WO2020262429A1 - Cancer biomarker and method for judging onset of cancer - Google Patents

Abstract

Provided is a cancer biomarker that comprises placental leucine aminopeptidase in urine. Also provided is a method for judging the onset of ovarian cancer, said method comprising a step for measuring the amount of placental leucine aminopeptidase or the activity of placental leucine aminopeptidase in the urine of a subject.

Description

Cancer biomarkers and methods for determining the onset of cancer

The present invention relates to a biomarker of cancer and a method for determining the onset of cancer.

For ovarian tumors, ultrasonography and, if necessary, MRI examinations are performed, and these diagnostic imagings are used to distinguish between ovarian tumors and other tumors, and the internal structure of the tumor is examined in detail to estimate whether it is benign or malignant. To do. Tumor marker tests are used along with diagnostic imaging to estimate benign or malignant, and if the value of the tumor marker is very high, the possibility of malignancy is high. However, tumor markers are also known to be elevated in benign endometriotic cysts (chocolate cysts) and the like. In addition, tumor markers often do not rise in the early stages of ovarian cancer, making them unsuitable for early detection of ovarian cancer. With the progress of research and technology, the clinical course of ovarian tumors, diagnostic imaging and evaluation of tumor markers will show a considerable degree of benign or malignant or intermediate (borderline malignant) ovarian tumors. Has become. However, the definitive diagnosis of ovarian cancer is still made by surgically removing the ovarian tumor and performing a histopathological examination.

In this way, ovarian cancer is one of the cancer types for which it is difficult to distinguish between benign and malignant cancer without surgery. In the case of cancer of other organs, a biopsy that takes a part of the tumor before surgery is often performed to determine whether it is malignant (cancer), but in the case of ovarian tumor, a needle stick is used. Preoperative biopsy has not been done because of the risk of cancer cells spreading into the abdomen. Therefore, there is a need for biomarkers that can diagnose benign, borderline malignant, or malignant ovarian tumors with high sensitivity and specificity prior to surgery.

Leucine aminopeptidase (LAP) is an enzyme having an activity of excising leucine from the amino terminus of a peptide, and three types of isozymes (cytoplasmic LAP, membrane-binding LAP, and placental LAP) are known. It has been reported that blood LAP is elevated in malignant tumors, and placental LAP (P-LAP) is reported to be elevated in renal cell carcinoma. Furthermore, it has been reported that the onset and prognosis of ovarian cancer can be evaluated by detecting P-LAP of ovarian tissue using an antibody (Patent Document 1).

Special table 2007-509313

An object of the present invention is to provide a biomarker contained in urine that can be collected non-invasively and capable of determining the onset and recurrence of cancer at an early stage. In particular, providing biomarkers for ovarian cancer that can distinguish between malignant ovarian tumors and non-malignant (benign and borderline malignant) ovarian tumors, and ovarian cancer that can determine the onset and recurrence of ovarian cancer at an early stage. An object of the present invention is to provide a method for determining the onset of cancer.

The present invention includes the following inventions in order to solve the above problems.
[1] A biomarker for cancer consisting of placental leucine aminopeptidase in urine.
[2] The biotechnology according to the above [1], wherein the cancer is ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, esophageal cancer, choriocarcinoma, or breast cancer. marker.
[3] The biomarker according to the above [1], wherein the cancer is ovarian cancer.
[4] A method for determining the onset of ovarian cancer, which comprises a step of measuring the amount of placental leucine aminopeptidase or the activity of placental leucine aminopeptidase in the urine of a subject.
[5] The method according to the above [4], wherein the subject is an individual having an ovarian tumor whose malignant or non-malignant determination is difficult in diagnostic imaging.
[6] The method according to the above [4], wherein the onset of ovarian cancer is recurrence after excision surgery.
[7] In the step of measuring the placental leucine aminopeptidase activity, the method of reacting the subject's urine with L-leucine-p-nitroanilide in the presence of methionine and measuring the amount of p-nitroanilide produced is used. The method according to any one of [4] to [6].

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a biomarker for cancer contained in urine that can be collected non-invasively. Since the biomarker of the present invention is more sensitive than conventional tumor markers and changes significantly, it is possible to distinguish between malignant tumors and non-malignant tumors. The biomarker of the present invention is very useful as a biomarker for ovarian cancer in which it is difficult to distinguish between a malignant tumor and a non-malignant tumor. In addition, by using the biomarker of the present invention, it is possible to provide a method for determining the onset of ovarian cancer, which can determine the onset and recurrence of ovarian cancer at an early stage.

