Prediction of thrombosis risk in cancer patients using soluble CLEC2

Abstract

The purpose of the present invention is to develop a biomarker which can reflect the clot formation in vivo more satisfactorily, and to provide a method which enables the evaluation of the risk for a cancer-related thrombosis in perioperative period in a cancer patient. The method includes a step for measuring the concentration of soluble CLEC2 occurring in blood collected from a cancer patient.

Description

[Technical Field]

[0001] The present invention relates to a method for predicting cancer-associated thrombosis using soluble CLEC2. [Background technology]

[0002] Blood clots formed in blood vessels are considered to be a life-threatening factor in a wide range of human diseases, and it is known that the risk of blood clot formation increases in various diseases, particularly acute diseases such as chronic obstructive pulmonary disease (COPD), infections, and sepsis, as well as progressive cancer, pregnancy, nephrotic syndrome, inflammatory bowel disease, and bone marrow hyperplasia. breeding Sexually transmitted diseases are said to have a high risk of thrombosis.

[0003] The occurrence of thrombosis worsens life prognosis. A retrospective clinical study in the United States found that the incidence of thrombosis in 1,874 cancer patients (2005-2012) was high at 16.4%. Of these, 2% had a history of thrombosis, and 14.4% developed the disease within three months before their cancer diagnosis.

[0004] Pancreatic cancer and brain tumors are particularly at high risk of thrombosis. A survey of the annual incidence of thrombosis showed that compared with 13.6 cases / 1000 people / year for all cancers, the incidence was 48 cases / 1000 people / year for brain tumors and 58.9 cases / 1000 people / year for pancreatic cancer (Non-Patent Document 2). The cause is thought to be that cancer cells express tissue factor (TF) and continuously release TF-positive microparticles (MPs) into the blood, activating the coagulation system.

[0005] Thus, cancer-bearing conditions are considered to be a thrombophilic predisposition, making thrombus formation more likely, leading to the development of venous thromboembolism (VTE) associated with cancer, also known as cancer-associated thrombosis (CAT). Venous thromboembolism (VTE) refers to the formation of a blood clot in a deep vein, while pulmonary thrombosis (PE) occurs when a deep vein clot breaks free, enters the bloodstream, travels through the right atrium and right ventricle, and becomes an embolism in the pulmonary artery, resulting in pulmonary embolism. Venous thromboembolism (VTE) is a disease concept that combines deep vein thrombosis (DVT) and pulmonary thrombosis (PE). However, in either case, DVT, especially cancer-associated CAT, significantly impacts prognosis.

[0006] The incidence of CAT varies depending on the type of cancer, and has been reported to be higher in intraperitoneal and thoracic cancers, brain cancers, and cancers of unknown cause. The incidence of CAT is also higher in cancer types with a high mortality rate, and it is believed that there is a correlation between the malignancy of cancer and the incidence of CAT.

[0007] The incidence of CAT is said to be high during the first three months after a cancer diagnosis, so particular attention is needed when starting cancer treatment. It has also been reported that cancer patients who have developed CAT are prone to CAT recurrence and bleeding during anticoagulant therapy, requiring careful management during treatment. Therefore, risk management involving many medical professionals is necessary during the so-called perioperative period.

[0008] Concomitant venous and arterial thrombosis is common in cancer patients (Non-Patent Document 1), and clinically, it is known that advanced disease stage, tumor burden, and length of hospital stay increase the risk of thrombosis.

[0009] Trousseau syndrome is also known as an example of hypercoagulability associated with cancer. Although Trousseau syndrome refers to a "hypercoagulable state associated with malignant tumors and associated migratory thrombophlebitis," it is not uncommon for the malignant tumor to be discovered only after the onset of cerebral infarction. For this reason, in Japan, it is increasingly understood as "systemic (especially cerebral) embolism caused by thrombosis associated with DIC and nonbacterial thrombotic endocarditis (NBTE) associated with malignant tumors." Hereinafter, in this specification, CAT will include Trousseau syndrome.

[0010] Excluding leukemia, the overwhelming majority of malignant tumors that cause CAT are adenocarcinomas such as lung cancer, pancreatic cancer, gastric cancer, and ovarian cancer (mucin-producing tumors). Head MRI often reveals multiple embolisms, and non-infectious thrombotic endocarditis (NBTE) is found in approximately half of cases, but the detection rate with transthoracic echocardiography is low, and transesophageal echocardiography is considered useful for diagnosis.

