Diagnostic blood tests

Abstract

The present application provides an in vitro method for determining extracellular matrix (ECM) degradation in a subject, the method comprising determining the expression product level of at least one gene selected from the group consisting of collagen type V α1 chain (COL5A1), transforming growth factor β-1 (TGFB1), integrin subunit α4 (ITGA4), integrin subunit β1 (ITGB1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9) and bone morphogenetic protein 1 (BMP1) in a sample isolated from the subject, wherein the at least one gene is optionally determined in combination with one or both of collagen type XI α1 chain (COL11A1) and collagen type V α2 chain (COL5A2), wherein when the expression product level is higher than a reference value, this indicates degraded ECM. A method for diagnosing and prognosing cancer and aneurysm is also provided. In addition, a reagent for determining the level of gene expression products in the above-mentioned diagnostic or prognostic method and a kit containing the reagent are also provided.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis, in particular the diagnosis of blood samples, and more particularly for the diagnosis and prognosis of cancer and aneurysm by detecting the degradation of the extracellular matrix. Background Art

[0002] Cancer represents a common and increasing cause of death. The World Health Organization estimates that during the next 20 to 30 years, cancer will be the leading cause of death worldwide. The most effective weapon against the increase in cancer mortality is early diagnosis. Many types of cancer can be effectively treated if detected in their early stages. This reality emphasizes the need to develop a set of simple, reliable and cost-effective molecular markers for detecting cancer in the initial stages of carcinogenesis. Another important strategy for effectively combating cancer is the early identification of patients at risk of developing metastasis so that they can be followed up more frequently and more aggressive therapeutic interventions can be applied in this patient subgroup.

[0003] Recent advances have also highlighted the importance of non-cellular components of the local microenvironment or niche, particularly the extracellular matrix (ECM), during cancer progression. The ECM is a mixture of extracellular molecules secreted by supporting cells that provide structural and biochemical support to surrounding cells. The ECM of mammals includes interstitial matrix and basement membrane. The interstitial matrix is ​​present between various mammalian cells (i.e., in the intercellular space). A gel of polysaccharides and fibrous proteins fills the interstitial space and acts as a compressive buffer against tensile stresses imposed on the ECM. The basement membrane is a sheet-like deposit of ECM on which various epithelial cells rest. Each type of connective tissue in mammals has a type of ECM: collagen fibers and bone minerals comprise the ECM of bone tissue; reticular fibers and matrix comprise the ECM of loose connective tissue; and plasma is the ECM of blood.

[0004] Although it is considered to be a stable structure that plays a major supporting role in maintaining tissue morphology for a long time, ECM is an important part of the resident cell environment, which is surprisingly dynamic and versatile, and affects the basic aspects of cell biology. Cell adhesion, intercellular communication and differentiation are common functions of ECM. This pleiotropic aspect of ECM function depends on the highly dynamic structure of ECM and its remodeling as an effective mechanism, which can regulate multiple cell behaviors. The main challenge in ECM biology is to understand how the role of ECM in normal development and the destruction of ECM dynamics can cause disease (such as cancer).

[0005] Another example of a disease that may be highly affected by ECM alterations is aortic aneurysm. In aortic aneurysm, ECM degradation of the aortic wall initially leads to dilation of the aorta and then to aneurysm formation.

[0006] Aortic aneurysm represents an important clinical entity that develops asymptomatically until rupture or dissection occurs. It is an important cause of mortality in developed countries. Rupture of an aortic aneurysm is often the first and last symptom, with a mortality rate of 75%. It is estimated that the incidence of aortic aneurysms will continue to increase worldwide in the coming years due to the aging of the general population. The pathogenesis and molecular mechanisms leading to aneurysm formation are under investigation, and there is currently no simple laboratory test that can reliably detect aortic aneurysms.

[0007] In view of the above epidemiological data, there is an urgent need to provide simple and reliable tests for early diagnosis of cancer and aortic aneurysm, as well as methods for effective follow-up of patients to early identify patients at risk of developing metastasis or recurrence. In addition, better strategies are generally needed to optimize treatment options for patients with cancer and aneurysms. DETAILED DESCRIPTION

[0008] The inventors surprisingly found that abnormal function or degradation of ECM can be accurately detected by measuring the expression levels of certain genes in peripheral blood. Overexpression of these specific gene groups in peripheral blood reveals the activation level of molecular mechanisms that promote ECM remodeling, which is related to cancer and aneurysm progression.

[0009] The fingerprint for detecting ECM degradation or abnormal function contains the genes listed in Table 1.

[0010] Table 1. Genetic fingerprints used to measure ECM degradation.

[0011] name symbol NCBI reference sequences Collagen type XI α1 chain COL11A1 NG_008033.1 Collagen V-type α2 chain COL5A2 NG_011799.2 Collagen V-type α1 chain COL5A1 NG_008030.1 Transforming Growth Factor Beta-1 TGFB1 NG_013364.1 Integrin subunit α4 ITGA4 NG_050623.1 Integrin subunit β1 ITGB1 NG_029012.1 Matrix metallopeptidase 2 MMP2 NG_008989.1 Matrix metallopeptidase 9 MMP9 NG_011468.1 Bone morphogenetic protein 1 BMP1 NG_029659.1

[0012] Therefore, a first aspect of the present invention provides an in vitro method for determining extracellular matrix (ECM) degradation in a subject, the method comprising determining the level of expression product of at least one gene selected from the group consisting of genes listed in Table 1 in a sample isolated from the subject, wherein, when the expression product level is above a reference value, this indicates a disordered / degraded ECM.

[0013] In the sense of the present invention, the expression "degradation of the ECM" is also to be understood as a reconstruction leading to a disordered ECM or abnormal ECM mechanical and dynamic properties.

[0014] The term "diagnosis" is known to those skilled in the art. "Diagnosis" as used herein is understood to be aware of a particular medical condition, complication; to determine the nature of a condition; or to distinguish the condition from another condition. It refers both to the process of attempting to determine or identify a possible condition and to the opinion reached by the process. In the sense of a diagnostic procedure, diagnosis can be considered an attempt to classify an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. Subsequently, diagnostic opinions are usually described in terms of the condition. "Prognosis" as used herein refers to predicting the likely progression and outcome of a disease and monitoring disease progression.

[0015] In the present invention, the term "expression product" of a gene should be understood to include mRNA products, full-length protein products or proteolytic fragments thereof, depending on the detection technology used. Therefore, when measuring "expression product level", it can refer to the level of mRNA, or to the level of the encoded full-length protein or to the level of its proteolytic fragments.

[0016] In the context of the present invention, the term "reference value" should be understood as a predetermined level of a gene expression product in a sample or sample group. This value is used as a threshold to distinguish between a subject in which the condition to be analyzed is present and a subject in which such a condition is not present. The sample is taken from a well-defined control subject or a control subject group that does not have a degraded ECM and its normal function, which also means that the control subject does not suffer from any condition associated with the abnormal function and / or degradation of the ECM. Those skilled in the art can select a subject or subject group that is more suitable for obtaining a reference value using common sense. The method for obtaining a reference value from a selected subject group is well known in the art. In one embodiment of the present invention, the reference value is determined by a subject or subject group that does not suffer from cancer or aneurysm. In a specific embodiment, the reference value is determined by a healthy subject or a healthy subject group.

[0017] In the sense of the present invention, "higher than reference value" expression should be understood as any increase in the expression product level, for example, at least 1.2 times or 1.5 times relative to the reference value expression product. In a specific embodiment, "higher than reference value" should be understood as at least 2 times relative to the reference value expression product.

[0018] In a specific embodiment of the method of the invention, the level of expression product of at least COL11A1 and / or COL5A2 is determined. In another specific embodiment, the method comprises determining the level of expression product of at least one gene selected from the group consisting of COL5A1, TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1, optionally in combination with one or both of COL11A1 and COL5A2, wherein when the expression product level is above a reference value, this indicates ECM degradation. In one embodiment, the expression products of both COL11A1 and COL5A2 are determined.

[0019] In one embodiment, the detection of ECM degradation is performed by measuring the expression product levels of at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine genes disclosed in Table 1. In a specific embodiment, the expression products of at least COL11A1, COL5A2 and MMP2 are measured. In other specific embodiments, the expression products of at least the following genes are determined: COL11A1, COL5A2, MMP2 and MMP9, or at least COL11A1, COL5A2, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, BMP4, MMP4, ITGB1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1 and COL5A1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1, COL5A1 and TGFB1. In another specific embodiment, the expression products of nine genes are determined.

[0020] Therefore, the inventors have found that the above-mentioned genome can fully analyze the molecular mechanism controlling ECM remodeling. Without wishing to be bound by theory, the inventors assume that the expression and synthesis of minor fibril-forming collagens (collagen Vα-2, collagen Vα-1 and collagen XIα-1) increase the formation of heterotypic fibrils of smaller size and diameter of the major fibril-forming collagens (collagen I and collagen III). The minor fibril-forming collagens can inhibit the assembly of the major fibril-forming collagens by the steric hindrance of the large globular amino-terminal domains that are partially retained in the final protein complex. In addition, the released large globular amino-terminal domain contains a well-characterized heparin binding domain that can interact with specific integrin receptors, which in turn controls the expression and activity of matrix metalloproteinases, which are responsible for the degradation of ECM components. This molecular mechanism leads to thinner, disordered and degraded ECM, and is therefore more sensitive to expansion and aortic aneurysm formation and cancer growth and metastasis.

[0021] Extensive research conducted by the inventors has also led to the identification of other genetic markers that can provide additional diagnostic information about ECM degradation. These genes are listed in Table 1bis. Therefore, in a specific embodiment of the present invention, the detection of ECM degradation is performed by additionally determining the level of expression products of at least one gene selected from the group consisting of genes in Table 1bis. In some embodiments, the detection of ECM degradation is performed by determining the level of expression products of at least one gene selected from the genes in Table 1bis and at least one gene disclosed in Table 1 or any combination thereof as defined above.

[0022] Table 1bis. Genetic fingerprints used to measure ECM degradation.

[0023] name symbol NCBI reference sequences Integrin subunit α3 ITGA3 NG_029107.2 Integrin subunit α6 ITGA6 NG_008853.1 Tissue inhibitor of matrix metallopeptidase 1 TIMP1 NG_012533.1 Collagen type I α1 chain COL1A1 NG_007400.1 Collagen type III alpha 1 chain COL3A1 NG_007404.1 Collagen type I α2 chain COL1A2 NG_007405.1

[0024] The biological sample separated from the subject can be any tissue or body fluid, such as blood, plasma, saliva, urine, cerebrospinal fluid or semen. However, in a preferred embodiment of the present invention, the biological sample is peripheral blood. This is important because it greatly accelerates and simplifies the detection method, and it is non-invasive. It is indeed surprising that the differential expression of the genome disclosed in Table 1 can be found in the peripheral blood of subjects with degraded ECM.

[0025] In one embodiment, the expression product of the gene determined in the context of the present invention is mRNA. In a preferred embodiment, the amount of mRNA in the subject being tested is quantified and compared to a reference value, which is the same mRNA amount in a control subject or the average mRNA amount in a group of control subjects. The known mRNA sequences of the genes comprising the fingerprint of the present invention are disclosed in Table 2, and the known protein sequences of the same genes are disclosed in Table 2p. It is noted that some genes may have several transcripts, such as COL11A1. However, the method of the present invention preferably measures all possible transcripts of a gene, so as to measure all transcribed mRNAs from a particular gene.

[0026] Table 2. mRNA sequences of genes in Table 1 and Table 1bis

[0027]

[0028]

[0029] Table 2p Protein sequences of genes in Table 1 and Table 1bis

[0030]

[0031]

[0032] The determination of mRNA amount can be carried out by any method known to those skilled in the art, as long as the method allows detection and quantification of mRNA in biological samples. Examples of these procedures include PCR, real-time quantitative PCR (QPCR), multiplex PCR, NASBA, LCR, RT-PCR, RNA sequencing, array hybridization or "Northern" transfer or a combination thereof. In most of the above-mentioned methods for detecting and quantifying RNA, RNA must be converted to complementary DNA (cDNA) before this procedure is performed. This conversion is completed by techniques known to those skilled in the art (e.g., reverse transcription, etc.).

