The authors acknowledge further work is needed to confirm this finding and to clarify whether it is the MPN subtype or grade of fibrosis that is associated with macrophage frequency.
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