It is a figure which shows the result of having measured the urinary P-LAP of the ovarian tumor patient (25 cases), and examined the correlation with the postoperative pathological diagnosis. It is a figure which shows the result of having measured the urinary p-LPA, the serum CA125 and the serum CA19-9 at the time of recurrence before the operation and after the operation of the ovarian cancer patient.

[Biomarker]
The present invention provides biomarkers for cancer. The cancer biomarker of the present invention (hereinafter referred to as "biomarker of the present invention") is a biomarker composed of placental leucine aminopeptidase (hereinafter referred to as "P-LAP") in urine. Patent Document 1 describes that the prognosis of cancer can be evaluated by measuring the amount of P-LAP in a cancer tissue isolated from an ovarian cancer patient. However, since P-LAP is a protein and urinary protein is negative if renal function is normal, those skilled in the art cannot have an incentive to use P-LAP in urine as a biomarker for ovarian cancer. Furthermore, the present inventors have confirmed that urinary P-LAP is more sensitive to ovarian cancer than serum P-LAP, and that the change depending on the presence or absence of ovarian cancer is remarkable (Example). reference). That is, it is not easy for a person skilled in the art to use urinary P-LAP as a biomarker for ovarian cancer, and the effect that urinary P-LAP is superior to serum P-LAP as a biomarker is effective. This is a particularly remarkable effect that cannot be easily predicted.

It has been reported that P-LAP is also expressed in cancer tissues in the following cancers. Therefore, urinary P-LAP is considered to be useful as a biomarker for these cancers.
・ Endometrial cancer (Shibata K et al. P-LAP / IRAP-induced cell proliferation and glucose uptake in endometrial carcinoma cells via insulin receptor signaling, BMC Cancer 2007, Jan 19; 7: 1)
・ Kidney cancer (Kuriyama M et al. Clinical evaluation of serum placental leucine aminopeptidase (P-LAP) activity in renal cell carcinoma, Nihon Hinyokika Gakkai Zasshi 1987 Jul; 78 (7): 1220-6.)
・ Prostate cancer (Lerman B et al. Oxytocin and cancer: An emaerging link. World Journal of Clinical Oncology. 2018 Sep 14; 9 (5): 74-82.)
・ Gastric cancer (Lerman B et al. Oxytocin and cancer: An emaerging link. World Journal of Clinical Oncology. 2018 Sep 14; 9 (5): 74-82.)
・ Pancreatic cancer (Lerman B et al. Oxytocin and cancer: An emaerging link. World Journal of Clinical Oncology. 2018 Sep 14; 9 (5): 74-82., Nagasaka T et al, Immunohistochemical localization of placental leucine aminopeptidase / oxytocinase in normal human placental, fetal and adult tissues, Reproduction Fertility and Development 1997; 9 (8): 747-53.)
・ Esophageal cancer (Lerman B et al. Oxytocin and cancer: An emaerging link. World Journal of Clinical Oncology. 2018 Sep 14; 9 (5): 74-82., Nagasaka T et al, Immunohistochemical localization of placental leucine aminopeptidase / oxytocinase in normal human placental, fetal and adult tissues, Reproduction Fertility and Development 1997; 9 (8): 747-53.)
・ Villous cancer (Nagasaka T et al, Immunohistochemical localization of placental leucine aminopeptidase / oxytocinase in normal human placental, fetal and adult tissues, Reproduction Fertility and Development 1997; 9 (8): 747-53., Ino K et al. Expression of placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in human normal and malignant invasive trophoblastic cells. Lab Invest. 2003 Dec; 83 (12): 1799-809.)
・ Breast cancer (Jose manuel Martinez-Martos et al. Kidney aminopeptidase activities are related to renal damage in experimental breast cancer, Journal of Clinical and Molecular Medicine. 2018 Doi: 10.15761 / JCMM.1000105)

The biomarker of the present invention, that is, urinary P-LAP can be measured by a known method. For example, an enzymatic method such as the L-leucine-p-nitroanilide method can be preferably used. In the L-leucine-p-nitroanilide method, p-nitro occurs when P-LAP in a sample acts on L-leucine-p-nitroanilide (substrate) to produce L-leucine and p-nitroanilide. The LAP activity value is determined by measuring the rate of increase in absorbance with the formation of anilide. When measuring urinary P-LAP by the L-leucine-p-nitroanilide method, a commercially available leucine aminopeptidase measurement kit can be used.

When measuring urinary P-LAP by the enzymatic method, the substrate and the sample may be reacted in the presence of methionine. This is because the presence of methionine can inhibit the activity of LAPs other than P-LAP (for example, LPA derived from cytoplasm, LAP derived from microsomes, etc.), and the activity based on P-LAP can be measured. The methionine concentration is not particularly limited, but may be, for example, 1 mM to 50 mM, 5 mM to 40 mM, 10 mM to 30 mM, or 15 mM to 25 mM.