[0033] In a specific embodiment of the present invention, by reverse transcription, then carry out real-time quantitative PCR to quantitative mRNA and measure the level of gene expression product.For this technology and many other technologies for detecting / quantitative gene expression, it is necessary to use amplification primers.In a preferred embodiment of the present invention, the primer sequence is derived from the transcript sequence of the gene disclosed in Table 2.In a specific embodiment, the primer for measuring the level of gene expression product (i.e. mRNA) is selected from those primers shown in Table 3.By reverse transcription, then carry out real-time quantitative PCR to measure the mRNA level of the above-mentioned gene and will be described in detail in the following examples.

[0034] Table 3. Primers used to detect mRNA of genes in Table 1 and Table 1bis

[0035]

[0036]

[0037] The present invention requires comparing the expression level of the gene expression product with a reference value. As mentioned above, the reference value is obtained from a control subject or a control subject group. The technician can use any available method to establish the comparison. For example, as a relative quantitative method, the 2 method of Livak and Schmittgen can be used. -ΔΔCt (Methods, 2001, Vol. 25, No. 4, pp. 402-408).

[0038] In another embodiment, a microarray comprising one or more probes corresponding to one or more biomarkers identified in Table 2 is used. The method results in hybridization patterns of labeled target nucleic acids produced on the array surface. The hybridization patterns of the resulting labeled nucleic acids can be visualized or detected in a variety of ways, wherein a specific detection method is selected based on the specific labeling of the target nucleic acid. Representative detection methods include scintillation counting, autoradiography, fluorescence determination, calorimetry, luminescence determination, light scattering, etc.

[0039] In other embodiments, the expression product of the gene measured in the context of the present invention is a full-length protein by the gene encoding or a fragment of the protein. In a specific embodiment of method provided by the invention, the level of protein markers or its fragment is measured by the quantitative test selected from the group consisting of immunoassay, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry. In some embodiments, protein to be measured is those protein shown in the table 2p.

[0040] In one embodiment, the levels of the encoded protein or fragments thereof are detected by mass spectrometry, such as by shotgun liquid chromatography mass spectrometry (LC-MS / MS) or multiple reaction monitoring (MRM) mass spectrometry.

[0041] In an alternative embodiment, expression levels are determined by immunochemistry.

[0042] The term "immunochemistry" as used herein refers to a variety of techniques for detecting antigens (in the present case any protein or antigenic fragment thereof encoded by the above-mentioned genes) in a sample by utilizing the principle of antibody specific binding to a target protein. Then, the visualization of antibody-antigen interactions can be achieved in a variety of ways, usually by conjugating the antibody to an enzyme (such as peroxidase) that can catalyze a color reaction or conjugating it to a fluorophore (such as fluorescein or rhodamine). Immunochemical techniques can be direct or indirect.

[0043] Suitable immunoassay procedures include enzyme-linked immunosorbent assay (ELISA, such as multiplex ELISA), enzyme immunospot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunochromatography or other immunoassay formats known to those skilled in the art (such as radioimmunoassay) and protein microarray formats. In one embodiment, the level of the protein is determined by immunoassay. In another embodiment, the expression level of the protein is determined by ELISA.

[0044] The term "antibody or fragment thereof capable of binding to a target protein" should be understood as any immunoglobulin or fragment thereof capable of selectively binding to a target protein as mentioned in aspects and embodiments of the present invention. It includes monoclonal antibodies and polyclonal antibodies. The term "fragment thereof" includes any part of an antibody having a size and conformation suitable for binding to an epitope of a target protein. Suitable fragments include F(ab), F(ab') and Fv. An "epitope" is the portion of an antigen that is recognized by the immune system (B cells, T cells or antibodies).

[0045] Another aspect of the invention relates to the use of a means for determining the level of an expression product of at least one gene selected from the group consisting of the genes of Table 1 in a method for detecting ECM degradation as defined above. In a specific embodiment, the means is used to determine the level of an expression product of at least one gene selected from the group consisting of COL11A1 and / or COL5A2. In another specific embodiment, the means is used to determine the level of an expression product of at least one gene selected from the group consisting of COL5A1, TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 (optionally in combination with one or both of COL11A1 and COL5A2). Preferably, the means is used for both COL11A1 and COL5A2. In a specific embodiment, the means is used to determine the level of an expression product of at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes, at least eight or at least nine genes selected from the group consisting of genes disclosed in Table 1. In other specific embodiments, the reagents include reagents for determining the levels of expression products of at least the following genes: COL11A1, COL5A2, and MMP2, or at least COL11A1, COL5A2, MMP2, and MMP9, or at least COL11A1, COL5A2, MMP2, MMP9, and BMP1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, and ITGA4, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, and ITGB1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1, and COL5A1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1, COL5A1, and TGFB1. In another embodiment, the reagent is used to determine the expression product levels of all genes disclosed in Table 1. In another embodiment, the reagent is used to determine the expression product levels of at least one gene disclosed in Table 1bis and at least one gene disclosed in Table 1 or any combination thereof as defined above.

[0046] In a specific embodiment, the reagent is used to measure mRNA. In one embodiment, the reagent comprises amplification primers. In a specific embodiment, the primers are those shown in Table 3 in each case.

[0047] In other embodiments, the reagent is a reagent for measuring a protein or a fragment thereof. In a specific embodiment, the reagent is an antibody or a fragment thereof that specifically binds to a target protein.

[0048] In another embodiment, the reagent forms part of a kit. In addition, the present invention also provides the use of a kit comprising reagents for determining the level of expression products as defined above for use in performing any of the methods provided herein. The kit may comprise the reagents and instructions for their use in detecting ECM degradation in a subject. The instructions may include information about threshold values ​​and / or reference values ​​for determining ECM degradation, the extent of such degradation.

[0049] Another aspect of the invention provides the use of an expression product of at least one gene selected from the group consisting of the genes of Table 1 in detecting ECM degradation in a subject. In some embodiments, at least COL11A1 and / or COL5A2 are selected biomarkers. In another embodiment, the expression product of at least one gene selected from the group consisting of COL5A1, TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 (optionally in combination with one or both of COL11A1 and COL5A2) is used as an in vitro biomarker for detecting ECM degradation in a subject. Preferably, both COL11A1 and COL5A2 are among the selected biomarkers.

[0050] In one embodiment, the biomarkers for detecting ECM degradation in a subject are expression products of at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine genes disclosed in Table 1. In a specific embodiment, at least COL11A1, COL5A2, and MMP2 are selected. In other specific embodiments, the following genes are selected: COL11A1, COL5A2, MMP2 and MMP9, or at least COL11A1, COL5A2, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, BMP4, MMP4, ITGB1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1 and COL5A1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1, COL5A1 and TGFB1. In another specific embodiment, nine genes are selected. In another embodiment, the biomarker for detecting ECM degradation in a subject is the expression product of at least one gene disclosed in Table 1bis and at least one gene disclosed in Table 1, or any combination thereof as defined above.

[0051] It has been found that degradation of ECM is associated with cancer, in particular with the malignant development and metastasis of tumors. It has also been found that degradation of ECM is closely associated with the growth and risk of rupture of aneurysms. Therefore, another embodiment of the present invention relates to a method for detecting ECM degradation in a subject as defined above, wherein degradation of ECM indicates that the patient suffers from cancer, aneurysm, or both cancer and aneurysm.

[0052] cancer

[0053] As shown in Example 1, the inventors were very surprised to find that some of the genes listed in Table 1 were significantly upregulated in the peripheral blood of cancer patients when compared with reference values, which allows for rapid and easy diagnosis of cancer. The inventors have shown that patients with non-small cell lung cancer and controls (subjects without any malignant tumors) can be distinguished with a sensitivity of 0.98 (95% confidence interval: 0.89-1.00, P < 0.001) and a specificity of 1.00 (95% confidence interval: 0.61-1.00, P < 0.001). These results show that the method of the present invention can accurately diagnose cancer from peripheral blood with high specificity and sensitivity.

[0054] The fingerprint for diagnosing cancer comprises the genes shown in Table 4 and Table 4bis.

[0055] Table 4. Genetic fingerprints for diagnosis of cancer.

[0056] name symbol NCBI reference sequences Collagen type XI α1 chain COL11A1 NG_008033.1 Collagen V-type α2 chain COL5A2 NG_011799.2 Collagen V-type α1 chain COL5A1 NG_008030.1 Integrin subunit α4 ITGA4 NG_050623.1 Integrin subunit β1 ITGB1 NG_029012.1 Matrix metallopeptidase 2 MMP2 NG_008989.1 Matrix metallopeptidase 9 MMP9 NG_011468.1 Bone morphogenetic protein 1 BMP1 NG_029659.1

[0057] Table 4bis. Genetic fingerprints for diagnosing cancer.

[0058]

[0059]

[0060] Therefore, another aspect of the present invention relates to a method for in vitro diagnosis of cancer in a subject, the method comprising determining the level of expression product of at least one gene selected from the group of genes listed in Table 4 in a sample isolated from the subject, wherein when the expression product level is higher than a reference value, this indicates that the subject has cancer.

[0061] "Reference value" and "above reference value" are understood as described above. In a preferred embodiment of the cancer-related aspects of the present invention, the reference value is obtained from a subject or subject group that does not suffer from any cancer malignancy. In a preferred embodiment of the cancer-related aspects of the present invention, "above reference value" is understood as the expression (overexpression) level of each gene expression product relative to the reference value increased by the following multiples:

[0062] At least 5-fold overexpression relative to the COL11A1 reference value,

[0063] At least 2-fold overexpression relative to the COL5A2 reference value,

[0064] At least 2-fold overexpression relative to the COL5A1 reference value,

[0065] At least 5-fold overexpression relative to the MMP2 reference value,

[0066] At least 7-fold overexpression relative to the MMP9 reference value,

[0067] At least 2-fold overexpression relative to the BMP1 reference value,

[0068] At least 1-fold overexpression relative to the ITGA4 reference value, or

[0069] At least 2-fold overexpression relative to ITGB1 reference value.

[0070] The present inventors have also found that there is a direct correlation between the mRNA levels in peripheral blood of the genes disclosed in Table 4 and the cancer stage.

[0071] Most types of cancer have 4 stages, numbered from I to IV. Stage I usually means the cancer is relatively small in size and contained to the organ where it started. Stage II usually means the tumor is larger than stage I, but the cancer has not yet begun to spread into surrounding tissues. Sometimes stage II means the cancer cells have spread to lymph nodes close to the tumor. This depends on the specific type of cancer. Stage III usually means the cancer is larger, it may have begun to spread into surrounding tissues, and there are cancer cells in lymph nodes in the area. Stage IV means the cancer has spread to another distant tissue or organ from where it started.

[0072] The correlation between the overexpression of the genes in Table 4 and the cancer stage is shown in Example 1. Although all the genes in Table 4 are overexpressed, when compared to the reference group, measured in terms of mRNA levels, it can be observed that the overexpression of the genes is lower in patients with stage I non-small lung cancer, although statistically significant, but steadily increases throughout stages II, III and IV. The present inventors have demonstrated that patients with advanced metastatic non-small cell lung cancer (stages III and IV) can be distinguished from patients with early stage non-small cell lung cancer (stages I and II) with a sensitivity of 0.95 (95% confidence interval: 0.78-0.99, P < 0.001) and a specificity of 0.96 (95% confidence interval: 0.80-0.99, P < 0.001).

[0073] Therefore, the present invention also relates to a method for in vitro differential diagnosis of a patient according to the stage of cancer of the patient, the method comprising determining the level of expression product of at least one gene selected from the group of genes listed in Table 4 in a sample isolated from a subject. An increase in the level of the expression product is associated with an increase in the stage of cancer. Patients with stage III or stage IV cancer are generally referred to as patients with advanced cancer. In particular, when the expression product has the following levels:

[0074] At least 10-fold overexpression relative to the COL11A1 reference value,

[0075] At least 5-fold overexpression relative to the COL5A2 reference value,

[0076] At least 5-fold overexpression relative to the COL5A1 reference value,

[0077] At least 8-fold overexpression relative to the MMP2 reference value,

[0078] At least 11-fold overexpression relative to the MMP9 reference value,

[0079] At least 5-fold overexpression relative to the BMP1 reference value,

[0080] At least 6-fold overexpression relative to the ITGA4 reference value, or

[0081] At least 8-fold overexpression relative to ITGB1 reference value.

