WO2004065632A1 - Method of judging tumor (cancer) or tumor (cancer) metastasis - Google Patents

Abstract

A method of conveniently evaluating a cancer disease and metastasis by examining the expression amount of a nucleic acid in blood cells. A method of judging caner by examining the expression of a nucleic acid. It is difficult to judge cancer by the conventional method which merely comprises assaying a protein in the serum. However, tumor and its metastasis can be conveniently judged by measuring the expression of a nucleic acid in monocytes or macrophages which are specific blood cell molecules in serum without examining the tumor per se by biopsy or the like. This judgment method is based on a finding that tumor and the resultant metastasis participate in blood cells, in particular, monocytes and macrophages and cause the expression of a nucleic acid. Since these factors are expressed in the state of tumor or its metastasis, diagnosis can be easily made by examining them.

Description

 Method for determining tumor (cancer) or metastasis of tumor (cancer)

Technical field

 Light

 The present invention relates to a method for determining or evaluating a tumor or a cancer by observing an increase or decrease in the expression level of an angiogenic factor in blood cells such as monocyte cells and Mactophage.

 book

BACKGROUND ART Cancer mortality is extremely high in men and women, and although much attention has been paid to cancer diseases, mortality has not declined significantly in recent years. Early detection of cancer is extremely important in determining survival and will be an important issue in the future. Cancer cells sometimes produce substances that are undetectable or found only at very low levels in healthy individuals. This feature could be used to develop innovative diagnostic techniques. Using these techniques, testing methods for substances produced by cancer cells have been developed. Tests aimed at detecting cancer-binding substances are also expected to be useful in discriminating between individuals with cancer in comparison to healthy individuals. As a representative example, one type of marker for metastatic staging of cancer is the presence of an oncogene 'allele. For example, genes associated with more than 30% of breast and ovarian cancers have been identified as neu / HER2 / c-erbB2 proto-oncogenes. The extent of the proto-oncogene and the degree of overexpression of its protein product may be attributed to the severity of the disease. They have been shown to correlate with prognostic signs (S'lamon et al., Science 244: 707-712 (1989)). Furthermore, abnormal alleles of the ras and myc gene families have been shown to be involved in human cancer progression, and some researchers speculate that they are useful as prognostic indicators ( Field. JK et al., Anticancer Res. 10: 1-22 (1990)). In addition, transfection of recipient cells with the H-ras family oncogene and the mutant p53 allele induces metastatic potential (Liotta, LA et al., Cell 64: 327-36 (1991). )) ₀ However, the main problem with the use of these oncogenes markers is that these factors are such a precursor to all types of tumors Les referred. On the contrary, these markers are specific to the tissue or tumor type in which they originated and are unlikely to have broad applicability to all types of metastatic cancer. These cancer detection methods use proteins characteristically produced by various cancer cells and the properties of cancer cells. An inspection needs to be performed. Sampling from living organisms to find each cancer or tumor is practically impossible. And that requires huge inspection costs and time. In other words, it is not practically possible to examine all of these tumor abilities, and at present the diagnosis using tumor markers has not been advanced. Therefore, a great deal of effort is currently being focused on identifying markers specific to the potential for metastasis of cancer.

Knowing the metastatic potential of a tumor is important in developing cancer treatments that maximize patient survival and quality of life. The most aggressive treatment regimen is rapid tumor growth It should be reserved for patients at very high risk of multiplication and metastasis.

 Recent studies have shown that a growing body of cancer is evidence that angiogenesis is essential for cancer progression. Angiogenesis is the development of new capillaries from pre-existing blood vessels. Normally, mammalian angiogenesis is limited to the reproductive system, embryogenesis, and repair following injury. However, angiogenesis can also occur in pathological conditions such as cancer, retinal neovascularization, neovascularization in atheroma plaques, hemangiomas, arthritis and psoriasis.