[0011] The American Society of Clinical Oncology (ASCO) guidelines list elevated platelet counts, elevated white blood cell counts, and low hemoglobin levels as biomarkers to watch out for in CAT, but there are no direct diagnostic indicators for CAT (Non-Patent Document 3). Domestic and international guidelines describe the management of CAT, recommending anticoagulant therapy as the initial treatment, and "no treatment" is not an option, except for asymptomatic peripheral deep vein thrombosis (DVT) discovered incidentally. When treating CAT, it is important to consider the susceptibility to both thrombus formation and bleeding, and that the influence of thrombophilia exists not only in venous but also in arterial thrombosis.

[0012] Podoplanin has attracted attention as a factor in cancer patients' thrombosis predisposition. Podoplanin is a membrane protein highly expressed on the surface of many cancer cells, including squamous cell carcinoma (lung, esophagus, cervix, etc.), mesothelioma, and brain tumors, and is involved in cancer invasion. Kunida et al. discovered that podoplanin promotes cancer metastasis through platelet aggregation (Non-Patent Document 4). It has also been reported that podoplanin is highly expressed in brain tumors and osteosarcomas and has high platelet aggregation ability. Furthermore, a new anti-podoplanin antibody was found to induce a high antitumor effect and suppress metastasis through its cytotoxic activity, demonstrating the usefulness of podoplanin as a therapeutic target for cancer metastasis.

[0013] C-type lectin-like receptor 2 (CLEC2), a receptor for podoplanin, was identified on platelets as a receptor for the platelet-activating snake venom rhodocytin. CLEC2 binding to podoplanin has various pathophysiological roles, including its ability to promote hematogenous tumor metastasis. Since CLEC2 is expressed almost exclusively in platelets and megakaryocytes in humans, it is thought to stabilize thrombi in the bloodstream by homophilically binding in a platelet activation-dependent manner (Non-Patent Document 5). [Prior art documents] [Patent documents]

[0014] [Patent Document 1] Special Publication No. 2008-544224 [Patent Document 2] Special Publication No. 2019-507345 [Non-patent literature]

[0015] [Non-Patent Document 1] Trousseau A. Phlegmasia alba dolens. Clinique Medicale de l'Hotel Dieu de Paris, Vol 3. Paris: Bailliere. 1865; 654-712 in French [Non-patent document 2] Freesia Horsted et al., PLoS Med. 2012 Jul; 9(7): e1001275 [Non-patent document 3] Lyman GH et al. J Clin Oncol 31(17):2189-2204, 2013 [Non-patent document 4] Kunita A et al. Am J Pathol. 170:1337-1347 (2007) [Non-Patent Document 5] Katsue Inoue et al., Journal of Thrombosis and Hemostasis 22(6):348-362(2011) Summary of the Invention [Problem to be solved by the invention]

[0016] The prognosis of widespread thrombosis is extremely poor, and early and accurate diagnostic methods are essential to implement appropriate treatment that significantly reduces mortality. Therefore, even in cancer patients, it is necessary to carefully manage the hypercoagulable state during the perioperative period to assess the risk of thrombosis and to strive for prevention and early treatment.

[0017] Known methods for diagnosing and risk stratifying venous thromboembolism include a method that combines multiple markers, including not only coagulation- and hemostasis-related markers but also markers for blood pressure regulation, inflammation, myocardial damage, and pulmonary damage (Patent Document 1), and a method that combines measurement of D-dimer with measurement of coagulation factor activity using fibrin formation as an indicator (Patent Document 2).However, none of these methods are easy to perform, and it is difficult to say that they accurately assess the risk of thrombus formation.As a result, there have been no markers that can be used for risk assessment or monitoring that can meet the needs of clinical settings.

[0018] Therefore, an object of the present invention is to develop a biomarker that better reflects thrombus formation in the body and to provide a method that enables risk assessment of CAT in cancer patients during the perioperative period. [Means for solving the problem]

[0019] The present inventors have conducted extensive research to solve the above problems and have found that the concentration of soluble CLEC2 (hereinafter sometimes referred to as "sCLEC2") in the plasma of cancer patients is significantly correlated with CAT, leading to the completion of the present invention. Furthermore, by measuring sCLEC2 concentrations over time in cancer patients and observing their fluctuations, we established a method for early, simple, and accurate risk assessment of CAT. We verified this method using samples from several dozen patients and demonstrated its usefulness for predicting and monitoring the risk of CAT in patients with pancreatic cancer and brain tumors.

