-1 (TSP-1) in primary myelofibrosis (PMF) - a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia Michaela Muth, Bianca M. Engelhardt, Nicolaus Kröger, Kais Hussein, Jérôme Schlué, Guntram Büsche, Hans H. Kreipe, Oliver Bock

To cite this version:

Michaela Muth, Bianca M. Engelhardt, Nicolaus Kröger, Kais Hussein, Jérôme Schlué, et al.. Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) - a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia. Annals of Hematology, Springer Verlag, 2010, 90 (1), pp.33-40. ￿10.1007/s00277-010-1024-z￿. ￿hal-00555318￿

HAL Id: hal-00555318 https://hal.archives-ouvertes.fr/hal-00555318 Submitted on 13 Jan 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Editorial Manager(tm) for Annals of Hematology Manuscript Draft

Manuscript Number: AOHE-D-10-00140R1

Title: Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) - a megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia

Article Type: Original Article

Keywords: biomarker; megakaryocytes; primary myelofibrosis;

Corresponding Author: Prof. Oliver Bock, M.D., Ph.D.

Corresponding Author's Institution: Hannover Medical School

First Author: Michaela Muth, Dipl.Biol.

Order of Authors: Michaela Muth, Dipl.Biol.; Bianca M Engelhardt; Nicolaus Kröger, M.D., Prof.; Jerome Schlue, M.D.; Guntram Büsche, M.D.; Hans H Kreipe, M.D., Prof.; Oliver Bock, M.D., Ph.D.

Abstract: Primary myelofibrosis (PMF) is a chronic myeloproliferative showing aberrant bone marrow remodelling with increased , progressive matrix accumulation and fibrosis development. Thrombospondins (TSP) are factors sharing pro-fibrotic and anti-angiogenic properties and have not been addressed in PMF before. We investigated the expression of TSP-1 and TSP-2 in PMF related to the stage of myelofibrosis (n = 51) and in individual follow-up biopsies by real-time PCR, immunohistochemistry and confocal laser scanning microscopy (CLSM). TSP-1 was significantly overexpressed (p < 0.05) in all stages of PMF when compared to a series of essential thrombocythemia (ET) and controls. Individual follow-up biopsies showed involvement of TSP-1 during progressive myelofibrosis. TSP-2 was barely detectable but 40% of cases with advanced myelofibrosis showed a strong expression. Megakaryocytes and interstitial proplatelet formations were shown to be the relevant source for TSP-1 in PMF. Stroma cells like endothelial cells and fibroblasts showed no TSP-1 labelling when double-immunofluorescence staining and CLSM were applied. Based on its dual function TSP-1 in PMF is likely to be a mediator within a pro-fibrotic environment which discriminates from ET cases. On the other hand TSP-1 is a factor acting (ineffectively) against exaggerated angiogenesis. Both features suggest TSP-1 to be a biomarker for monitoring a PMF targeted therapy.

Response to Reviewers: Reviewer #1: We appreciate this important comment. In order to expand findings on TSP-1 expression level in other myeloproliferative states, we included a series of Philadelphia- positive, BCR-ABL positive CML (n = 10) and CMML (n = 11). TSP-1 mRNA expression was not really higher in CML (median 2.5, range 0.3 - 8.6) and CMML (median 1.5, range 0.6 - 6.3) compared to control cases. Apart from some outliers which in CML might be attributable to the increase of the population of smaller (micro) megakaryocytes, we could underline the finding of a PMF-restricted overexpression of TSP-1. Since ET also shows megakaryocytes which are increased in size and numbers the high TSP-1 expression by megakaryocytes in PMF could stand for a relevant pathobiological feature. We added a revised Figure 1 and added some points to the Discussion section.

Reviewer #2: We are grateful for this reviewers comments and aimed to address the issues appropriately. 1. We correlate the individual TSP-1 expression with the following hematological parameters: number of platelets and splenomegaly Interestingly, we found that in PMF with manifest fibrosis (mf2/3) cases with a TSP-1 expression higher than the median also showed higher number of platelets. This phenomenon was restricted to manifest PMF although other PMF stages with higher TSP-1 expression also had have more outliers with a tendency to higher platelet numbers. Moreover, as expected PMF mf 0 showed significantly (p < 0.001) higher platelet counts than advanced PMF (mf 2/3). However, this significance was only demonstrable for the "=" subgroup. Due to the higher platelet counts advanced PMF with high TSP-1 mRNA level ("+" subgroup) did not significantly differ from PMF mf 0. We added a new Figure 4 and some points to the Discussion section. The presence of splenomegaly was not associated with a given TSP-1 mRNA expression level in PMF.

2. We highlighted the finding of TSP-1 mRNA increase in PMF as a discriminating feature from ET by the conclusion (2nd issue) at the end of the discussion section.

Authors' Response to Reviewers' Comments Click here to download Authors' Response to Reviewers' Comments: Comments to reviewer comments June 2010.doc

Dear Professor Barbui, Dear Sirs !

Thank you very much for consideration of our study entitled

“Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) - A megakaryocyte-derived biomarker which largely discriminates PMF from essential thrombocythemia”

We are grateful for the opportunity to revise and improve the initial version based on the valuable comments raised by the reviewers. Accordingly, we expanded the analysis on TSP-1 expression by inclusion of 2 other myeloproliferative conditions, i.e. CML and CMML (requested by reviewer #1) and we correlated TSP-1 expression with selected clinical/hematological characteristics of the patients (requested by reviewer #2). Table 1 was revised due to inclusion of clinical parameters of the CML and CMML groups. Moreover, we added some points to the Discussion section dealing with the options and limitations of TSP-1 as a biomarker.