An immunoassay method such as an ELISA method can be preferably used as a method for measuring P-LAP in urine. In the immunoassay method, the amount of P-LAP in urine can be measured by contacting the sample with the anti-P-LAP antibody and detecting the complex of P-LAP and the anti-P-LAP antibody in the sample. ..

[Method of determining the onset of ovarian cancer]
The present invention provides a method for determining the onset of ovarian cancer or a method for assisting the determination of the onset of ovarian cancer. The method of the present invention can assist a physician in diagnosing ovarian cancer. Ovarian tumors are classified into benign tumors, borderline malignant tumors (having properties intermediate between benign and malignant) and malignant tumors (ovarian cancer). It is very difficult to distinguish without inspection. The method of the present invention is very useful because it can discriminate between malignant tumors (ovarian cancer) and non-malignant tumors with high specificity. In addition, the method of the present invention is very useful in that not only the onset of ovarian cancer can be determined at an early stage, but also the recurrence of ovarian cancer after resection surgery can be determined at an early stage.

According to the "Ovarian Tumor Handling Regulations" (2016), ovarian tumors are classified as I. Epithelial tumor, II. Sexual cord stromal tumor, III. Embryo cell tumor, IV. It is classified into other tumors, each of which includes benign tumors, borderline malignancies and malignant tumors. Benign tumors of epithelial tumors include serous cystadenoma, mucinous cystadenoma, endometrioid adenoma, clear cell adenoma, adenoma, superficial papilloma, Brenner tumor, endometriotic cyst, etc. Be done. Borderline malignancies of epithelial tumors include serous borderline malignancies, mucinous borderline malignancies, intimal borderline malignancies, clear cell borderline malignancies, borderline malignant Brenner tumors and the like. Malignant tumors of epithelial tumors include low-grade serous cancer, high-grade serous cancer, mucinous cancer, endometrioid cancer, clear cell cancer, malignant Brenner tumor, serous mucinous cancer, undifferentiated cancer, and microscopic Examples include serous borderline malignancies with papillary patterns. Benign tumors of sex cord interstitial tumors include thecoma cell tumor, Sertri / interstitial tumor (well-differentiated type), sclerosing interstitial tumor, fibroma, Rydik cell tumor, and sex cord tumor with cricoid tubules. And so on. Borderline malignancies of sex cord stromal tumors include juvenile granulosa cell tumors and sertoly / stromal cell tumors (moderately differentiated). Examples of malignant tumors of sex cord stromal tumors include fibrosarcoma, Sertri / stromal cell tumor (poorly differentiated type), malignant steroid cell tumor, and adult granulosa cell tumor. Benign tumors of embryonic cell tumors include mature teratomas, benign ovarian thyroid tumors, and sebaceous adenomas. Malignant tumors of germ cell tumors include mature teratomas with malignant transformation, oval sac tumors, multiple germinoma, embryonic cancer, undifferentiated germinoma, choriocarcinoma, malignant ovarian thyroid tumor, and immature teratoma (G1 to G3). ), Cartinoid tumors and the like. Benign tumors of other tumors include ovarian reticular adenomas. Borderline malignancies of other tumors include gonadal blastoma (pure type). Examples of malignant tumors of other tumors include small cell carcinoma, malignant lymphoma, and secondary (metastatic) tumors.

The method of the present invention may include a step of measuring the amount of P-LAP or P-LAP activity in the urine of a subject. The subject is not limited as long as it is a mammal capable of developing ovarian cancer, and examples thereof include humans, monkeys, chimpanzees, dogs, cats, cows, horses, pigs, rabbits, mice, and rats. It is preferably human. The subject may be an individual suspected of having an ovarian tumor, an individual having an ovarian tumor, or an individual having an ovarian tumor whose malignant or non-malignant determination is difficult to determine by diagnostic imaging. Good.

In the method of the present invention, as a method for measuring the amount of P-LAP or P-LAP activity in urine, an enzyme method such as the L-leucine-p-nitroanilide method and an immunoassay method such as the ELISA method are preferably used. Can be done.

Whether or not a subject has ovarian cancer is determined, for example, with respect to the P-LAP value measured by the L-leucine-p-nitroanilide method, the P-LAP value of the malignant tumor group and the non-malignant tumor group. A determination method in which a cutoff value is set based on the P-LAP value of the above and whether or not the cutoff value is exceeded is used as a determination criterion. The cutoff value may be set in the range of 10 to 30 mU / mL, in the range of 10 to 20 mU / mL, or in the range of 10 to 15 mU / mL.

