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Advances in dermatology and venereology Acta Dermato-Venereologica
Fibrinogen-like Protein 2 Activity as a Potential Biomarker for Diagnosis of Early Mycosis Fungoides
Shany SHERMAN 1, 2, 7 #, Esther RABIZADEH 2, 5 #, Lilach MOYAL 1, 7, Tami LIVNAT 3, Esther ZIV 2, 5, Izhack CHERNY 2, 5, Aida INBAL 2, 4, 6 * and Emmilia HODAK 1, 4, 7
1
Laboratory for Molecular Dermatology and 2 Laboratory of Hemato-Oncology, Felsenstein Medical Research Center, Tel Aviv University, Petach Tikva, 3 National Hemophilia Center and Thrombosis Unit, Sheba Medical Center, Tel Hashomer, Ramat Gan, 4 Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel, 5 Laboratory of Hematology, 6 Thrombosis and Hemostasis Unit, and 7 Department of Dermatology, Beilinson Hospital, Rabin Medical Center, Beilinson Hospital, Petach Tikva 4941492, Israel. * E-mail: Aidainbal @ hotmail. com
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These authors contributed equally to this work. Accepted Aug 16, 2016; Epub ahead of print Aug 18, 2016
Fibrinogen-like protein 2( FGL-2)/ fibroleukin( also known as FGL-2 prothrombinase) is a serine protease, similar to activated coagulation factor X( Xa), capable of directly cleaving prothrombin into thrombin, an essential factor in the coagulation cascade( 1). FGL-2 is expressed on the surface of monocytes / activated macrophages and endothelial cells and secreted by peripheral blood CD4 + and CD8 + T cells( 2, 3).
Studies have reported an increase in FGL-2 protein and mRNA expression in various types of solid tumours( 4), suggesting a role of FGL-2 in tumour progression. These findings were supported by our recent study showing increased FGL-2 activity in peripheral blood mononuclear cells( PBMC) of patients with B-cell lymphoma( 5). The proposed mechanisms by which FGL-2 affects tumour development are enhancement of tumour cell proliferation, induction of angiogenesis promotion of immune suppression( 6), and generation of thrombin leading to thrombin-mediated tumourigenesis( 4).
Mycosis fungoides( MF) is the predominant subtype of primary cutaneous T-cell lymphoma( 7).“ Early MF” is characterized by patches and plaques in the absence of evidence of extracutaneous spread, and“ late MF” is characterized by tumours and erythroderma. The majority of affected patients have early MF, and an estimated 20 – 30 % progress to late MF( 8). MF often poses a diagnostic challenge, especially in the early patch / plaque stage, owing to its overlapping features with benign dermatoses, such as psoriasis and atopic dermatitis( 9). The histopathological diagnosis of early MF is one of the most controversial issues in dermatopathology( 10, 11), and there is no non-invasive diagnostic biomarker that may assist clinicians in distinguishing early MF from inflammatory dermatoses.
In the present work we studied FGL-2 activity in PBMC of patients with MF and compared it with controls.
MATERIALS AND METHODS
The study group consisted of 20 consecutive patients with MF, 14 early-stage( I – IIA) and 6 late-stage( IIB – IVB) between April 2013 and February 2015. Findings were compared with 2 control groups: 101 healthy volunteers( medical personnel) and 21 patients diagnosed with an inflammatory T-cell-mediated dermatoses( psoriasis vulgaris or atopic dermatitis).
Staging of MF was determined according to the criteria of the International Society for Cutaneous Lymphomas( ISCL)/ European Organization for Research and Treatment of Cancer( EORTC)( 7, 8, 12).
Blood samples were collected from all participants following written informed consent and approval by the ethics committee of the hospital.
FGL-2 prothrombinase activity was measured in PBMC( isolated as described in reference 5) by thrombin generation( TG) assay( 5) with some modifications: PBMC extract containing 6.5 × 10 5 cells was suspended in a 500 µ l reaction buffer( 5) and homogenized by sonication for 1 min in a W385 sonicator( Labotal, Jerusalem, Israel). The sample containing approximately 62,000 cells was mixed with purified human prothrombin( Stago, Asnieres, France) to a final concentration of 5 µ M. The reaction mixture underwent further incubations and centrifugations( as in( 5)) and transferred to a flat-bottomed 96-well plate. A fluorogenic substrate( ZGGR-7-amido-4-methylcoumarin, ZGGR-AMC)( Stago, Asnieres, France) was added to a final concentration of 0.35 nM, and a fluorescent signal was measured by Fluoroskan Ascent ®( Thermo Labsystems, Helsinki, Finland) at 400 – 460 nm. Thrombin generation was calculated as described previously( 5). Quantitative RT-PCR analysis of fgl-2 mRNA in PBMC was performed as published before( 5).
Analysis of variance( ANOVA) was used to assess differences in FGL-2 activity and mRNA( expressed as mean ± SEM), followed by post hoc analysis. A multivariate linear regression analysis was conducted to adjust for possible confounders. A receiver operating characteristic( ROC) curve was generated to measure performance of the FGL-2 activity assay. Statistical analyses were performed using SPSS ver. 21.0 and GraphPad prism v. 6. A p-value of < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
The background characteristics of the 3 groups of participants and the treatment modalities used are shown in Table SI 1. There were no differences among the groups with regard to age, sex, percent lymphocytes, systemic treatment or phototherapy, although percent monocytes showed a trend for a statistical significance( p = 0.059).
FGL-2 activity was increased almost 1.5-fold in patients with MF compared with normal controls( Fig. 1a; 147 ± 11 % vs 100 ± 3 %, p < 0.0001) and 1.4-fold compared with patients with inflammatory dermatoses( Fig. 1a; 147 ± 11 % vs. 104 ± 10 %, p = 0.001). There was no significant difference in FGL-2 activity between patients with early and late MF( Fig. 1b; 148 ± 12 % vs.
1 https:// www. medicaljournals. se / acta / content / abstract / 10.2340 / 00015555-2514 doi: 10.2340 / 00015555-2514 Acta Derm Venereol 2017; 97: 370 – 372
This is an open access article under the CC BY-NC license. www. medicaljournals. se / acta Journal Compilation © 2017 Acta Dermato-Venereologica.