Please find below a point-by-point list highlighting the changes/additional experiments requested by the reviewers. All changes or additional parts are highlighted in RED.

We would be happy if this revised version will be acceptable for publication in your Journal “Annals of Hematology”.

Sincerely yours, on behalf of all authors Oliver Bock

Reviewer #1: We appreciate this important comment. In order to expand findings on TSP-1 expression level in other myeloproliferative states, we included a series of Philadelphia-chromosome positive, BCR-ABL positive CML (n = 10) and CMML (n = 11). TSP-1 mRNA expression was not really higher in CML (median 2.5, range 0.3 – 8.6) and CMML (median 1.5, range 0.6 – 6.3) compared to control cases. Apart from some outliers which in CML might be attributable to the increase of the population of smaller (micro) megakaryocytes, we could underline the finding of a PMF-restricted overexpression of TSP-1. Since ET also shows megakaryocytes which are increased in size and numbers the high TSP-1 expression by megakaryocytes in PMF could stand for a relevant pathobiological feature. We added a revised Figure 1 and added some points to the Discussion section.

Reviewer #2: We are grateful for this reviewers comments and aimed to address the issues appropriately. 1. We correlate the individual TSP-1 expression with the following hematological parameters: number of platelets and splenomegaly Interestingly, we found that in PMF with manifest fibrosis (mf2/3) cases with a TSP-1 expression higher than the median also showed higher number of platelets. This phenomenon was restricted to manifest PMF although other PMF stages with higher TSP-1 expression also had have more outliers with a tendency to higher platelet numbers. Moreover, as expected PMF mf 0 showed significantly (p < 0.001) higher platelet counts than advanced PMF (mf 2/3). However, this significance was only demonstrable for the “=” subgroup. Due to the higher platelet counts advanced PMF with high TSP-1 mRNA level (“+” subgroup) did not significantly differ from PMF mf 0. We added a new Figure 4 and some points to the Discussion section. The presence of splenomegaly was not associated with a given TSP-1 mRNA expression level in PMF.

2. We highlighted the finding of TSP-1 mRNA increase in PMF as a discriminating feature from ET by the conclusion (2nd issue) at the end of the discussion section. Manuscript Click here to download Manuscript: Muth et al TSPs in PMF._Revised June 2010.doc Click here to view linked References

Muth et al. REVISED

1 2 3 Thrombospondin-1 (TSP-1) in primary myelofibrosis (PMF) 4 5 6 – a megakaryocyte-derived biomarker which largely discriminates 7 8 9 PMF from essential thrombocythemia 10 11 Michaela Muth1, Bianca M. Engelhardt1, Nicolaus Kröger#, Kais Hussein, 12 13 14 Jérôme Schlué, Guntram Büsche, Hans H. Kreipe, Oliver Bock* 15 16 Institute of Pathology, Hannover Medical School, 30625 Hannover, Germany 17 18 # Department of Stem Cell Transplantation, University Medical Center 19 20 21 Hamburg-Eppendorf, 20246 Hamburg, Germany 22 23 1These authors contributed equally to this work. 24 25 26 27 28 Category: Original article 29 30 31 Word count: Abstract: 205, Text: 3207 (w/o References) 32 33 Running title: Thrombospondins in primary myelofibrosis 34 35 36 37 38 The authors disclose any conflict of interest. 39 40 41 42 43 *Corresponding author: 44 45 Oliver Bock, M.D. 46 Professor 47 48 Institute of Pathology 49 Hannover Medical School 50 Carl-Neuberg-Strasse 1 51 30625 Hannover 52 53 Germany 54 Tel.: 0049-511-532-4501 55 Fax: 0049-511-532-5799 56 [email protected] 57 58 59 60 61 62 63 64 1 65 Muth et al. REVISED

Abstract 1 2 3 Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm showing 4 5 aberrant bone marrow remodelling with increased angiogenesis, progressive matrix 6 7 8 accumulation and fibrosis development. Thrombospondins (TSP) are factors sharing 9 10 pro-fibrotic and anti-angiogenic properties and have not been addressed in PMF 11 12 before. 13 14 15 We investigated the expression of TSP-1 and TSP-2 in PMF related to the stage of 16 17 myelofibrosis (n = 51) and in individual follow-up biopsies by real-time PCR, 18 19 20 immunohistochemistry and confocal laser scanning microscopy (CLSM). 21 22 TSP-1 was significantly overexpressed (p < 0.05) in all stages of PMF when 23 24 25 compared to a series of essential thrombocythemia (ET) and controls. Individual 26 27 follow-up biopsies showed involvement of TSP-1 during progressive myelofibrosis. 28 29 TSP-2 was barely detectable but 40% of cases with advanced myelofibrosis showed 30 31 32 a strong expression. Megakaryocytes and interstitial proplatelet formations were 33 34 shown to be the relevant source for TSP-1 in PMF. Stroma cells like endothelial cells 35 36 37 and fibroblasts showed no TSP-1 labelling when double-immunofluorescence 38 39 staining and CLSM were applied. 40 41 42 Based on its dual function TSP-1 in PMF is likely to be a mediator within a pro-fibrotic 43 44 environment which discriminates from ET cases. On the other hand TSP-1 is a factor 45 46 acting (ineffectively) against exaggerated angiogenesis. Both features suggest TSP-1 47 48 49 to be a biomarker for monitoring a PMF targeted therapy. 50 51 52 53 54 Keywords: biomarker, megakaryocytes, primary myelofibrosis, thrombospondins 55 56 57 58 59 60 61 62 63 64 2 65 Muth et al. REVISED

