Erythropoierin And Erythropoietin Receptor Expression In The Skin Lesions Of Patients With Mycosis Fungoides

ObjectivesErythropoietin (Epo), a glycoprotein cytokine, acts via erythropoietin receptor (EpoR) to stimulate proliferation and differentiation of RBC precursors. Expression of EpoR have recently been demonstrated in several nonhematopoietic tissues except on RBC precursors, which suggests Epo's biological functions are not confined to hematopoietic cells. In fact recent evidence have shown that Epo is a pleiotropic cytokine and has broader roles. Surprisingly, recent studies have found that Epo and EpoR expression on many types of malignant tumor cells and derived cell lines, and further studies found that the paracrine and (or) autocrine Epo-EpoR signaling existing in malignant tumors is associated with the transformation of biological behaviors of malignant cells including promotion of proliferation, inhibition of apoptosis and decreased chemo-radiation sensitivity. However expression of Epo and EpoR in the skin lesions of mycosis fungoides is rarely reported, we aim to study the issue.Methods1,Clinical samples: biopsy specimens of 16 cases of mycosis fungoides (including 12 cases of early stage and 4 cases of tumor stage) and 15 cases of inflammatory skin disorders (composed of 6 cases of psoriasis, 5 cases of lichen planus and 4 cases of eczema) were selected from the dermatopathology files of China Medical University and divided into 2 groups. Hematoxylin and eosin (HE)-stained slides of all cases were reviewed and the diagnosis were confirmed. All cases included had no visceral dysfunction and abnormal peripheral blood RBC counts and hemoglobin levels. Cases in 2 groups were comparable in age and sex structure. 2,Immunohistochemistry (S-P): Immunohistochemical assays were performed on formalin-fixed paraffin-embedded sections. Five-micrometer-thick sections were cut and deparaffinized in xylene and rehydrated in graded alcohols. All slides were steamed in 0.01 M sodium citrate buffer (PH 6.0) for 20 minutes for antigen retrieval. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 20 minutes and ready to be stained. The main agents including antibodies against Epo (rabbit polyclonal, H-162, 1:200 dilution; Santa Cruz Biotechnologies ) , antibodies against EpoR (rabbit polyclonal, C-20, 1:400 dilution; Santa Cruz Biotechnologies), immunohistochemical staining kit (SP-9000, American Zymed Biotechnologies) and diaminobenzidine chromogen (Beijing Zhongshan Biotechnologies) were all used according to the protocols. Then slides were counterstained with hematoxylin.3,Control: Slides of aggressive malignant melanoma was regarded as positive control; absence of primary antibodies and substituted by PBS were used as negative control.4,Interpretation of Immunohistochemical Stains: Immunohistochemical stains for Epo and EpoR were interpreted semiquantitatively by assessing the intensity and extent of staining on the entire tissue sections present on the slides according to a four-tiered (0-3) scale by 3 dermatopathologists blindly. For Epo, cytoplasmic;for EpoR, cytoplasmic and/or membrane-immunoreactivity was considered as positive. Then the percentage of: 1) weakly, 2)moderately, and 3)strongly staining cells was determined, so that sum of these categories equated with the overall percentage of positivity. A staining score was then calculated as follows: score=1×percentage of weak+2×percentage of moderate+3×percentage of strong staining. H&E and immunohistochemical stained slides of all cases were read by 3 dermatopathologists simultaneously via microscope (Olympus BX50).5,Statistical analysis software SPSS 11.5 was used and statistical significance was established if the two-sided P value of a test was less than 0.05. ResultsMycosis fungoides group (n=16) demonstrated significantly higher expression levels of Epo and EpoR than inflammatory dermatosis group (n=15), p=0.005, 0.003 respectively. Early stage mycosis fungoides group (n=12) also showed stronger expression of Epo and EpoR compared with inflammatory dermatosis group (n=15), p=0.021, 0.013 respectively.ConclusionEpo-EpoR signaling may be involved in the pathogenesis process of mycosis fungoides.