 In the absence of angiogenesis, tumors can remain in place as small asymptomatic lesions for many years. Perfusion provides a large supply of oxygen and nutrients to tumors that lead to angiogenesis. Thus, angiogenic tumors can grow and proliferate. Tumors must constantly stimulate the growth of new capillaries to keep growing. In addition, angiogenesis causes tumor cells to enter the host animal's circulatory system. New blood vessels provide a way for tumor cells to enter the circulation and metastasize to distant sites.

In fact, the extent of neovascularization is strongly correlated with metastasis in primary breast, bladder, prostate, non-small cell lung, cutaneous melanoma, and cervical cancer. In these studies, tumor specimens were analyzed histologically. ■ The extent of tumor mass angiogenesis is an independent predictor of metastatic potential and has been shown to be more reliable than other prognostic markers, but its number is easy to measure given any cancer or tumor Not something. However, the value of markers for this metastasis also varies greatly depending on the type of tumor, making it difficult to determine at present. That is why many people die from reoccurrence due to metastasis It is the current situation.

Disclosure of the invention

 There is a need for rapid and objective techniques that can be commonly used in the clinic to predict tumor metastasis by assessing tumor angiogenesis. Therefore, in order to solve the problem by using as few tests as possible to determine cancer, we focus on blood cells flowing through the whole body and examine the response of the blood cells to cancer cells. Diagnosis of metastasis and diagnosis of metastasis were continued, and as a result, monocytes, granulocytes, leukocytes, and macrophages were found to be involved in tumors, particularly metastases. , Fit-1, tek / tie -2, FC γ receptor, insulin growth factor (IGF), platelet-derived growth factor, colony stimulating factor, ゥ • Kinase-type plasminogen activator, plasminogen activator The present inventors have found that cancer or tumor can be determined from the expression levels of nucleic acids by the beta inhibitor, collagenase, ΜΜΡ (matrix meta-oral protease) and ゥ -kinase factor genes.

Of these factors, those most commonly found to be associated with cancer or tumor and metastasis are basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), CD147 / EMMPRIN, insulin growth factor 2, platelet-derived growth factor, and colony stimulating factor. Other factors that are candidates for angiogenesis metastasis markers include oral kinase-type plasminogen activator and plasminogen activator inhibitor 1, as well as various collagenases and Perkinase _n To select markers that are generally applicable to angiogenesis and metastasis, it is important to identify key factors in this process. At present, the factor or factors that cause tumor angiogenesis have not yet been determined, but bFGF and VEGF are presumed to be synergistic. bFGF levels were elevated in urine in approximately 37% of a wide range of cancer patients. Similarly, bFGF was elevated in the serum of 10% of such patients. The highest levels of bFGF were observed in patients with metastatic disease. bFGF is abnormally high in cerebrospinal fluid in children with brain tumors, and these high levels correlate with microvessel density in tumor specimens.

 Angiogenesis in cancer tissue is difficult to determine because of the complex relationship between a wide variety of neogenesis factors and other components. In order to perform the procedure on cancerous tissue, it is necessary to remove the cancerous tissue, and the method is limited. It is therefore an object of the present invention to provide an objective judgment tool for characterizing and staging the progression of a disease state using the specific properties of a cancer or tumor.

The present invention takes advantage of the abnormal growth of cells due to cancer or tumors that triggers initiation of angiogenesis and the like. In embodiments, the condition of the cancer is detected and determined by examining the expression of a nucleic acid by a specific gene from blood cells of the subject. The information obtained by this diagnostic tool should be used by physicians to plan treatment protocols to treat and manage these disease states. This expression indicates that the level of VEGF could be a molecular marker for the progression of the cancer state, and we have now found that this expression of VEGF also occurs in monocytes. In another embodiment of the present invention, the expression of receptor proteins on endothelial cells associated with tumors that respond to VEGF and other factors can also be detected and used to diagnose.

 These receptor proteins include, but are not limited to, KDR / flk-1, fit-1, CD147 / EMMPRIN and / or tek / tie-2. Co-expression of VEGF and any of these receptor proteins is also indicative of a cancer or tumor where growth and metastasis may be increased. Identification of specific receptors involved in the angiogenesis pathway is important in determining which treatments are most effective and selective at inhibiting angiogenesis.