Introduction 1 2 3 Primary myelofibrosis (PMF) is a chronic myeloproliferative neoplasm with features 4 5 like clustering of atypical and enlarged megakaryocytes, exaggerated formation of 6 7 8 new vessels, abnormal progenitor cell trafficking, and the high risk to develop 9 10 manifest myelofibrosis [1]. 11 12 Megakaryocytes are known to harbour large amounts of different biologically active 13 14 15 factors [2]. Megakaryocytes in PMF are clonal and apparently responsible for a 16 17 majority of the reactive changes demonstrable in the aberrant bone marrow 18 19 20 architecture [3]. The bone marrow in PMF is thought to be pervade by numerous 21 22 megakaryocyte-derived growth factors and cytokines lastly leading to a reactive pro- 23 24 25 angiogenic and pro-fibrotic environment [4]. 26 27 Thrombospondins (TSP) were identified in -stimulated platelets and are 28 29 expressed by a variety of cells such as endothelial cells, fibroblasts and smooth 30 31 32 muscle cells [5]. TSP are so-called matricellular , i.e. they can interact with 33 34 cell surface receptors and with components of the extracellular matrix thereby 35 36 37 mediating cell-matrix interactions [6]. TSP-1 and -2 are trimers of about 145 kDa and 38 39 belong to one subgroup. TSP-3,-4,-5 are much smaller with about 100 kDa in size. 40 41 42 The latter are not fully investigated yet and their roles might be distinct from TSP-1 43 44 and TSP-2. 45 46 An important pro-angiogenic factor in PMF is VEGF [7, 8] whereas factors with anti- 47 48 49 angiogenic properties in PMF have not been investigated yet. Two members of the 50 51 TSP family, i.e. TSP-1 and TSP-2, are mediators of an anti-angiogenic response. 52 53 54 TSP-1 was shown to prevent the formation of new vessels by inhibition of VEGF and 55 56 stromal cell derived factor 1 (SDF-1), [9]. However, there is some experimental 57 58 59 evidence suggesting that TSP-1 can also promote angiogenesis in vitro. This 60 61 62 63 64 3 65 Muth et al. REVISED

promotion could be correlated with the action of active matrix-metalloproteinase 9 1 2 (MMP-9) and might depend on its concentration and the in vitro model used [10]. 3 4 5 Interestingly, TSP-1 is also known as an activator of TGF-1 which is the most 6 7 relevant inducer of fibroblast activation and matrix synthesis in the fibrotic response 8 9 10 and in PMF [11, 12]. TSP-1 also inhibits the activity of matrix-metalloproteinases 11 12 (MMPs) which are primarily involved in proteolysis of collagens and other matrix 13 14 15 components. From this point of view, any aberrant expression could contribute to a 16 17 particular hallmark in PMF, i.e. increased angiogenesis in case of TSP-1 down- 18 19 regulation and development of myelofibrosis when TSP-1 action is abrogated. 20 21 22 TSP are also involved in proper megakaryocyte development and platelet function, 23 24 i.e. TSP-2 deficiency was shown to impair the connection of megakaryocyte 25 26 27 proplatelet formations with bone marrow sinuses which led to exaggerated interstitial 28 29 fragmentation [13]. This study revealed that TSP-2 in megakaryocytes was almost 30 31 32 entirely the result of an uptake from the bone marrow microenvironment rather than 33 34 de novo synthesis. 35 36 37 We were interested in the expression level and cellular sources of TSP-1 and TSP-2 38 39 in PMF and aimed to get insights in the role of TSP in PMF-related changes of the 40 41 bone marrow architecture. In addition, TSP expression in essential thrombocythemia 42 43 44 (ET) as a MPN with very low risk of fibrosis development should be compared with 45 46 PMF and controls. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 4 65 Muth et al. REVISED

Material and Methods 1 2 3 Bone marrow study group 4 5 Formalin-fixed and paraffin-embedded (FFPE) bone marrow trephines with PMF 6 7 8 (n = 51), ET (n = 10), chronic myelomonocytic leukemia (CMML; n = 11) and chronic 9 10 myelogenous leukemia (CML; n = 10) diagnosed according to the World Health 11 12 Organization (WHO) criteria 2008 [14] were retrieved from the bone marrow registry 13 14 15 of the Institute of Pathology, Hannover Medical School. Three PMF groups were 16 17 established according to the presence and degree of myelofibrosis (MF) as 18 19 20 determined by silver impregnation (Gomori) according standard procedures [15]. 21 22 Accordingly, PMF cases were assigned to a group of hypercellular, prefibrotic PMF 23 24 25 (MF0, n = 20), a group showing demonstrable increase of reticulin fibres (MF1, n = 26 27 12), cases with advanced myelofibrosis and osteosclerosis (MF2/3, n = 19). 28 29 Individual PMF follow-up biopsies (n = 5, up to 4 core biopsies per case, median: 3 30 31 32 biopsies) ranging from 1 to 7 years (median 3 years) were also analyzed. Criteria of 33 34 selection for follow-up biopsies was that the first diagnostic biopsy should either show 35 36 37 no fibrosis (MF 0, n = 3) or already demonstrable myelofibrosis (MF 2-3, n = 2). 38 39 Biopsies of the control group (n = 12) were clinically indicated to exclude an 40 41 42 involvement by peripheral lymphoma (n = 7), metastasis of small cell lung carcinoma 43 44 (n = 1) or because of splenomegaly (n = 1), Budd-Chiari syndrome (n = 1) or 45 46 leukocytopenia (n = 2). All biopsies showed no evidence for a neoplasm or any other 47 48 49 disease and were diagnosed as “haematopoiesis in line with age”. 50 51 Quantification of potential mutant allele burden of JAK2 (V617F) and MPL (W515L/K) 52 53 54 in PMF was performed using Pyrosequencing® assays as described [16]. CML cases 55 56 all showed presence of BCR-ABL fusion . Clinical parameter of patients and 57 58 59 controls including molecular status of JAK2 and MPL are shown in Table 1. 60 61 62 63 64 5 65 Muth et al. REVISED