[0082] This indicates that the subject has stage III or IV metastatic cancer (advanced cancer).

[0083] The method of the present invention is also used for the in vitro differential diagnosis of patients with advanced cancer (stages III and IV) and patients with early stage cancer (stages I and II), wherein, when the expression product has a level as defined above, this indicates that the patient is in the advanced cancer stage, and when the expression product has a level below the threshold value defined above, this indicates that the patient is in the early cancer stage.

[0084] The III and IV stages of cancer are also often considered to mean a high risk of metastasis. In the sense of the present invention, "metastasis" is generally understood in the art as a process in which cancer cells spread to new areas of the body that are different from the primary cancer site (usually through the lymphatic system or bloodstream). The tumor formed by the cells that have spread is called a secondary tumor. Cancer may have spread to the area near the primary site (regional metastasis), or spread to a more distant part of the body (distant metastasis). The method of the present invention provides a reliable test for identifying a high risk of metastasis or an early stage cancer patient in the metastatic process. This is very advantageous for the clinical management of cancer patients, who can receive the most appropriate treatment according to their progress, and when the risk of metastasis is high, a strict follow-up plan can be carried out if necessary.

[0085] Therefore, the present invention also provides a method for determining the risk of cancer metastasis in a subject, the method comprising determining the level of expression product of at least one gene selected from the group of genes listed in Table 4 in a sample isolated from the subject. High levels of expression products indicate a high risk of metastasis. In particular, when the expression product has the following levels:

[0086] At least 10-fold overexpression relative to the COL11A1 reference value,

[0087] At least 5-fold overexpression relative to the COL5A2 reference value,

[0088] At least 5-fold overexpression relative to the COL5A1 reference value,

[0089] At least 8-fold overexpression relative to the MMP2 reference value,

[0090] At least 11-fold overexpression relative to the MMP9 reference value,

[0091] At least 5-fold overexpression relative to the BMP1 reference value,

[0092] At least 6-fold overexpression relative to the ITGA4 reference value, or

[0093] At least 8-fold overexpression relative to ITGB1 reference value.

[0094] This indicates that the subject has a high risk of cancer metastasis.

[0095] According to all of the above, therefore, by using the method of the present invention, cancer patients can be classified according to their cancer stages. Therefore, another aspect of the present invention relates to an in vitro method for classifying cancer patients according to their cancer stages, the method comprising determining the expression product level of at least one gene selected from the group consisting of the genes listed in Table 4 in a sample isolated from a subject, and correlating the expression product level with the cancer stage.

[0096] The method can also provide early information about the risk of relapse in patients who have been treated for cancer and have overcome the disease. As generally understood in the art, "relapse" is a deterioration in someone's health status after a temporary improvement. This is very important in clinical terms because early detection and subsequent management of relapse in cancer patients can greatly improve the prognosis of patients suffering from relapse.

[0097] Therefore, another aspect of the present invention relates to an in vitro method for detecting recurrence in a subject who has been treated for cancer, the method comprising determining the level of an expression product of at least one gene selected from the group consisting of the genes in Table 4 in a sample isolated from the subject, wherein, when the expression product level is above a reference value, this indicates that the patient is at high risk of suffering from recurrence.

[0098] Therefore, another aspect of the present invention relates to an in vitro method for the prognosis of a cancer patient, the method comprising determining the level of an expression product of at least one gene selected from the group consisting of the genes listed in Table 4 in a sample isolated from the patient. A high level of the expression product indicates a poor prognosis. In particular, when the expression product has the following levels:

[0099] At least 10-fold overexpression relative to the COL11A1 reference value,

[0100] At least 5-fold overexpression relative to the COL5A2 reference value,

[0101] At least 5-fold overexpression relative to the COL5A1 reference value,

[0102] At least 8-fold overexpression relative to the MMP2 reference value,

[0103] At least 11-fold overexpression relative to the MMP9 reference value,

[0104] At least 5-fold overexpression relative to the BMP1 reference value,

[0105] At least 6-fold overexpression relative to the ITGA4 reference value, or

[0106] At least 8-fold overexpression relative to ITGB1 reference value.

[0107] This indicates a poor prognosis.

[0108] This method is not limited to a specific type of cancer. The overall mechanism of cancer progression and diffusion associated with ECM degradation causing metastasis is universal to a certain extent for almost all types of cancer. When compared with non-small cell lung cancer patient controls, changes in gene expression levels occurring at the tissue level were clearly detected in peripheral blood with a sensitivity of 98% and a specificity of 100%, and when early stage (I and II stage) lung cancer patients were compared with late metastatic stage (III and IV stage) patients, the sensitivity was 95% and the specificity was 96%. We have confirmed similar tissue expression patterns in female patients diagnosed with breast cancer. Therefore, since these detected changes actually reflect changes in the ECM, they can be used to distinguish patients with other types of cancer, particularly those patients with the ability to metastasize through ECM degradation.

[0109] In specific embodiments, the cancer is non-small cell lung cancer, breast cancer, colon cancer, rectal cancer, small intestine cancer, prostate cancer, small cell lung cancer, mesothelioma, kidney cancer, pancreatic cancer, stomach cancer, esophageal cancer, laryngeal cancer, oropharyngeal cancer, liver cancer, bile duct cancer, gallbladder cancer, bladder cancer, thyroid cancer, endometrial cancer, ovarian cancer, vaginal cancer, urethral cancer, testicular cancer, bone cancer, brain cancer, skin cancer, melanoma, sarcoma, angiosarcoma, liposarcoma, etc. In specific embodiments, the cancer is non-small cell lung cancer or breast cancer, such as non-small cell lung cancer.

[0110] While providing a reliable and early diagnosis of cancer, including categorizing patients according to the progression and metastasis of their disease, the present diagnostic method is useful to a clinician because it enables him / her to make the most appropriate decision to treat the patient. Since anticancer treatment options may be highly dependent on the stage of the cancer, in particular, whether there is metastasis or a high risk of metastasis, the clinician can recommend the most appropriate (conservative or aggressive) anticancer therapy based on the differential diagnosis performed as described above.

[0111] Therefore, in another aspect, the present invention relates to an in vitro method for recommending an anti-cancer therapy in a subject, the method comprising: (a) diagnosing whether the subject has cancer or determining a poor prognosis for a subject with cancer by a method as defined above, and (b) if the subject is diagnosed with cancer or a poor prognosis for cancer, recommending an anti-cancer therapy. This aspect may also be considered as a method for treating a cancer patient, the method comprising (a) diagnosing whether the subject has cancer or determining a poor prognosis for a subject with cancer by a method as defined above, and (b) if the subject is diagnosed with cancer or a poor prognosis for cancer, administering an anti-cancer therapy to the patient. If the patient is not diagnosed with cancer, the clinician may recommend a follow-up visit for the subject.

[0112] In some embodiments, the method is for recommending a metastatic cancer therapy in a subject when the diagnosis indicates that the subject has metastatic cancer.

[0113] Anticancer therapy includes surgery, chemotherapy, radiotherapy, immunotherapy, targeted therapy and hormone therapy. Most cancer patients take combined therapy according to the type of cancer and the degree of its progress at the time of diagnosis. Preferably, anticancer therapy is selected from the above options based on cancer type and stage, clinical trial results and histopathological findings. The treatment regimen for treating metastatic cancer depends on the type of primary cancer, the site of spread, the treatment used in the past and the general health of the patient. In most cases, the treatment regimen for treating metastatic cancer includes a combination of at least two therapies selected from the most aggressive forms of surgery, chemotherapy, radiotherapy, immunotherapy, targeted therapy and hormone therapy. Although some types of metastatic cancer can be cured with current treatment, most cannot be cured. In most metastatic cancers, the goal of these treatments is to stop or slow the growth of cancer or relieve symptoms (palliative therapy), and in some cases, the treatment of metastatic cancer can help prolong life. It is also important to mention that in most cases, the therapeutic intervention for metastatic cancer includes chemotherapy and / or radiotherapy with significant side effects, which cannot be tolerated by a considerable number of patients before the treatment cycle is completed, therefore, it is prominent that early diagnosis is required even in the case of metastasis.

[0114] In addition, according to the present invention, the cancer patient that receives specific treatment can be monitored to ensure the effectiveness of therapeutic intervention or warn the early stage of recurrence or the generation of metastatic disease.Present method allows convenient follow-up and improved cancer patient management, thereby avoids unnecessary pain and / or minimizes side effect.Successful therapeutic intervention will for example cause the expression level of these genes to approach control, and as long as there is no recurrence or metastatic disease, this effect should be kept.

[0115] Therefore, another aspect of the present invention provides an in vitro method for determining the response of a cancer patient to an anti-cancer therapy, the method comprising determining the level of an expression product of at least one gene selected from the group consisting of the genes in Table 4 in a sample isolated from the patient, and comparing the level of the expression product with the level of the expression product of the same gene determined for the same patient before the start of the therapy or at an earlier stage of the therapy, wherein a decrease in the expression product of the gene relative to the start of the therapy or the earlier stage of the therapy indicates a good response.

[0116] Another aspect can be defined as a kind of in vitro method for recommending alternative and / or complementary therapy in cancer patients, the method comprises determining the expression product level of at least one gene in the group consisting of the genes of table 4 in the sample separated from the patient, and comparing the expression product level with the expression product level of the same gene determined for the same patient before the start of the therapy or at the earlier stage of the therapy, wherein, when the expression product of the gene increases relative to the start of therapy or the earlier stage of the therapy, this indicates that alternative and / or complementary therapy is recommended. This can also be formulated into a method for treating cancer patients who are unresponsive to anticancer therapy, the method comprises determining the expression product level of at least one gene in the group consisting of the genes of table 4 in the sample separated from the patient, and comparing the expression product level with the expression product level of the same gene determined for the same patient before the start of the therapy or at the earlier stage of the therapy, and when the expression product of the gene increases relative to the start of therapy or the earlier stage of the therapy, giving alternative and / or complementary therapy. Sometimes, when the expression product of the gene does not change relative to the start of therapy or the earlier stage of the therapy, the clinician can even recommend or give alternative and / or complementary therapy.

[0117] In all the above methods relating to cancer, preferably the level of expression products of at least COL11A1 and / or COL5A2 is determined. In a specific embodiment, the method comprises determining the level of expression products of at least one gene selected from the group consisting of COL5A1, ITGA4, ITGB1, MMP2, MMP9 and BMP1, optionally in combination with one or both of COL11A1 and COL5A2, wherein when the level of the expression product is above a reference value, this indicates that the subject suffers from cancer. Preferably, the expression products of both COL11A1 and COL5A2 are determined. In other embodiments, the method comprises determining the level of expression products of at least three, at least four, at least five, at least six, at least seven or at least eight genes disclosed in Table 4. In a specific embodiment, the expression products of at least COL11A1, COL5A2 and COL5A1 are determined. In other specific embodiments, the expression products of at least the following genes are determined: COL11A1, COL5A2, COL5A1 and MMP2, or at least COL11A1, COL5A2, COL5A1, MMP2 and MMP9, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9, BMP1, ITGA4 and ITGB1. In another specific embodiment, the expression products of eight genes are determined. In other embodiments of the present invention, the expression product level of at least one gene selected from the genes in Table 4bis is additionally determined. In some embodiments, the above methods related to cancer comprise determining the level of an expression product of at least one gene selected from the genes in Table 4bis and at least one gene disclosed in Table 4, or any combination thereof as defined above.

[0118] As disclosed above, the biological sample obtained from the patient can be any tissue or body fluid, such as blood, plasma, saliva, urine, cerebrospinal fluid or semen, preferably peripheral blood. The diagnostic test based on peripheral blood samples is very simple, less invasive and cost-effective for the widespread use of the general population. More specifically, elderly patients with limited access to tertiary diagnosis and treatment centers will be able to utilize simple and cost-effective blood tests to achieve diagnosis and monitoring. In clinical settings, after therapeutic intervention, it is well known to monitor cancer patients with computed tomography every six months in the first two years and then annually. Similarly, the diagnostic blood test of the present invention has the promising potential for reliable monitoring of cancer patients after therapeutic intervention. Because blood tests are relatively simpler, convenient and cost-effective, and there is no adverse effect of the radiation carried by serial computed tomography, it can be used more frequently. The diagnostic tests of the present invention can be used every three months in order to detect early recurrence of malignancies and also before scanning the patient with a computed tomography scan, in such a way that if they show low or normal levels of expression of genes associated with malignancies and metastatic disease, they may decide to postpone the computed tomography scan to a future point in time when its contribution to the potential diagnosis of recurrence can be fully demonstrated.

