Lech Zimnoch

Expression of Prothrombin Fragment 1+2 in Cancer Tissue as an Indicator of Local Activation of Blood Coagulation

Immunohistochemistry was applied to AMeX-fixed tissue sections of 12 adenocarcinomas of the stomach (seven intestinal adenocarcinomas and five diffuse carcinomas), 12 adenocarcinomas of the pancreas (nine ductal adenocarcinomas and three signet ring carcinomas), and 12 squamous cell carcinomas of the larynx obtained at surgical resection to examine the possibility of extravascular activation of blood coagulation in cancer tissues by exploring the in loco patterns of distribution of fibrinogen, a final product of blood coagulation, fibrin, and a by-product of coagulation reactions (prothrombin fragment 1+2). Gastric, pancreatic, and laryngeal cancers exhibited fibrinogen antigen in abundance throughout the tumor stroma. Fibrin was detected along the edges of nests of carcinoma cells and at the host-tumor interface. Prothrombin fragment 1+2 was present in the blood vessels in areas of neoangiogenesis at the host-tumor interface (gastric and pancreatic cancer tissues) and on the tumor cell bodies (pancreatic and laryngeal cancer tissues). The presence of prothrombin fragment 1+2 in cancer tissues appears to be a good indicator of coagulation activation and thrombin generation at the tumor burden.

Expression of protein C (PC), protein S (PS) and thrombomodulin (TM) in human colorectal cancer☆

INTRODUCTION Colorectal cancer (CRC) is often complicated by thromboembolic episodes. It has been recognized that blood coagulation proteins play a role in cancer progression. An important inhibitory mechanism is provided by the protein C (PC) system consisting of PC, protein S (PS) and thrombomodulin (TM). Recently, novel biological activities have been ascribed to the PC system that do not relate to their hemostatic functions, eg. in angiogenesis, apoptosis and inflammation. OBJECTIVES The purpose of the study was to elucidate the solid phase interactions between CRC tissue and components of the PC system that may contribute to tumor progression. MATERIAL AND METHODS CRC tissues were obtained at surgical resection during treatment of 66 patients. Immunohistochemical studies were performed using polyclonal antibodies against PC, PS and TM. A semiquantitative analysis of the protein expression was also performed. RESULTS Weak expression of PC was observed in cancer cells of two-thirds of the specimens examined, while in 3/66 cases there was no staining for PC in cancer cells. One fourth of CRCs exhibited strong expression of PC. The presence of PS was demonstrated in 64/66 cases of CRC. However, its expression was irregular in terms of intensity of staining and percentage of cancer cells exhibiting protein expression. Weak expression of TM was demonstrated in two thirds of the cases examined, while a strong TM staining was revealed in one third of colon cancers. CONCLUSION Heterogeneous expression of the PC system components in CRC tissue may point to their biological activity modulating tumor growth.