Real-time RT-PCR 1 2 RNA extracted from bone marrow core biopsies [17] was transcribed into the 3 4 5 complementary DNA by means of the High Capacity cDNA Reverse Transcription Kit 6 7 (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR was performed on 8 9 10 an ABI PRISM 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, 11 12 CA, USA) using the individual TaqMan Assays for thrombospondin 13 14 1 (THBS-1, Hs00962908_m1, 59bp amplicon), thrombospondin 2 (THBS-2, 15 16 17 Hs01568065_m1, 63bp amplicon) and the reference -GUS (-Glucuronidase, 18 19 GUSB, Hs99999908_m1, 81bp amplicon) and RNA polymerase 2A (POLR2A: 20 21 22 Hs00172187_m1, 61bp amplicon); all assays were purchased from Applied 23 24 Biosystems, Foster City, CA, USA. 25 26 27 28 29 Statistics 30 31 32 The sample- and detector-specific evaluation of amplification curves of the ABI 33 34 PRISM 7500 Fast Real-time PCR System were statistically analyzed with Prism 5.0 35 36 37 (GraphPad Software, San Diego, CA, USA) by applying the nonparametric Kruskal- 38 39 Wallis test followed by Dunn's post-test. P values ≤ 0.05 were displayed as 40 41 statistically significant. 42 43 44 45 46 47 Immunohistochemistry 48 49 50 Immunohistochemistry was performed using the ZytoChem-Plus HRP Polymer-Kit 51 52 and visualization was performed with the DAB Substrate Kit High Contrast kit (both 53 54 55 Zytomed Systems GmbH, Berlin, Germany). Counterstaining was accomplished with 56 57 haematoxylin. Bone marrow sections (2-3µm) were deparaffinised in xylol including 58 59 60 incubation with 3% H2O2 for 10 min, pre-treated in a pressure cooker (3 min, 125°C, 61 62 63 64 6 65 Muth et al. REVISED

in 10 mmol/l pH 6.0 citrate buffer) and incubated for 1 hour with a monoclonal mouse 1 2 anti-human Thrombospondin 1 antibody (1:100 dilution, [6], ab1823, abcam, 3 4 5 Cambridge, UK) and a monoclonal mouse anti-human Thrombospondin-2 antibody 6 7 (1:200 dilution, MAB1635, R&D systems). Kidney tissue was used as the TSP-1 8 9 10 positive control. TSP-2 stained colon carcinoma cells in a hepatic metastasis which 11 12 was used as the positive control. 13 14 15 Double-immunofluorescence staining 16 17 18 For double-immunofluorescence staining FFPE sections (2-3μm) were processed as 19 20 21 shown for conventional immunohistochemistry with the TSP-1 antibody and by 22 23 application of a CD105/ antibody (1:50 dilution, RB-9291-P, Thermo 24 25 Scientific, Fremont, CA, USA). Visualization was achieved by incubation of the 26 27 TM 28 secondary antibody for 30 minutes (1:100 dilution, Cy 2-conjugated AffiniPure F 29 30 (ab') Fragment Donkey Anti Mouse IgG (H+L), 715-226-150, dianova, Hamburg, 31 2 32 TM 33 Germany; 1:100 dilution, Cy 3-conjugated AffiniPure F(ab')2 Fragment Donkey Anti 34 35 Rabbit IgG (H+L), 711-166-152, dianova, Hamburg, Germany). Nuclear 36 37 38 counterstaining was achieved by staining with Hoechst blue. An isotype control 39 40 monoclonal/ polyclonal antibody (1:100 dilution, negative control for mouse IgG1 Ab- 41 42 43 1, clone NCG01, DLN-05791, dianova, Hamburg, Germany; 1:100 dilution, negative 44 45 control for rabbit IgG Ab-1, DLN-13121, dianova, Hamburg, Germany) was used as 46 47 negative control in lieu of the primary antibody. 48 49 50 51 Confocal laser-scan microscopy (CLSM) 52 53 54 Bone marrow sections were investigated by using a confocal laser scanning 55 56 microscope (Leica DM IRB with a TCS SP2 AOBS scan head, Leica, Heidelberg, 57 58 Germany, provided by the core facility for laser microscopy at the Hannover Medical 59 60 61 School; MHH). The microscope is equipped with a 405nm laser for excitation of blue 62 63 64 7 65 Muth et al. REVISED

