JP2009011249A - Method for diagnosing malignant alteration of alimentary canal interstitial tumor originated from stomach as primary lesion - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining malignant change of alimentary canal interstitial tumor which originates from stomach as a primary lesion. <P>SOLUTION: This method for determining whether the alimentary canal interstitial tumor, originating from the stomach as the primary lesion, is malignant or not, is constituted of (a) a process of determining whether a dipeptidylpeptidase IV gene is expressed or not in the alimentary canal interstitial tumor originated from the stomach as the primary lesion, obtained from a patient; and (b) a process of determining that the tumor is malignant, when expressing the dipeptidylpeptidase IV in the process (a). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to a method for diagnosing malignant transformation of gastrointestinal stromal tumor having a primary origin in the stomach and a pharmaceutical composition for treating the tumor.

BACKGROUND ART GIST (gastrointestinal stromal tumor) occupies most mesenchymal tumors in the gastrointestinal tract, and c-kit receptor tyrosine kinase, similar to interstitial cells of Cajal (ICC). And CD34 are expressed, indicating the possibility of being an ICC-derived tumor. GIST frequently has a gain-of-function mutation in the KIT or PDGFRα gene that encodes c-kit, and molecular target therapy with a tyrosine kinase receptor activity inhibitor (eg, imatinib mesylate; trade name: Gleevec) It is said to be effective for GIST patients.

  However, GIST has a broad spectrum, ranging from benign to healing only by surgical resection to malignant with poor prognosis, and responsiveness to Gleevec is also observed only in some tumors, so it is not always uniform clinically. Absent. Therefore, a new biomarker that can appropriately predict the prognosis of GIST is required.

  In addition, the response rate of imatinib mesylate in c-Kit positive cases was not 46.5%, and in addition, relatively mild side effects occurred in almost 100% of cases, and myelosuppression and bleeding (brain, hard) Serious side effects such as submembrane, gastrointestinal tract), tumor bleeding / gastrointestinal perforation, interstitial pneumonia / pulmonary fibrosis, hepatic dysfunction, severe skin symptoms, paralytic ileus also occur at a frequency of 5-10% It has been reported. Furthermore, there have been reports of cases in which resistance is acquired by secondary mutations in the KIT gene in long-term administration cases. Therefore, development of another molecular targeted therapeutic agent having a different mechanism of action from imatinib mesylate is expected.

  Dipeptidyl peptidase IV (hereinafter referred to as “DPP IV”) (EC.3.4.14.5) is a transmembrane glycoprotein (serine dipeptidyl peptidase) that is strongly expressed in T cells and certain cancer cells It is. Dipeptidyl peptidase IV has been confirmed in leukemia cells and solid cancer cell lines such as prostate cancer and renal cell cancer, and is not only involved in tumor cell motility and invasiveness, but also in the cell cycle. (Non-patent document 1: Blood / Immunity / Tumor, Vol. 6, No. 3, 2001-7, pp. 61 (283) -64 (286)). Furthermore, there is a report that administration of anti-DPP IV antibody suppressed the growth of DPP IV-positive leukemia cells and showed long-term survival by administration of anti-DPP IV antibody in SCID mice transplanted with leukemia cell lines (Non-patent document 2 : Clinical Cancer Research, Vol. 7, pp.2031-2040, 2001).

  In addition, since DPP IV inhibitors inhibit the enzyme that degrades glucagon-like peptide-1 (GLP-1), a hormone that enhances insulin secretion, various DPP IV inhibitors (for example, SYR-322, TA-6666, KRP) -104) has already been developed and used clinically as a therapeutic agent for diabetes.

Blood / Immunity / Tumor, Vol.6, No. 3, 2001-7, pp.61 (283) -64 (286) Clinical Cancer Research, Vol. 7, pp.2031-2040, 2001

Summary of the Invention

  In the gastrointestinal stromal tumor (GIST) whose primary site is the stomach, the present inventors have significantly increased the expression of the dipeptidyl peptidase IV gene in malignant GIST with a poor prognosis compared to other GIST. I found out. The present invention is based on this finding.

  Therefore, an object of the present invention is to provide a method for determining malignant transformation of a gastrointestinal stromal tumor having a primary origin in the stomach, and a pharmaceutical composition for treating the tumor.

