JP7313005B2 - Cancer biomarkers and methods for determining cancer development - Google Patents

Description

The present invention relates to cancer biomarkers and methods for determining cancer development.

For ovarian tumors, ultrasound examination and, if necessary, MRI examination are performed. Based on these diagnostic imaging, it is possible to distinguish between ovarian tumors and other tumors. To estimate benign or malignant tumors, tumor marker tests are used together with diagnostic imaging, and when tumor marker values are extremely high, the possibility of malignancy is high. However, tumor markers are known to be elevated even in benign endometriotic cysts (chocolate cysts). In addition, tumor markers often do not rise in the early stages of ovarian cancer, making it unsuitable for early detection of ovarian cancer. With advances in research and technology, the clinical course, imaging, and tumor marker evaluation of ovarian tumors has provided a great deal of insight into whether ovarian tumors are benign, malignant, or intermediate (borderline). However, the definitive diagnosis of ovarian cancer is still made by surgically removing the ovarian tumor and conducting histopathological examination.

Thus, ovarian cancer is one of the cancer types in which it is difficult to determine whether it is benign or malignant without surgery. In the case of cancers of other organs, it is often the case that a histopathological examination is performed by taking a biopsy of a part of the tumor before surgery to determine whether it is malignant (cancer) or not. However, in the case of ovarian tumors, there is a risk that cancer cells will spread into the peritoneal cavity if a needle is inserted into the tumor, so biopsy is not performed before surgery. Therefore, there is a need for biomarkers that can diagnose benign, borderline, or malignant ovarian tumors preoperatively with high sensitivity and specificity.

Leucine aminopeptidase (LAP) is an enzyme that has the activity of cleaving leucine from the amino terminus of peptides, and three isozymes (cytoplasmic LAP, membrane-bound LAP, placental LAP) are known. There is a report that blood LAP is elevated in malignant tumors, and there is a report that placental LAP (P-LAP) is 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 in ovarian tissue using an antibody (Patent Document 1).

Special table 2007-509313

An object of the present invention is to provide a biomarker that is contained in urine that can be collected noninvasively and that enables early determination of onset and recurrence of cancer. In particular, the object is to provide an ovarian cancer biomarker capable of distinguishing between malignant ovarian tumors and non-malignant (benign and borderline malignant) ovarian tumors, and to provide a method for determining the onset of ovarian cancer that enables early determination of the onset and recurrence of ovarian cancer.

The present invention includes the following inventions in order to solve the above problems.
[1] A cancer biomarker consisting of placental leucine aminopeptidase in urine.
[2] The biomarker of [1] above, wherein the cancer is ovarian cancer, endometrial cancer, renal cancer, prostate cancer, gastric cancer, pancreatic cancer, esophageal cancer, choriocarcinoma, or breast cancer.
[3] The biomarker according to [1], wherein the cancer is ovarian cancer.
[4] A method for determining the onset of ovarian cancer, comprising the step of measuring the placental leucine aminopeptidase amount or placental leucine aminopeptidase activity in the urine of a subject.
[5] The method of [4] above, wherein the subject is an individual with an ovarian tumor that is difficult to determine whether it is malignant or non-malignant in diagnostic imaging.
[6] The method according to [4] above, wherein the onset of ovarian cancer is recurrence after surgical excision.
[7] The method according to any one of [4] to [6], wherein, in the step of measuring placental leucine aminopeptidase activity, the urine of the subject is reacted with L-leucine-p-nitroanilide in the presence of methionine, and the amount of p-nitroanilide produced is measured.

INDUSTRIAL APPLICABILITY According to the present invention, a cancer biomarker contained in urine that can be collected noninvasively can be provided. Since the biomarkers of the present invention are more sensitive than conventional tumor markers and their changes are more pronounced, they can 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 malignant tumors and non-malignant tumors. In addition, by using the biomarker of the present invention, it is possible to provide a method for determining the onset of ovarian cancer that enables early determination of onset and recurrence of ovarian cancer.

FIG. 10 is a diagram showing the results of measuring the urinary P-LAP measurement of ovarian tumor patients (25 cases) and examining the correlation with postoperative pathological diagnosis. FIG. 2 shows the results of measuring urinary p-LPA, serum CA125 and serum CA19-9 in ovarian cancer patients before surgery, after surgery, and at recurrence.