dyes, a 543nm laser for red dyes and a 488nm laser for excitation of green dyes. 1 2 Images were recorded in a resolution of a 40x objective and analysed by using the 3 4 5 Leica Confocal Software (LCS-Lite 2.61; Leica, Heidelberg, Germany). 6 7 8 9 10 Results 11 12 13 Overexpression of TSP-1 in all stages of disease 14 15 TSP-1 mRNA was overexpressed in all stages of disease independently of the 16 17 18 degree of myelofibrosis. Cases showing no demonstrable fibre increase 19 20 overexpressed TSP-1 by up to 39.0-fold (median 4.0, range 0.8 – 39.0), PMF with 21 22 23 increase of reticulin (MF1) showed overexpression by up to 48.0-fold (median 3.9, 24 25 range 0.6 – 48.0). PMF with manifest myelofibrosis showed TSP-1 overexpression by 26 27 up to 32.0-fold (median 3.7, range 0.6 – 31.7). All MF stages in PMF showed 28 29 30 significantly higher TSP-1 mRNA (p < 0.05) compared to haematopoiesis in line with 31 32 age (median 1.2, range 0.2 – 6.0), Figure 1. An underlying correlation of TSP-1 33 34 35 expression with the molecular status, i.e. presence of JAK2 (V617F) and MPL 36 37 (W515L) could not be found. Apart from some outliers CMML (median 1.5, range 0.6 38 39 40 – 6.3) and CML (median 2.5, range 0.3 – 8.6) showed no demonstrable TSP-1 41 42 overexpression. There was also no significant difference compared to 43 44 haematopoiesis in line with age. 45 46 47 48 49 TSP-2 is overexpressed in advanced PMF 50 51 52 TSP-2 mRNA was overexpressed only in PMF cases in the advanced stage 53 54 (n = 15, median 9.1, range 1.4 – 84.0) compared to hypercellular, prefibrotic PMF 55 56 57 (n = 16, median 2.1, range 0.1 – 4.9, p < 0.01), ET (n = 9, median 1.2, range 58 59 0.2 – 6.9, p < 0.01) and control cases (n = 9, median 1.3, range 0.1 – 6.0, p < 0.01), 60 61 62 Figure 2. All in all the absolute expression level of TSP-2 (displayed in threshold 63 64 8 65 Muth et al. REVISED

cycles by real-time PCR) was rather weak in PMF and controls. No correlation of 1 2 TSP-2 expression with the presence of JAK2 (V617F) and MPL (W515L) could be 3 4 5 demonstrated. 6 7 We also tested CML and CMML for TSP-2 mRNA expression. However, the yield of 8 9 10 detectable signals was low, i.e. only 20% of the cases showed a notable signal. We 11 12 therefore resigned from further investigation and illustration of results of TSP-2 13 14 mRNA in CML and CMML (data not shown). 15 16 17 18 19 Individual courses in PMF showed involvement of TSP-1 in progressive 20 21 22 myelofibrosis 23 24 We monitored the expression level of TSP-1 mRNA in 5 individual courses of PMF 25 26 27 showing progressive remodeling of the bone marrow, i.e. changes in the degree of 28 29 myelofibrosis. All courses showed considerable TSP-1 dynamics during development 30 31 of myelofibrosis (#3, #4, #5) or ongoing matrix remodeling (#2), respectively (Figure 32 33 34 3). Follow-up #1 started with an exaggerated TSP-1 overexpression of 29-fold at 35 36 MF 0 and showed a 12-fold overexpression when MF 2 was demonstrable. Of note, 37 38 39 this case rapidly evolved to fibrosis within 1 year. #5 showed an increase of up to 5- 40 41 fold but in the end of only 2-fold during development of MF 1 in the 7 year follow-up. 42 43 44 All other courses lastly retained TSP-1 overexpression of up to 3-fold (#2) and 13-fold 45 46 (#3, #4, respectively) when compared to the control group. 47 48 49 50 51 Clinical correlates showed the association of increased TSP-1 mRNA level with 52 53 higher platelet counts in advanced PMF 54 55 56 Depending on the degree of myelofibrosis PMF cases with TSP-1 mRNA expression 57 58 level above the median (+) were separated from cases which expressed TSP-1 59 60 61 similar to the median or below (=). In advanced PMF high TSP-1 mRNA level were 62 63 64 9 65 Muth et al. REVISED

found to be associated with likewise higher platelet counts (median 366, range 241 - 1 2 1348 in the + group versus 161, range 94 - 427 in the = group). Apart from higher 3 4 5 outliers the + groups in other mf degrees (mf 0, median 978, range 587 – 1936; mf 1, 6 7 median 605, range 373 - 1275) showed no such demonstrable difference when 8 9 10 compared to the respective = group (mf 0, median 968, range 605 – 1305; mf 1, 11 12 median 573, range 108 – 697). As expected PMF mf 0 showed significantly higher 13 14 platelet counts than PMF mf 2/3 did (p < 0.001) but this only hold true for the 15 16 17 subgroup with similar or lower TSP-1 mRNA level (=) but not for the + group, Figure 18 19 4. 20 21 22 23 24 The expression of TSP-1 in PMF is restricted to megakaryocytes, proplatelet 25 26 27 formations and platelet deposits 28 29 Immunohistochemistry showed that the entire megakaryocytic lineage was strongly 30 31 immunoreactive for TSP-1 in PMF. In contrast to normal bone marrow which also 32 33 34 showed TSP-1 positive megakaryocytes adjacent to sinusoids and endothelial cells 35 36 (Figure 5 E) PMF displayed high numbers of interstitially located TSP-1 positive 37 38 39 megakaryocytes and platelet deposits (Figure 5 A). Of note, long pseudopodia and 40 41 large proplatelet formations of PMF megakaryocytes were nicely demonstrable by 42 43 44 TSP-1 staining (Figure 5 C). ET showed TSP-1 positive megakaryocytes but failed to 45 46 exhibit likewise positive proplatelet formations and deposition (Figure 5 D). No TSP-1 47 48 49 labeling was demonstrable in endothelial cells and in fibroblasts in areas of matrix 50 51 accumulation in PMF (Figure 5 A + B, respectively). 52 53 Double-immunofluorescence staining using the TSP-1 antibody and a 54 55 56 CD105/endoglin antibody specific for endothelial cells confirmed that TSP-1 and 57 58 CD105/endoglin did not co-localize in endothelial cells (Figure 6). TSP-1 was 59 60 61 62 63 64 10 65 Muth et al. REVISED