  The determination method according to the present invention is a method for determining whether or not a gastrointestinal stromal tumor having a stomach as a primary lesion is malignant, and (a) a stomach obtained from a patient as a primary lesion. A step of determining whether a dipeptidyl peptidase IV gene is expressed in a gastrointestinal stromal tumor, and (b) if the dipeptidyl peptidase IV gene is expressed in step (a), the tumor is malignant It is a method including the process of determining with being.

  Furthermore, the pharmaceutical composition according to the present invention is a pharmaceutical composition for treating a gastrointestinal stromal tumor having a primary stomach, comprising a dipeptidyl peptidase IV inhibitor and a pharmaceutically acceptable carrier.

  According to the present invention, it is possible to predict the prognosis of a gastrointestinal stromal tumor whose primary site is the stomach and select an appropriate treatment method. Also provided are means of treating the tumor even if it is malignant. In particular, since dipeptidyl peptidase IV inhibitors are administered to patients for a long time as a therapeutic agent for diabetes, they have the advantage of fewer side effects.

Detailed description of the invention

  It has been demonstrated herein that dipeptidyl peptidase IV expression is a poor prognostic factor for gastrointestinal stromal tumors originating in the stomach. Therefore, by examining the presence or absence of dipeptidyl peptidase IV expression in tumors or biopsy specimens removed from patients, it is possible to determine whether the tumor is malignant, for example, the possibility of recurrence and / or metastasis Is possible. From the above knowledge, according to the present invention, a method for determining or diagnosing whether or not a gastrointestinal stromal tumor having a stomach as a primary lesion is malignant is provided.

  As used herein, “gastrointestinal stromal tumor” is a mesenchymal tumor (GIMT) that occurs in the gastrointestinal tract and refers to a solid tumor mainly composed of spindle-shaped cells. This gastrointestinal stromal tumor is a tumor that forms on the wall of the gastrointestinal tract such as the esophagus, stomach, small intestine, and large intestine, and is a type of tumor that constitutes a submucosal tumor.

  As used herein, the term “malignant” has the same meaning as used for neoplasms such as tumors, and generally the tumor has local invasiveness, destructive growth or metastasis. Means. According to a preferred embodiment of the present invention, “malignant” means that the tumor is recurrent or metastatic.

  The determination method according to the present invention includes a step of determining whether or not the dipeptidyl peptidase IV gene is expressed in a gastrointestinal stromal tumor obtained from a patient and having the stomach as the primary focus. As a result, when the dipeptidyl peptidase IV gene is expressed, it is determined (diagnosed) that the tumor is malignant.

  The gastrointestinal stromal tumor to be determined may be a surgical sample removed from a patient by a surgical operation or a biopsy sample obtained by an endoscope or the like. For example, it is advantageous to use a biopsy sample from a patient as a determination target in examining a treatment plan including determination of whether or not a gastrointestinal stromal tumor should be surgically removed.

  Standard methods known in the art can be used to determine the presence or absence of expression of the dipeptidyl peptidase IV gene. The sequence of the dipeptidyl peptidase IV gene and the amino acid sequence encoded thereby are well known in the art. For example, human-derived dipeptidyl peptidase IV shown in SEQ ID NO: 1 and SEQ ID NO: 2 (NCBI access number: X60708) Of the sequence. One skilled in the art can determine the presence or absence of the gene expression using standard methods by referring to these sequences.

  According to a preferred embodiment of the invention, determining the presence or absence of gene expression comprises detecting dipeptidyl peptidase IV protein in the tumor. Dipeptidyl peptidase IV protein can be detected by methods well known in the art. For example, the amino acid sequence of dipeptidyl peptidase IV protein can be exemplified by that represented by SEQ ID NO: 2, and those skilled in the art can appropriately detect the protein by referring to this sequence.

  Dipeptidyl peptidase IV protein can be suitably detected, for example, by using an antibody that binds to the protein. Such an antibody is preferably an antibody specific for the dipeptidyl peptidase IV protein. The antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Monoclonal antibodies specific for dipeptidyl peptidase IV protein are commercially available. Detection of dipeptidyl peptidase IV protein is preferably performed by immunohistochemical staining using such specific antibodies. Alternatively, an immunoblot method in which a protein extract obtained from a tumor cell is electrophoresed and then detected using a specific antibody may be used.

