Examination method for determining ovarian cancer platinum preparation resistance by RNA modification in extracellular vesicle

Abstract

Provided is a biomarker useful for diagnosis of platinum preparation resistance. Also provided is an examination method for determining platinum preparation resistance using the biomarker.　Attention is paid to an EV released from an excised tissue of a cancer site as an EV derived from only a lesion site reflecting the in vivo environment, and it was found that the EV released from the ovarian cancer tissue has a characteristic RNA modification different from that of the EV released from the normal ovarian tissue.

Description

Testing method for determining platinum drug resistance in ovarian cancer by RNA modification in extracellular vesicles

[0001] The present invention relates to a testing method that assists in identifying platinum resistance. This application claims priority to Japanese Patent Application No. 2023-147093, which is incorporated herein by reference.

[0002] Ovarian cancer is a malignant tumor that develops in the ovaries and has the highest mortality rate among female genital cancers. Although the initial treatment response rate for ovarian cancer is high, more than half of cases will recur.

[0003] The primary treatment for ovarian cancer is usually chemotherapy using anticancer drugs including platinum agents. Platinum agents have severe side effects that place a great physical burden on the patient, and platinum agent resistance, which is a resistance to platinum agents, has become a problem.

[0004] In cases where recurrence is resistant to platinum agents (platinum-resistant recurrence), platinum agents are ineffective, and treatment without platinum agents is selected. On the other hand, in cases where recurrence is sensitive to platinum agents (platinum-sensitive recurrence), maintenance therapy is switched to a platinum agent or a combination with bevacizumab or olaparib. Chemotherapy sensitivity information is important for postoperative adjuvant chemotherapy and treatment selection for recurrent cancer, but it is not possible to obtain this information before treatment.

[0005] Patent Literature 1 discloses genes specifically expressed in cisplatin-resistant ovarian cancer cell lines as cisplatin resistance markers for ovarian tumors. Patent Literature 2 discloses methods for predicting and overcoming resistance to chemotherapy in ovarian cancer and for predicting the occurrence of colon cancer. However, these are gene expression information characteristic of anticancer drug-resistant tumor tissues obtained by analysis using established ovarian cancer cell lines.

[0006] Studies using cultured cell lines and mouse models have revealed that extracellular vesicles (EVs) released by cancer cells affect the tumor microenvironment and metastasis, and the application of mRNA, miRNA, proteins, and other molecules contained in body fluid-derived EVs as cancer biomarkers has attracted attention. Non-Patent Document 1 reported that EV proteins are highly expressed in tumor-derived EVs through proteomic analysis of EVs isolated from human renal cell carcinoma tissue. However, biomarkers for platinum drug resistance based on RNA modifications characteristically contained in EVs released directly from ovarian cancer tissue are unknown.

[0007] International Publication No. 2007 / 126076 Japanese Patent No. 5285012

[0008] Int J Cancer, 2018, 142(3), 607-617

[0009] Previous research into extracellular vesicles (EVs) in cancer has focused on functional analysis of EVs derived from the cell culture supernatant of cancer cell lines, or biomarker discovery using EVs derived from body fluids such as blood and urine. Monolayer culture of cancer cell lines misses out the influence of various in vivo cell interactions and humoral factors, and does not take into account the influence of the in vivo environment. EVs derived from body fluids contain a mixture of EVs released by the various cells that make up the body, making it difficult to obtain EVs derived exclusively from the lesion site. Furthermore, comparisons with control normal cells have not been conducted sufficiently.

[0010] The present invention relates to providing a biomarker useful for diagnosing platinum-resistant disease, and further aims to provide a testing method that uses the biomarker to assist in determining platinum-resistant disease.

[0011] As a result of extensive research to solve the above problems, the inventors focused on EVs released from resected cancer tissue as EVs derived exclusively from diseased sites, reflecting the in vivo environment. They discovered that EVs released from ovarian cancer tissue have RNA modification characteristics that differ from those of EVs released from normal ovarian tissue, leading to the completion of the present invention.