[0119] The expression product level of gene is measured as described above. In some embodiments, the expression product is mRNA, and preferably measured by reverse transcription and then real-time quantitative PCR. Amplification primers are derived from transcript mRNA sequences of genes disclosed in Table 4 and Table 4bis (as shown in Table 2), and suitable primers for amplifying transcript sequences are provided in Table 3. In other embodiments, the expression product is a coded protein, and measured by mass spectrometry or immunochemistry as described above.

[0120] Another aspect of the present invention relates to the use of an agent for determining the level of expression products of at least one gene selected from the group consisting of the genes of Table 4 in a method related to cancer as defined above. In a specific embodiment, the agent is used to determine the level of expression products of at least one gene selected from the group consisting of COL11A1 and / or COL5A2. In another specific embodiment, the agent is used to determine the level of expression products of at least one gene selected from the group consisting of COL5A1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 (optionally in combination with one or both of COL11A1 and COL5A2). Preferably, the agent is used to determine both COL11A1 and COL5A2. In a specific embodiment, the agent is used to determine the level of expression products of at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes or at least eight genes selected from the group of genes disclosed in Table 4. In other specific embodiments, the reagents include reagents for determining the expression product levels of at least the following genes: COL11A1, COL5A2 and COL5A1, or at least COL11A1, COL5A2, COL5A1 and MMP2, or at least COL11A1, COL5A2, COL5A1, MMP2 and MMP9, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9, BMP1, ITGA4 and ITGB1. In another embodiment, the reagents are used to determine the expression product levels of all genes disclosed in Table 4. In another embodiment, the reagent is used to determine the level of expression products of at least one gene disclosed in Table 4bis and at least one gene disclosed in Table 4 or any combination thereof as defined above.

[0121] In a specific embodiment, the reagent is used to measure mRNA. In one embodiment, the reagent comprises an amplification primer. In a specific embodiment, the primers are those shown in Table 3 in each case. In other embodiments, the reagent is used to measure a protein or a fragment thereof. In a specific embodiment, the reagent is an antibody or a fragment thereof that specifically binds to a target protein.

[0122] In another embodiment, the reagent forms part of a kit. The kit of the present invention may comprise the reagent for determining the level of the expression product and instructions for cancer diagnosis / prognosis / risk of metastasis / classification according to the stages defined above. The instructions may include information about thresholds and / or reference values ​​for determining cancer diagnosis / prognosis / risk of metastasis / classification according to the stages defined above.

[0123] On the other hand, the present invention also provides the use of the expression product of at least one gene selected from the group consisting of genes listed in Table 4 as a biomarker in the in vitro diagnosis of cancer in a subject. The present invention also relates to the use of the biomarker in the in vitro differential diagnosis of cancer patients according to the cancer stage of the cancer patient. Some embodiments relate to the use of the biomarker in the differential diagnosis of cancer patients with cancer in stage III or stage IV. Other embodiments relate to the use of the biomarker in the differential diagnosis of patients with advanced cancer (stages III and IV) and patients with early stage cancer (stages I and II). Specific embodiments relate to the use of the biomarker in diagnosing a high risk of cancer metastasis in a subject. On the other hand, the use of the expression product of at least one gene selected from the group consisting of genes listed in Table 4 as a biomarker in diagnosing the recurrence of patients who have received cancer treatment intervention is provided.

[0124] Another aspect of the present invention provides the use of the expression product of at least one gene selected from the group consisting of genes listed in Table 4 as a biomarker in the in vitro prognosis of cancer in a subject. Another aspect provides the use of the expression product of at least one gene selected from the group consisting of genes listed in Table 4 as a biomarker in recommending anti-cancer therapy to a subject with cancer. In one embodiment, the use is for recommending metastatic cancer therapy to a subject with a high risk of metastasis.

[0125] Another aspect provides the use of an expression product of a gene selected from the group consisting of genes listed in Table 4 as a biomarker in determining the response of a cancer patient to a specific anti-cancer therapy.

[0126] In some embodiments of the above aspects, at least COL11A1 and / or COL5A2 are selected biomarkers. Another embodiment provides the use of an expression product of at least one gene selected from the group consisting of COL5A1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 (optionally in combination with one or both of COL11A1 and COL5A2) as an in vitro biomarker. Preferably, both COL11A1 and COL5A2 are among the selected biomarkers. In other embodiments, the selected biomarkers are expression products of at least three, at least four, at least five, at least six, at least seven or at least eight genes disclosed in Table 4. In a specific embodiment, the expression products of at least COL11A1, COL5A2 and COL5A1 are selected. In other specific embodiments, the expression products of at least the following genes are selected: at least COL11A1, COL5A2, COL5A1 and MMP2, or at least COL11A1, COL5A2, COL5A1, MMP2 and MMP9, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, COL5A1, MMP2, MMP9, BMP1, ITGA4 and ITGB1. In another specific embodiment, the expression products of eight genes are selected. In other embodiments, the biomarker is the expression product of at least one gene disclosed in Table 4bis and at least one gene disclosed in Table 4 or any combination thereof as defined above.

[0127] Aneurysm

[0128] It was also found that the expression levels of some genes disclosed in Table 1 were closely related to aneurysms. The genetic fingerprint for diagnosing aneurysms comprises the genes shown in Table 5 and Table 5bis.

[0129] As shown in Example 2, the inventors were very surprised to find that some of those genes were significantly upregulated in the peripheral blood of patients with thoracic aortic aneurysms when compared to reference values, which allows for rapid and easy diagnosis of this condition. The inventors have also shown that patients with thoracic aortic aneurysms can be distinguished from controls (subjects without thoracic aortic aneurysms or cancer) with a sensitivity of 0.95 (95% confidence interval: 0.89-1.00, P < 0.001) and a specificity of 0.92 (95% confidence interval: 0.78-1.00, P < 0.001). These results show that the method of the present invention can accurately diagnose aneurysms from peripheral blood samples.

[0130] Table 5. Genetic fingerprints for diagnosis of aneurysms.

[0131] name symbol refer to Collagen type XI α1 chain COL11A1 NG_008033.1 Collagen V-type α2 chain COL5A2 NG_011799.2 Transforming Growth Factor Beta-1 TGFB1 NG_013364.1 Integrin subunit α4 ITGA4 NG_050623.1 Integrin subunit β1 ITGB1 NG_029012.1 Matrix metallopeptidase 2 MMP2 NG_008989.1 Matrix metallopeptidase 9 MMP9 NG_011468.1 Bone morphogenetic protein 1 BMP1 NG_029659.1

[0132] Table 5bis. Genetic fingerprints for diagnosis of aneurysms.

[0133]

[0134]

[0135] Therefore, another aspect of the present invention relates to a method for in vitro diagnosis of an aneurysm in a subject, the method comprising determining the level of an expression product of at least one gene selected from the group consisting of the genes disclosed in Table 5 in a sample isolated from the subject, wherein when the expression product level is higher than a reference value, this indicates that the subject suffers from an aneurysm.

[0136] "Reference value" and "above reference value" are understood as described above. In a preferred embodiment of the aneurysm-related aspect of the present invention, the reference value is obtained from a subject or subject group that does not suffer from any aneurysm. In a preferred embodiment of the aneurysm-related aspect of the present invention, "above reference value" is understood as the expression (overexpression) level of each gene expression product relative to the reference value increased by the following fold:

[0137] At least 5-fold overexpression relative to the COL11A1 reference value,

[0138] At least 1.5-fold overexpression relative to the COL5A2 reference value,

[0139] At least 3-fold overexpression relative to the TGFB1 reference value,

[0140] At least 3-fold overexpression relative to the MMP2 reference value,

[0141] At least 2.5-fold overexpression relative to the MMP9 reference value,

[0142] At least 5-fold overexpression relative to the BMP1 reference value,

[0143] At least 1.5-fold overexpression relative to the ITGA4 reference value, or

[0144] At least 3-fold overexpression relative to ITGB1 reference value.

[0145] Furthermore, as shown in the results of Example 2, the present inventors have found that there is a direct correlation between the mRNA levels in peripheral blood of the genes shown in Table 5 and the size of thoracic aortic aneurysms.

[0146] Aneurysms are usually classified according to their size and symptoms. An aneurysm of any blood vessel in the body is usually defined as an increase in the external vessel diameter greater than 50% of the normal diameter of a healthy individual based on normal values ​​for gender and age. The normal diameter of the thoracic aorta in an adult is 2-3 cm. A thoracic aorta with a diameter of 4.5 cm (50% increase compared to 3 cm) is considered an aortic aneurysm. It is estimated that the risk of rupture or dissection of a thoracic aortic aneurysm is significantly higher when the aortic diameter is greater than 5 cm, and the risk of rupture becomes even higher in larger diameters. From a clinical point of view, thoracic aortic aneurysms with a diameter between 5 cm and 6 cm should be considered for intervention. In larger thoracic aortic aneurysms (6-7 cm in diameter) or even in very large thoracic aortic aneurysms (>7 cm in diameter), the need for intervention is considered urgent and emergency, respectively.

[0147] The correlation between the overexpression of the genes in Table 5 and the size of the aortic aneurysm is shown in Example 2. It can be observed that the overexpression of the genes measured by mRNA expression levels in patients with smaller aortic aneurysms (i.e., aortic diameter 5-6 cm vs. aortic diameter 6-7 cm vs. aortic diameter>7 cm) is lower, although statistically significant. In contrast, the overexpression of the genes is significantly increased in patients with very large aortic aneurysms (aortic outer diameter greater than 7 cm). The inventors have demonstrated that, using the method of the present invention, patients with relatively large aortic aneurysms (aortic diameter equal to or greater than 6 cm) can be distinguished from patients with relatively small aortic aneurysms (aortic diameter between 4.5 cm and 6 cm) with a sensitivity of 0.95 (95% confidence interval: 0.86-1.00, P<0.001) and a specificity of 0.86 (95% confidence interval: 0.71-1.00, P<0.001).

[0148] Therefore, in one embodiment of the method of the present invention, the patient is differentiated according to the size of the aneurysm, and the method comprises determining the expression product level of at least one gene selected from the group of genes listed in Table 5 in a sample isolated from a subject. An increase in the level of the expression product is associated with an increase in the size of the aneurysm. In another embodiment, the method is used to differentiate between patients diagnosed with larger aneurysms. In another embodiment, the method is used to differentiate between patients diagnosed with larger aneurysms and patients with smaller aneurysms. In particular, when the expression product has the following levels:

[0149] At least 15-fold overexpression relative to the COL11A1 reference value,

[0150] At least 5-fold overexpression relative to the COL5A2 reference value,

[0151] At least 10-fold overexpression relative to the TGFB1 reference value,

[0152] At least 10-fold overexpression relative to the MMP2 reference value,

[0153] At least 12-fold overexpression relative to the MMP9 reference value,

[0154] At least 10-fold overexpression relative to the BMP1 reference value,

[0155] At least 5-fold overexpression relative to the ITGA4 reference value, or

[0156] At least 8-fold overexpression relative to ITGB1 reference value.

[0157] This indicates that the patient has a larger aneurysm, such as a larger aortic aneurysm (equal to or greater than 6 cm in diameter). When the expression product level is higher than the reference value but lower than these thresholds, this indicates that the patient's aneurysm is a relatively small aneurysm, especially for an aortic aneurysm with a diameter less than 6 cm.

