The Bone Marrow Stroma in Myeloproliferative Neoplasms Dr Bridget S Wilkins Consultant Haematopathologist St Thomas Hospital, London
Normal BM Stromal Components - - Cells - - Mesenchymal origin: Adipocytes (Myo)fibroblasts Endothelium Osteoblasts Osteoclasts Osteocytes Haemopoietic origin: Macrophages Mast cells Plasma cells Lymphocytes
Normal BM Stromal Components - - Fibres and Matrix - - Fibres: Reticulin Collagen Osteoid Non-fibre matrix: Proteoglycans Tenascin N-CAM (CD56)
Stromal Macrophages CD68R (PG-M1)
CD68R (PG-M1)
CD68R (PG-M1)
Normal Stromal Reticulin
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)
Stromal oedema and gelatinous change
Stromal reticulin patterns in trephine sections Grade 1 = WHO 0 Grade 2 = WHO 0 or 1 Grade 3 = WHO 2 Grade 4 = WHO 3
PT-1: Overall survival by reticulin 1.00 grade Overall survival 0.95 0.90 0.85 0.80 Reticulin 0-1 Reticulin 2 Reticulin 3-4 p=0.10 Multivariate 0.75 0 1 2 3 4 5 6 Time from trial entry (years)
PT-1: Myelofibrotic transformation by reticulin grade Myelofibrosis-free survival 1.00 0.95 0.90 0.85 0.80 0.75 Reticulin 0-1 Reticulin 2 Reticulin 3-4 0 1 2 3 4 5 6 Time from trial entry (years) p=0.0007 Multivariate
L-NGFR looks like Retculin G3
Reticulin (Gomori) looks G3/G4 mix
Normal Trabecular Bone
Fibrous tunnelling of trabeculae: renal osteodystrophy
Neo-osteogenesis (interstitial) Giemsa ph 5.0
Neo-osteogenesis: appositional on devitalised bone
Prominent Trabecular Cement Lines
Neo-osteogenesis (appositional) Giemsa ph 5.0
Osteoblasts and an Osteoclast
Stromal Lipid Granuloma
Pericapillary Plasmacytosis CD138
What is the stroma like in PV?
PV L-NGFR
PV CD34
What does it look like in ET?
What does it look like in PMF?
Advanced PMF
Pre-fibrotic PMF
Pre-fibrotic PMF L-NGFR
Established (fibrotic) PMF
Fibrotic PMF L-NGFR
Fibrotic PMF L-NGFR
Fibrotic PMF Reticulin
Fibrotic PMF CD34
Fibrotic PMF CD34
Megakaryocytes and Platelet Production CD61
How Platelets are Produced CD61
How Platelets are Produced
How Platelets are Produced CD61
Platelet Release Endothelium
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
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?
Why don t platelets normally cause bone marrow fibrosis? Platelets are created as streamers of inactive pro-platelets
Why don t platelets normally cause bone marrow fibrosis? Pro-platelets fragment into active platelets in the bloodstream
Why do platelets cause bone marrow fibrosis in MPN? Pro-platelets are released and/or activated prematurely in the marrow stroma
Platelet Storm in PMF
PMF - CD61
PV - CD61
What Happens to These? CD61
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)
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 2010 34 1424-9.
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
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, 150-157
Osteomyelofibrosis as End Stage of PMF (or post-et/pv MF) Why isn t this self-limiting?
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
The End!