[0020] That is, the present invention provides the following: [1] A method for assessing the risk of cancer-related thrombosis in cancer patients during the perioperative period, comprising measuring the concentration of soluble CLEC2 in blood collected from the cancer patient. [2] A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising: (1) providing a sample from a patient who may have cancer-associated thrombosis or who has been diagnosed with cancer-associated thrombosis; (2) determining the concentration of soluble CLEC2 in the sample; (3) correlating the soluble CLEC2 concentration with the presence or absence of cancer-associated thrombosis or the likelihood of outcome in the patient; The method of [1] includes: [3] A method for assessing the risk of cancer-related thrombosis in cancer patients during the perioperative period, wherein the step of correlating the soluble CLEC2 concentration with the presence or absence of cancer-related thrombosis or the likelihood of outcome in the patient comprises assessing whether the patient is at risk of cancer-related thrombosis based on changes in the soluble CLEC2 concentration. [4] A method for assessing the risk of cancer-related thrombosis in cancer patients during the perioperative period, comprising using at least one coagulation- and hemostasis-related marker in addition to the soluble CLEC2 concentration. [5] A method for assessing the risk of cancer-related thrombosis in cancer patients during the perioperative period, according to any one of [1] to [4], characterized in that the soluble CLEC-2 concentration is replaced by a value obtained by dividing the soluble CLEC-2 concentration by the platelet count. [6] A method for assessing the risk of cancer-related thrombosis in cancer patients during the perioperative period, comprising providing samples from the cancer patient over time from before surgery to 30 days after surgery, and continuously monitoring the risk assessment to predict the effectiveness of an antiplatelet agent. [7] Any of the methods [1] to [6], wherein the cancer is selected from the group consisting of pancreatic cancer, squamous cell carcinoma (lung, esophageal, cervical, etc.), mesothelioma, brain tumor, advanced cancer, and myeloproliferative disorder. [8] The method according to any one of [1] to [7], wherein the cancer-associated thrombosis is Trousseau syndrome. [Effects of the Invention]

[0021] The method of the present invention, which measures the sCLEC2 concentration in the blood of cancer patients, enables early, simple, and accurate risk assessment of CAT. Furthermore, it is expected that the prediction accuracy will be improved in perioperative monitoring of CAT development in cancer patients, including the period after surgery, chemotherapy, and other treatments, compared with conventional tests using blood markers or platelet aggregation tests. [Brief explanation of the drawings]

[0022] [Figure 1] 1 shows a standard curve prepared using hsCLEC2 protein as a standard. [Figure 2] FIG. 1 is a graph comparing the plasma concentration of sCLEC2 between pancreatic cancer patients and healthy individuals. [Figure 3] FIG. 10 is a graph comparing the plasma concentration of sCLEC2 in subjects with and without blood abnormalities. [Figure 4] FIG. 10 is a graph comparing the plasma concentrations of sCLEC2 and D-dimer in subjects with and without blood abnormalities. [Figure 5] FIG. 10 is a graph comparing the plasma concentration of sCLEC2 in brain tumor patients with and without DVT. [Figure 6]FIG. 1 is a graph showing the relationship between the presence or absence of DVT onset and C2PAC values. [Figure 7] FIG. 1 is a graph comparing the temporal changes of various markers in perioperative cases of glioblastoma. DETAILED DESCRIPTION OF THE INVENTION

[0023] Although the embodiment of the present invention will be described in detail below, the mode of use is not limited to this. For example, the present invention includes: a method for assessing the risk of CAT, comprising measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the number of platelets); A method for assisting risk assessment of CAT, comprising measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the platelet count); A method for measuring (or determining) the soluble CLEC-2 concentration in a sample (or the value obtained by dividing the soluble CLEC-2 concentration by the platelet count) for risk assessment of CAT; an in vitro risk assessment method for CAT, comprising measuring (or determining) the concentration of soluble CLEC-2 in a sample (or the value obtained by dividing the concentration of soluble CLEC-2 by the number of platelets); Use of an antibody capable of detecting soluble CLEC2 concentration in the manufacture of a kit for risk assessment of CAT; A method for measuring (or determining) the soluble CLEC-2 concentration (or the value obtained by dividing the soluble CLEC-2 concentration by the platelet count) in a sample to provide information necessary for risk assessment of CAT. Includes:

[0024] As used herein, "cancer" refers to a group of diseases characterized by uncontrolled cell proliferation, infiltrating and spreading cells from the site of origin, i.e., the primary site, to other parts of the body, and is not limited to cancers that originate in epithelial cells. It is anticipated that the present invention is suitable for cancers in which podoplanin or its receptor CLEC2, which will be described later, is known to be involved. Generally, cancers are often classified according to the organ, tissue, shape, etc. in which they originate. Examples of cancers include squamous cell carcinoma, which develops from the malignant proliferation of cells called epidermal keratinocytes present in the epidermis; basal cell carcinoma, which develops from cells that make up the underlying basal layer or hair follicles; and myeloproliferative disorders. In addition, when classified by organ, cancers include, for example, brain tumors, tongue cancer, laryngeal cancer, thyroid cancer, esophageal cancer, stomach cancer, colon cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, lung cancer, and ovarian cancer. leather These include, but are not limited to, breast cancer, ovarian cancer, cervical cancer, uterine cancer (uterine cancer), renal cell carcinoma, renal pelvis and ureter cancer, prostate cancer, bladder cancer, skin cancer, bone and soft tissue tumors, leukemia, malignant lymphoma, and childhood cancer, and also include advanced cancers of these.

