The Bone Marrow Stroma in Myeloproliferative Neoplasms. Dr Bridget S Wilkins Consultant Haematopathologist St Thomas Hospital, London

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1 The Bone Marrow Stroma in Myeloproliferative Neoplasms Dr Bridget S Wilkins Consultant Haematopathologist St Thomas Hospital, London

2 Normal BM Stromal Components - - Cells - - Mesenchymal origin: Adipocytes (Myo)fibroblasts Endothelium Osteoblasts Osteoclasts Osteocytes Haemopoietic origin: Macrophages Mast cells Plasma cells Lymphocytes

3 Normal BM Stromal Components - - Fibres and Matrix - - Fibres: Reticulin Collagen Osteoid Non-fibre matrix: Proteoglycans Tenascin N-CAM (CD56)

4 Stromal Macrophages CD68R (PG-M1)

5 CD68R (PG-M1)

6 CD68R (PG-M1)

7 Normal Stromal Reticulin

8 Reactive Changes in BM Stroma Oedema Gelatinous change Increased reticulin fibres Increased plasma cells Increased mast cells Increased phagocytic activity in stromal macrophages Increased/decreased remodelling of existing trabecular bone New bone formation (neo-osteogenesis)

9 Stromal oedema and gelatinous change

10 Stromal reticulin patterns in trephine sections Grade 1 = WHO 0 Grade 2 = WHO 0 or 1 Grade 3 = WHO 2 Grade 4 = WHO 3

11 PT-1: Overall survival by reticulin 1.00 grade Overall survival Reticulin 0-1 Reticulin 2 Reticulin 3-4 p=0.10 Multivariate Time from trial entry (years)

12 PT-1: Myelofibrotic transformation by reticulin grade Myelofibrosis-free survival Reticulin 0-1 Reticulin 2 Reticulin Time from trial entry (years) p= Multivariate

13 L-NGFR looks like Retculin G3

14 Reticulin (Gomori) looks G3/G4 mix

15 Normal Trabecular Bone

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18 Fibrous tunnelling of trabeculae: renal osteodystrophy

19 Neo-osteogenesis (interstitial) Giemsa ph 5.0

20 Neo-osteogenesis: appositional on devitalised bone

21 Prominent Trabecular Cement Lines

22 Neo-osteogenesis (appositional) Giemsa ph 5.0

23 Osteoblasts and an Osteoclast

24 Stromal Lipid Granuloma

25 Pericapillary Plasmacytosis CD138

26 What is the stroma like in PV?

27 PV L-NGFR

28 PV CD34

29 What does it look like in ET?

30 What does it look like in PMF?

31 Advanced PMF

32 Pre-fibrotic PMF

33 Pre-fibrotic PMF L-NGFR

34 Established (fibrotic) PMF

35 Fibrotic PMF L-NGFR

36 Fibrotic PMF L-NGFR

37 Fibrotic PMF Reticulin

38 Fibrotic PMF CD34

39 Fibrotic PMF CD34

40 Megakaryocytes and Platelet Production CD61

41 How Platelets are Produced CD61

42 How Platelets are Produced

43 How Platelets are Produced CD61

44 Platelet Release Endothelium

45 How Platelets are Produced Cytoplasm is compartmentalised rather like segments of an orange Within each segment, platelet granules, contractile proteins and membrane components are assembled Transported to cell edge by microtubules Demarcation membranes develop at edge creating packets of components: Pro-platelets

46 Platelets and Bone Marrow Fibrosis Platelet-derived growth factors and cytokines are believed to be essential in development of stromal fibrosis in myelofibrosis PDGF, TGF-beta, FGFs, VEGF, PDEGF Platelets in MPN are fairly normal in composition and function, so Why doesn t normal bone marrow become fibrotic?

47 Why don t platelets normally cause bone marrow fibrosis? Platelets are created as streamers of inactive pro-platelets

48 Why don t platelets normally cause bone marrow fibrosis? Pro-platelets fragment into active platelets in the bloodstream

49 Why do platelets cause bone marrow fibrosis in MPN? Pro-platelets are released and/or activated prematurely in the marrow stroma

50 Platelet Storm in PMF

51 PMF - CD61

52 PV - CD61

53 What Happens to These? CD61

54 Why don t platelets normally cause bone marrow fibrosis? Normal stroma protects itself actively against fibrosis Fibroblasts and macrophages produce matrix metalloproteinases and other proteases that digest collagen fibres We currently know little about how these protective mechanisms are overwhelmed in MPN (e.g., absolute/relative increase in TIMPs)

55 Proplatelets in ET and PMF Visualised by immunohistochemistry plus confocal microscopy, proplatelet density is increased compared with normal bone marrow. Proplatelet density is greater in overtly fibrotic PMF than in pre-fibrotic PMF and ET Suggestion of more in pre-fibrotic PMF but not statistically significant Muth M, Büsche G, Bock O, Hussein K, Kreipe H. Leukemia Research

56 JAK2-V716F allele burden in different haemopoietic lineages Lineage penetrance of V617F in +ve patients varies between MPN subtypes Microdissection studies have confirmed that erythroid, granulocytic and megakaryocytic lineages may be involved in all subtypes but combinations vary Lineage penetrance is least in ET, but MK still involved in many cases Kreft A, et al. Virchows Archiv 459, 521-7

57 Angiogenesis in MPN Stroma Conventional studies of CD34+ve microvessel density show increases in MPN associated with increased VEGF expression and with extent of fibrosis Not correlated with JAK2-V617F status CD105+ve MVD correlated more closely with extent of VEGF expression and fibrosis, particularly in patients with high allele burden Medinger M, Skoda R, Gratwohl A,et al. Br J Haem 146,

58 Osteomyelofibrosis as End Stage of PMF (or post-et/pv MF) Why isn t this self-limiting?

59 Osteogenesis in Osteomyelofibrosis Not a simple linear progression of disease Cellular Fibrotic New bone Hypercellular PMF can have abundant new bone formation End-stage fibrotic disease may have none Sometimes accompanies aplastic marrow with extensive fat replacement of stroma Anecdotal examples of reversal posttransplantation

60 The End!