Protein Z is present in human breast cancer tissue

Thromboembolic complications are common findings in breast cancer patients [1, 2]. Hemostatic system components play a role in tumor progression, independent of their role in hemostasis [1, 2]. Since the report of multifactorial activation of factor X in cancer patients [1], inhibitors which control the activity of this factor have attracted attention. Protein Z (PZ), a vitamin K-dependent plasma nonpeptidase glycoprotein [3, 4], is involved in one mechanism responsible for the direct inhibition of factor Xa. PZ circulates in plasma complexed with PZ-dependent protease inhibitor (ZPI) and enhances the rate of ZPImediated factor Xa inhibition [5, 6]. The presence of ZPI in breast cancer tissue was previously reported [7], while information on PZ in tumor malignant tissue is lacking. The purpose of the present study was to evaluate the localization of PZ (both protein and mRNA) at the primary site of human breast cancer. Tumor fragments were obtained during the surgical treatment of 13 previously untreated breast cancer patients (clinical stage T1-2N0M0). Studies were performed on 4 cases of G2 and 9 cases of G3 invasive ductal carcinomas. Immunohistochemical studies (IHC) were performed (Vectastain Kits, Vector Laboratories, Burlingame, CA, USA) [7] employing a polyclonal antibody against homogeneous, plasma-derived human PZ (prepared in rabbits, and purified from immune sera by protein A-Sepharose chromatography). The antibody did not cross-react with all other purified human plasma vitamin K-dependent proteins including prothrombin, protein C, protein S, factor VII, factor X, and factor IX by ELISA. Controls consisted of omission of the primary antibody from the procedure. The control fragments of normal breast tissues obtained from neoplasm-free surgical margins were processed simultaneously as well. Antigen staining was detected by the dark brown reaction product. A semiquantitative analysis of PZ IHC expression in cancer cells (based on both the percentage of cancer cells with PZ positive staining and the intensity of staining) was performed [7]. Immunoreactive score (IRS) values of 1–4 were interpreted as weak, 5–8 as medium, and 9–12 as strong PZ expression. In situ hybridization (ISH) method employed a biotin-labeled 25-nucleotide single-stranded DNA probe (probe sequence: 50 Biotin-CGTCATACCGCATGTGCA CATGGAC-Biotin 30) specific for PZ mRNA. The probe was synthesized by Sigma-Aldrich, Poznan, Poland. The ISH protocol of R&D Systems (R&D Systems, Minneapolis, MN, USA) was followed. Hybrids were detected with rabbit anti-biotin monoclonal antibodies according to the IHC ABC technique associated with ImmunoMax amplification technique described in detail elsewhere [7]. Negative controls included hybridization without addition of the molecular probe and incubation of slides in a RNase A solution (R&D Systems, Minneapolis, MN, USA) before hybridization. Reaction results appeared as dark brown staining. E. Sierko M. Z. Wojtukiewicz (&) Department of Oncology, Medical University, 12 Ogrodowa St, Bialystok, Poland e-mail: mzwojtukiewicz@gmail.com

Protein Z-dependent protease inhibitor (ZPI) is present in loco in human breast cancer tissue.

Protein Z-dependent protease inhibitor (ZPI) is present in loco in human breast cancer tissue -

Co-localization of Protein Z, Protein Z-Dependent protease inhibitor and coagulation factor X in human colon cancer tissue: implications for coagulation regulation on tumor cells.

INTRODUCTION Several hemostatic system components, including factor X (FX), contribute to cancer progression. The Protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex directly inhibits factor Xa proteolytic activity. The aim of this study was to determine the antigenic distribution of ZPI and PZ, in relation to FX, as well as indicators of blood coagulation activation (F1+2 and fibrin) in human colon cancer tissue. MATERIALS & METHODS Studies were performed on human colon cancer fragments. Immunohistochemical (IHC) ABC procedures and double staining method employed polyclonal antibodies against PZ, FX, F1+2 and monoclonal antibodies against ZPI and fibrin. In-situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either FX, PZ or ZPI mRNAs. RESULTS Expression of FX, PZ and ZPI in association with colon cancer cells was observed by IHC. Moreover, the presence of both F1+2 and fibrin in association with colon cancer cells was found, which indicates that blood coagulation activation proceeds extravascularly at the tumor site. Furthermore, expression of FX and PZ was visualized in association with endothelial cells. In turn, colon cancer-associated macrophages were characterized by FX , PZ and ZPI presence. The double staining studies revealed strong FX/PZ, FX/ZPI, as well as PZ/ZPI co-localization on colon cancer cells. ISH studies revealed the presence of FX mRNA, PZ mRNA and ZPI mRNA in colon cancer cells indicating induced synthesis of these proteins. CONCLUSIONS The localization of PZ/ZPI and FX in colon cancer cells indicates that PZ/ZPI may contribute to anticoagulant events at the tumor site. Strong co-localization of PZ/ZPI and FX in cancer cells, and the presence of the mRNAs encoding the proteins, suggests their role in the tumors biology. However, the presence of F1+2 and fibrin at the colon cancer site also suggests that the regulation of FXa by the PZ/ZPI complex at this site is incomplete.