[Biomarker]
The present invention provides cancer biomarkers. The cancer biomarker of the present invention (hereinafter referred to as "the biomarker of the present invention") is a biomarker consisting of urinary placental leucine aminopeptidase (hereinafter referred to as "P-LAP"). Patent Document 1 describes that cancer prognosis can be evaluated by measuring the amount of P-LAP in cancer tissue isolated from ovarian cancer patients. However, since P-LAP is a protein and normal renal function is negative for proteinuria, those skilled in the art have no motivation to use urinary P-LAP 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 there is a marked change depending on the presence or absence of ovarian cancer (see Examples). That is, the use of urinary P-LAP as a biomarker for ovarian cancer is not something that a person skilled in the art can easily do, and the effect that urinary P-LAP is superior to serum P-LAP as a biomarker is a remarkable effect that cannot be easily predicted.

P-LAP is also reported to be expressed in cancer tissues of the following cancers. Therefore, urinary P-LAP is considered 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)
・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.)
・Lerman B et al. Oxytocin and cancer: An emaerging link. World Journal of Clinical Oncology. 2018 Sep 14; 9(5):74-82. ):747-53.)
・Lerman B et al. Oxytocin and cancer: An emaerging link. World Journal of Clinical Oncology. 2018 Sep 14; 9(5):74-82. ):747-53.)
Choriocarcinoma (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, enzymatic methods such as the L-leucine-p-nitroanilide method can be preferably used. In the L-leucine-p-nitroanilide method, 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 associated with the production of p-nitroanilide. When measuring urinary P-LAP by the L-leucine-p-nitroanilide method, a commercially available leucine aminopeptidase assay kit can be used.

When measuring urinary P-LAP by an enzymatic method, the substrate and sample may be reacted in the presence of methionine. This is because the presence of methionine can inhibit the activity of LAP other than P-LAP (eg, cytoplasm-derived LPA, microsome-derived LAP, 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 suitably used as a method for measuring urinary P-LAP. In the immunoassay method, the amount of urinary P-LAP can be measured by contacting a sample with an anti-P-LAP antibody and detecting a complex of P-LAP and anti-P-LAP antibody in the sample.

[Method for Determining Ovarian Cancer Onset]
The present invention provides a method for determining the onset of ovarian cancer or a method for assisting in determining the onset of ovarian cancer. The methods of the invention can assist physicians in diagnosing ovarian cancer. Ovarian tumors are classified into benign tumors, borderline malignant tumors (with properties intermediate between benign and malignant), and malignant tumors (ovarian cancer), but it is very difficult to distinguish between malignant tumors (ovarian cancer) and non-malignant tumors without performing histopathological examination. 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 extremely useful in that it not only enables early determination of the onset of ovarian cancer, but also early determination of recurrence after ovarian cancer resection surgery.

According to the "Ovarian Tumor Treatment Code" (2016), ovarian tumors are classified as I.M. epithelial tumors, II. sex cord stromal tumors, III. germ cell tumors IV. It is classified into other tumors, each of which includes benign, borderline and malignant tumors. Benign epithelial tumors include serous cystadenoma, mucinous cystadenoma, endometrioid adenoma, clear cell adenoma, adenofibroma, superficial papilloma, Brenner tumor, endometriotic cyst, and the like. Borderline epithelial tumors include serous borderline malignancy, mucinous borderline malignancy, endometrioid borderline malignancy, clear cell borderline malignancy, and borderline malignant Brenner tumor. Examples of malignant epithelial tumors include low grade serous carcinoma, high grade serous carcinoma, mucinous carcinoma, endometrioid carcinoma, clear cell carcinoma, malignant Brenner's tumor, serous mucinous carcinoma, undifferentiated carcinoma, and serous borderline malignant tumor with micropapillary pattern. Benign sex cord-stromal tumors include capsiocytoma, Sertoli-stromal tumor (well-differentiated), sclerosing stromal tumor, fibroma, Leydig's cell tumor, sex cord tumor with circular tubules, and the like. Borderline malignant sex cord-stromal tumors include juvenile granulosacytoma and Sertoli-stromal cell tumor (moderately differentiated). Malignant tumors of sex cord-stromal tumors include fibrosarcoma, Sertoli stromal cell tumor (poorly differentiated), malignant steroid cell tumor, and adult granulosa cell tumor. Benign germ cell tumors include mature teratomas, benign ovarian thyroid tumors, and sebaceous adenomas. Examples of malignant germ cell tumors include mature teratoma accompanied by malignant transformation, yolk sac tumor, polyembryoma, embryonal cancer, dysgerminoma, choriocarcinoma, malignant ovarian thyroid tumor, immature teratoma (G1-G3), and carcinoid tumor. Other benign tumors include ovarian reticadenomas. Other borderline malignant tumors include gonadal blastoma (pure type). Other malignant tumors include small cell carcinoma, malignant lymphoma, secondary (metastatic) tumors, and the like.