[0025] As used herein, "cancer-associated thrombosis (CAT)" is understood as a general term for thrombosis associated with cancer. Thrombosis that may occur in cancer patients includes venous thromboembolism associated with cancer itself, such as blood flow stagnation due to cancer, dehydration, bed rest, and increased coagulation by cancer cells; venous thromboembolism associated with cancer treatment, such as vascular damage and increased coagulation due to chemotherapy, vascular damage and congestion due to catheters, portal vein congestion due to portal hypertension, and portal vein vascular disorder; and venous thromboembolism caused by additional risks other than cancer, such as a history of venous thromboembolism, obesity, old age, long-term bed rest, lower limb paralysis, cast immobilization, thrombophilia (antithrombin deficiency, protein C / S deficiency, antiphospholipid antibody syndrome, etc.), estrogen treatment, varicose veins, congestive heart failure, respiratory failure, and severe infection.

[0026] Furthermore, in this specification, "Trousseau syndrome," which is positioned as one aspect of CAT, is not limited to a pathological condition of CAT in which stroke symptoms are present due to hypercoagulation, but also refers to thrombosis associated with DIC associated with malignant tumors and systemic embolism caused by nonbacterial thrombotic endocarditis (NBTE).

[0027] In this specification, the term "perioperative period" refers to the period from outpatient visit when surgery is decided to take place to hospitalization, anesthesia, surgery, postoperative recovery, discharge, and rehabilitation before and after surgery.

[0028] Furthermore, as used herein, "CLEC2" refers to a platelet-activating receptor belonging to the C-type lectin family, which is normally present on the platelet membrane and is released into the blood upon platelet activation. As used herein, the term "soluble CLEC2 (sCLEC2)" refers to such CLEC2 or a molecule derived from CLEC2 that is released from platelets and detected in the blood (or in the buffer when incubated in a buffer).

[0029] sCLEC2 includes proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The proteins with molecular weights of approximately 40 kDa and approximately 32 kDa are present on the platelet membrane surface and are presumed to be released in a state contained in microparticles produced upon platelet activation. These proteins are thought to have glycosylation. On the other hand, the protein with molecular weight of approximately 25 kDa is thought to be cleaved by proteases upon platelet activation and released from the platelets. In the present invention, the amount of sCLEC2 as described above is measured. Regarding sCLEC2, the proteins with molecular weights of approximately 40 kDa, approximately 32 kDa, and approximately 25 kDa may be detected together, or only the protein with molecular weight of approximately 25 kDa may be detected.

[0030] The sCLEC2 concentration used in the present invention may be the sCLEC2 concentration alone, or the value obtained by dividing the sCLEC2 concentration by the platelet count. Unless otherwise specified, the sCLEC2 concentration herein is interpreted as including both the sCLEC2 concentration and the sCLEC2 concentration divided by the platelet count.

[0031] Although the sample used for the measurement is preferably derived from a human, samples derived from animals other than humans may be used to understand the pathology of experimental animals, etc. Experimental animals are not particularly limited, and examples thereof include guinea pigs, rats, mice, chinchillas, etc.

[0032] The method of the present invention is also suitable for use in testing for thrombotic hemostatic disorders. As used herein, "hemostasis" refers to the effective and appropriate halting of blood flow or bleeding through the cooperation of platelets and coagulation factors. As used herein, "thrombotic hemostatic disorders" includes, but is not limited to, conditions and diseases involving excessive bleeding or abnormal blood coagulation. In particular, the method is suitable for use in predicting the risk of venous thromboembolism (VTE) and cancer-associated thrombosis (CAT), which is VTE associated with cancer.

[0033] For example, if the sCLEC2 concentration is higher than that of healthy individuals or those without thrombotic hemostatic diseases, it can be said that the patient is likely to have or is at high risk of developing a CAT thrombotic hemostatic disease. Based on such a comparison, the sCLEC2 concentration can be compared before and after surgery and used to predict the risk of thrombosis.

