JP2012013504A - Prognostic determination method of corpus uteri endometrioid adenocarcinoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly precise and specific prognostic determination method of cancer in a prognostic determination of corpus uteri endometrioid adenocarcinoma.SOLUTION: The prognostic determination method of the corpus uteri endometrioid adenocarcinoma comprises: measuring development patterns of a molecule, carbonyl reductase, which is bound to SCC antigen and is well known as a tumor marker of the epidermoid cancer, in a carcinoma tissue by an immune structure dyeing method using an antibody specifically bound to the carbonyl reductase; scoring the acquired development pattern; and, when the result is negative or weak positive, determining it as an adverse prognosis.

Description

  The present invention relates to a method for determining the prognosis of endometrial endometrioid adenocarcinoma, and more specifically, the expression status of carbonyl reductase (CR) in endometrial endometrioid adenocarcinoma tissue in which no specific tumor marker exists. It is related with the method of determining the prognosis of endometrial endometrioid adenocarcinoma by scoring. Furthermore, the present invention relates to an antibody that specifically binds to CR, a peptide for producing the antibody, and a test kit for detecting CR using the antibody.

  In recent years, endometrial cancer is rapidly increasing in Japan. In Europe and the United States, endometrial cancer is the most common gynecological malignancy, and is the fourth most common female malignancy after breast cancer, lung cancer, and colon cancer. In Japan, further increase in endometrial cancer is feared. In fact, the prevalence of 100,000 endometrial cancer populations has been reported to increase from 1.4 to 4.5 when comparing 1975 and 1998. The reasons for the increase are obesity due to westernization of eating habits, late marriage and declining birthrate. In such a situation, the clinical importance of endometrial cancer is considered to be increasingly higher.

  Current well-known tumor markers for detecting endometrial cancer include CA125, CA19-9, CA72-4, CEA and the like. However, the positive rate representing detection is 21.1-43.0% for CA125, 24.0-31.0% for CA19-9, 21.4-33.3% for CA72-4, and 2 for CEA. .1-14% and low (Non-Patent Document 1). Thus, it cannot be said that there is a tumor marker with high specificity for endometrial cancer.

  In the current situation, endometrial cancer undergoes radical surgery, and the prognosis is predicted from the results of histopathological search of the removed organ. Therefore, the need for adjuvant therapy such as postoperative chemotherapy or radiation therapy cannot be determined until the surgical operation is performed. A method for predicting and determining the prognosis of cancer that can reduce the number of patients receiving unnecessary treatment is desired.

  So far, there have been several reports on how to determine the prognosis of cancer. Regarding the prognosis of bladder cancer, a determination method by detecting the presence or absence of centrosome duplication abnormality of cells existing in urine has been clarified (Patent Document 1). : Activated leukocyte-cell adhesion molecule (activated leukocyte adhesion molecule) is detected from the blood, and a prognostic situation is determined according to the detection result (Patent Document 2). In addition, as a method for predicting a high risk of bone metastasis from cancer such as breast cancer, lung cancer, prostate cancer, or stomach cancer, γ-GTP (γ-glutamyl transpeptidase) level in urine and cancer cells is measured, and the γ -A method for predicting that the risk of bone metastasis is high when the GTP level is higher than a set value is disclosed (Patent Document 3). Regarding esophageal squamous cell carcinoma, a prognostic diagnosis method by a method for detecting a specific structural change of a specific chromosome has been disclosed (Patent Document 4). The present inventors previously classified the expression pattern of CR at the primary site by immunohistochemical staining for stage I and II of cervical squamous cell carcinoma and clarified a prognostic determination method (Patent Document 5). .

  Regarding CR, in vitro studies, it has been reported that in cells having high metastatic potential, the expression is decreased and the metastatic potential is decreased due to overexpression (Non-patent Document 2). From this, it is presumed that CR is involved in the malignancy and metastasis of cancer, but there is no knowledge that disclosed a prognosis determination method for endometrial endometrioid adenocarcinoma patients occupying most of endometrial cancer.

JP 2006-275989 A (Table 2) Japanese Patent No. 3779294 (FIG. 2) JP 2006-153499 A (FIG. 2) JP 2002-272497 A (Tables 2 and 3) JP 2008-182987 A (FIGS. 6-8, Table 2)

Akihiro Murakami et al. Women's outpatient care manual Obstetrics and gynecology and tumor marker Obstetrics and gynecology treatment 94: 619-623, 2007 Ismail, E et al. Cancer Res 60: 1173-1176,2000

  For endometrial endometrioid adenocarcinoma, there is no known postoperative determination method that can predict the prognosis of cancer and reduce the number of patients receiving unnecessary treatment. The main object of the present invention is to provide a highly accurate and highly specific cancer prognosis determination method.