[0158] Aneurysms (particularly those of larger diameter) are considered to be at risk of rupture. Rupture of a blood vessel (such as the aorta) causes massive internal bleeding, and shock and death may occur unless treated immediately. If the size of the aneurysm reaches a specific diameter (i.e., a diameter greater than 5 cm in the ascending thoracic aorta or a diameter greater than 6 cm in the descending thoracic aorta) and / or its rapid growth (greater than 0.5 cm per year), surgery is recommended to avoid rupture. To date, the most cost-effective screening test to determine whether a patient has an aneurysm at risk of rupture is performed by computed tomography studies. The method of the present invention constitutes a reliable, convenient and more cost-effective test for identifying patients at risk of rupture. After simple blood sampling, in view of the expression pattern of the genes in Table 5, clinicians can recommend appropriate medical interventions, such as surgery. In addition, if necessary, strict follow-up can be performed on the patient. Therefore, the present invention also relates to a method for in vitro diagnosis of the risk of aneurysm rupture in a subject, the method comprising determining the expression product level of at least one gene selected from the group consisting of the genes in Table 5 in a sample separated from the subject. A high level of the expression product indicates a risk of rupture. In particular, when the expression product has the following levels:

[0159] At least 15-fold overexpression relative to the COL11A1 reference value,

[0160] At least 5-fold overexpression relative to the COL5A2 reference value,

[0161] At least 10-fold overexpression relative to the TGFB1 reference value,

[0162] At least 10-fold overexpression relative to the MMP2 reference value,

[0163] At least 12-fold overexpression relative to the MMP9 reference value,

[0164] At least 10-fold overexpression relative to the BMP1 reference value,

[0165] At least 5-fold overexpression relative to the ITGA4 reference value, or

[0166] At least 8-fold overexpression relative to ITGB1 reference value.

[0167] This indicates a high risk of aneurysm rupture.

[0168] Overexpression of the genes in Table 5 is proportional to the size of the aneurysm. Therefore, by using the method of the present invention, patients with aneurysms can be classified according to the size of the aneurysm or according to having a high risk of rupture or a low risk of rupture. Therefore, another aspect of the present invention relates to an in vitro method for classifying patients with aneurysms according to the size of the aneurysm, the method comprising determining the expression product level of at least one gene selected from the group consisting of the genes in Table 5 in a sample isolated from a subject, and correlating the level with the size and risk of rupture of the aneurysm. Suitable thresholds for this classification are defined above.

[0169] Therefore, another aspect of the present invention relates to an in vitro method for the prognosis of aneurysm patients, the method comprising determining the level of expression product of at least one gene selected from the genes in Table 5 in a sample isolated from the patient, wherein the level of the expression product is directly correlated with a poor prognosis. Therefore, a high level of the expression product indicates a poor prognosis. In particular, when the expression product has a risk of rupture at a level as defined above, this indicates a poor prognosis.

[0170] The present method is not limited to a specific type of aneurysm. The overall mechanism of aneurysm progression associated with ECM degradation is common to all aneurysms. In a specific embodiment, the aneurysm is selected from an aortic aneurysm, which can be a thoracic (ascending thoracic or arch or descending thoracic aortic aneurysm) or an abdominal or thoracoabdominal aortic aneurysm. Other blood vessels that experience aneurysmal disease with a poor prognosis in the late stages are the cerebral arterial vessels, the iliac arteries, and the subclavian arteries. In a preferred embodiment, the aneurysm is a thoracic aortic aneurysm.

[0171] While providing a reliable and early diagnosis of aneurysms, including classifying patients according to the progression of their disease and risk of rupture, the present diagnostic method is useful to the clinician because it enables him / her to make the most appropriate decision to treat the patient. Since the treatment regimen may depend on the size of the aneurysm, and in particular whether there is a risk of rupture, the clinician can recommend the most appropriate therapy (including surgical intervention) based on the differential diagnosis performed as described above. In the case where a smaller diameter aneurysm exhibits a high level of the molecular indicators proposed by the present invention, surgical intervention may be recommended even in a smaller diameter aneurysm, taking into account the fact that, although rare, there are smaller aneurysms that are prone to rupture, and there are sporadic cases of ruptured aneurysms with relatively small diameters. The biomarkers of the present invention are generally good indicators of prognosis regarding the risk of aneurysm rupture. Therefore, in sporadic cases, smaller aneurysms may result in high levels of expression products of the disclosed markers, which will however indicate a poor prognosis and a high risk of rupture, thus providing very useful information for the clinical management of a particular patient.

[0172] Thus, in another aspect, the present invention relates to an in vitro method for recommending a treatment regimen for an aneurysm in a subject, the method comprising: (a) diagnosing whether the subject suffers from an aneurysm or determines a poor prognosis by a method as defined above, and (b) recommending a treatment regimen for the aneurysm if the subject is diagnosed with an aneurysm or determined to have a poor prognosis. This aspect may also be contemplated as a method for treating a patient suffering from an aneurysm, comprising (a) diagnosing whether the subject suffers from an aneurysm or determines a poor prognosis by a method as defined above, and (b) administering a treatment regimen for treating the aneurysm to the patient if the method indicates that the subject suffers from an aneurysm or has a poor prognosis. If the patient is not diagnosed as suffering from an aneurysm, the clinician may recommend a follow-up visit for the subject.

[0173] Treatment options for treating aneurysms include replacement of the aneurysm with a synthetic graft or endovascular approaches and stent-graft procedures in an attempt to isolate the aneurysmal portion of the vessel from the blood circulation. Preferably, the treatment option for the aneurysm is selected based on the anatomy and location of the aneurysm (there are certain anatomical limitations that completely preclude the possibility of an endovascular approach) and the age and general condition of the patient.

[0174] In some embodiments, the method is used to recommend an appropriate treatment regimen in patients whose diagnosis indicates a high risk of vascular rupture. Typically, in these cases, the appropriate therapy is surgical intervention, open surgery, or endovascular therapy with stent graft implantation in a minimally invasive manner. Other adjunctive (but not curative) treatments for pharmacological interventions include administration of statins, beta blockers, and aggressive antihypertensive agents.

[0175] In addition, according to the present invention, patients with aneurysms receiving specific treatments can be monitored to ensure the effectiveness of the therapeutic intervention or to warn of early risk of diseased blood vessel rupture. The present method allows for easy follow-up and improved management of patients with aneurysms, thereby avoiding unnecessary suffering and / or minimizing side effects. For example, successful therapeutic intervention will result in expression levels of these genes close to the control.

[0176] Therefore, another aspect of the present invention provides an in vitro method for determining the response of a patient suffering from an aneurysm to an aneurysm treatment regimen, the method comprising determining the level of an expression product of at least one gene selected from Table 5 in a sample isolated from the patient, and comparing the level of the expression product with the level of the expression product of the same gene determined for the same patient before the start of the therapy or at an earlier stage of the therapy, wherein a decrease in the expression product of the gene relative to the start of the therapy or the earlier stage of the therapy indicates a good response.

[0177] Another aspect may be defined as an in vitro method for recommending an alternative and / or supplemental treatment regimen in a patient with an aneurysm, the method comprising determining the level of an expression product of at least one gene selected from Table 5 in a sample isolated from the patient, and comparing the level of the expression product with the level of the expression product of the same gene determined for the same patient before the start of the therapy or at an earlier stage of the therapy, wherein an increase in the expression product of the gene relative to the start of therapy or at an earlier stage of the therapy indicates a need for an alternative and / or supplemental treatment regimen. This may also be formulated as a method for treating a patient with an aneurysm who is not responsive to a treatment regimen for the aneurysm, particularly in the case where the aneurysm remains within the intravascular approach in vivo, the method comprising determining the level of an expression product of at least one gene selected from Table 5 in a sample isolated from the patient, and comparing the level of the expression product with the level of the expression product of the same gene determined for the same patient before the start of the therapy or at an earlier stage of the therapy, and when the expression product of the gene increases relative to the start of therapy or at an earlier stage of the therapy, administering an alternative and / or supplemental aneurysm treatment regimen. Sometimes, the clinician may even recommend or administer an alternative and / or complementary therapy when the expression product of the gene has not changed relative to the start of therapy or an earlier stage of therapy.

[0178] The present method can also provide early information about the risk of recurrence in patients who have already had an aneurysm treated. Successful therapeutic intervention will result in expression levels of the gene close to the control. However, a patient who has already had an aortic aneurysm is still at risk of having another aortic aneurysm in another part of the native aorta. Early detection and subsequent management of recurrence can greatly improve the prognosis of patients with aneurysms.

[0179] Therefore, another aspect of the present invention relates to an in vitro method for detecting recurrence in a subject who has been treated for aneurysm, the method comprising determining the level of expression product of at least one gene selected from the group consisting of genes in Table 5 in a sample isolated from the subject, wherein, when the expression product level is higher than a reference value, this indicates that the subject is at high risk of suffering from recurrence.

[0180] In all the above methods relating to aneurysms, preferably the level of expression products of at least COL11A1 and / or COL5A2 is determined. In a specific embodiment, the method comprises determining the level of expression products of at least one gene selected from the group consisting of TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1, optionally in combination with one or both of COL11A1 and COL5A2, wherein when the level of the expression product is above a reference value, this indicates that the subject suffers from an aneurysm. Preferably, the expression products of both COL11A1 and COL5A2 are determined. In other embodiments, the method comprises determining the level of expression products of at least three, at least four, at least five, at least six, at least seven or at least eight genes disclosed in Table 5. In a specific embodiment, the expression products of at least COL11A1, COL5A2 and MMP2 are determined. In other specific embodiments, the expression products of at least the following genes are determined: COL11A1, COL5A2, MMP2 and MMP9, or at least COL11A1, COL5A2, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4 and ITGB1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1 and TGFB1. In another specific embodiment, the expression products of eight genes are determined. In other embodiments of the present invention, the expression product level of at least one gene selected from the genes in Table 5bis is additionally determined. In some embodiments, the above methods involving aneurysms comprise determining the expression product level of at least one gene selected from the genes in Table 5bis and at least one gene disclosed in Table 5 or any combination thereof as defined above.

[0181] As already disclosed above, the biological sample obtained from the patient can be any tissue or body fluid, such as blood, plasma, saliva, urine, cerebrospinal fluid or semen. Preferably, the sample is peripheral blood.

[0182] In the clinical setting, it is well known to monitor patients with aneurysms, for example, by scanning the aortic aneurysm with computed tomography every six months before surgery (until the aortic diameter reaches the point of surgical intervention), and every six months in the first year after the surgical intervention and annually thereafter. Similarly, the diagnostic blood test of the present invention has a promising potential for reliably monitoring patients with aneurysms, for example, aortic aneurysms after therapeutic intervention (also including cases of endovascular stent intervention). In the case of endovascular intervention, there are many cases where the diameter of the aortic aneurysm continues to expand, despite its isolation from the circulation and the influence of blood pressure, still leading to devastating complications (such as stent migration). The blood diagnostic test of the present invention may also become a good prognostic indicator for complications of endovascular surgery. Because the blood test is relatively more convenient and cost-effective, without the adverse effects of radiation carried by serial computed tomography scans, it can be used more frequently. The blood diagnostic test of the present invention could be used every three months to detect early changes in aortic diameter and also before scanning the patient with a computed tomography scan, so that if they show low or normal levels of gene expression associated with aortic aneurysm dilation, they might decide to postpone the computed tomography scan to a future point in time when its contribution to the potential diagnosis of a larger aortic aneurysm can be fully demonstrated.

[0183] As above-mentioned measure the expression product level of gene.In some embodiments, expression product is mRNA, and preferably by reverse transcription then real-time quantitative PCR measure.Amplification primers are derived from the transcript mRNA sequence of the gene disclosed in Table 5 and Table 5bis (as shown in Table 2), and suitable primers for amplifying transcript sequence are provided in Table 3.In other embodiments, expression product is the protein of coding, and by mass spectrometry or immunochemical measure as mentioned above.

[0184] Another aspect of the present invention relates to the use of an agent for determining the level of expression products of at least one gene selected from the group consisting of the genes of Table 5 in a method related to aneurysms as defined above. In a specific embodiment, the agent is used to determine the level of expression products of at least one gene selected from the group consisting of TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 (optionally in combination with one or both of COL11A1 and COL5A2). Preferably, the agent is used to determine both COL11A1 and COL5A2. In a specific embodiment, the agent is used to determine the level of expression products of at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes or at least eight genes selected from the group of genes disclosed in Table 5. In a specific embodiment, the expression products of at least COL11A1, COL5A2 and MMP2 are selected. In other specific embodiments, the expression products of at least the following genes are selected: COL11A1, COL5A2, MMP2 and MMP9, or at least COL11A1, COL5A2, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4 and ITGB1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1 and TGFB1. In another embodiment, the reagent is used to determine the expression product levels of all genes disclosed in Table 5. In another embodiment, the reagent is used to determine the expression product levels of at least one gene disclosed in Table 5bis and at least one gene disclosed in Table 5 or any combination thereof as defined above.

