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Macrophage abundance in the bone marrow of patients with myeloproliferative neoplasms

Jan 26, 2021
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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.

Methods

The study analyzed BM biopsies from 61 patients, comprising:

  • 44 patients with Ph MPN, including 18 with PMF, 12 with PV, and 14 with ET
  • Nine patients with CML
  • Eight patients who underwent BM biopsy for other reasons (non-neoplastic controls)

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.

Patient characteristics

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
 (n = 18)

PV
(n = 12)

ET
(n = 14)

CML
(n = 9)

Control BM
(n = 8)

 

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
(7.7–15.8)

16.2
(14.2–19.5)

14.0
(11.6–16.3)

10.8
(7.3–14.2)

11.6
(9.0–14.1)

 

Mean platelets, ×109/L (range)

692.9
(95.0–2,023.0)

477.3
(236.0–1,107.0)

862.2
(600.0–1,465.0)

232.0
(27.0–374.0)

108.0
(82.0–618.0)

 

Mean WBC, ×109/L (range)

14.3
(2.1–36.2)

15.6
(4.9–32.5)

10.0
(4.1–20.0)

146.5
(47.2–368.0)

9.8
(3.5–14.2)

 

Results

Immunohistochemistry

As summarized in Table 2, staining with anti-CD68 and anti-CD163 revealed the following:

  • Significantly increased frequencies of CD68-positive and CD163-positive cells were seen in PMF samples when compared with PV, ET, CML, and control BM.
  • Significantly higher frequencies of CD68-positive cells in PV samples compared with ET, CML, and control BM samples.

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.

Confocal multiplex microscopy

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

Correlation with grade of myelofibrosis

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).

Key findings

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.

Conclusion

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.

  1. Molitor DCA, Boor P, Buness A, et al. Macrophage frequency in the bone marrow correlates with morphologic subtype of myeloproliferative neoplasm. Ann Hematol. 2021;100(1):97-104. DOI: 1007/s00277-020-04304-y

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