  It has been found by the present inventors that CR is a molecule that binds to an SCC antigen well known as a tumor marker for squamous cell carcinoma (Murakami, A et al. Int J Oncol 34: 1395-). 1400, 2010). The expression pattern of CR is not the same in cancer tissues, and there are strong and weak cells. Furthermore, CR immunohistochemical staining in the primary tissue resulted in a poor prognosis in cases with reduced expression. The present inventors focused on that point and found a close relationship between the expression pattern and the prognosis of endometrial endometrioid cancer, and completed the present invention.

  That is, the present invention provides the following (1) to (4).

  (1) A method for determining the prognosis of cancer, which is a method for determining the prognosis of endometrial endometrial adenocarcinoma using the expression pattern of carbonyl reductase in cancer tissue as an index.

  (2) The expression pattern of carbonyl reductase in cancer tissue is scored by the ratio of staining intensity and staining positive cells, and when negative or weakly positive, it is determined that the prognosis is poor (1) ) Prognosis determination method as described in.

(3) The expression pattern of carbonyl reductase in cancer tissue is measured by immunohistochemical staining using an antibody obtained based on any of the following peptides that specifically bind to carbonyl reductase: The prognosis determination method according to any one of (1) and (2).
(A) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing;
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing.

  (4) A kit for carrying out the method according to any one of (1) to (3) above, comprising an antibody that specifically binds to carbonyl reductase, reagents for immunohistochemical staining, and instruments. Prognostic kit.

  According to the present invention, it is possible to provide a cancer prognosis determination method, particularly a highly accurate and specific prognosis determination method for endometrial endometrioid adenocarcinoma, and further provide a determination kit for simple determination. be able to.

It is a figure which shows the detection about the localization of CR which strongly expresses in a normal endometrium. It is a figure which shows the expression pattern of CR by a change immunohistochemical staining in the change of the estrous cycle in a normal endometrium (A, B; proliferation phase, C, D; secretion phase). It is a figure which shows the negative detection result in the expression pattern of CR by immunohistochemical staining in endometrial endometrioid adenocarcinoma. It is a figure which shows the detection result of weak positive by the expression pattern of CR by the immunohistochemical staining in endometrial endometrioid adenocarcinoma. It is a figure which shows the detection result of strong positive by the expression pattern of CR by immunohistochemical staining in endometrial endometrioid adenocarcinoma. It is a figure which shows the expression pattern of CR according to an advanced stage in endometrial endometrioid adenocarcinoma. It is a figure which shows PFS according to CR staining score in patients with endometrial endometrioid adenocarcinoma (A: study in 3 groups of strong positive, weak positive, and negative, B: study in 2 groups of strong positive, weak positive + negative ). It is a figure which shows OS according to CR staining score in patients with endometrial endometrioid adenocarcinoma (A: study in 3 groups of strong positive, weak positive, negative, B: study in 2 groups of strong positive, weak positive + negative ).

  Among endometrial cancers, endometrioid adenocarcinoma is the most frequently observed histologically. The present invention is a prognostic determination method for endometrial endometrioid adenocarcinoma, wherein a peptide for use in determination is prepared and an antibody against the peptide is prepared. Next, the prognosis of the cancer is determined by measuring the CR expression pattern in the cancer tissue using the antibody and analyzing the CR expression pattern in the cancer tissue.

  CR is purified from human brain and has a molecular weight of about 30 kDa and is composed of 277 amino acids (Wermuth, B. J Biol Chem 256: 1206-1213, 1981, Wermuth, B. et al J Biol Chem 263: 16185-16188, 1988). The gene is located on chromosome 21 long arm 22.12 and has been found to have 82% homology with the mouse gene (Forrest, GL. Et al. Biochim Biophys Acta 1048: 149-155. 1990). CR is an NADPH-dependent enzyme localized in the cytoplasm using a carbonyl compound as a substrate. It is known that it has the action of catalyzing the reduction of prostaglandins and steroids in the brain, and is involved in the detoxification action in the liver.

  Information on the CR gene is known as human CR, and is registered under the accession number J04056 in the NCBI gene database. According to it, it turns out that it is polypeptide which consists of DNA and amino acid sequence which are shown to sequence number 1 of a sequence table.