[0034] For example, if the sCLEC2 concentration is measured in patients with pancreatic cancer or brain tumors, and the ratio is high, it can be determined that platelet activation has occurred in the body, and antiplatelet drugs such as aspirin can be administered as a primary preventative measure. Pi sCLEC2 concentrations can be measured in patients taking antiplatelet drugs such as Dogrel, and if the levels are high, it may be possible to consider increasing the dose of the antiplatelet drug, switching to a different type of antiplatelet drug, or administering additional drugs.

[0035] The method for detecting the presence of sCLEC2 is not particularly limited, but an immunological method using an antibody that recognizes sCLEC2 (hereinafter, this may be referred to as an "anti-sCLEC2 antibody") is preferred. Immunological protein detection can be performed using any commonly known method, such as immunoassays using labeled antibodies, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, fluorescent antibody assay, radioimmunoassay, and immunochromatography, or Western blotting, latex agglutination, and immunoturbidimetry. Among these, immunoassays using labeled antibodies are preferred in terms of ease of operation and measurement accuracy. For intraoperative diagnosis, rapid results are desired, and therefore chemiluminescent immunoassays, immunochromatography, and the like are particularly preferred.

[0036] A sample is collected from a subject (especially a patient) using, for example, a blood collection tube for collecting plasma. Considering the need to count platelets, blood collection tubes containing EDTA are preferred, but those containing heparin or citrate can also be used. Those skilled in the art can select and use an appropriate blood collection tube from these options. Samples for measuring plasma sCLEC2 concentration and platelet count may be obtained using a single blood collection tube, or, if blood is collected simultaneously, separate tubes may be used. Plasma sCLEC2 concentration is measured using plasma centrifuged at 2000 g for approximately 20 minutes, for example. However, the centrifugation conditions are not limited to these, and whole blood may also be used for the measurement. The following explanation uses, but is not limited to, the measurement of plasma sCLEC2 concentration. Blood platelet count is measured using whole blood containing an anticoagulant such as EDTA.

[0037] Regarding the correlation between the measured sCLEC2 concentration in samples from cancer patients and the presence or absence of CAT or the likelihood of outcome, a threshold may be appropriately set by comparing the sCLEC2 concentration in samples from cancer patients with the sCLEC2 concentration in samples from healthy individuals, or a risk assessment of CAT may be performed if a significant fluctuation in sCLEC2 concentration is detected from a time-course record of sCLEC2 concentration in the same patient, such as comparing pre-operative and post-operative data or comparing the first day after surgery with several days after surgery.

[0038] When sCLEC2 concentration is divided by platelet count to predict the risk of CAT, platelet count is usually measured using an automated hemocytometer, but it can also be counted using a hemocytometer and a microscope.

[0039] The sCLEC2 concentration in plasma is expressed in pg / mL, for example, and the platelet count in blood is expressed in 1000 / mm 3 The sCLEC2 concentration can be expressed in any unit, such as ng / mL or ng / L, and the platelet count can be expressed in 10,000 / mm3. 3 Any unit can be used, but a consistent unit should be used for comparison. Although the sCLEC2 concentration / platelet count can take various values ​​depending on the unit used, they are essentially the same concept.

[0040] The ratio is calculated using the values ​​obtained from the clinical testing device that measures sCLEC2 concentration and the values ​​obtained from the hemocytometer that measures platelet count. In routine clinical practice, this calculation is preferably performed automatically on a system such as a hospital testing system, hospital system, or electronic medical record system that is connected to both measuring devices. However, it is also possible to construct a system that connects the data from the two measuring devices, or to construct a machine that can simultaneously measure sCLEC2 concentration and platelet count. Alternatively, the ratio can be calculated manually using both sets of data.

[0041] Furthermore, the sCLEC2 concentration in plasma, or the correlation between the sCLEC2 concentration / platelet count and the degree of platelet activation or various diseases, may be used, for example, as a threshold for determination, or as original data or statistically processed data for calculating a threshold for determination.

[0042] sCLEC2 is released into the blood upon platelet activation. Existing platelet activation markers, such as PF4 and βTG, have the problem of nonspecific release due to granule stimulation caused by the physical pressure of blood collection. However, sCLEC2 is released via a signaling-dependent mechanism that triggers platelet activation, making it a potentially more accurate marker of in vivo platelet activation. Furthermore, because CLEC2 expression is almost exclusively limited to the platelet / megakaryocyte lineage in humans, it is a specific marker with few false positives. Therefore, measuring sCLEC2 allows for early diagnosis of platelet activation status and can be used as a method for predicting thrombosis risk.