Protein Z/protein Z-dependent protease inhibitor system in human non-small-cell lung cancer tissue.

INTRODUCTION NSCLC progression is often associated with VTE. Activation of factor X is an important step in blood coagulation activation in cancer patients. PZ)/ZPI contribute to direct factor Xa inhibition, and ZPI - attenuates factors IXa and XIa activity. The role of the PZ/ZPI in NSCLC is obscure. The aim of the study was to localize ZPI and PZ in NSCLC tissue in relation to factors X, IX and XI, as well as indicators of blood coagulation activation: prothrombin fragment F1+2 (F1+2) and fibrin. MATERIAL & METHODS Immunohistochemical studies were performed on surgical NSCLC specimens employing antibodies against ZPI, PZ, coagulation factors X, IX, XI, as well as fibrinogen, F1+2 and fibrin. A semiquantitative analysis (acc. to immunoreactive score-IRS) was conducted. RESULTS Medium expression of ZPI(IRS=6.5), together with weak expression of PZ(IRS=4), was observed in cancer cells. Strong or medium staining for factors IX, X, and XI(IRS=8-9) was revealed in cancer cells. Fibrinogen(IRS=10) and fibrin(IRS=8) were demonstrated in tumor stroma and cancer cells. F1+2(IRS=10) was localized in NSCLC cells. Endothelial cells (ECs) and tumor infiltrating macrophages (TAMs) were characterized by a positive staining for ZPI and PZ. CONCLUSIONS ZPI and PZ expression in NSCLC cells, ECs and TAMs may suggest a role for PZ/ZPI in the anticoagulant mechanisms at the tumor site. The presence of F1+2 and fibrin, along with a disproportional expression of ZPI and PZ, might point to impaired function of the coagulation inhibitory system in NSCLC tissue.

Protein Z/protein Z-dependent protease inhibitor system in loco in human gastric cancer

In gastric cancer, hemostatic system components contribute to cancer progression, as activation of factor X (FX) was observed. The protein Z (PZ)/protein Z-dependent protease inhibitor (ZPI) complex inhibits factor Xa proteolytic activity. The purpose of this study was to determine the distribution of ZPI and PZ in relation to FX, and prothrombin fragment (F1 + 2), a standard marker for blood coagulation activation, in human gastric cancer tissue. ABC procedures and a double staining method employed polyclonal antibodies against PZ, FX, and F1 + 2 and a monoclonal antibody against ZPI. In situ hybridization (ISH) methods employed biotin-labeled 25-nucleotide single-stranded DNA probes directed to either PZ or ZPI mRNAs. FX and components of PZ/ZPI coagulation inhibitory system were observed in cancer cells. F1 + 2 was observed in gastric cancer cells as well. Double staining studies revealed FX/PZ, FX/ZPI, and PZ/ZPI co-localization on gastric cancer cells. ISH studies demonstrated the presence of PZ mRNA and ZPI mRNA in gastric cancer cells indicating induced synthesis of these proteins. The co-localization of PZ/ZPI and FX in gastric cancer cells indicates in loco that these proteins may play a role in anticoagulant events at the tumor tissue.

Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

BACKGROUND Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). OBJECTIVES The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. MATERIAL AND METHODS G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). RESULTS Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. CONCLUSIONS The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

Evaluation of Urokinase-Type Plasminogen Activator and Its Receptor in Neointima of Polyester Vascular Grafts

The aim of the study was to assess the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in neointima of polyester vascular grafts. Anastomotic areas were examined at 1, 4 and 12 months after prosthesis implantation in dogs, as well as in human vascular grafts. Immunohistochemistry was performed for uPA and uPAR. Graft neointima in dogs was positively stained for uPA with increased intensity at 4 and 12 months, whereas uPAR expression appeared at 4 and its intensity was increased at 12 months. Intensive uPA and positive uPAR labeling was shown in human grafts. The results demonstrated that in the early period of the healing process of polyester vascular grafts only uPA is present in the neointima in the region of the graft to adjacent artery anastomosis, whereas in healed grafts in dogs and humans uPAR is found as well.