exclusively demonstrable in megakaryocytes and their progenies. CD105/endoglin 1 2 stained the exaggerated angiogenesis in PMF. 3 4 5 TSP-2 expression was heterogeneous with regard to staining pattern and 6 7 intensity. TSP-2 staining in PMF and controls showed megakaryocytes which 8 9 10 exhibited a weak labeling and others which were unlabelled. Since this pattern was 11 12 demonstrable on the same slide staining failures can be excluded. A prominent 13 14 difference as shown for TSP-1 staining was not demonstrable by TSP-2 15 16 17 immunohistochemistry (therefore not shown). 18 19 20 21 22 23 24 25 Discussion 26 27 Megakaryocytes and platelets normally produce large amounts of pro-angiogenic 28 29 30 factors allowing proliferation of endothelial cells in vitro and in vivo. However, 31 32 platelets also produce factors capable for counteraction against the formation of new 33 34 35 vessels such as platelet-factor 4 or thrombospondins, i.e. most notably TSP-1. A 36 37 physiologically fine tuned pro- and anti-angiogenic balance apparently ensures the 38 39 40 homeostasis essential for proper vascularization. 41 42 TSPs were shown to exhibit potent anti-angiogenic properties, i.e. cells deficient in 43 44 TSP-1 and -2 stimulate angiogenesis to a much greater extent than their normal 45 46 47 counterparts [9]. The onset of angiogenesis in often involves down-regulation 48 49 of endogenous TSP-1 by ras mutated cancer cells and also in adjacent non- 50 51 52 transformed fibroblasts but not in endothelial cells [18]. 53 54 Studies with TSP-1 knock out mice revealed that after myelosuppression these 55 56 57 animals quickly recovered from thrombocytopenia compared to wild-type animals. 58 59 This fact might be attributable to the concomitantly increased angiogenesis and 60 61 62 vessel density in the bone marrow which presented a 2-fold higher number of 63 64 11 65 Muth et al. REVISED

megakaryocytes in the knock out model. Subsequently, platelet numbers increased 1 2 more rapidly and thrombopoiesis was restored also more quickly. Another study 3 4 5 using TSP-2 knout out mice also showed that the pro-angiogenic effects in this model 6 7 were mediated by enhanced MMP-9 action [19]. 8 9 10 A hallmark in bone marrow pathology of PMF is the population of enlarged and 11 12 clustered megakaryocytes most likely responsible for progressive myelofibrosis and 13 14 exaggerated angiogenesis. The latter is already overt in early prefibrotic stages and 15 16 17 apparently contributes to an easy escape of progenitor cells into the periphery to 18 19 colonize distant organs such as spleen. 20 21 22 We hypothesized that TSP might be aberrantly expressed in PMF, i.e. decreased 23 24 expression could explain a contribution to 2 typical PMF features, the high number of 25 26 27 megakaryocytes and the high vessel density in PMF bone marrow. 28 29 On the other side, TSP-1 was shown to activate latent TGF-1 [20], the most 30 31 32 effective pro-fibrotic factor involved in the development of myelofibrosis and other 33 34 fibrotic states [21]. In addition, TSP-1 up-regulates TIMP-1 [22], the tissue inhibitor of 35 36 37 metalloproteinases, whose overexpression might favor matrix accumulation and 38 39 fibrosis. 40 41 We recently found by an explorative low density array profiling that TSP-1 (THBS1) 42 43 44 was overexpressed in total bone marrow cells of advanced stage PMF [23]. The 45 46 interesting action profile attracted us to study the TSP in PMF in more detail. 47 48 49 In the present study, TSP-1 was significantly overexpressed by bone marrow cells in 50 51 PMF independently of the stage of disease, i.e. the degree of myelofibrosis. This 52 53 54 feature discriminated from ET cases which also showed megakaryocytes increased 55 56 in size and numbers but lacked up-regulated TSP-1. 57 58 To test for TSP-1 in other MPN or associated MPN/MDS, we selected CML and 59 60 61 CMML cases. Apart from some outliers neither CML nor CMML showed 62 63 64 12 65 Muth et al. REVISED

demonstrable higher or lower levels when compared to PMF, ET and controls. The 1 2 overall stronger TSP-1 mRNA expression in CML might be attributable to the 3 4 5 increase of (micro-) megakaryocytes demonstrable in some of these cases. 6 7 When we looked for clinical parameters and TSP-1 mRNA expression level in PMF 8 9 10 subgroups we found that interestingly in advanced stages those cases with high 11 12 TSP-1 mRNA expression (labeled as “+” in Figure 4) also had have higher platelet 13 14 counts. As expected PMF mf 0 showed significantly higher platelet counts than 15 16 17 advanced stages did. However, this only hold true for the “=” subgroup of mf 2/3 18 19 cases with only moderately increased TSP-1 mRNA level. 20 21 22 TSP-1 protein was exclusively present in megakaryocytes in PMF including large 23 24 proplatelet formations and platelet aggregates within the bone marrow interstitium. 25 26 27 These proplatelet formations in our study group showed no association with vessels 28 29 or endothelial gaps. This accumulation apart from vessels suggests a potential key 30 31 role in myelofibrosis development because of the likelihood that due to fragmentation 32 33 34 high amounts of pro-fibrotic factors could accumulate in the bone marrow as well. By 35 36 contrast, a sufficient anti-angiogenic effect through TSP-1 is hard to imagine because 37 38 39 exaggerated angiogenesis is overt in all stages and TSP-1 is apparently not acting 40 41 on endothelial cells, at least TSP-1 was not detectable by 2 different morphological 42 43 44 and staining approaches. By double-immunofluorescence labeling TSP-1 was only 45 46 present in the megakaryocytes and progenies such as large proplatelet formations 47 48 49 but not in endothelial cells (CD105 positive). TSP-1 positive proplatelets are in good 50 51 agreement with a previous study which detected TSP-1 in the demarcation 52 53 membrane of thrombin-stimulated human megakaryocytes [24]. The demarcation 54 55 56 membrane is the intracellular precursor formation of pseudopodia which later mediate 57 58 the continuity with the extracellular space and also delivery of platelets [25]. 59 60 61 62 63 64 13 65 Muth et al. REVISED