 VEGF is a heparin-binding homodimeric glycoprotein of about 45 kD. VEGF has been isolated from cell lines of various origins and its genes have been cloned from cells of other species as well as human cells. VEGF gene transcripts are spliced into any of at least four different molecular species consisting of 121, 165, 189, and 206 amino acids. VEGF 165 is the most common form.

Release of VEGF by the tumor mass stimulates angiogenesis in adjacent endothelial cells. Normally, capillary endothelial cells alternate very slowly (over thousands of days) and remain dormant by contact with specialized cells called perivascular cells. When VEGF is expressed by the tumor mass, endothelial cells in proximity to VEGF + tumor cells upregulate expression of the VEGF receptor molecules KDR / flk-1 and / or fit-1. KDR / flk-1 and flt-1 are both receptor tyrosine kinase proteins with high specificity for VEGF. Those ligands bind These receptors then dimerize and transmit intracellular signals by tyrosine phosphorylation.

 Binding of VEGF to its' cognate VEGF receptor in endothelial cells initiates a chemotactic effect in these cells through signaling. New capillaries emerge from the endothelial vessels towards the VEGF + tumor cells. During angiogenesis, endothelial cells proliferate rapidly by dividing at most every 5 days. Other receptors observed during angiogenesis, although their role in such processes remains unclear, are fll-4 receptors, fibroblast growth factor receptors, platelet-derived growth factor receptors, and epithelium. Growth factor receptor, and the Met oncogene.

The present invention can evaluate not only the cancer or tumor but also the metastatic stage of the cancer, and can examine the course of treatment in a method for devising an appropriate treatment. If it is an indicator of improvement, it is very effective in preventing recurrence due to metastasis of cancer that seems to have been cured once. In particular, the key to the method of the present invention lies in the discovery that expression of genes related to cancer or tumors is caused by blood cells. In one embodiment of the invention, a sample of blood cells is molecularly characterized for the presence of VEGF messenger RNA or protein using the techniques described below. Using a similar technique, the sample is also characterized for expression of factors HIF-1, KDR / flk-1, fit-1 and / or tek / tie-2. The presence of additional angiogenic factors and receptors, as well as the presence of oncogene alleles, can also be assessed. To date, no attempt has been made to detect cancer by monitoring the expression of nucleic acids by such blood cells or by the genes of monocytes, granulocytes, leukocytes or macrophages. Determination of cancer or tumor or cancer or To determine the metastasis of a tumor, whole blood cells, or monocytes, granulocytes, leukocytes, or macula phages may be separated and used by one or more of them.

 This finding allows tumors or cancer cells to be "switched" to a phenotype that requires angiogenesis, etc., and monocytes, granulocytes, leukocytes or macrophages capture these factors as if to help them work. This indicates that metastasis can occur. The presence of additional receptor tyrosine kinases involved in angiogenesis will provide further information about the angiogenesis stage caused by the tumor and potential targets for therapeutic treatment. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the expression of VEGF and CD147 / EMMPRIN by ΡΤ-PCR amplification expressed on monocytes.

 FIG. 2 shows the expression levels of VEGF, CD147 / EMMPRIN, and MMP by PT-PCR amplification expressed in monocytes, with the intensity of GAPDH being 100.

 Fig. 3 shows the expression levels of various factors by the expressed PT-PCR. Best mode for carrying out the invention

ABL, ERBB-1, NEU GTP ヽ GSP MYC, L-MYC, H-RAS, K-RAS, N-RAS.RET, ROS, K -The expression of any of a number of oncogenes known to be involved in cancer, including SAM, SIS, SRC, and TPK, can also be analyzed. In addition, tumors such as RB1, p53, WT1, DCC, NFL1, FAP, and MEN-1 The expression of the suppressor gene can also be assessed.