[0043] Since plasma sCLEC2 concentrations tend to be high in individuals with many platelets and low in individuals with few platelets, the plasma sCLEC2 concentration is affected by the number of platelets in the blood and does not necessarily represent platelet activation. However, taking advantage of the fact that the sCLEC2 concentration is positively correlated with the number of platelets in the blood, the value obtained by dividing the plasma sCLEC2 concentration by the number of platelets in the blood may be used.

[0044] Diagnosing thrombotic diseases by dividing the plasma sCLEC2 concentration by the blood platelet count to calculate the amount of sCLEC2 released per platelet is preferable because it allows evaluation of the degree of platelet activation independently of the blood platelet count. Specifically, for example, if the plasma sCLEC2 concentration is expressed in pg / mL (A) and the blood platelet count is 1000 / mm3, 3 The number obtained by dividing A by B can be used as an index of platelet activation.

[0045] To predict the risk of postoperative thrombosis in cancer patients who have undergone surgical treatment, blood samples are taken over time, plasma sCLEC2 concentrations are measured, and changes in the concentration can be observed. For example, by appropriately setting the sample collection period based on the patient's background information, a more detailed profile can be obtained, allowing for monitoring of the condition and risk assessment. Patients who show a significant increase in plasma sCLEC2 levels compared to preoperative levels can be predicted as being at risk for CAT. This method can also be used to monitor the condition of patients diagnosed with CAT after drug administration.

[0046] CAT has a high recurrence rate, and many cases are thought to require long-term anticoagulant therapy. However, anticoagulant therapy for CAT may increase the risk of bleeding, and anticancer drugs may cause hematotoxicity, resulting in a decrease in white blood cells (especially neutrophils), red blood cells, and platelets. Therefore, the long-term treatment strategy for CAT must be determined taking into account the risk of bleeding and prognosis. Therefore, although determining the duration of anticoagulant therapy for CAT is considered difficult, no clear criteria have been established for the strategy of anticoagulant therapy. Therefore, using the present invention to evaluate a patient's risk of CAT may be helpful in determining an appropriate treatment strategy.

[0047] The frequency of concentration measurements used for monitoring can be set appropriately according to the background information, including the treatment history of each patient, and the treatment plan. In this case, risk assessment can be performed by collecting samples at regular intervals from before surgery until 30 days after surgery. However, since it is expected that the situation will differ depending on the patient's background, the monitoring period and timing can be set appropriately, for example, by periodically observing between 7 and 10 days after surgery, when particular attention is generally required for the occurrence of CAT.

[0048] In the present invention, sCLEC2 concentration may be used in combination with biomarkers currently reported to be useful for assessing the risk of CAT, with the aim of supporting risk assessment using sCLEC2 concentration. Using these biomarkers in combination is preferable because it allows for more accurate risk assessment of CAT. For example, D-dimer and other biomarkers are sometimes used for postoperative monitoring, as in the present invention. However, as in the examples described below, the inventors have found that in cases of pancreatic cancer diagnosed with CAT, despite fluctuations in sCLEC2 concentration, there are cases where D-dimer concentration remains unchanged, making it impossible to assess the risk of CAT. This is thought to be due to the mechanism of thrombus formation. Specifically, by monitoring fibrin-mediated thrombus formation using D-dimer and platelet-mediated thrombus formation using sCLEC2 over time, more accurate risk assessment of CAT may be possible. In other words, platelet-dominant thrombus formation can be predicted.

[0049] The present invention may also be used in combination with other findings such as imaging evaluation. In clinical practice, CAT is generally diagnosed by imaging diagnosis of patients suspected of CAT, and therefore, using this information in combination is expected to improve accuracy, making it preferable.

[0050] In general, the treatment of thrombosis involves administering antiplatelet drugs such as aspirin in cases of arterial thrombosis, such as myocardial infarction, atherothrombotic cerebral infarction, and arteriosclerosis obliterans, where platelet activation and aggregation lead to coagulation. In cases of thrombosis, such as deep vein thrombosis, atrial fibrillation, cardiogenic cerebral embolism, and pulmonary embolism, where coagulation is initiated by activation of the coagulation system in veins or atria, anticoagulants such as warfarin and DOACs are administered. Furthermore, while the present invention is intended for use in CAT, a cancer-related thrombosis, anticoagulants are currently used as standard treatment. It is conceivable that the risk assessment of platelets-based thrombosis using the present invention may enable the selection of antiplatelet drugs. For these reasons, monitoring sCLEC2 levels in postoperative patients using the present invention is highly significant, as it enables rapid, reliable, and cost-effective postoperative risk stratification, allowing the need for postoperative care and treatment measures to be reliably assessed and estimated through monitoring. For example, the method of the present invention can provide significant benefits, since it allows for rapid and reliable assessment of therapeutic efficacy and risk prediction, enabling the selection of drugs with lower bleeding risks. [Example]