  According to another preferred embodiment of the invention, the step of determining the presence or absence of gene expression comprises detecting dipeptidyl peptidase IV mRNA in said tumor. For example, the mRNA of dipeptidyl peptidase IV includes a nucleotide sequence represented by SEQ ID NO: 1 (where t in the sequence is converted to u). Thus, it is possible to detect dipeptidyl peptidase IV mRNA appropriately. A specific method for detection includes, for example, a method of performing electrophoresis after amplification by RT-PCR using a specific primer pair.

  In order to carry out the determination / diagnosis method according to the present invention as described above, necessary reagents can be put together into a kit. Therefore, according to the present invention, there is provided a kit for determining whether or not a gastrointestinal stromal tumor that originates in the stomach is malignant, and the kit detects the expression of a dipeptidyl peptidase IV gene. A reagent capable of The detection reagent contained in the kit according to the present invention is selected according to the specific method to be carried out. As such a detection reagent, a reagent for detecting dipeptidyl peptidase IV protein in a tumor, for example, an antibody (preferably a monoclonal antibody) that specifically binds to dipeptidyl peptidase IV protein can be suitably used. . Alternatively, such a detection reagent is a reagent for detecting dipeptidyl peptidase IV mRNA in a tumor, for example, a specific primer and RT-PCR used in RT-PCR using dipeptidyl peptidase IV mRNA as a template. It may be a reagent for use. The kit according to the present invention may further contain other reagents, reaction containers, instructions, etc., depending on the specific method to be carried out.

  In the present specification, as described above, it has been demonstrated that the expression of dipeptidyl peptidase IV is a poor prognostic factor for gastrointestinal stromal tumors originating in the stomach. Furthermore, it has been reported that a monoclonal antibody against CD26 containing dipeptidyl peptidase IV has an antitumor effect against anaplastic large cell T cell lymphoma (Clinical Cancer Research, Vol. 7, pp. 2031-2040, 2001). Considering these findings together, dipeptidyl peptidase IV is an essential protein in the malignant transformation of gastrointestinal stromal tumors with the stomach as the primary lesion, and therefore, the stomach has the primary lesion by inhibiting dipeptidyl peptidase IV. It is thought that malignant transformation of gastrointestinal stromal tumors can be prevented and treated.

  Therefore, according to the present invention, a method for treating or preventing a gastrointestinal stromal tumor originating in the stomach comprising administering to a subject a therapeutically effective amount of a dipeptidyl peptidase IV inhibitor Is provided. Furthermore, the present invention provides the use of a dipeptidyl peptidase IV inhibitor in the manufacture of a medicament for treating gastrointestinal stromal tumors originating in the stomach.

  A gastrointestinal stromal tumor with the stomach as the primary lesion may or may not be malignant, but is preferably a malignant tumor. Tumor treatment also includes, for example, prevention of malignancy, recurrence or metastasis of the tumor. Furthermore, the subject to be treated or prevented is preferably a mammal, such as a human or non-human mammal.

  According to a preferred embodiment of the invention, the dipeptidyl peptidase IV inhibitor is a small molecule such as a drug. Such small molecules have already been developed as anti-diabetic drugs, and examples include SYR-322 (Takeda Pharmaceutical), TA-6666 (Tanabe Seiyaku), KRP-104 (Kyorin Pharmaceutical) and the like. Such small molecules can also be obtained by screening by methods known in the art using the inhibitory activity of dipeptidyl peptidase IV as an index.

  According to a preferred embodiment of the present invention, the dipeptidyl peptidase IV inhibitor is an antibody that specifically binds to a dipeptidyl peptidase IV protein. The antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody. Monoclonal antibodies specific for dipeptidyl peptidase IV protein are commercially available.

  According to a preferred embodiment of the present invention, the dipeptidyl peptidase IV inhibitor is an antisense nucleic acid molecule that specifically suppresses expression of the dipeptidyl peptidase IV gene. The antisense method is a well-known technique for suppressing the expression of a specific gene. In one embodiment, the antisense nucleic acid molecule is an antisense RNA having a length of about 10 to 40 bases designed based on the sequence of the 5 'coding region of the dipeptidyl peptidase IV gene. In another embodiment, the antisense nucleic acid molecule is a DNA oligonucleotide designed to be complementary to the sequence of a region involved in transcription of the dipeptidyl peptidase IV gene. Such an antisense nucleic acid molecule can be easily designed based on the sequence of the dipeptidyl peptidase IV gene as shown in SEQ ID NO: 1.