 The level of gene expression can be determined by using nucleic acid from the cell as a starting point for such an assay technique, which can be isolated according to standard nucleic acid preparation procedures well known to those skilled in the art. For example, RNA of blood cells suspected of expressing the VEGF gene can be isolated and tested using hybridization or PCR techniques as described below. Isolated cells can be derived from cell culture or from patients, but generally can be easily separated from blood. Granulocytes, leukocytes, and macrophages can also be easily separated from conventional methods. One or more labeled nucleic acid reagents, including recombinant DNA molecules or in vitro transcribed antisense RNA probes, from RNA obtained from a sample or nucleic acid such as cDNA generated from RNA, and these reagents Contacting and incubating under conditions suitable for specifically annealing to the complementary sequence within the gene can be included.

 Preferably, these nucleic acid reagents are at least 15 to 30 nucleotides in length. After incubating, remove any unannealed nucleic acids from the nucleic acid: VEGF molecule hybrid. The presence of the hybridized nucleic acid is then detected if such a molecule is present.

Diagnostic methods using patient blood cells or monocytes, granulocytes, leukocytes, and macrophages include, for example, PCR (Mullis, KB, 1987, an experimental embodiment described in US Pat. No. 4,683,202) after amplification of those molecules. Detecting the amplified molecule using techniques well known to those skilled in the art. To determine whether the expression of the nucleic acid by the gene was up-regulated, the resulting wide sequence was こ と が Can be compared to what would be expected if the broadened nucleic acid contained only normal levels of gene transcript.

 In one embodiment of such a detection scheme, cDNA is synthesized from the RNA of interest (eg, by reverse transcribing an RNA molecule to cDNA). Next, a nucleic acid amplification reaction such as a PCR amplification reaction is performed using the sequence in the cDNA as a type III. In the reverse transcription step and the nucleic acid amplification step of this method, the nucleic acid reagent used as a synthesis initiation reagent (for example, a primer) is selected from the nucleic acid sequences of the respective factors. The preferred length of such a nucleic acid reagent is at least 9 to 30 nucleotides. For the detection of amplification products, nucleic acid amplification can be performed using radioactively or non-radioactively labeled nucleotides. In addition, sufficient amplification product can be produced that can be visualized using standard bromide staining or other suitable nucleic acid staining methods.

Example

Reverse transcriptase reactions and PCR amplification were performed in a Perkin Elmer 960 PCR machine (Erneryville, CA) without interruption. 20 // 1 0 £? (Jechirupi mouth carbonate) 40 011 _§ 1 1 in treated water? Immediately before adding the R reagent, it was placed in a water bath at 65 ° C for 5 minutes, and then quickly cooled on ice. The total PCR volume of 50 μl is 2.5 units of Taq Holimerase, Perkin Elmer, Emeryvi I 1e, CA), and 2 units of AMV reverse transcriptase (Boehringer Mannheim, I ndianapolis, IN), dCTP, dATP, dGTP and dTTP (Perkin Elmer, Emerryvi 1) le, CA), 18 pM of each primer, 10 mM Tris-HCI) 50 mM KCI) 2 mM MgC12 (Perkin Elmer) Emeryvi 11 e, CA). The PCR conditions were as follows. Cycle 1 is for 15 minutes at 42 ° C, then 197 ° C for 15 seconds (1 cycle): Cycle 2 is for 1 minute at 9.5 ° C, then 1 minute at 60 ° C. 2 ° C for 30 seconds (15 cycles): Cycle 3 is 95 ° C for 1 minute, followed by 60 ° C for 1 minute and 72 ° C for 1 minute (10 cycles): Cycle 4 is 9 5 ° C for 1 minute, followed by 60 ° C for 1 minute and 72 ° C for 2 minutes (8 cycles): Cycle 5 is 72 ° C for 15 minutes (1 cycle): The final cycle is when the sample is mechanical. It was stored at 4 ° C until it was removed from the container.

The PCR product of 501 was concentrated to 10 μl by vacuum centrifugation, and the whole sample was electrophoresed in a thin 3% trisodium borate-EDTA containing ethyl bromide (含 む ェ). All specimens were analyzed at least twice to confirm positive or negative results.

