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US-12618841-B2 - Methods of diagnosing malignant diseases

US12618841B2US 12618841 B2US12618841 B2US 12618841B2US-12618841-B2

Abstract

A method for diagnosing a malignant proliferative disease or disorder in a subject, and/or for following up, monitoring or prognosticating the therapy of a malignant proliferative disease or disorder in a subject is disclosed. The method is based on measurement of platelet-mediated fibrinogen-like protein 2 (FGL2) activity in a sample essentially comprising platelets obtained from the subject. In accordance with the disclosed method, platelet-mediated FGL2 activity level higher than control is indicative of the presence of a malignant proliferative disease or disorder in a subject.

Inventors

  • Esther Rabizadeh

Assignees

  • ST INNOVATIVE DIAGNOSTICS LTD

Dates

Publication Date
20260505
Application Date
20221204

Claims (16)

  1. 1 . A kit for diagnosis, follow-up or prognosis of a malignant proliferative disease or disorder, the kit comprising: (a) means for measuring platelet-mediated FGL2 activity level in a biological sample containing platelets of a subject to be tested; said means comprising (1) factor X deficient plasma; (2) tissue factor or an artificial tissue factor surrogate; (3) FGL2 activity level predetermined calibration curve; (b) instructions for measuring platelet-mediated FGL2 activity level in said sample comprising measuring coagulation time of said sample being suspended in said factor X deficient plasma and transforming coagulation time into FGL2 activity level using said predetermined calibration curve; and, optionally, (c) at least one means for collecting a biological sample containing platelets of a subject to be tested; and/or (d) at least one control sample.
  2. 2 . The kit according to claim 1 , designed for assessing platelet-mediated FGL2 coagulation activity level.
  3. 3 . The kit according to claim 1 , designed for assessing platelet-mediated FGL2 units or thrombin concentration.
  4. 4 . The kit according to claim 1 , comprising FGL2 substrate.
  5. 5 . The kit according to claim 1 , comprising FGL2 substrate prothrombin, an active segment thereof, a derivative thereof, a homolog thereof, or any chromogenic substrate suitable for the determination of serine proteases.
  6. 6 . The kit according to claim 1 , comprising Factor X deficient plasma (FXDP), calcium and optionally phospholipids.
  7. 7 . The kit according to claim 1 , further comprising at least one of a buffer, a detectable moiety, an enzyme substrate, a color reagent and any combinations thereof.
  8. 8 . The kit according to claim 1 , comprising means for collecting a sample to be tested.
  9. 9 . The kit according to claim 1 , wherein the control sample is platelet-mediated FGL2 activity sample in a subject not affected by a malignant proliferative disease.
  10. 10 . The kit according to claim 1 , wherein said platelet-mediated FGL2 level is activity is fibrin formation or coagulation induced by addition of Factor X deficient plasma (FXDP) and tissue factor.
  11. 11 . The kit according to claim 1 , wherein said platelet-mediated FGL2 activity level is thrombin generation or prothrombinase activity induced by addition of an FGL2 prothrombinase substrate.
  12. 12 . The kit according to claim 1 , wherein said biological sample is pure platelets, a platelet-rich plasma (PRP) or peripheral blood cells (PBC) supplemented with platelets obtained from the subject.
  13. 13 . The kit according to claim 1 , wherein said malignant proliferative disease or disorder is a solid tumor or a non-solid tumor of circulating cells.
  14. 14 . The kit according to claim 1 , wherein said malignant proliferative disease or disorder is selected from mycosis fungoides pancreatic cancer, breast cancer, squamous cell carcinoma, multiple myeloma, prostate cancer, Langerhans cell sarcoma, thyroid papillary cancer, esophageal cancer, endometrial sarcoma, mammary gland cancer, mediastinal large cell lymphoma, Hodgkin lymphoma, small cell lung cancer or non-small-cell lung carcinoma, kidney cancer, uterus cancer, bladder cancer, colon cancer, ovarian cancer, mixed tumors of salivary gland, tumors in lip and oral cavity, carcinoma of the eyelid and carcinoma of the conjunctiva, pharynx cancer, larynx cancer, paranasal sinuses cancer, adenomas, adenocarcinomas, sarcomas, Ewing's tumor, testicular cancer, retinoblastoma, Wilms' tumor, neuroblastoma, mesothelioma, skin cancer, malignant melanomas, carcinoma of the lacrimal gland, sarcoma of the orbit, brain cancer, spinal cord cancer, vascular system cancer, hemangiosarcoma, malignant lymphoma, Kaposi's sarcoma, mycosis fungoides, Sézary syndrome, myeloid leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute myelogenous leukemia with maturation, acute promyelocytic leukemia, acute non-lymphocytic leukemia, acute non-lymphocytic leukemia with increased basophils, acute monocytic leukemia, acute myelomonocytic leukemia with eosinophilia, lymphocytic leukemia, and myelo-proliferative diseases.
  15. 15 . The kit according to claim 1 , wherein said malignant proliferative disease or disorder is mycosis fungoides (MF) and the Sézary syndrome.
  16. 16 . The kit according to claim 1 , wherein said malignant proliferative disease or disorder is a solid tumor or a hematological malignancy.