  According to a preferred embodiment of the present invention, the dipeptidyl peptidase IV inhibitor is an siRNA nucleic acid molecule that specifically suppresses expression of the dipeptidyl peptidase IV gene. As used herein, “siRNA nucleic acid molecule” means not only siRNA itself but also a longer double-stranded RNA molecule capable of introducing siRNA into a target cell. siRNA nucleic acid molecules are well-known tools that can suppress the expression of specific genes by RNA interference (RNAi) (Elbashir, S.M. et al., Nature 411, 494-498, 2001). The siRNA typically comprises a 19-21 base pair nucleotide sequence that is homologous to a sequence specific to the target gene mRNA. The double-stranded RNA molecule described above typically comprises a longer nucleotide sequence that is homologous to a sequence specific for the mRNA of the target gene. Such siRNA nucleic acid molecules can be easily designed based on the sequence of the dipeptidyl peptidase IV gene as shown in SEQ ID NO: 1. Further, the siRNA nucleic acid molecule can be expressed by an appropriate vector delivered into the cell. Therefore, the dipeptidyl peptidase IV inhibitor may be a vector that expresses an siRNA nucleic acid molecule that specifically suppresses the expression of the dipeptidyl peptidase IV gene. Such vectors can be readily constructed by standard procedures well known in the art (Bass, BL, Cell 101, 235-238, 2000; Tavernarakis, N. et al., Nat. Genet 24, 180-183, 2000; Malagon, F. et al., Mol. Gen. Genet. 259, 639-644, 1998; Parrish, S. et al., Mol. Cell 6, 1077-1087, 2000) .

  The dipeptidyl peptidase IV inhibitor can be administered by any suitable route such as topical, intravenous, subcutaneous, intramuscular, oral, rectal, mucosal. The therapeutically effective amount is appropriately determined by a doctor or veterinarian according to the severity of symptoms, the age of the subject, the effectiveness of the specific drug used, the route of administration, the frequency of administration, and the like. Generally, the therapeutically effective amount is about 0.001 to about 1000 mg / kg body weight, preferably about 0.01 to about 10 mg / kg body weight, more preferably about 0.01 to about 1 mg / kg per day. Weight is kg.

  The dipeptidyl peptidase IV inhibitor can be administered with a pharmaceutically acceptable carrier. Therefore, according to the present invention, there is provided a pharmaceutical composition for treating gastrointestinal stromal tumors originating in the stomach, comprising a dipeptidyl peptidase IV inhibitor and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers such as vehicles, excipients, diluents and the like are appropriately selected by those skilled in the art depending on the route of administration, the properties of the specific drug used, and the like.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these do not limit the present invention.

Example 1: Relationship between the malignancy of gastrointestinal stromal tumors originating in the stomach and dipeptidyl peptidase IV expression Samples of gastrointestinal stromal tumors originating in the stomach from 1972 to 2005 There were 152 cases that were surgically excised at the Central Hospital. The breakdown was 83 cases for men and 69 cases for women. The age at the first visit was 28 to 83 years (average 59 years), and the follow-up period was 4 to 352 months with an average of 117 months. Using formalin-fixed paraffin sections of excised specimens from these cases, immunostaining was performed by the ABC method using the Avidin-Biotin Complex method. As the primary antibody, DPP IV antibody (Anti-human Dipeptidyl Peptidase IV Antibody. Catalog number: AF1180) manufactured by R & D Systems was used. The determination of immunostaining was performed using macrophages and vascular endothelium present in the tumor as an internal control.

  The results of immunohistochemical staining are shown in FIG. In FIG. 1, the upper row shows HE staining, and the lower row shows DPP IV immunohistochemical staining (peroxidase method) of each case. The two cases on the left are positive for DPP IV expression, and strong staining is observed in the cell membrane and cytoplasm of both spindle-shaped cells and epithelial cells. One case on the right is DPP IV negative. Inflammatory cells (dotted arrows) and vascular endothelium (solid arrows) in the same specimen are positive for DPP IV, and it can be evaluated that the staining procedure was appropriate. As a result of immunohistochemical staining for all samples, 50 of the 152 cases (33%) were positive for DPP IV and the remaining 102 cases (67%) were negative.

