JP-7855199-B2 - Methods for diagnosing bladder cancer

JP7855199B2JP 7855199 B2JP7855199 B2JP 7855199B2JP-7855199-B2

Inventors

康東天
内海健
伊神恒

Assignees

株式会社ＬＳＩメディエンス
国立大学法人九州大学

Dates

Publication Date: 20260508
Application Date: 20210806
Priority Date: 20200806

Claims (7)

A method to assist in determining whether a patient has bladder cancer by concentrating microvesicles with a diameter of 200 nm to 1 μm contained in the urine of a target patient, and determining whether the target patient has bladder cancer based on the amount of marker protein present in the microvesicles, The method is characterized in that the observation of the amount of marker protein in the microvesicle is performed by detecting the amount of at least one of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicle.
The method according to claim 1, characterized in that the amount of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicles is observed by calculating the ratio (A/B) of microvesicles (A) expressing CD66a, CD66b, CD66c, CGM2, and/or CD66e to microvesicles (B) expressing CD10, CD13, and/or CD26.
The method according to claim 1, characterized in that the amount of CD66a, CD66b, CD66c, CGM2, or CD66e in the microvesicles is observed by calculating the ratio (A/C) of microvesicles (A) expressing CD66a, CD66b, CD66c, CGM2, and/or CD66e to microvesicles (C) expressing CD10, CD13, CD26, and/or MUC1.
The method according to any one of claims 1 to 3, wherein the amount or ratio of marker protein in urinary microvesicles derived from the target patient is higher than the amount or ratio of marker protein in urinary microvesicles derived from the control, indicating that the target patient has bladder cancer.
A method for assisting the determination of whether or not a person has bladder cancer, comprising the steps of: measuring the amount of CD66a/b/c/e or CGM2 present in microvesicles with a diameter of 200 nm to 1 μm contained in urine; and determining that the urine originates from a bladder cancer patient if the measured amount of CD66a/b/c/e or CGM2 is greater than that of a control.
A method according to any one of claims 1 to 5, for assisting in determining whether a patient has bladder cancer by comparing the amount of protein present in a first sample taken from the patient with the amount of protein present in a second sample taken from the patient after the treatment period, for observing the course of treatment for bladder cancer, or for determining the effectiveness of treatment for bladder cancer.
The method according to any one of claims 1 to 6, further comprising the step of correlating the findings as indicating superficial bladder cancer, invasive stage 1 bladder cancer, or invasive stage 2-3 bladder cancer.

Description

This invention relates to the detection of cancer cell-specific extracellular vesicles (medium/large extracellular vesicles) present in the urine of bladder cancer patients and to a method for diagnosing bladder cancer using these vesicles. Cancer, recognized as a leading cause of death in Japan since the 1980s, is also among the leading causes of death worldwide. Despite advances in medical diagnostic and treatment techniques, it is still difficult to say that diagnostic or treatment methods are fully established. Among these, bladder cancer is particularly prevalent, with an estimated hundreds of thousands of cases diagnosed worldwide each year, and 20,000 new cases diagnosed in Japan. Approximately one-third of these cases are thought to be invasive or metastatic at the time of diagnosis, and it has the highest incidence and mortality rates among various urinary tract cancers (renal pelvis, ureter, bladder) (Non-patent Literature 1). Bladder cancer is caused by the malignant transformation of bladder tissue, including urothelium, and histologically, urothelial carcinoma accounts for 90% of all cases. Of these cases, 70% are superficial and non-invasive. Approximately 75% of patients diagnosed with transitional cell carcinoma present with superficial tumors that can be treated with transurethral resection. The recurrence rate of superficial disease exceeds 60%, and less than 30% of recurrent bladder tumors progress to invasive disease (Non-Patent Literature 2). Therefore, in addition to the importance of early detection of patients with or suspected of having bladder cancer, lifelong surveillance is considered crucial for treatment success. The vast majority of patients with bladder cancer seek medical attention after experiencing macroscopic or microscopic hematuria, or other concerning urinary symptoms such as frequent urination and dysuria. However, there is no accurate and easy way to identify the presence of early-stage bladder cancer, and a definitive diagnosis of bladder cancer requires a combination of procedures. As an initial screening method, urine cytology, which involves microscopic examination of cells derived from a urine sample, may be used. Urine cytology allows for the examination of detached cells for the presence of specific cell surface antigens, nuclear morphology, gene expression, or other biological markers. While useful for cases of high-grade malignant tumors, it has limitations in its sensitivity to detect other low-grade malignant tumors (Non-Patent Literature 3). Furthermore, the accuracy of urine cytology is problematic because the degree to which bladder cancer cells are released into the urine varies greatly depending on the timing of urine collection and the stage of the disease. In addition, subjectivity cannot be ruled out in the interpretation of the results, and the results can fluctuate. Therefore, cytology is not ideal for screening and surveillance of bladder cancer. The most reliable test performed on patients exhibiting the above symptoms and suspected of having bladder cancer is a cystoscopy under local or general anesthesia, followed by a solid tissue biopsy if necessary, to confirm the diagnosis. However, these symptoms are often caused by conditions such as urinary tract infections or benign prostatic hyperplasia, and such invasive tests that place a significant burden on patients must be performed even on those without bladder cancer. Therefore, there is a need for less invasive screening tests using highly accurate biomarkers. Commonly known methods for measuring tumor markers for urological cancers include NMP22 (Nuclear Matrix Protein 22), BTA (Bladder Tumor Antigen), and cytokeratin 8 and 18. However, while these methods have relatively higher sensitivity than cytology, they have been reported to produce a high rate of false positives (Non-Patent Literature 4, Non-Patent Literature 5, Non-Patent Literature 6). Therefore, methods for detecting bladder cancer patients using existing biomarkers are not sufficient as diagnostic markers for bladder cancer, and in particular, they do not provide guidance for the early diagnosis of bladder cancer. Consequently, a bladder cancer diagnostic marker that enables early diagnosis and has high sensitivity and specificity has yet to be found. In recent years, it has been proposed to use micromembrane fractions separated from cells as liquid biopsies, a non-invasive diagnostic material, to determine disease status and the presence or absence of disease (Patent Documents 1 and 2). Such micromembrane fractions are broadly classified into exosomes, microvesicles, etc., depending on their size, and their use is attracting attention. Among these, microvesicle extracellular vesicles are known to play an extremely important role in tumor invasion (Non-Patent Document 7), and extracellular vesicles present in urine have been reported to be involved in diseases such as diabetic nephropathy (Non-Patent Document 5), suggesting thei