  Human CR can be adjusted by a known method. As revealed by Wermuth et al., There is a method of producing clones from a library of cDNA obtained from human placenta (Wermuth, B. J Biol Chem 256: 1206-1213, 1981). Alternatively, it can be expressed in a host cell such as Escherichia coli as a fusion with a GST (glutathione S-transferase) protein and purified using a glutathione column or a nickel column.

  The expression pattern of CR is measured using a peptide having the same immunogenicity as CR and having an amino acid sequence of at least 8 amino acids, preferably 10 amino acids or more. As long as the peptide has the desired immunogenicity, the amino terminus and / or carboxy terminus may be modified. Examples thereof include peptides having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing. be able to. Alternatively, the peptide may be a peptide in which one or more amino acid residues are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. Such a variant has an amino acid sequence having at least 70% or more, preferably 80% or more, more preferably 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing. It may be a thing.

  The peptide of SEQ ID NO: 2 regards the amino acid sequence site from the 184th histidine (His) to the 195th glycine (Gly) of the amino acid sequence of SEQ ID NO: 1 as the antigen-determining site. Is chemically synthesized by adding cysteine to the amino acid sequence of CHQKEGWPSSAYG.

  Further, the peptide of SEQ ID NO: 3 has an amino acid sequence site from the 99th aspartic acid (Asp) acid to the 113th threonine (Thr) of the amino acid sequence of SEQ ID NO: 1 as another antigenic determination site. It is assumed that cysteine was added after threonine on the C-terminal side and chemically synthesized, and has an amino acid sequence of DTPPFHIQAEVTMKTC.

  There are several methods for producing the peptide. For example, based on CR gene information, a marker gene or a partial gene thereof is incorporated into an expression vector, introduced into an appropriate host cell, a transformant is prepared, the transformant is cultured, and a recombinant protein is obtained. Examples thereof include a method of expressing and purifying the expressed recombinant protein from a culture or culture supernatant, or a method of chemically synthesizing an oligopeptide based on the amino acid sequence information of CR. Furthermore, the CR protein can also be obtained by cleaving with an appropriate protein cleaving enzyme.

  The antibody used in the present invention is an antibody that recognizes the above-mentioned peptide, is an antibody that specifically binds to CR in cancer tissue and can measure the expression pattern of CR. The antibody can be prepared using a known method, and may be a polyclonal antibody or a monoclonal antibody. Moreover, as long as the characteristic which recognizes CR is not lost, antibody fragments, such as a low molecular weight antibody and a modified antibody, may be sufficient.

  For example, a polyclonal antibody is a peptide having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing having the above antigenic property, or the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence listing. Alternatively, a peptide in which a plurality of amino acid residues are deleted, substituted or added is administered subcutaneously or intraperitoneally to a mammal such as a rabbit, rat, mouse, monkey or sheep using an appropriate adjuvant as necessary. The blood is collected from the sensitized animal, and serum is obtained from this blood by a known method. As the polyclonal antibody, the separated serum can be used as it is. For example, SDS-PAGE (SDS-polyacrylamide gel electrophoresis) or CNBr-activated Sepharose 4B (manufactured by Amersham) is used. It can also be purified and used by the Affinity method.

  The measurement of CR expression pattern using the above antibody can be performed according to a known protein measurement method. For example, Western blotting method, dot blot method, immunoprecipitation method, enzyme immunoassay method (EIA; enzyme-linked immunosorbent assay), radioimmunoassay method (RIA), fluorescence Examples thereof include immunological measurement methods such as an antibody method (FIA) and immunohistochemical staining.

  In the measurement method, an antibody that specifically binds to CR is labeled with a detectable substance. Examples of the label include radioactive isotopes, affinity labels such as biotin and avidin, enzyme labels such as horseradish peroxidase and alkaline phosphatase, fluorescent labels such as FITC and rhodamine, chemiluminescence labels using ECL (Enhanced Chimi Luminescence), and the like. It is done. Such labels may be combined, and the labeling method is performed by a method already well known.

  Examples of immunological measurement methods include competitive assay methods and sandwich methods. In the ELISA method of the sandwich method, a substance serving as an enzyme substrate is added to a bound enzyme-labeled antibody to cause color development or light emission, and then a reaction stop solution is added to measure the intensity or pattern of color development or light emission. Since an intensity or pattern proportional to the antigen concentration in the sample is obtained, the expression status of the antigen in the sample can be grasped based on comparison with the control substance.

  For the CR expression analysis using the antibody of the present invention, a human tissue staining kit can also be used. When using Histofine (manufactured by Nichirei Co., Ltd.), an immunohistochemical staining kit, a biotin-labeled anti-rabbit IgG antibody is bound as a secondary antibody to an antibody bound to CR, and peroxidase-labeled strepto which is an enzyme reagent. Bind avidin. Subsequently, the expression pattern of CR in cancer tissue can be observed by staining peroxidase with DAB (3,3-Diaminobenzidine).

