Absence of Tissue Factor Expression by Neoplastic Plasma Cells in Multiple Myeloma

ORIGINAL ARTICLE Absence of tissue factor expression by neoplastic plasma cells in multiple myeloma

G Cesarman-Maus1, E Braggio2, H Maldonado1 and R Fonseca2

Thrombosis portends a poor prognosis in individuals with solid tumors. Constitutive expression of tissue factor (TF) by cancer cells is a key in triggering activation of coagulation and promoting aggressive tumor behavior. Though multiple myeloma (MM) is associated with a high frequency of thrombosis in the context of thalidomide and lenalidomide therapy, prognosis is not affected by its occurrence. We sought to determine the expression of TF in MM. F3 (TF gene) expression proﬁling was analyzed in 55 human MM cell lines (HMCL) and in 223 solid tumor cell lines obtained from GlaxoSmithKline (GSK) Cancer Cell Line Genomic Proﬁling Dataset. TF was not expressed in any of the 55 HMCLs studied, in sharp contrast to solid tumors, 90% of which showed TF expression. F3 expression was also absent in tumor samples from 239 MM patients. Immunohistochemistry for TF was negative, with either no or focal (1 þ ) staining in 70/73 MM patients. Only three marrow biopsies were moderately (2 þ ) positive either focally or diffusely, suggesting that in rare cases bone marrow microenvironment may support TF expression. General lack of TF expression by neoplastic plasma cells may explain why thrombosis is not predictive of poor outcome, and why aspirin prophylaxis is often effective in MM.

Leukemia (2012) 26, 1671--1674; doi:10.1038/leu.2012.43 Keywords: myeloma; thrombosis; tissue factor

INTRODUCTION prevented by the use of prophylactic aspirin.10 Here, we sought to study the gene expression status of F3 (encoding the The occurrence of venous thromboembolism in individuals with TF protein) by microarray analysis in human myeloma cell lines solid epithelial tumors has been associated with a poor prognosis (HMCL) and bone marrow from MM patients, as well as the TF with more than three-fold higher risk of early death, as compared 1,2 protein expression in myeloma plasma cells in bone marrow with cancer patients without thrombosis. This detriment in biopsies, and to compare the results with solid tumor cell lines. survival is not only attributable to death from thrombotic complications such as pulmonary embolism. Thrombosis is itself, for many patients, an indicator of an underlying aggressive tumor behavior. There are several molecular links between the pro- MATERIALS AND METHODS thrombotic state and oncogenic events such as malignant Gene expression levels were analyzed in 223 solid tumor cell lines obtained transformation, angiogenesis, in vivo tumor growth and metas- from GlaxoSmithKline (GSK) Cancer Cell Line Genomic Profiling Dataset tasis. These include the upregulation of cyclo-oxigenase-2, (https://array.nci.nih.gov/caarray/project/woost-00041) and in 55 HMCLs plasminogen activator inhibitor 1, and tissue factor (TF) genes (Supplementary Table 1) using the Affymetrix HG-U133_Plus_2 array. The and proteins.3 In solid epithelial tumors, the expression of TF on expression level of F3 (the gene that encodes for TF) was transformed the cell surface membrane of cancer cells and on circulating using Plier16 algorithm and the probe intensities were log2 median- microparticles shed from these neoplastic as well as from non- normalized across all cell lines. Additionally, samples from 239 MM neoplastic cells has a key role in thrombosis and oncogenesis.4,5 patients were also studied for gene expression (data available at The mechanisms of thrombosis in individual with hematological www.broadinstitute.org/mmgp/home). All patients provided appropriate malignancies may, however, differ from those with solid tumors. informed consent. Thrombosis is a frequent feature in individuals with multiple Immunohistochemistry (IHC) staining for TF was performed using standard myeloma (MM), particularly those receiving treatment with oral procedures on bone marrow biopsy tissue microarrays (TMA) for 73 patients immunomodulatory drugs such as thalidomide or lenalidomide (in triplicate), using TF positive (nasopharyngeal epithelium, squamous cell (so-called IMIDs), concomitantly with anthracyclines or dexa- carcinoma) and CD138 positive (myeloma) controls. Connective tissue from methasone, in whom up to a third may develop venous nasopharyngeal epithelium was used as negative control. We have used these thromboembolism if not given prophylaxis.6,7 The lack of impact TMA slides in previous publications.11,12 Briefly, slides were deparaffinized of thrombosis on survival in MM has recently been reported in using xylene-free dewaxing reagent (72 1C, 4 min, EZprep; Ventana Medical large US and European multicenter studies in both, newly Systems, Tucson, AZ, USA) and stained for IHC using a BenchMark ULTRA diagnosed and relapsed refractory patients.8,9 This finding automated slide stainer (Ventana). Following CC1 alkaline antigen retrieval suggests that activation of coagulation and malignant behavior (95 1C, 8 min.), TMAs were incubated with primary rabbit polyclonal in MM may not be driven by TF on neoplastic plasma cells. anti-human TF antibody (FL-295, dilution 1:75, 36 1C, 4 min, Santa Cruz Furthermore, a puzzling clinical observation has been that Biotechnology, Santa Cruz, CA, USA). Ventana ultraView Universal polymer- thrombosis in the context of IMID-based therapy may be based diaminobenzidine detection kit was used for visualization of antibody