Interestingly, in a previous study with mice knocked out for TSP-2 these animals 1 2 failed to show a proper interaction of megakaryocytic pseudopodia and bone marrow 3 4 5 sinuses to deliver proplatelets and platelets [13]. In our study TSP-2 mRNA was only 6 7 barely detectable except for some high outliers in advanced PMF. TSP-2 protein 8 9 10 detection was only demonstrable in some megakaryocytes in both PMF and normal 11 12 haematopoiesis but technical issues can not be excluded with certainty as the reason 13 14 for this heterogeneity. However, it was found that megakaryocytes in vitro show an 15 16 17 up-take of extracellular TSP-2 but no intracellular de novo synthesis [13]. If so, TSP-2 18 19 positive megakaryocytes in PMF and controls are biologically different in their current 20 21 22 state of TSP-2 up-take and heterogeneous staining pattern was not artificial. 23 24 Nevertheless, TSP-2 in our study did not sufficiently reveal its secrets in PMF 25 26 27 pathology. 28 29 In summary, the abundance of TSP-1 in PMF has presumably 3 relevant issues: 30 31 1. A pro-fibrotic effect is likely, and explainable by a megakaryocytic-derived local 32 33 34 accumulation with subsequent activation of latent TGF-1, increased matrix synthesis 35 36 37 and inhibition of MMP activity. 2. The strictly morphological based diagnosis of 38 39 hypercellular, prefibrotic PMF versus ET could be supported by TSP-1 expression 40 41 analysis when larger cohorts will be successfully tested for reliability of this marker in 42 43 44 the diagnostic setting, 3. The proposed but functionally complex effect of TSP-1 on 45 46 endothelial cells might be aberrant or insufficient in PMF due to overweight of other 47 48 49 pro-angiogenic factors. However, TSP-1 could nevertheless be an excellent indicator 50 51 of increased angiogenesis. Accordingly, our data fit to the recent observation that in 52 53 54 tumor bearing mice TSP-1 is obviously a biomarker for the increased tumor 55 56 angiogenesis indicating the individuals attempt to counteract against new vessel 57 58 formation [26]. We conclude that TSP-1 is a promising factor in PMF useful to be 59 60 61 considered as a biomarker for monitoring potentially targeted therapy. 62 63 64 14 65 Muth et al. REVISED

1 2 3 Acknowledgements 4 5 The authors are indebted to Ms. Sabine Schröter for her skillful technical assistance. 6 7 8 The funding and grant support by the Deutsche Forschungsgemeinschaft 9 10 (BO1954/1-2; O.B., H.K.) and the Bundesministerium für Bildung und Forschung – 11 12 13 (O.B.) is gratefully acknowledged. 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 15 65 Muth et al. REVISED

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22. John, A.S., Hu, X., Rothman, V.L., Tuszynski, G.P. (2009) Thrombospondin-1 1 2 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) 3 4 5 production in human tumor cells: exploring the functional significance in tumor 6 7 cell invasion. Exp Mol Pathol 87(3):184-8. doi:10.1016/j.yexmp.2009.09.002 8 9 10 23. Bock, O., Muth, M., Theophile, K., Winter, M., Hussein, K., Busche, G., 11 12 Kroger, N., Kreipe, H. (2009) Identification of new target molecules PTK2, 13 14 TGFBR2 and CD9 overexpressed during advanced bone marrow remodelling 15 16 17 in primary myelofibrosis. Br J Haematol 146(5):510-20. doi: 10.1111/j.1365- 18 19 2141.2009.07808.x 20 21 22 24. Cramer, E.M., Masse, J.M., Caen, J.P., Garcia, I., Breton-Gorius, J., Debili, N., 23 24 Vainchenker, W. (1993) Effect of thrombin on maturing human 25 26 27 megakaryocytes. Am J Pathol 143(5):1498-508. 28 29 25. Schulze, H., Korpal, M., Hurov, J., Kim, S.W., Zhang, J., Cantley, L.C., Graf, T. 30 31 and Shivdasani, R.A. (2006) Characterization of the megakaryocyte 32 33 34 demarcation membrane system and its role in thrombopoiesis. Blood 35 36 107(10):3868-75. doi: 10.1182/blood-2005-07-2755. 37 38 39 26. Zaslavsky, A., Baek, K.H., Lynch, R.C., Short, S., Grillo, J., Folkman, J., 40 41 Italiano, J.E. Jr., Ryeom, S. (2010) Platelet-derived thrombospondin-1 (TSP-1) 42 43 44 is a critical negative regulator and potential biomarker of angiogenesis. Blood 45 46 in press, doi: 10.1182/blood-2009-09-242065. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 19 65 Muth et al. REVISED