  When the cancer prognosis is determined using the tissue of a cancer patient, the cancer tissue of the patient from whom informed consent was obtained is used. The advanced stage classification of endometrial cancer type endometrial adenocarcinoma patients can be classified into four stages from advanced stage I to IV according to the criteria of FIGO (International Federation of Gynecology and Obstetrics). Advanced stage I is cancer that is confined to the uterine body, advanced stage II is cancer that extends to the uterine body and neck, advanced stage III is cancer that extends outside the uterus but crosses the small pelvic cavity No, or with regional lymph node metastasis, advanced stage IV, is defined as cancer that has crossed the small pelvic cavity or apparently affects the bladder or intestinal mucosa.

  In order to determine the prognosis of endometrial endometrioid adenocarcinoma of the present invention, the expression of CR in cancer tissue is achieved by contacting the antibody composition of the present invention with a sample derived from the cancer tissue obtained from a patient. Can be detected by staining the immunoconjugate, and the prognosis of the cancer can be determined by measuring the expression pattern of CR. The evaluation method can be performed, for example, according to the method of Umemoto et al. (Br. J. Cancer 85: 1032-1036, 2001). That is, the expression pattern of CR is not stained at all with staining intensity (High: +3, Moderate: +2, Weak: +1, Negative: +0) and the ratio of staining positive cells (over 50%: +2, less than 50%: +1). : +0)), and the sum is divided into three patterns (A) strong positive, (B) weak positive, and (C) negative. From each pattern, (1) progression-free survival (PFS: progression free survival, period from the date of surgery until clinical recurrence is confirmed), and (2) overall survival (OS: overall survival, death from the date of surgery) Period) until the prognosis is determined by statistical analysis.

  When the expression pattern of CR in cancer tissue is (A) strong positive, PFS and OS are extended and it can be predicted that the prognosis is good, and the expression pattern of CR is (B) weakly positive, or (C) If negative, PFS and OS are low and can be predicted as poor prognosis.

  The present invention also provides a kit in which reagents for highly accurate and specific prognosis determination of endometrial endometrioid adenocarcinoma are combined in advance. In addition to the antibody, the kit can also contain an immobilization carrier, a labeling substance, a substrate compound used for detection of the label, and the like.

  Hereinafter, examples will be given to describe the present invention in more detail, but the present invention is not limited thereto.

<Synthesis of peptides>
Information on the amino acid sequence of CR shown in SEQ ID NO: 1 was analyzed, and the antigen determining site of CR was searched. As a result, the amino acid sequence region from the 184th histidine (His) to the 195th glycine (Gly) of the amino acid sequence of SEQ ID NO: 1 was found to be an excellent antigen determination site. Therefore, in order to prepare an antibody of CR, a synthetic peptide CHQKEGWPSSAYG (SEQ ID NO: 2 in the sequence listing) in which cysteine was added before histidine on the N-terminal side was prepared.

<CR antibody production>
The synthetic peptide prepared in Example 1 was immunized by preparing an emulsion using Freund's adjuvant and injecting the emulsion subcutaneously into a rabbit. For immunization, 0.15 mg of the synthetic peptide was administered at the first time, 0.3 mg after 2 weeks, and then 4 times in total every 2 weeks. Blood samples were collected three times before the administration of the synthetic peptide, one week after administration of the synthetic peptide three times, and one week after the administration of the fourth administration, three times in total. The antibody titer of CR in each test blood sample was measured by ELISA. One week after the final immunization, the whole blood was collected from the immunized rabbit. The final blood sample was subjected to affinity purification with CNBr-activated Sepharose 4B (manufactured by Amersham Pharmacia) to obtain a CR polyclonal antibody.

