M.Sc Thesis


M.Sc StudentChoshha Lisa
SubjectThe Role of Endothelial Protein C Receptor (EPCR) in
Diffuse Large B Cell Lymphoma
DepartmentDepartment of Medicine
Supervisor DR. Galit Sarig
Full Thesis textFull thesis text - English Version


Abstract

Thrombotic complications and hyper-coagulation in cancer are associated with tumor progression and poor patient outcome. The endothelial protein C receptor (EPCR) is an important part of the protein C anticoagulant pathway which also plays major roles in cytoprotective and tumorigenesis mechanisms. The role and clinical relevance of the EPCR A6936G polymorphism in malignancies, especially in hematological malignancies has not been clarified yet.

The aim of the current study was to evaluate the association between EPCR A6936G polymorphism with clinical parameters of diffuse large B cell lymphoma (DLBCL), and with pattern of EPCR expression in DLBCL cells. 

Clinical data of 249 newly diagnosed DLBCL patients was collected from the electronic medical records and DNA was extracted from patients' bone marrow (BM) or peripheral blood samples. The EPCR A6936G polymorphism was analyzed using Fluorescent Resonance Energy Transfer (FRET) real-time PCR.

EPCR expression level was evaluated in two DLBCL cell lines (OCI-LY18 and U-2946), 38 DLBCL patient archival formalin-fixed paraffin-embedded sections, and in BM samples from 5 DLBCL patients with BM disease involvement.

Measuring the EPCR expression levels in DLBCL cell lines enable us to study the pattern of expression in pure DLBCL cells, isolated from other diverse cells which are also located in the DLBCL patient biopsies.

Levels of EPCR mRNA were studied using quantitative reverse transcription PCR (qRT-PCR), and protein levels were evaluated using immunohistochemically (IHC) staining and flow-cytometry analysis.

No association between EPCR A6936G polymorphism and overall survival, progression free survival or any other clinical parameter was observed in the current cohort of DLBCL patients.

Low to very low EPCR mRNA levels were documented in the DLBCL U-2946 (∆Ct = 2.97) and OCI-LY18 (∆Ct = 9.44) cell lines compared with the primary HUVECs (∆Ct = (-3.66)).

In addition, low EPCR protein levels were observed in both DLBCL cell lines using IHC staining and flow-cytometry analysis. Flow cytometry analysis revealed that 10% - 20% of both DLBCL cell lines express cytoplasmic and surface EPCR. 

No statistical difference was observed in EPCR mRNA levels between biopsies from patients with the EPCR 6936 AG/GG and AA polymorphisms. Compering mRNA levels of EPCR (∆Ct (-0.855)) to the endothelial marker ERG mRNA (∆Ct 1.33) and to the B-lymphocyte marker CD20 (∆Ct (-3.66)), suggests a possible low EPCR mRNA expression level in patient's DLBCL cells.

Out of the 38 IHC stained biopsies, only in two sections (5.3%), from patients with the EPCR 6936 AG polymorphism, the DLBCL cells were positively stained with anti-EPCR antibodies.

Flow-cytometry analysis of DLBCL cells from 5 BM samples demonstrated that 24% of the cells express cytoplasmic EPCR with MFI ratio of 3.3, but no surface EPCR was documented on those DLBCL cells.

In conclusion, EPCR A6936G polymorphism was not found to have a clinical relevance in DLBCL patients. DLBCL cells probably express low to very low EPCR levels, however the association with the EPCR A6936G polymorphism and the clinical parameters are not clear yet. Further studies on the role of EPCR in DLBCL and other hematological malignancies might enlighten this query.