1Department of Pathology, NCI, Mexico City, Mexico and 2Department of Hematology --Oncology, Mayo Clinic, Scottsdale, AZ, USA. Correspondence: Dr R Fonseca, Department of Hematology --Oncology, Mayo Clinic, Collaborative Research Building, 1-105, Scottsdale, AZ 85259-5494, USA. E-mail: [email protected] Received 9 January 2012; revised 31 January 2012; accepted 2 February 2012; accepted article preview online 15 February 2012; advance online publication, 9 March 2012 Tissue factor expression and multiple myeloma G Cesarman-Maus et al 1672 localization. In order to obtain a double stain with anti-CD138 (positive in indicator of an underlying biological advantage to cancer growth plasma cells), the slides were then incubated with primary mouse monoclonal and metastasis1,2 TF, a cell-surface membrane glycoprotein that anti-human CD138 (Dako, Carpinteria, CA, USA, Clone MI15, dilution 1:150), triggers the activation of coagulation by binding to factor VII, followed by polymer-based alkaline phosphatase detection. TMAs were is the most likely candidate responsible for the molecular washed thoroughly in between steps using reaction buffer and manually link between thrombosis and malignant tumor behavior, and counterstained with Harris haematoxylin, and finally mounted with ultimately for the impact on poor clinical outcome of individuals non-aqueous medium. TF expression by plasma cells was scored with solid tumors who develop thrombosis.4,5 Genetic events semi-quantitatively for staining intensity as negative, 1 þ (weak), 2 þ affecting oncogenes (RAS and MET) and tumor suppressor genes (moderate) and 3 þ (strongly positive). The percentage of positive cells (TP53 and PTEN) have been shown to directly enhance TF was recorded. expression by neoplastic cells.3 TF-dependent fibrin generation on the surface of circulating neoplastic cells confers an advantage for metastasis.13 Activated coagulation factors Xa and thrombin as RESULTS AND DISCUSSION well as TF itself, are able to induce receptor-mediated intracellular We found F3 to be commonly expressed in a wide variety of cell signals through protease-activated receptors (PAR1 and PAR 2), lines originated from solid tumors (except for renal cancer cells). which promote invasion and angiogenesis as well as protection Conversely, F3 was absent or minimally expressed in all HMCLs from apoptosis.14,15 Although TF does not affect in vitro (Figure 1) and in all 239MM patient samples analyzed (data not proliferation, it is required for adequate tumor vascularization shown). Protein expression by IHC from 73 triplicate patient TMA and growth in murine models in vivo.13 Furthermore, tumor cell TF cores was negative in 80% of the samples, with the rest showing has been shown to be pro-angiogenic independently of its weak focal cytoplasmic TF in plasma cells. Only three patients had procoagulant activity by upregulating the generation of vascular moderate (2 þ ) focal or diffuse cytoplasmic heterogeneous endothelial growth factor.16 These data suggest that tumors staining, all having at least one negative core. In these cases, expressing TF are associated with an underlying procoagulant none of the plasma cell membranes stained for TF. No samples state intimately linked to malignant tumor behavior. with strong (3 þ ) plasma cell staining were seen. TF was present in In the present study, we found that HMCLs do not express F3 or non-plasma cells with diffuse cytoplasmic staining in megakar- have a very low level of expression compared with solid tumors. yocytes and neutrophils and granular staining within macro- This, however, does not rule out a role for TF from other cellular phages, whereas endothelial cells showed both intracytoplasmic sources in the development of thrombosis. The potential source as well as membrane staining (Figure 2). of circulating TF is broad and may be different in different Hemostasis, inflammation, angiogenesis and oncogenesis are disease states. Many cell types, particularly activated endothelial intricately connected and share many of the same proteins, cells and monocytes, can synthesize and express cell enzyme products, receptors and cell signaling pathways. surface-bound TF, or shed microparticles bearing TF (MP-TF).17 Therefore, venous thrombosis has been regarded as a clinical Although Negaard et al.19 argue against a major role of soluble

Figure 1. Gene expression analysis. Results clearly show absence or very low expression of F3 in HMCLs in contrast to diverse solid tumor cell lines. Renal cell carcinoma does not express F3.