 The risk of possible false positives arising from cross-contamination was determined by performing RT-ΡCR in a single tube without interruption and using a filtered tip. The use of large amounts of Taq polymerase greatly extended the elongation time, and enhanced sensitivity by analyzing all 501 PCR products on a thin agarose gel containing ethidium bromide.

Figures 1 and 2 show the expression of nucleic acids in blood cells of cancer patients. Patient 1 Before treatment for cancer patients, Patient 2 Treatment for cancer patients 3 months, Patients Treatment for cancer patients 6 months, N healthy individuals, P cancer patients 2. Higher levels of VEGF and CD147 / EMMPRIN expression were found in monocytes of cancer patients compared to healthy N (Patient 1, P). It was also found that its expression gradually weakened during the treatment of cancer (Patient 2, 3). VEGF in plasma was in the normal range of 25.3 pg / ml before treatment (less than 38.3 _Pg / ml), and after treatment, it was 34.4 pg / ml, indicating that the condition of cancer could not be determined. Did not. However, VEGF expression in the blood cells decreased with each treatment, and it was in fact healing. Examining gene expression can be a convenient means of determining cancer or metastasis of cancer that was not revealed by plasma serum. In addition, blood cells are fractionated into monocytes, granulocytes, leukocytes, and macrophages by a conventional method, and bFGF, VEGF, CD147 / EMMPRIN, KDR / flk-1, fit-1, tek / tie-2, FC y receptor, insulin growth factor (IGF), platelet-derived growth factor, colony stimulating factor, perokinase-type plasminogen activator, plasmaminogen activator inhibitor, collagenase, MMP (matrix The expression of nucleic acids by the respective genes in healthy subjects and cancer patients was examined for T. metastasis protease and oral kinase factor, respectively, while keeping the number of blood cells constant. As a result, it was found that, in healthy subjects, the expression level of nucleic acids by the genes of these factors was small, but in cancer patients, they were all increased at a high level (Fig. 3). As a result, it was confirmed that cancer or tumor could be determined by examining the expression of the nucleic acid by the gene in one or more of these factors. Industrial applicability

Using the information obtained from the methods of the invention detailed above, a physician can design an appropriate treatment tailored to the molecular stage of the patient's cancer state. The present invention Using the method to measure the molecular profile of a tumor allows physicians to plan appropriate treatments to optimize both cancer treatment efficacy and patient quality of life. If the blood cells isolated from the patient are found to have increased VEGF expression using the diagnostic methods of the invention, the patient is a candidate for treatment for cancer or tumor. This leads to earlier detection of cancer or tumor, and early detection of cancer or tumor due to metastasis that once recurs. It is expected that early detection will save more people.

Claims

The scope of the claims  A method of determining cancer or metastasis of cancer by examining the expression level of a nucleic acid in a spherical cell.  2. The method for determining cancer or metastasis of cancer according to claim 1, wherein the expression level of a nucleic acid by a gene in one or more of monocytes, granulocytes, leukocytes, and macrophages is examined.  Scope of claim 1 or 2 described in Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), CD147 / EMMPRIN, 'KDR / flk-l, flt- l, a method for determining cancer or cancer metastasis by examining the expression level of nucleic acid by a gene in one or more of tek / tie-2 3. The method according to claim 1, wherein the FCy receptor, insulin growth factor (IGF), platelet-derived growth factor, colony stimulating factor, perkinase-type plasminogen activator, plasminogen activator. A method for determining cancer or cancer metastasis by examining the expression level of nucleic acid by one or more of the following inhibitors, collagenase, MMP (matrix meta-oral protease), and oral kinase factor. Claims 1 and 2, wherein the factors described in Claims 3 and 4 were prepared using a primer sequence complementary to RNA or cDNA or DNA that is a nucleic acid expressed by a gene. A method for determining cancer and cancer metastasis, using a primer. ' 5. The method according to claim 5, wherein when the expression is increased as compared to a healthy person, the cancer or metastasis is determined.