[0051] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0052] Example 1: Measurement of sCLEC2 in human plasma The plasma sCLEC2 concentration was measured according to Example 6 of Japanese Patent No. 4961595. A sandwich ELISA system was constructed using mouse anti-human sCLEC2 antibody. Purified 1-11D5 antibody (F(ab)'2) was diluted to 10 μg / mL in 0.05 mol / L carbonate buffer (pH 9.5) and added to an immunoplate (Maxisorp; NUNC) at 100 μL per well. After overnight incubation at 4°C, the plate was washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 and blocked by adding 200 μL of PBS containing 1% bovine serum albumin (BSA) to each well. Next, a dilution series of human sCLEC2 (hsCLEC2) protein was prepared as a standard using 10% SuperBlock (Thermo Fisher Scientific), 0.1% sodium octanoate, and 0.14 mol / L sodium chloride in phosphate buffer (PB). Human plasma was diluted at least five-fold in the same buffer. 100 μL of each antibody was added to each well and incubated at 37°C for 1.5 hours. After incubation, the plate was washed three times in the same manner. Next, the prepared biotin-labeled 3-11E6 antibody (F(ab)'2-biotin) was diluted to 1.0 μg / mL with 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride in PB, and added to each well at a concentration of 100 μL. After incubation at 37°C for 1 hour, the plate was washed three times in the same manner. Next, AMDEX streptavidin-conjugated horseradish peroxidase (AMDEX streptavidin-conjugated horseradish peroxidase; GE Healthcare) was diluted with 10% SuperBlock, 0.1% sodium octanoate, and 0.14 mol / L sodium chloride in PB, and added to each well at a concentration of 100 μL. After incubation at 37°C for 1 hour, the plates were washed five times in the same manner, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) solution was added to each well. After incubation at room temperature for approximately 20 minutes, the reaction was stopped with 1 mol / L sulfuric acid solution. The absorbance at 450 nm (-620 nm) was measured using a plate spectrophotometer (BIO-TEK INSTRUMENTS / EL312e). Figure 1 shows a standard curve prepared using hsCLEC2 protein as a standard.

[0053] Example 2: Measurement of sCLEC2 in plasma samples from pancreatic cancer patients Plasma samples from 14 pancreatic cancer patients and 10 healthy controls purchased from BiolVT (Westbury, NY) were diluted with buffer and assayed using the method described in Example 1. The results for the pancreatic cancer patients are shown in Table 1. The mean ± SE values ​​for sCLEC2 concentrations in the healthy controls and pancreatic cancer patients were calculated and shown in Figure 2. The sCLEC2 concentration was significantly elevated (p<0.05) compared with the healthy controls. This demonstrated that platelets are activated by the effects of cancer in pancreatic cancer patients. Next, the pancreatic cancer patients were classified according to the presence or absence of hematological abnormalities. As shown in Figure 3, patients with hematological abnormalities showed higher sCLEC2 levels than those without hematological abnormalities. Among these, the sCLEC2 level in patients diagnosed with an abnormal coagulation profile was 1382.2 pg / mL, approximately eight times higher than the mean value in healthy controls, indicating that patients at high risk of thrombosis exhibited higher levels.

[0054] VTE monitoring typically involves D-dimer analysis in addition to imaging assessment. Therefore, D-dimer levels in 14 pancreatic cancer patient plasma samples purchased from BiolVT were measured using the LPIA Genesis D-dimer reagent (LSI Medience) and compared with the sCLEC2 concentrations measured in Example 2 (Figure 4). Two patient groups were observed: one with elevated D-dimer levels relative to sCLEC2 levels, and the other with elevated sCLEC2 levels relative to D-dimer levels. Thrombosis is broadly classified as venous thrombosis and arterial thrombosis. Venous thrombosis is considered to be primarily fibrin-based, while arterial thrombosis is primarily platelet-based. D-dimer, a fibrin degradation product, is commonly used as a biomarker for fibrin-based venous thrombosis, whereas no commonly used biomarker exists for platelet-based arterial thrombosis, such as Trousseau syndrome. In this example, the finding that there were groups in which sCLEC2 levels were elevated but not D-dimer levels indicated that sCLEC2 captures the pathology of platelet-mediated arterial thrombosis, and demonstrates that sCLEC2 is useful for testing for Trousseau syndrome, which was not apparent with conventional D-dimer tests.