[0185] In a specific embodiment, the reagent is used to measure mRNA. In one embodiment, the reagent comprises amplification primers. In a specific embodiment, the primers are in each case those shown in Table 3. In another embodiment, the reagent forms part of a kit. The kit of the present invention may comprise the reagent for measuring the level of expression product and instructions for aneurysm diagnosis / prognosis / rupture risk / size classification as defined above. The instructions may include information about thresholds and / or reference values ​​for determining aneurysm diagnosis / prognosis / rupture risk / size classification as defined above.

[0186] On the other hand, the present invention also provides the use of the expression product of at least one gene selected from the group consisting of the genes in Table 5 as a biomarker in the in vitro diagnosis of aneurysms in a subject. In some embodiments, the biomarker is used to distinguish and diagnose patients according to the size of the patient's aneurysm. In other embodiments, the biomarker is used to distinguish and diagnose patients with larger aneurysms, such as in the case of aortic aneurysms, with a size greater than 6 cm. In another embodiment, the method is used to diagnose patients with aneurysms at risk of rupture. In another embodiment, the method is used to distinguish and diagnose patients with larger aneurysms from patients with smaller or medium-sized aneurysms, such as for aortic aneurysms, distinguishing and diagnosing patients with aneurysms with a diameter greater than 6 cm from patients with aneurysms with a diameter less than 6 cm.

[0187] Another aspect of the present invention provides the use of the expression product of at least one gene selected from the group consisting of genes in Table 5 as a biomarker in the in vitro prognosis of patients with aneurysms. Another aspect of the present invention provides the use of the expression product of at least one gene selected from the group consisting of genes in Table 5 as a biomarker in determining the response of patients with aneurysms to a treatment regimen for the aneurysm.

[0188] In some embodiments of the above aspects, at least COL11A1 and / or COL5A2 are selected biomarkers. Another embodiment provides the use of an expression product of at least one gene selected from the group consisting of TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 (optionally in combination with one or both of COL11A1 and COL5A2) as an in vitro biomarker. Preferably, both COL11A1 and COL5A2 are among the selected biomarkers. In other embodiments, the selected biomarkers are expression products of at least three, at least four, at least five, at least six, at least seven or at least eight genes disclosed in Table 5. In a specific embodiment, the expression products of at least COL11A1, COL5A2 and MMP2 are selected. In other specific embodiments, the expression products of at least the following genes are selected: COL11A1, COL5A2, MMP2 and MMP9, or at least COL11A1, COL5A2, MMP2, MMP9 and BMP1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1 and ITGA4, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4 and ITGB1, or at least COL11A1, COL5A2, MMP2, MMP9, BMP1, ITGA4, ITGB1 and TGFB1. In another specific embodiment, the expression products of eight genes are selected. In other embodiments, the biomarker is the expression product of at least one gene disclosed in Table 5bis and at least one gene disclosed in Table 5 or any combination thereof as defined above.

[0189] The in vitro method of the present invention provides diagnosis, prognosis and / or response to treatment (monitoring) information. In one embodiment, the method of the present invention further comprises the steps of (i) collecting diagnosis, prognosis and / or response to treatment (monitoring) information, and (ii) storing the information in a data carrier.

[0190] In the sense of the present invention, "data carrier" should be understood as any means (e.g., paper) containing meaningful information data for diagnosing and / or prognosing ECM degradation, cancer, or aneurysm in a subject. Such means can be considered as a carrier. The carrier can also be any entity or device capable of carrying prognostic data. For example, the carrier can contain a storage medium, such as a ROM (e.g., a CD ROM or a semiconductor ROM), or a magnetic recording medium, such as a floppy disk or a hard disk. In addition, the carrier can be a transmittable carrier, such as an electrical or optical signal, which can be transmitted via a cable or optical cable or by radio or other means. When the diagnosis / prognosis / response to treatment data is embodied in a signal that can be transmitted directly by a cable or other device or apparatus, the carrier can be composed of such a cable or other device or apparatus. Other carriers are associated with USB devices and computer archives. Examples of suitable data carriers are paper, CD, USB, computer archives in a PC, or sound recordings with the same information.

[0191] Finally, another aspect of the present invention provides an algorithm for performing any of the methods for diagnosis, prognosis and / or response to treatment as defined in the above aspects. In the sense of the present invention, the term "algorithm" is also synonymous with a collection or decision diagram, predictor and combination of data for correctly classifying individual samples.

[0192] According to aspects and embodiments of the present invention, diagnosis, prognosis and / or response to treatment of ECM degradation, cancer or aneurysm can be performed using a mathematical algorithm that evaluates detectable levels of biomolecules, proteins, protein fragments, antibodies and / or mRNA comprising one or more biomarkers as defined above, in combination with or independently of other clinical parameters, to correctly classify an individual sample as being derived from a healthy patient, a patient with degraded ECM, cancer (including a specific cancer stage and risk of metastasis) or an aneurysm (including the size and risk of rupture of the aneurysm).

[0193] The classification algorithm can be as simple as determining whether the amount of a specific biomarker or a subset of biomarkers measured is above or below a specific threshold (reference value). When multiple biomarkers are used, the classification algorithm can be a linear regression formula. Alternatively, the classification algorithm can be the product of any one of a variety of learning algorithms. In the case of a complex classification algorithm, it may be necessary to use a computer (e.g., a programmable digital computer) to execute the algorithm on the data to determine the classification. In either case, the state can then be recorded on a tangible medium, such as in a computer-readable format (e.g., a memory drive or disk) or simply printed on paper. The results can also be reported on a computer screen. The algorithm is used as a diagnostic and / or prognostic method, and it is particularly a part of a kit for implementing the method disclosed in the aforementioned aspects.

[0194] Throughout the specification and claims, the word "comprising" and variations of the word are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprising" includes the case of "consisting of ...". Unless otherwise specified, all terms used herein in this application should be understood with ordinary meanings known in the art. Other more specific definitions of certain terms used in this application are as described above, and are intended to be uniformly applied throughout the specification and claims, unless otherwise explicitly listed definitions provide a broader definition.

[0195] Other objects, advantages and features of the present invention will become apparent to those skilled in the art after reading the specification, or may be learned through practice of the present invention. The following examples and accompanying drawings are provided as illustrations, and they are not intended to limit the present invention. In addition, the present invention includes all possible combinations of the specific and preferred embodiments described herein. Sequence Listing <110> I. Tumplis <120> Diagnostic blood tests <130> P4048PC00 <160> 32 <170> PatentIn Version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> COL1A1 forward primer <400> 1 ctctgactgg aagagtggag agta 24 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> COL1A1 reverse primer <400> 2 ttggtggttt tgtattcaat cact 24 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> COL1A2 forward primer <400> 3 catcccagcc aagaactggt 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> COL1A2 reverse primer <400> 4 actgggccaa tgtccacaaa 20 <210> 5 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> COL3A1 forward primer <400> 5 agtgaccgac aaaattccag ttat 24 <210> 6 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> COL3A1 reverse primer <400> 6 cttttactgg tgagcacagt catt 24 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> COL5A1 forward primer <400> 7 ttcaagcgtg ggaaactgct 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> COL5A1 reverse primer <400> 8 gggagaagcc ttcactgtcc 20 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> COL5A2 forward primer <400> 9 tgagttgtgg agctgactct aatc 24 <210> 10 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> COL5A2 reverse primer <400> 10 taacagaagc atagcacctt tcag 24 <210> 11 <211> 25 <212> DNA <213> Artificial sequence <220> <223> COL11A1 forward primer <400> 11 gaaattgtac cttggtgcca ccaac 25 <210> 12 <211> 25 <212> DNA <213> Artificial sequence <220> <223> COL11A1 reverse primer <400> 12 ggatggatga gaatgagcac catat 25 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ITGA3 forward primer <400> 13 acaaggatga ctgtgagcgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ITGA3 reverse primer <400> 14 ctgcctacct gcatcgtgta 20 <210> 15 <211> 25 <212> DNA <213> Artificial sequence <220> <223> ITGA4 forward primer <400> 15 gtctttgtca ctaaaatgtt cccca 25 <210> 16 <211> 18 <212> DNA <213> Artificial sequence <220> <223> ITGA4 reverse primer <400> 16 cagcaagagc ggacctga 18 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ITGA6 forward primer <400> 17 gttgggaggg tggttcaaca 20 <210> 18 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> ITGA6 reverse primer <400> 18 cgaatcccat tgctttggca c 21 <210> 19 <211> 19 <212> DNA <213> Artificial sequence <220> <223> ITGB1 forward primer <400> 19 atcagacgcg cagaggagg 19 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ITGB1 reverse primer <400> 20 tgctgttcct ttgctacggt 20 <210> twenty one <211> twenty one <212> DNA <213> Artificial sequence <220> <223> MMP2 forward primer <400> twenty one cgcatctggg gctttaaaca t 21 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <220> <223> MMP2 reverse primer <400> twenty two ctgtctgggg cagtccaaag 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> MMP9 forward primer <400> twenty three ttcagggaga cgcccatttc 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <220> <223> MMP9 reverse primer <400> twenty four tcgctggtac aggtcgagta 20 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <220> <223> TIMP1 forward primer <400> 25 cttctggcat cctgttgttg 20 <210> 26 <211> 20 <212> DNA <213> Artificial sequence <220> <223> TIMP1 reverse primer <400> 26 ggtataaggt ggtctggttg 20 <210> 27 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> BMP1 forward primer <400> 27 ccatgacaac aagcacgact g 21 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> BMP1 reverse primer <400> 28 gccacaatga cccactcaca 20 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <220> <223> TGFB1 forward primer <400> 29 gagcctgagg ccgactacta 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <220> <223> TGFB1 reverse primer <400> 30 gggttcaggt accgcttctc 20 <210> 31 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> ACTB forward primer <400> 31 agcattgctt tcgtgtaaat tatg 24 <210> 32 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> ACTB reverse primer <400> 32 gtgtgcactt ttattcaact ggtc 24 BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figure 1 Real-time quantitative RT-PCR reactions in cancer patients. Panels A to F show the real-time quantitative amplification curves of collagen XIα-1, collagen Vα-2, collagen Vα-1, collagen Iα-1, collagen Iα-2, and collagen IIIα-1.

[0197] Figure 2 Real-time quantitative RT-PCR reactions in cancer patients. Panels A to D show the real-time quantitative amplification curves of integrin receptor alpha-4, integrin receptor beta-1, integrin receptor alpha-3, and integrin receptor alpha-6.

[0198] Figure 3Real-time quantitative RT-PCR reactions in cancer patients. Panels A to F show the real-time quantitative amplification curves of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, bone morphogenetic protein-1, transforming growth factor β-1, and β-actin.

[0199] Figure 4 Real-time quantitative RT-PCR reactions in patients with aneurysms. Panels A to F show the real-time quantitative amplification curves of collagen XIα-1, collagen Vα-2, collagen Vα-1, collagen Iα-1, collagen Iα-2, and collagen IIIα-1.

[0200] Figure 5 Real-time quantitative RT-PCR reactions in patients with aneurysms. Panels A to D show the real-time quantitative amplification curves of integrin receptor subunit α-4, integrin receptor subunit β-1, integrin receptor subunit α-3, and integrin receptor subunit α-6.

[0201] Figure 6 Real-time quantitative RT-PCR reactions in patients with aneurysms. Panels A to F show the real-time quantitative amplification curves for matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, bone morphogenetic protein-1, transforming growth factor-β-1, and β-actin.

[0202] Example

[0203] Example 1. Expression patterns in cancer patients

[0204] method: Total RNA was extracted from peripheral blood samples from patients with non-small cell lung cancer as confirmed by computed tomography (46 patients in total: 13 patients stage I, 11 patients stage II, 13 patients stage III and 9 patients stage IV) and from patients without any malignancy (controls, n=6).

[0205] DNA was removed by in-column recombinant DNA enzyme treatment. Total RNA was eluted in RNase-free water and stored at -80°C until further use. RNA concentration was determined in a Qubit 1.0 fluorometer (Invitrogen / Thermo Fisher, USA) by Quant-iT RNA assay kit, which uses a dye specific for RNA rather than DNA. All RNAs were in sufficient quantity.