Leukemia (2012) 1671 --1674 & 2012 Macmillan Publishers Limited Tissue factor expression and multiple myeloma G Cesarman-Maus et al 1673

Figure 2. IHC staining for tissue factor in bone marrow biopsies: representative samples. Positive controls for TF (a) nasopharingeal epithelium (b) squamous cell carcinoma, and negative control (c) reactive lymph node, are shown. Negative connective tissue staining in nasopharyngeal connective tissue was also used as control. Multiple myeloma bone marrow biopsies show characteristic negative plasma cell TF staining. Dual staining for TF (brown) and the plasma cell antigen CD138 (red) (d, e) shows TF only in non-myeloma cells, admixed with cells that are negative for both, and a rare case (f) with focal plasma cells moderately positive for TF. An additional marrow (g) shows moderate diffuse cytoplasmic TF in neoplastic plasma cells (only anti-TF was used). TF is present in the bone marrow in non-myeloma cells such as (h) megakaryocytes and (i) endothelium. non-microparticle-bound TF in plasma in MM and other hemato- capable, like neutrophils, of fusion with TF-bearing microparticles logical malignancies, a recently published study by Auwerda derived from other cell types is unknown. Our finding of rare et al.18 showed MP-TF activity levels are significantly higher in patient samples with moderate TF staining suggests that bone untreated MM patients compared with normal healthy donors. marrow microenvironment may have a role in either promoting The authors found that MP-TF activity remains elevated in patients expression of TF or possibly supporting direct TF transfer to who develop venous thromboembolism, but decreases in those malignant plasma cells. that do not. Our findings show that although TF may be relevant Finally, the fact that TF is not expressed by neoplastic plasma to thrombosis in some MM patients, it is most likely not derived cells may explain why heparin, which works in part by inhibiting from the neoplastic cells. The moderate expression of TF in both TF,24 is not better than aspirin for DVT prophylaxis of patients with bone marrow megakaryocytes and endothelial cells (Figure 2) MM.25 In support of our findings, a recent study by Pal et al.26 suggests that it is these cells, which are the origin of at least part shows that promyelocyte-derived cathepsin G may be a more of the circulating TF-bearing microparticles. As thrombosis does important mediator of thrombosis in the setting of IMID therapy in not affect the outcome of most patients with MM,8,9 we propose MM. We speculate that the intravascular nature of MM and other that it is the TF produced by the neoplastic cells themselves hematopoietic malignancies makes it less likely for them to that drives malignant behavior, and that TF derived from non- depend on pro-coagulant factors for disease spread. Something neoplastic cell sources as a result of host response to cancer clearly of benefit to solid tumor clones. (acute phase reaction, inflammation and necrosis) or to known prothrombotic treatment regimens,20 does not affect oncogenesis though it may still be important for the development of CONFLICT OF INTEREST thrombosis. The fact that the vast majority of neoplastic plasma cells do not RF is a consultant for Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine and Lilly (all o$10 000). RF receives research support from Cylene, Onyx and Celgene. express TF is not surprising, as, unlike monocytes, lymphocytes The remaining authors declare no conflict of interest. do not express TF under physiologic conditions. There is in vitro evidence that phorbolmyristate acetate (PMA)-stimulated activated B lymphocytes can express TF, but this has not been described for plasma cells.21 MM plasma cells have rarely been ACKNOWLEDGEMENTS shown to contain TF. A single case report shows TF detected by We would like to acknowledge GSK for making publicly available the Cancer Cell Line IHC in plasma cells from a patient with MM and DVT.22 TF found in Genomic Profiling Data used in this study. cells may not necessarily mean that TF is being transcribed by the cell itself. In an elegant study, Egorina et al.23 have shown that AUTHOR CONTRIBUTIONS monocytes are the only source of TF in whole blood, and provide GCM designed and analyzed research and wrote paper, EB designed and analyzed solid experimental evidence for a direct transfer of TF from research and wrote paper, HM designed and analyzed research and RF designed and monocytes to granulocytes. Whether neoplastic plasma cells are analyzed research and wrote paper.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

Leukemia (2012) 1671 --1674 & 2012 Macmillan Publishers Limited