[0055] [Table 1]

[0056] Example 3: Measurement of sCLEC2 in plasma samples from brain tumor patients Plasma samples were collected from patients admitted to the Department of Neurosurgery (NNS) during general blood collection 7 to 10 days after surgery for brain tumors, primarily malignant gliomas, and from healthy volunteers who provided consent. sCLEC2 concentrations were measured using the method described in Example 1. The results for the samples are shown in Tables 2 and 3. Figure 5 shows sCLEC2 concentrations in patients with DVT / PE (n = 10), patients without DVT / PE (n = 48), and healthy volunteers (n = 15). The sCLEC2 concentrations 7 to 10 days after surgery tended to be higher in DVT / PE patients compared with patients without DVT / PE (p = 0.239). Table 4 also shows the mean, standard deviation, and median values ​​for patients with DVT / PE and patients without DVT / PE. DVT was identified on average 10 days after surgery by screening based on D-dimer levels and clinical symptoms. This suggests the possibility of platelet activation in patients who developed DVT / PE during the perioperative period for brain tumor surgery. Furthermore, because CLEC2 is expressed in platelets and may be affected by platelet count, the sCLEC2 concentration was compared between the two groups by dividing it by the platelet count (sCLEC2 concentration pg / mL divided by platelet count 1000 / μL; hereafter referred to as C2PAC). This showed a significant increase in cases with DVT / PE compared to cases without DVT / PE (p=0.032, Table 2, Table 3, Figure 6).

[0057] DVT monitoring is generally performed using D-dimer in addition to imaging evaluation. Therefore, we evaluated the D-dimer, sCLEC2, and C2PAC levels in a patient (71-year-old female with glioblastoma) from whom blood samples could be taken over time (Figure 1). 7In this case, DVT was identified 7 days after surgery, and anticoagulant therapy was initiated. sCLEC2 and C2PAC levels peaked on the 7th day, when DVT was identified, and then gradually decreased due to the effects of anticoagulant therapy. Meanwhile, D-dimer levels remained relatively high not only at the time of DVT identification but also 14 days after the initiation of anticoagulant therapy, but no significant changes were observed. sCLEC2 and C2PAC levels responded more sensitively to treatment, suggesting that sCLEC2 and C2PAC levels are the most useful tools for monitoring the effectiveness of anticoagulant therapy in this case.

[0058] [Table 2]

[0059] [Table 3]

[0060] [Table 4] [Industrial Applicability]

[0061] As described above, by applying the method for predicting thrombosis risk of the present invention by measuring blood sCLEC2, it is possible to predict a patient's risk of thrombosis before the onset of thrombosis, enabling prompt initiation of appropriate treatment. Therefore, the method for predicting thrombosis risk of the present invention can be applied in a wide range of fields, such as medicine and biology, and is particularly useful in the field of clinical testing.

Claims

1. A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising measuring the concentration of soluble CLEC2 in blood collected from the cancer patient, The method, wherein the cancer-associated thrombosis is Trousseau syndrome.

2. A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising: (1) determining the concentration of soluble CLEC2 in a sample collected from a patient suspected of having cancer-associated thrombosis or diagnosed with cancer-associated thrombosis; (2) correlating the soluble CLEC2 concentration with the presence or absence of cancer-associated thrombosis or the likelihood of outcome in the patient; The method of claim 1 , comprising:

3. A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising: The method of claim 1 or 2, wherein the step of correlating the soluble CLEC2 concentration with the presence or absence of cancer-associated thrombosis or the likelihood of outcome in the patient comprises assessing whether the patient is at risk of cancer-associated thrombosis by comparing the soluble CLEC2 concentration with a predetermined decision threshold.

4. A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising: The method according to any one of claims 1 to 3, comprising using at least one coagulation and hemostasis-related marker in addition to the concentration of soluble CLEC2.

5. A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising: The method according to any one of claims 1 to 4, wherein a value obtained by dividing the soluble CLEC2 concentration by the platelet count is used instead of the soluble CLEC2 concentration.

6. A method for assessing the risk of cancer-related thrombosis in a cancer patient during the perioperative period, comprising: The method according to any one of claims 1 to 5, characterized in that the provision of samples derived from the cancer patient is carried out over time from before surgery to 30 days after surgery, and risk assessment is continuously monitored.

7. The method according to any one of claims 1 to 6, wherein the cancer is selected from the group consisting of pancreatic cancer, squamous cell carcinoma, mesothelioma, brain tumor, advanced cancer, and myeloproliferative disorder.

Images