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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
#
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 .