  Next, a survival curve of overall survival and disease-free survival was created by the Kaplan-Meier method, and examined by the Log-rank test. The result is shown in FIG. In each graph shown on the left and right of FIG. 2, the survival curves of the DPP IV expression negative group (102 cases, upper line) and the positive group (50 cases, lower line) are compared. The graph on the left represents overall survival and the graph on the right represents disease free survival. Both graphs show that the positive group has a poor prognosis with a statistically significant difference compared to the DPP IV negative group (p <0.00001 and p <0.00001).

  Furthermore, the correlation between DPP IV staining and tumor clinical and pathological parameters was examined by Fisher's exact test. The results are shown in Table 1.

  In Table 1, the MIB-1 index (proliferation index), which is also used for classification of histological grade and risk group, is a standard method (for example, Hum. Pathol., Vol. 28, pp. 160-165, 1997). As shown in Table 1, the results of DPP IV staining were significantly correlated with patient age, tumor size, histological grade and risk group.

FIG. 1 is a photomicrograph showing the expression of dipeptidyl peptidase IV in a gastrointestinal stromal tumor whose primary site is the stomach. FIG. 2 is a graph showing the relationship between the expression of dipeptidyl peptidase IV and the life prognosis of patients with gastrointestinal stromal tumors that originate in the stomach.

Claims (19)

A method for determining whether a gastrointestinal stromal tumor originating in the stomach is malignant,
(A) a step of determining whether or not a dipeptidyl peptidase IV gene is expressed in a gastrointestinal stromal tumor obtained from a patient and having a stomach as a primary lesion; and (b) dipeptidyl in step (a). Determining the tumor is malignant if the peptidase IV gene is expressed.   The method of claim 1, wherein step (a) comprises detecting dipeptidyl peptidase IV protein in the tumor.   The method according to claim 2, wherein the dipeptidyl peptidase IV protein comprises the amino acid sequence represented by SEQ ID NO: 2.   The method of claim 2, wherein the detection of dipeptidyl peptidase IV protein is performed by immunohistochemical staining with a specific antibody.   2. The method of claim 1, wherein step (a) comprises detecting dipeptidyl peptidase IV mRNA in the tumor.   The method according to claim 5, wherein the mRNA of dipeptidyl peptidase IV comprises a nucleotide sequence represented by SEQ ID NO: 1, wherein t is converted to u.   The method of claim 1, wherein the tumor is a surgical sample or biopsy sample from a patient.   A kit for determining whether or not a gastrointestinal stromal tumor originating in the stomach is malignant, comprising a reagent capable of detecting the expression of a dipeptidyl peptidase IV gene.   The kit according to claim 8, wherein the reagent is a reagent for detecting dipeptidyl peptidase IV protein in the tumor.   The kit according to claim 9, wherein the reagent for detecting dipeptidyl peptidase IV protein is an antibody that specifically binds to dipeptidase IV protein.   The kit according to claim 8, wherein the reagent is a reagent for detecting mRNA of dipeptidyl peptidase IV in the tumor.   A pharmaceutical composition for treating gastrointestinal stromal tumors originating in the stomach, comprising a dipeptidyl peptidase IV inhibitor and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 12, wherein the tumor is a malignant tumor.   13. The pharmaceutical composition according to claim 12, wherein the treatment of the tumor comprises prevention of malignancy, recurrence or metastasis of the tumor.   13. A pharmaceutical composition according to claim 12, wherein the dipeptidyl peptidase IV inhibitor is a small molecule.   13. The pharmaceutical composition according to claim 12, wherein the dipeptidyl peptidase IV inhibitor is an antibody that specifically binds to dipeptidyl peptidase IV protein.   The pharmaceutical composition according to claim 12, wherein the dipeptidyl peptidase IV inhibitor is an antisense nucleic acid molecule that specifically suppresses expression of the dipeptidyl peptidase IV gene.   The pharmaceutical composition according to claim 12, wherein the dipeptidyl peptidase IV inhibitor is an siRNA nucleic acid molecule that specifically suppresses expression of a dipeptidyl peptidase IV gene.   The pharmaceutical composition according to claim 12, wherein the dipeptidyl peptidase IV inhibitor is a vector that expresses an siRNA nucleic acid molecule that specifically suppresses expression of a dipeptidyl peptidase IV gene.