The expression of P-LAP is also observed in various cancer tissues other than ovarian cancer. Therefore, the method of the present invention can be applied to cancers other than ovarian cancer. Specifically, for example, uterine body cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, esophageal cancer, choriocarcinoma, breast cancer and the like.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[Example 1: Measurement of P-LAP in urine of a patient with ovarian tumor]
1-1 Materials and methods (1) Samples Urine was collected with the consent of ovarian tumor patients (25 patients) whose benign or malignant diagnosis was difficult by diagnostic imaging, and used as samples.
(2) Urinary P-LAP measurement method A leucine aminopeptidase measurement kit (Cerotech Co., Ltd.) was used. Specifically, 160 μL of the first reagent and 40 μL of the second reagent were added to the tube, L-methionine was added to a final concentration of 20 mM, 4 μL of urine was added, and the mixture was incubated at 37 ° C. for 5 minutes. The absorbance at 405 nm was measured using a spectrophotometer.
(3) Pathological diagnosis The above 25 patients with ovarian tumor were subsequently operated on, and the ovarian tumor was removed and histopathological examination was performed to make a benign or malignant diagnosis.

(4) Results The results are shown in Fig. 1 and Table 1. Of the 25 cases, 10 were malignant and 15 were benign to borderline malignant (non-malignant). The urinary P-LAP (mean value: 43.4 mU / mL) of malignant tumor patients was significantly higher than that of urinary P-LAP (mean value: 6.8 mU / mL) of non-malignant tumor patients. When the cutoff value was set to 15 mU / mL, the sensitivity was 90% and the specificity was 100%. The breakdown of 15 cases of benign to borderline malignancy is chocolate cyst 7 cases, mature teratoma 2 cases, mucinous cystadenoma 2 cases, fibroma 1 case, mucinous borderline malignant tumor 2 cases, serous borderline malignant tumor 1 case. Met. The breakdown of 10 malignant tumors was clear cell cancer in 4, intimal cancer in 2, mucinous cancer in 1, serous cancer in 2, and undifferentiated cancer in 1.

(5) Serum P-LAP measurement In 5 of the above 25 cases (benign 2 cases, malignant 3 cases), serum P-LAP was measured. The measuring method is the same as (2) above. The results are shown in Table 2. There was no difference in serum P-LAP levels between malignant tumor patients and benign tumor patients.

[Example 2: Transition of urinary P-LPA before, after, and at the time of recurrence of ovarian cancer patients]
Urinary p-LPA was measured before, after, and at the time of recurrence of ovarian cancer patients. At the same time, tumor markers serum CA125 and serum CA19-9 were measured. This patient underwent surgery on May 9, 2018, measured after surgery on May 22, 2018, and was discharged on the following day. After that, recurrence was confirmed on May 31, the same year.

The results are shown in Fig. 2. Urinary P-LPA was 163 mU / mL before the operation, 8 mU / mL after the operation, and 152 mU / mL at the time of recurrence. Conventional tumor markers CA125 and CA19-9 also decreased in concentration after surgery and increased in concentration at the time of recurrence, but the rate of decrease after surgery and the rate of increase at the time of recurrence were more remarkable in urinary P-LPA. there were. In other words, it was shown that urinary P-LPA can more sensitively reflect the presence of cancer cells than conventional tumor markers. From this result, it was clarified that urinary P-LPA is useful as a marker for determining the recurrence of surgically resected ovarian cancer.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all the academic and patent documents described in the present specification are incorporated herein by reference.

Claims (7)

1. A cancer biomarker consisting of placental leucine aminopeptidase in urine.
2. The biomarker according to claim 1, wherein the cancer is ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, stomach cancer, pancreatic cancer, esophageal cancer, choriocarcinoma, or breast cancer.
3. The biomarker according to claim 1, wherein the cancer is ovarian cancer.
4. A method for determining the onset of ovarian cancer, which includes a step of measuring the amount of placental leucine aminopeptidase or the activity of placental leucine aminopeptidase in the urine of a subject.
5. The method according to claim 4, wherein the subject is an individual having an ovarian tumor whose malignant or non-malignant determination is difficult in diagnostic imaging.
6. The method according to claim 4, wherein the onset of ovarian cancer is recurrence after excision surgery.
7. In the step of measuring the placental leucine aminopeptidase activity, a method of reacting a subject's urine with L-leucine-p-nitroanilide in the presence of methionine and measuring the amount of p-nitroanilide produced is used. The method according to any one of 6.

Images