<Detection of CR in normal endometrial epithelium>
Using the antibody prepared in Example 2, the expression of CR in normal endometrial epithelial tissues collected with the consent of the patient was confirmed by immunostaining. In the experimental method, immunohistological staining was performed according to the procedures (1) to (8) using the purified polyclonal antibody and the Histofine SAB-PO (R) kit (Nichirei) according to the protocol. (1) A formalin-fixed paraffin-embedded tissue is cut out to a thickness of 5 μm, immersed in xylene three times, and then immersed in 100% ethanol to perform a deparaffinization operation. (2) Immerse the tissue section in methanol containing 0.3% hydrogen peroxide for 50 minutes to inactivate endogenous peroxidase. (3) After washing the tissue section three times with PBS, in order to prevent non-specific adsorption of the prepared anti-CR antibody, 10% at room temperature using 10% normal goat serum attached to the Histofine SAB-PO (R) kit. Block for a minute. (4) As a primary antibody reaction, the anti-CR polyclonal antibody adjusted to a concentration of 2 μg / ml with PBS containing 1% bovine albumin is reacted overnight at 4 ° C. (5) The tissue section is washed 3 times with PBS, and then reacted with the secondary antibody biotin-labeled goat anti-rabbit IgG antibody included in the kit at room temperature for 10 minutes. (6) After completion, the tissue section is washed 3 times with PBS, and the peroxidase-labeled streptavidin solution contained in the kit is added. Allow to react for 5 minutes at room temperature. (7) After completion, the tissue section is washed 3 times with PBS and immersed in 0.02% DAB solution (30 mg DAB is dissolved in 130 ml of 0.05 M Tris HCl buffer and 20 ml of 0.05 M Trisma base) for 1 minute. . After completion, rinse with running water for 5 minutes. (8) Stain hematoxylin, wash with running water for 10 minutes, and enclose.

  From the above results, it is clear that CR is localized in the cytoplasm of normal endometrial epithelium (FIG. 1), and is expressed more strongly in the secretory phase in the normal endometrial epithelium than in the proliferative phase. (FIGS. 2A-D).

<Detection of CR in endometrial endometrial adenocarcinoma tissue>
Tissue collection and examination were performed on 109 patients with endometrial adenocarcinoma of the uterine corpus that were not treated before surgery with informed consent and could be followed for prognosis. Table 1 shows the number of cancer patients in each advanced stage.

  Immunohistochemical staining was performed according to the procedures (1) to (8) in the same manner as in Example 3 using the cancer tissue collected from the first surgical case of the patient. The expression pattern of CR by immunohistochemical staining is shown in FIG. 3, FIG. 4, and FIG. CR expression patterns were scored according to the method of Umemoto et al. (Br. J. Cancer 85: 1032-1036, 2001) and classified into three types. That is, by the sum of the staining intensity (High: +3, Moderate: +2, Weak: +1, Negative: +0) and the percentage of staining positive cells (50% or more: +2, less than 50%: +1, no staining: +0), As shown in Table 2, it was classified by scoring. When classified into two groups, a negative + weak positive group in which CR expression is reduced and a positive group in which CR expression is maintained, as shown in FIG. The proportion of the group increased.

<Prognosis by CR expression pattern>
The results of examining the PFS and OS in each staining pattern (strong positive, weak positive, negative) for the cancer patient who provided the cancer tissue in Example 4 were followed for a maximum prognosis of 175 months. It was shown to. In the group showing weak positive and negative, a decrease in PFS was observed, and a decrease in OS was also observed. In addition, even when compared between the two groups, a strong positive group and a weak positive + negative group in which expression was reduced, a decrease was observed in both PFS and OS. As is clear from the P value (statistical significance comparing the population), recurrence and death occur in weakly positive and negative cases that show attenuated CR expression in the primary endometrial adenocarcinoma The prognosis was poor. From the above results, it was revealed that CR can be a new index in determining prognosis of endometrial endometrioid adenocarcinoma and examining individualization of treatment.

  The prognosis determination method for endometrial endometrioid adenocarcinoma using the CR expression pattern in cancer tissue as an index according to the present invention performs the cell status macroscopically, thus enabling accurate determination, and cancer patients On the other hand, it can provide important information on prognosis and can be used as a basis for judging the necessity of postoperative adjuvant therapy. In addition, it is considered that screening for drug discovery that leads to cancer treatment or suppression of cancer metastasis can be performed using the CR expression pattern as an index.

Claims (4)

A method for determining the prognosis of cancer, comprising determining the expression pattern of carbonyl reductase in cancer tissue as an index. The expression pattern of carbonyl reductase in a cancer tissue is scored by the staining intensity and the ratio of staining positive cells, and when negative or weakly positive, it is determined that the prognosis is poor. Prognosis determination method. The expression pattern of carbonyl reductase in cancer tissue is measured by immunohistochemical staining using an antibody obtained based on any of the following peptides that specifically bind to carbonyl reductase: The prognosis determination method according to any one of 2 above.
(A) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing;
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2 or 3 in the sequence listing. A kit for carrying out the method according to any one of claims 1 to 3, comprising an antibody that specifically binds to carbonyl reductase, a reagent for immunohistochemical staining, and instruments. .