Table 1 Study group – clinical and molecular status 1 2 3 PMF MF 0 PMF MF 1 PMF MF ET Ph+ - Control 4 # 5 2/3 CML CMML group 6 20 12 19 10 10 11 12 7Group size 8 9 JAK2 (V617F) n = 10 n = 9 n = 13 n = 4 n.d. n.d. none 10 (50%) (75%) (68%) (40%) 11frequency 12 13 JAK2 (V617F) 29.4 33.7 53 18.7 - - - 14 (14.8-47.1) (7.1-63.3) (33.8-79.3) (11.7-28.5) 15mutant allele burden 16in % (median, range) 17 18 19MPL (W515L) n = 1 none n = 3 none n.d. n.d. none 20frequency 21 22 23MPL mutant allele (33.9) - (97.2, - - - - 47.1, 24burden (%) 45.3) 25 26 27Erythrocytes (106/µL) 4.6 4.6 3.9 3.5 4.3 3.4 4.2 (3.2-6.5) (2.8-7.2) (2.5-5.5) (2.3-4.7) (2.6-5.5) (2.3-4.3) (3.1-4.8) 28 29 30Hemoglobin (g/d L) 13.7 11.7 11 9.4 12.6 10.9 13.5 (10.3-16.8) (8.6-14.6) (7-15) (7.4-14) (6.8-16.2) (7.4-12.6) (13-14.3) 31 32 33Hematocrit (%) 41.3 38 32.5 30.7 36.8 32.4 40 (31.1-50.8) (28.2-51) (22-44.6) (19.8–43) (20.8-46.7) (21.6-38.8) (38.7-41.3) 34 35 36Leukocytes (103/µL) 10.6 11.2 14.9 13 37.4 8 6.3 (5-20) (5.2-16.2) (5-40) (3–22.2) (11.5-83.7) (4-18) (3.5-7.9) 37 38 39Platelets (103/µL) 962.6 590 320.5 498 572 157 243.4 (587-1936) (108-1275) (11.8-1348) (294-1081) (296-1500) (85-243) (58-352) 40 41 42Splenomegaly * 7/10/3 6/4/2 14/2/3 -/-/10 1/-/9 3/1/7 1/5/6 43(yes/no/not) 44 45Gender (M/F) 8/12 7/5 12/7 7/3 6/4 7/4 5/7 46 47 48Age (y) 68 68 72 70 55 66 53 (47-81) (48-78) (48-88) (67-74) (17-90) (39-80) (23-73) 49 50 51 52 F = Female; M = male; y = years; * = defined as clinically palpable. 53 Clinical data are given as mean with range in brackets. JAK2 (V617F) is given as median. 54 MPL (W515K) was not detectable in the study group. # = BCR-ABL positive as determined 55 by multiplex RT-PCR. 56 57 58 59 60 61 62 63 64 20 65 Muth et al. REVISED

Figure legends 1 2 3 Figure 1 4 5 TSP-1 mRNA was significantly overexpressed in all stages of PMF (MF0, MF1, MF2- 6 7 8 3) when compared with control bone marrow. Horizontal black bars represent median 9 10 of expression. Expression level in the control group was arbitrarily set to 1. * p < 0.05. 11 12 Figure 2 13 14 15 TSP-2 mRNA was overexpressed in the advanced stage of PMF (MF2-3) when 16 17 compared to PMF-MF0 (** p < 0.01), ET (** p < 0.01) and the control group (** p < 18 19 20 0.01). Horizontal white or black bars represent median of expression. The expression 21 22 level in the control group was arbitrarily set to 1. 23 24 25 Figure 3 26 27 Follow-ups of TSP-1 mRNA level in PMF during individual courses of disease. MF 28 29 dynamics are indicated by the arrow. Initial TSP-1 mRNA level at time point of 30 31 32 diagnosis was calculated relative to the mean expression level in the control group 33 34 (Figure 1). Different symbols represent individual follow-ups ranging from 1 to 7 35 36 37 years. 38 39 Figure 4 40 41 42 PMF cases with TSP-1 mRNA level above median were labeled as (+) and those 43 44 with TSP-1 mRNA level similar or below the median as (=). Based on the degree of 45 46 myelofibrosis the 2 groups (+, =) were tested for a potential relation of platelet counts 47 48 49 and TSP-1 mRNA level. Advanced PMF cases with higher TSP-1 mRNA level also 50 51 had higher platelet counts. 52 53 54 55 56 57 58 59 60 61 62 63 64 21 65 Muth et al. REVISED

Figure 5 1 2 Immunohistochemistry (DAB brown) with a monoclonal antibody against TSP-1 3 4 5 showed labeling of normal megakaryocytes in control bone marrow (5 E). In PMF 6 7 clustered megakaryocytes (5 A, * indicate sinuses) like streamed megakaryocytes 8 9 10 and large deposits of interstitially located proplatelets (5 B) showed strong staining. 11 12 TSP-1 positive pseudopodia and long megakaryocytic protrusions (5 C, white arrow) 13 14 were exclusively demonstrable in PMF bone marrows. ET showed marked TSP-1 15 16 17 labeling in megakaryocytes but lacked notable deposition of proplatelets and 18 19 pseudopodia formation (5 D). Stroma cells such as fibroblasts and endothelial cells 20 21 22 showed no TSP-1 reactivity in all slides under investigation. 23 24 25 26 27 Figure 6 28 29 Merged figure from confocal laser scanning microscopy and double- 30 31 immunofluorescence staining revealed TSP-1 (green) restricted to megakaryocytes 32 33 34 and platelets and CD105/endoglin (red) restricted to endothelium. No TSP-1 could be 35 36 detected in endothelial cells. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 22 65 Entire figure set

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