[0206] cDNA was synthesized in a 20 μL total volume in a thermal cycler Primus 25 (MWG-Biotech, Germany) from 1 μg of total RNA and random hexamers according to the RT2 First Strand kit (Qiagen, Germany). The RT2 First Strand kit includes a proprietary genomic DNA elimination step to remove any residual contamination in the RNA sample before reverse transcription, thereby eliminating false positive signals. An RNA negative control (blank) was also used. The cDNA concentration was determined in a qubit1.0 fluorometer (Invitrogen / Thermo Fisher, USA) by a Quant-iT DNA assay kit, which uses a dye specific for DNA rather than RNA. The cDNA samples were then stored at -20°C until real-time quantitative PCR analysis was performed.

[0207] To investigate the mRNA expression levels of COL11A1 (all variants), COL5A2, COL5A1 (all variants), TGFB1, ITGA4 (all variants), ITGB1 (all variants), MMP2 (all variants), MMP9, BMP1 (all variants), ITGA3, ITGA6 (all variants), TIMP1, COL1A1, COL3A1 and COL1A2, the real-time RT-qPCR assay was validated in a Rotor-Gene Q MDX (Qiagen, Germany) real-time thermal cycler using appropriate RT2 qPCR primer assays (Qiagen, Germany) and RT2 SYBR Green Mastermix (Qiagen, Germany) in a total volume of 20 μL. All transcripts (variants) of each gene were assayed. Primers were those disclosed in Table 3 above. β-actin was used as a reference gene. For amplification, the manufacturer's instructions were followed.

[0208] The size and purity of all products were checked by electrophoresis on 2% w / v agarose gel and by melting point analysis. In each run, one gene per cDNA sample was amplified at a time. Also for our internal laboratory quality control, random cDNA samples were selected to be included in all runs. The identification of the genes studied is as follows:

[0209] 1. COL11A1 [homo sapiens], also referred to herein as collagen XIα-1

[0210] Gene ID: 1301

[0211] Location: 1p21.1

[0212] Exon count: 71

[0213] 2. COL5A2 [homo sapiens], also referred to herein as collagen Vα-2

[0214] Gene ID: 1290

[0215] Location: 2q32.2

[0216] Exon count: 55

[0217] 3. COL5A1 [homo sapiens], also referred to herein as collagen Vα-1

[0218] Gene ID: 1289

[0219] Location: 9q34.3

[0220] Exon count: 68

[0221] 4. COL3A1 [homo sapiens], also referred to herein as collagen III alpha-1

[0222] Gene ID: 1281

[0223] Location: 2q32.2

[0224] Exon count: 51

[0225] 5. COL1A1 [homo sapiens], also referred to herein as collagen I alpha-1

[0226] Gene ID: 1277

[0227] Location: 17q21.33

[0228] Exon count: 51

[0229] 6. COL1A2 [homo sapiens], also referred to herein as collagen I alpha-2

[0230] Gene ID: 1278

[0231] Location: 7q21.3

[0232] Exon count: 52

[0233] 7. ITGA3 [homo sapiens], also referred to herein as integrin receptor subunit alpha-3

[0234] Gene ID: 3675

[0235] Location: 17q21.33

[0236] Exon count: 26

[0237] 8. ITGA4 [homo sapiens], also referred to herein as integrin receptor subunit alpha-4

[0238] Gene ID: 3676

[0239] Location: 2q31.3

[0240] Exon count: 29

[0241] 9. ITGA6 [homo sapiens], also referred to herein as integrin receptor subunit alpha-6

[0242] Gene ID: 3655

[0243] Location: 2q31.1

[0244] Exon count: 28

[0245] 10. ITGB1 [homo sapiens, human], also referred to herein as integrin receptor subunit beta-1

[0246] Gene ID: 3688

[0247] Position: 10p11.22

[0248] Exon count: 18

[0249] 11. MMP2 [homo sapiens, human], also referred to herein as integrin receptor subunit beta-1 matrix metallopeptidase 2

[0250] Gene ID: 4313

[0251] Location: 16q12.2

[0252] Exon count: 17

[0253] 12. MMP9 [homo sapiens], also referred to herein as matrix metallopeptidase 9

[0254] Gene ID: 4318

[0255] Location: 20q13.12

[0256] Exon count: 13

[0257] 13.TIMP1 [homo sapiens], also referred to herein as TIMP metallopeptidase inhibitor 1

[0258] Gene ID: 7076

[0259] Location: Xp11.3

[0260] Exon count: 6

[0261] 14. BMP1 [homo sapiens], also referred to herein as bone morphogenetic protein 1

[0262] Gene ID: 649

[0263] Position: 8p21.3

[0264] Exon count: 25

[0265] 15.TGFB1 [homo sapiens], also referred to herein as transforming growth factor β1

[0266] Gene ID: 7040

[0267] Location: 19q13.2

[0268] Exon count: 7

[0269] 16. ACTB [homo sapiens], also referred to herein as actin β

[0270] Gene ID: 60

[0271] Position: 7p22.1

[0272] Exon count: 6

[0273] The NCBI reference sequence of the ACTB gene is NG_007992.1 (its transcript is NG_007992.1)

[0274] To prepare the calibrant (standard) for the β-actin gene assay, several PCR products were combined and then purified by the PureLink PCR purification kit (Invitrogen / Thermo Fisher), followed by measuring the concentration by the Quant-iT dsDNA broad range assay kit in a qubit 1.0 fluorometer (Invitrogen / Thermo Fisher, USA). The number of copies / μL was calculated as previously described (Kroupis C. et al., Clin Biochem. 2005, vol. 38, issue 1, p. 50-57). The highly concentrated calibrant was serially diluted and a standard curve for the two genes was obtained. As a relative quantification method, the 2-fold quantification method of Livak and Schmittgen (supra) was used. -ΔΔCt (RQ = 2- ΔΔCt, where RQ is mRNA expression).

[0275] Statistical methods The sensitivity and specificity of the qRT-PCR of the gene combination were calculated with 95% confidence intervals. The sensitivity / specificity results were measured as transcriptional biomarkers that were upregulated in the patient group with non-small cell lung cancer (n=46, sensitivity) and transcriptional biomarkers that were downregulated in the control patients (n=6, specificity). In addition, the sensitivity / specificity results were measured as transcriptional biomarkers that were upregulated in the patient subgroup with advanced metastatic stage non-small cell lung cancer (n=22, patients in stages III and IV-sensitivity) and transcriptional biomarkers that were downregulated (or less upregulated than controls) in the patient subgroup with early stage non-small cell lung cancer (n=24, patients in stages I and II-specificity). P values ​​are given by Fisher's exact test, which compares the proportions between the compared subgroups.

[0276] result:

[0277] Figure 1 The representative real-time RT-PCR curves of various types of collagen from the patient suffering from non-small cell lung cancer are shown. Clearly show that, compared with the control, the mRNA expression level of XIα-1, Vα-2 and Vα-1 type collagen is higher in the patient suffering from non-small cell lung cancer. It is also shown that the expression level of these minor fibril-forming collagens is even more overexpressed in the late stage and metastatic stage (III and IV stage) of non-small cell lung cancer. Table 6 shows the multiple change expression pattern relative to the reference group (non-cancer patient).

[0278] Table 6. Expression of various types of collagen in patients with non-small cell lung cancer relative to a reference group (non-cancer patients). Fold change expression pattern

[0279]

[0280] Figure 2 Representative real-time RT-PCR curves of various types of integrin receptors from patients with non-small cell lung cancer are shown. It is clearly shown that the mRNA expression levels of integrin receptors α-4, β-1, α-3 and α-6 are higher in patients with non-small cell lung cancer compared to controls. It is worth noting that the expression levels of these integrin receptors are higher in stage II and even more overexpressed in the late and metastatic stages (stages III and IV) of non-small cell lung cancer. Table 7 shows the expression pattern of fold changes relative to the reference group (non-cancer patients).

[0281] Table 7. Various types of integrins in patients with non-small cell lung cancer relative to the reference group (non-cancer patients) Fold-change expression patterns of receptors

[0282]

[0283] Figure 3 Representative real-time RT-PCR curves of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, bone morphogenetic protein-1, transforming growth factor beta-1 and reference gene beta-actin from patients with non-small cell lung cancer are shown. It is clearly shown that compared with the control, the mRNA expression levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, bone morphogenetic protein-1, transforming growth factor beta-1 in patients with non-small cell lung cancer are higher. It is also shown that the expression levels of these mRNAs that are controlling ECM remodeling are even more overexpressed in the late and metastatic stages (stages III and IV) of non-small cell lung cancer. In the control and non-small cell lung cancer patients of different stages, the expression level of beta-actin did not change. Table 8 shows the expression pattern of fold changes relative to the reference group (non-cancer patients).

[0284] Table 8. The expression of various types of matrix metals in patients with non-small cell lung cancer relative to the reference group (non-cancer patients). Fold change expression patterns of proteases, bone morphogenetic protein-1, transforming growth factor β-1 and reference gene β-actin Mode

[0285]

[0286] All products of the RT-PCR reactions were tested in agarose electrophoresis gels. All amplified products were represented by a single product at the expected molecular weight position. Further DNA sequencing analysis confirmed the expected sequence of the amplified PCR products.

[0287] In summary, it was found that the expression pattern of collagen XIα-1, collagen Vα-2, collagen Vα-1, integrin receptor α-4, integrin receptor β-1, matrix metalloproteinase-2, matrix metalloproteinase-9 and bone morphogenetic protein-1 genes provides reliable diagnostic information for cancer in peripheral blood, in this case particularly non-small cell lung cancer. There is also a correlation between the overexpression of these genes and the stage of cancer. As mentioned above, this group of genes can fully analyze the new molecular mechanisms that control ECM remodeling / degradation. More specifically, by using these 8 core genes, patients with non-small cell lung cancer and controls can be distinguished with a sensitivity of 0.98 (95% confidence interval: 0.89-1.00, P < 0.001) and a specificity of 1.00 (95% confidence interval: 0.61-1.00, P < 0.001). Finally, the expression pattern of the eight genes could distinguish patients with advanced metastatic NSCLC (stages III and IV) from patients with early-stage NSCLC (stages I and II) with a sensitivity of 0.95 (95% confidence interval: 0.78-0.99, P < 0.001) and a specificity of 0.96 (95% confidence interval: 0.80-0.99, P < 0.001).

[0288] This group of genes not only showed high sensitivity and specificity in distinguishing controls from cancer patients and distinguishing early stage cancer patients from late metastatic patients, but also, in addition, could quantify early and metastatic stages. More specifically, since it can be seen from Tables 6-8 and Figure 1-3 , thus providing fold changes in gene expression levels for quantitative purposes between early and metastatic stages (Table 9).

[0289] Table 9. Quantification of mRNA expression in peripheral blood of patients with non-small cell lung cancer based on fold change in gene expression level (mRNA) relative to controls. Cell lung cancer in patients at early and advanced stages (metastatic stage).

[0290] Gene Early stages of cancer Advanced stage of cancer (metastatic stage) Collagen XIα-1 >5-fold change >10-fold change Collagen Vα-2 >2-fold change >5-fold change Collagen Vα-1 >2-fold change >5-fold change Matrix metalloproteinase-2 >5-fold change >8-fold change Matrix metalloproteinase-9 >7-fold change >11-fold change Bone morphogenetic protein-1 >2-fold change >5-fold change Integrin receptor alpha-4 >1-fold change >6-fold change Integrin receptor beta-1 >2-fold change >8-fold change

[0291] This group of genes reflects ECM remodeling, and ECM remodeling is essential in the monitoring and follow-up of patients, especially after therapeutic intervention. Successful therapeutic intervention will result in the expression level of these genes close to the control. In the above-mentioned experimental measurements, as described in detail, it was found that the level of minor fibril-forming collagen and the gene level (confirmed by computer tomography) involved in ECM degradation in cancer-free patients were expressed at significantly lower levels in peripheral blood. Although the above-mentioned experiments were carried out in patients with non-small cell lung cancer, we have confirmed similar expression patterns in female patients with breast cancer diagnosis. Therefore, since these detected changes actually reflect changes in ECM, they can potentially be used to distinguish patients with other types of cancer, particularly patients with the ability to transfer by ECM degradation.