Description

FIELD AND BACKGROUND OF THE INVENTION The present invention relates, at least in part, to methods of diagnosing and monitoring a malignant disease based on fibrinogen-like protein 2 activity. The fibrinogen like protein 2 (FGL2), also known as fibroleukin is a member of the fibrinogen-related protein superfamily that shares high conservation among different species. The human gene is approximately 7 kb in length with 2 exons. From the nucleotide sequence of the human gene, a 439-amino acid long protein is predicted. Thrombosis and inflammation are regulated by an assortment of proteins and cells that contribute to the pathophysiology of the pathways. Fibrinogen-like protein 2 is considered a prominent player in this axis, exhibiting multiple functions that are involved in normal and pathological processes, thereby serving as an important mediator of immune coagulation and inflammation. Fibrinogen-like protein 2 encompasses two structurally and functionally distinct forms. A soluble form FGL2 (sFGL2), expressed and secreted by regulatory T cells (peripheral blood CD4+ and CD8+ T cells). Soluble FGL2 lacks the N-terminal region (˜50 kDa) and possesses significant immunosuppressive properties while considered to be deficient of procoagulant activity. The membrane-bound form of FGL2 (mFGL2), also referred to herein as “FGL2 prothrombinase”, contains the N-terminal region (˜65 kDa) and possesses procoagulant activity. It is a serine protease capable of directly cleaving prothrombin into thrombin while bypassing the canonical coagulation route. The mFGL2 form has been reported to be mainly expressed on the surface of macrophages, dendritic cells, endothelial cells, epithelial cells and cancer cells. Interestingly, mFGL2 procoagulant activity has been reported to be exerted mainly in response to pathologic stimuli, while the soluble form (sFGL2) is expressed and secreted constitutively by lymphocytes. Membrane-bound FGL2 does not need to be cleaved to be activated, it leads to fibrin deposition in the absence of factor VII or factor X, and triggers thrombosis, however, it still requires Factor Va, calcium and phospholipids to reach its maximal enzymatic activity. The importance of mFGL2 is manifested by the range of functions and applications that were ascribed to it. This protein plays a key role in various biological processes including embryonic development and miscarriage, microthrombosis, inflammatory immuno-response, viral infection and fulminant hepatitis, ischemia/reperfusion injury, allograft rejection and tumorigenesis. An increase in FGL2 protein and FGL2 mRNA expression has been observed in various types of solid tumors, while the normal tissue surrounding the tumor did not display overexpression of FGL2 as observed in the tumor itself, and it has been suggested the FGL2 has a role in tumor progression (Su, K. et al. (2008) World J. Gastroenterol. 14(39): 5980-5989). Fibrinogen-like protein 2 in tumor cells and milieu may affect tumor development via several proposed possible mechanisms, such as enhancement of tumor cell proliferation, induction of angiogenesis and metastasis, activation of MAPK pathway, promotion of immune suppression, and generation of thrombin leading to thrombin-mediated tumorigenesis. Despite the biological availability of active FGL2 prothrombinase in the blood, there is no published data regarding the precise origin and significance of this active procoagulant form in peripheral blood cells (PBC). Platelets have multipurpose roles and a significant influence on hemostasis, inflammation, infection, immunity and malignant diseases. Hemostasis is considered the primary role of platelets. Beyond aggregation, activated platelets provide the phosphatidylserine phospholipid surfaces upon which the complex reactions of the blood coagulation cascade are localized. Platelets further store and secret coagulation factors and co-factors including factors V, XI, XIII, prothrombin (in their inactive forms), high molecular weight kininogens and polyphosphates. The pathophysiologic role of platelets in tumor development is well recognized (see, for example, Franco et al., Blood, 126:582-588, 2015; Golebiewska and Poole, Blood Rev., 29:153-162, 2015). SUMMARY This disclosure is directed, at least in part, to a method for diagnosing a malignant proliferative disease or disorder in a subject, comprising the steps of: (a) obtaining a biological sample containing platelets of the subject; (b) inducing platelet-mediated fibrinogen-like protein 2 (FGL2) activity in the biological sample; (c) measuring the FGL2 activity level in the sample; and (d) comparing the measured FGL2 activity level with that of a control value, whereby a sample with platelet-mediated FGL2 activity level higher than control is indicative of the presence of a malignant proliferative disease or disorder in a subject. This disclosure is further directed to a method for prognosis, follow up or monitoring the therapy of a malignant p