The method of the present invention may include the step of measuring the amount of P-LAP or P-LAP activity in the urine of a subject. Subjects are not limited as long as they are mammals that can develop ovarian cancer, and include humans, monkeys, chimpanzees, dogs, cats, cows, horses, pigs, rabbits, mice, rats, and the like. Humans are preferred. 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 that is difficult to determine whether it is malignant or non-malignant in diagnostic imaging.

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

Determination of whether or not a subject has developed ovarian cancer can be performed, for example, by setting a cutoff value based on the P-LAP value of the malignant tumor group and the P-LAP value of the non-malignant tumor group for the P-LAP value measured by the L-leucine-p-nitroanilide method, and determining whether or not the cutoff value is exceeded. The cut-off value may be set in the range of 10-30 mU/mL, may be set in the range of 10-20 mU/mL, or may be set in the range of 10-15 mU/mL.

Expression of P-LAP is also observed in various cancer tissues other than ovarian cancer. Therefore, the method of the present invention is applicable to cancers other than ovarian cancer. Specifically, for example, endometrial cancer, renal cancer, prostate cancer, gastric cancer, pancreatic cancer, esophageal cancer, choriocarcinoma, breast cancer, and the like.

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these.

[Example 1: Measurement of urinary P-LAP in ovarian tumor patients]
1-1 Materials and Methods (1) Samples With the consent of 25 patients with ovarian tumors whose diagnosis of benign or malignant was difficult by diagnostic imaging, urine was collected 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. Absorbance at 405 nm was measured using a spectrophotometer.
(3) Pathological Diagnosis The 25 ovarian tumor patients described above underwent surgery thereafter, and the ovarian tumor was excised and diagnosed as benign or malignant by histopathological examination.

(4) Results The results are shown in FIG. 1 and Table 1. Ten of the 25 cases were malignant and 15 were benign to borderline malignant (non-malignant). Urinary P-LAP in malignant tumor patients (mean value: 43.4 mU/mL) was significantly higher than urinary P-LAP in non-malignant tumor patients (mean value: 6.8 mU/mL). Setting the cut-off value at 15 mU/mL gave a sensitivity of 90% and a specificity of 100%. The 15 cases of benign to borderline malignancy included 7 chocolate cysts, 2 mature teratomas, 2 mucinous cystadenomas, 1 fibroma, 2 mucinous borderline malignant tumors, and 1 serous borderline malignant tumor. The 10 malignant tumors consisted of 4 clear cell carcinomas, 2 endometrial carcinomas, 1 mucinous carcinoma, 2 serous carcinomas, and 1 undifferentiated carcinoma.

(5) Serum P-LAP Measurement Serum P-LAP was measured for 5 of the above 25 cases (2 benign, 3 malignant). The measuring method is the same as in (2) above. Table 2 shows the results. No difference was observed in serum P-LAP values between patients with malignant tumors and patients with benign tumors.

[Example 2: Transition of urinary P-LPA in ovarian cancer patients before surgery, after surgery, and at recurrence]
Urinary p-LPA was measured in ovarian cancer patients before surgery, after surgery, and at recurrence. At the same time, tumor markers serum CA125 and serum CA19-9 were measured. This patient underwent surgery on May 9, 2018, had postoperative measurements on May 22, 2018, and was discharged from the hospital on the following day, 23rd. Later, on May 31, 2011, a recurrence was confirmed.

The results are shown in FIG. Urinary P-LPA was 163 mU/mL before surgery, 8 mU/mL after surgery, and 152 mU/mL at recurrence. The concentrations of CA125 and CA19-9, which are conventional tumor markers, also decreased after surgery and increased at the time of recurrence. In other words, it was shown that urinary P-LPA can more sensitively reflect the presence of cancer cells than conventional tumor markers. These results demonstrate 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 are possible within the scope of the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments are also included in the technical scope of the present invention. In addition, all scientific and patent documents mentioned in this specification are incorporated herein by reference.

Claims (5)

A biomarker for ovarian cancer consisting of placental leucine aminopeptidase in urine. A method for determining the onset of ovarian cancer, comprising the step of measuring placental leucine aminopeptidase amount or placental leucine aminopeptidase activity in the urine of a subject. 3. The method of claim 2 , wherein the subject is an individual with an ovarian tumor that is difficult to determine whether it is malignant or non-malignant in diagnostic imaging. 3. The method of claim 2 , wherein the onset of ovarian cancer is recurrence after resection surgery. 5. The method according to any one of claims 2 to 4, wherein the step of measuring placental leucine aminopeptidase activity includes reacting the subject's urine with L-leucine-p-nitroanilide in the presence of methionine and measuring the amount of p-nitroanilide produced.