[0292] Example 2. Expression patterns in aneurysm patients

[0293] method: Total RNA was extracted from peripheral blood samples from patients with thoracic aortic aneurysms (i.e., ascending thoracic aorta) as confirmed by computed tomography (42 patients in total: 21 patients with thoracic aortic aneurysms with aortic diameters between 5 and 6 cm, 13 patients with thoracic aortic aneurysms with aortic diameters between 6 and 7 cm, and 8 patients with thoracic aortic aneurysms with aortic diameters greater than 7 cm) and from patients without aortic aneurysms (controls, n=13).

[0294] This method is the same as that in Example 1.

[0295] result:

[0296] Figure 4Representative real-time RT-PCR curves of various types of collagen from controls and patients with small (aortic diameter of 5-6 cm), medium (aortic diameter of 6-7 cm) and large-sized thoracic aortic aneurysms (aortic diameter>7 cm) are shown. Clearly showing that compared with controls, the mRNA expression levels of XIα-1, Vα-2 and Vα-1 type collagens are higher in patients with thoracic aortic aneurysms. It is also shown that the expression levels of these minor fibril-forming collagens are even more overexpressed in large-sized thoracic aortic aneurysms (aortic diameter>7 cm). Table 10 shows the expression pattern of fold changes relative to the reference group (patients with normal thoracic aortic diameter, ranging between 2.5 cm and 3.0 cm in diameter).

[0297] Table 10. In patients with thoracic aortic aneurysm relative to a reference group (patients with normal thoracic aortic diameter) Fold change expression patterns of various types of collagen

[0298]

[0299] Figure 5 Representative real-time RT-PCR curves of various types of integrin receptors from patients with thoracic aortic aneurysms are shown. It is clearly shown that the mRNA expression levels of integrin receptor subunits α-4, β-1, α-3 and α-6 are higher in patients with thoracic aortic aneurysms compared to controls. It is worth noting that increased expression levels of these integrin subunit receptors are shown in relatively small-sized aneurysms (aortic diameter of 5-6 cm) and are even more overexpressed in larger-sized thoracic aortic aneurysms (aortic diameter of 6-7 cm and >7 cm). Table 11 shows the fold change expression pattern relative to the reference group (patients with normal thoracic aortic diameter).

[0300] Table 11. In patients with thoracic aortic aneurysm relative to a reference group (patients with normal thoracic aortic diameter) Fold-change expression patterns of various types of integrin receptors

[0301]

[0302] Figure 6Representative real-time RT-PCR curves of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, bone morphogenetic protein-1, transforming growth factor beta-1, and reference gene beta-actin from patients with thoracic aortic aneurysms are shown. This shows that compared with the control, the mRNA expression levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, bone morphogenetic protein-1, and transforming growth factor beta-1 are higher in patients with thoracic aortic aneurysms. It also shows that the expression levels of these mRNAs that are controlling ECM remodeling are even more overexpressed in thoracic aortic aneurysms with larger diameters. There is no change in the expression level of beta-actin in the control and patients with thoracic aortic aneurysms of different sizes. Table 12 shows the fold change expression pattern relative to the reference group (patients with normal thoracic aortic diameter).

[0303] Table 12. In patients with thoracic aortic aneurysm relative to a reference group (patients with normal thoracic aortic diameter) Various types of matrix metalloproteinases, bone morphogenetic protein-1, transforming growth factor β-1 and the reference gene β-actin Fold change expression pattern

[0304]

[0305] All products of the RT-PCR reactions were tested in agarose electrophoresis gels. All amplified products were represented by a single product at the expected molecular weight position. Further DNA sequencing analysis confirmed the expected sequence of the amplified PCR products.

[0306] In conclusion, it was found that the expression pattern of collagen XI alpha-1, collagen V alpha-2, integrin receptor alpha-4, integrin receptor beta-1, matrix metalloproteinase-2, matrix metalloproteinase-9, transforming growth factor beta-1, and bone morphogenetic protein-1 genes provides reliable diagnostic information for aneurysms in peripheral blood, in this case particularly thoracic aortic aneurysms. There was also a correlation between the overexpression of these genes and the size of the aneurysm. This set of genes allows for the full analysis of the proposed new molecular mechanisms controlling ECM remodeling. More specifically, by using these genes, all of which were significantly upregulated in patients with thoracic aortic aneurysms, and larger diameter thoracic aortic aneurysms were significantly upregulated, it was possible to distinguish thoracic aortic aneurysm patients from controls with a sensitivity of 0.95 (95% confidence interval: 0.89-1.00, P < 0.001) and a specificity of 0.92 (95% confidence interval: 0.78-1.00, P < 0.001). Finally, by using these genes, patients with larger aortic aneurysms (diameter greater than 6 cm) could be distinguished from patients with smaller aortic aneurysms (diameter between 5 cm and 6 cm) with a sensitivity of 0.95 (95% confidence interval: 0.86-1.00, P < 0.001) and a specificity of 0.86 (95% confidence interval: 0.71-1.00, P < 0.001).

[0307] This group of genes not only showed high sensitivity and specificity in distinguishing controls from patients with thoracic aortic aneurysms and distinguishing patients with relatively small-sized aortic aneurysms from patients with relatively large-sized thoracic aortic aneurysms, but also, in addition, could be quantified between small-sized (aortic diameter of 5-6 cm) and larger-sized thoracic aortic aneurysms (aortic diameter>6 cm) compared to controls. More specifically, since it can be seen from Tables 10-12 and Figure 4-6 The results are presented in Table 13, and therefore the fold changes in gene expression levels can be provided for quantitative purposes between small and larger sized thoracic aortic aneurysms (Table 13).

[0308] Table 13. Quantification of small size based on fold change in gene expression level (mRNA) in peripheral blood relative to control and larger thoracic aortic aneurysms.

[0309] Gene Aneurysm 5-6 cm Aneurysm ≥ 6 cm Collagen XIα-1 >5-fold change >15-fold change Collagen Vα-2 >1.5-fold change >5-fold change Matrix metalloproteinase-2 >3-fold change >10-fold change Matrix metalloproteinase-9 >2.5-fold change >12-fold change Bone morphogenetic protein-1 >5-fold change >10-fold change Integrin receptor alpha-4 >1.5-fold change >5-fold change Integrin receptor beta-1 >3-fold change >8-fold change Transforming Growth Factor Beta-1 >3-fold change >10-fold change

[0310] The selected genes of this group reflect ECM remodeling / degradation, and ECM remodeling is necessary in the monitoring and follow-up of patients, especially before and after therapeutic intervention. A patient who has already suffered from aortic aneurysm is still at risk of suffering from another aortic aneurysm in another part of the aorta. Successful therapeutic intervention will result in the expression level of these genes close to the control. In experimental measurements, it was found that the level of minor fibril-forming collagen and the gene level (confirmed by computer tomography) involved in ECM degradation in patients without aneurysms were expressed at significantly lower levels in peripheral blood. Although the above results were obtained from patients with thoracic aortic aneurysms, similar expression patterns can be detected in patients with abdominal or thoracoabdominal aortic aneurysms. Therefore, the differential expression of the above genes can potentially be used to distinguish patients with other types of aneurysms.

[0311] Finally, and very importantly, in our clinical series, it has been confirmed by other large clinical data sets that approximately 19% of patients diagnosed with thoracic aortic aneurysm had a history of treated malignancy, which usually included one of the following malignancies: non-small cell lung cancer, colon cancer, and prostate cancer in male patients, and non-small cell carcinoma, colon cancer, and breast cancer in female patients. Conversely, in patients diagnosed with malignancy, aortic aneurysms (mainly thoracic aortic aneurysms) were also detected on computed tomography in a percentage of approximately 24%. The coexistence of malignancies with aortic aneurysms suggests that these diseases do share common molecular mechanisms of ECM remodeling and vice versa, since in both cases, their progression (aortic enlargement or metastatic disease) is based on alterations in the composition and physiological / biological properties of the ECM.

[0312] Citation List

[0313] Non-patent literature:

[0314] Livak and Schmittgen, "Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method". Methods, 2001, Volume 25, Issue 4, Pages 402-8

[0315] Kroupis C. et al, “Development and applications of a real-time quantitative RT-PCR method (QRT-PCR) for BRCA1 mRNA”. Clin Biochem, 2005, Vol. 38, No. 1, pp. 50-57.

Claims

1. Use of a reagent for determining the mRNA level of at least COL11A1, COL5A2, TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 in peripheral blood in the preparation of a kit for diagnosing aneurysm in a subject.

2. Use of a reagent for determining the mRNA level in peripheral blood of at least COL11A1, COL5A2, TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 in the preparation of a kit for determining the risk of aneurysm rupture in a subject.

3. Use of a reagent for determining the mRNA level in peripheral blood of at least COL11A1, COL5A2, TGFB1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 in the preparation of a kit for determining the response of a patient with an aneurysm to an aneurysm treatment regimen.

4. Use of an agent according to claim 1 in the preparation of a kit for diagnosing an aneurysm in a subject, use of an agent according to claim 2 in the preparation of a kit for determining the risk of aneurysm rupture in a subject, or use of an agent according to claim 3 in the preparation of a kit for determining the response of a patient with an aneurysm to an aneurysm treatment regimen, wherein the agent is a primer for determining mRNA levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

5. Use of the reagent according to claim 4 in preparing a kit for diagnosing an aneurysm in a subject, in preparing a kit for determining the risk of aneurysm rupture in a subject, or in preparing a kit for determining the response of a patient with an aneurysm to an aneurysm treatment regimen, wherein for the COL11A1 gene, the primers are the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12; for the COL5A2 gene, the primers are the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10; for the TGFB1 gene, the primers are the nucleotide sequences of SEQ ID NO: 29 and SEQ ID NO: 30; for the ITGA4 gene, the primers are the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16; for the ITGB1 gene, the primers are the nucleotide sequences of SEQ ID NO: 19 and SEQ ID NO: 20; for the MMP2 gene, the primers are the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 22; for the MMP9 gene, the primers are the nucleotide sequences of SEQ ID NO: 23 and SEQ ID NO:

24. NO: 24; and for the BMP1 gene, the primers are the nucleotide sequences of SEQ ID NO: 27 and SEQ ID NO:

28.

6. Use of a reagent for determining the mRNA level in peripheral blood of at least COL11A1, COL5A2, COL5A1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 in the preparation of a kit for diagnosing cancer in a subject, wherein the cancer is non-small cell lung cancer.

7. Use of a reagent for determining the mRNA level in peripheral blood of at least COL11A1, COL5A2, COL5A1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 in the preparation of a kit for determining the risk of cancer metastasis in a subject, wherein the cancer is non-small cell lung cancer.

8. Use of a reagent for determining the mRNA level in peripheral blood of at least COL11A1, COL5A2, COL5A1, ITGA4, ITGB1, MMP2, MMP9 and BMP1 in the preparation of a kit for determining the response of a patient with cancer to a cancer treatment regimen, wherein the cancer is non-small cell lung cancer.

9. Use of an agent according to claim 6 in the preparation of a kit for diagnosing cancer in a subject, use of an agent according to claim 7 in the preparation of a kit for determining the risk of cancer metastasis in a subject, or use of an agent according to claim 8 in the preparation of a kit for determining the response of a patient suffering from cancer to a cancer treatment regimen, wherein the agent is a primer for determining mRNA levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

10. Use of the agent according to claim 9 in preparing a kit for diagnosing cancer in a subject, in preparing a kit for determining the risk of cancer metastasis in a subject, or in preparing a kit for determining the response of a patient suffering from cancer to a cancer treatment regimen, wherein for the COL11A1 gene, the primers are the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 12; for the COL5A2 gene, the primers are the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 10; for the COL5A1 gene, the primers are the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8; for the ITGA4 gene, the primers are the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 16; for the ITGB1 gene, the primers are the nucleotide sequences of SEQ ID NO: 19 and SEQ ID NO: 20; for the MMP2 gene, the primers are the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 22; for the MMP9 gene, the primers are the nucleotide sequences of SEQ ID NO: 23 and SEQ ID NO:

24. NO: 24; and for the BMP1 gene, the primers are the nucleotide sequences of SEQ ID NO: 27 and SEQ ID NO: 28.

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