[0012] That is, the present invention comprises the following: 1. A testing method for assisting in the identification of platinum drug resistance, characterized by detecting modified nucleosides specific to platinum drug resistance in RNA contained in extracellular vesicles in a sample collected from a subject. 2. The method according to the preceding item 1, comprising a step of comparing the abundance ratio of modified nucleosides specific to platinum drug resistance relative to unmodified nucleosides with a cutoff value. 3. The method according to the preceding item 1 or 2, wherein the modified nucleosides specific to platinum drug resistance are one or more selected from the group consisting of 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm) and 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um), and dihydrouridine (D). 4. 4. The method according to any one of the preceding items 1 to 3, wherein the sample is a tissue-derived sample from an ovarian cancer patient. 5. The method according to any one of the preceding items 1 to 4, wherein the cutoff value is a value set based on an ROC curve based on the abundance ratio of modified nucleosides specific to platinum drug resistance to unmodified nucleosides. 6. The method according to any one of the preceding items 1 to 5, wherein the modified nucleosides specific to platinum drug resistance are a combination of 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um), and dihydrouridine (D).

[0013] The present invention provides a testing method that helps determine platinum resistance using RNA modifications encapsulated in extracellular vesicles as an indicator. Information on the presence or absence of platinum resistance in ovarian cancer patients significantly influences treatment strategies, leading to the determination of an appropriate postoperative chemotherapy strategy.

[0014] Schematic diagram of modified nucleoside analysis of extracellular vesicles (EVs) derived from ovarian cancer postoperative cancer tissue (Example 1). The ratio of 5-carboxyhydroxymethyluridine (chm5U) to uridine (U) (chm5U / U), the ratio of 2'-O-methylcytidine (Cm) to cytidine (C) (Cm / C), and the ratio of 1-methylinosine (m1I) to inosine (I) (m1I / I) contained in normal ovarian tissue EVs and ovarian cancer tissue EVs are shown. (Example 1) The abundance ratio of 5-carboxyhydroxymethyluridine (chm5U) to uridine (U) (chm5U / U), the abundance ratio of 2'-O-methylcytidine (Cm) to cytidine (C) (Cm / C), and the abundance ratio of 1-methylinosine (m1I) to inosine (I) (m1I / I) contained in ovarian cancer tissue EVs from patients diagnosed with platinum-sensitive and platinum-resistant ovarian cancer tissue EVs are shown. (Example 1) ROC curves based on the abundance information of each modified nucleoside (chm5U, Cm, m1I) contained in ovarian cancer tissue EVs are shown. (Example 1) The results of comparing the rates of platinum-sensitive and platinum-resistant ovarian cancers in high and low chm5U, Cm, and m1I inclusion groups are shown. (Example 1) The ROC curve obtained by multiple logistic regression analysis using the measured values ​​of three modified nucleosides (chm5U, Cm, m1I) contained in ovarian cancer tissue EVs is shown.(Example 1) The abundance ratios of each modified nucleoside (5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um), dihydrouridine (D)) to unmodified nucleosides (uridine (U), cytidine (C), inosine (I), adenosine (A)) contained in ovarian cancer tissue EVs from patients diagnosed with platinum-sensitive ovarian cancer and those diagnosed with platinum-resistant ovarian cancer are shown. These ratios are chm5U / U, Cm / C, m1I / I, m5U / U, m6A / A, m6,6A / A, m5Um / U, and D / U. (Example 2) The ROC curve (left) for each modified nucleoside (chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, D) contained in ovarian cancer tissue EVs is shown, along with the ROC curve (right) obtained by multiple logistic regression analysis using the total modified nucleoside measurements. (Example 2) The results of comparing the rates of platinum sensitivity and platinum resistance in the high and low chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, and D inclusion groups are shown. (Example 2)

[0015] The present invention relates to a testing method that assists in determining platinum drug resistance, characterized by detecting modified nucleosides specific to platinum drug resistance in RNA contained in extracellular vesicles in a sample collected from a subject (hereinafter, sometimes referred to as the "testing method of the present invention").

[0016] As used herein, "platinum-resistant" refers to a recurrent case of ovarian cancer that exhibits resistance to platinum agents in the treatment of ovarian cancer, specifically, a case in which recurrence occurs less than six months after the completion of chemotherapy including a platinum agent is deemed to be platinum-resistant. As used herein, "platinum-sensitive" refers to a recurrent case of ovarian cancer that does not exhibit resistance to platinum agents in the treatment of ovarian cancer, specifically, a case in which recurrence occurs six months or more after the completion of chemotherapy including a platinum agent is deemed to be platinum-sensitive.

[0017] RNA is an organic polymer in which nucleosides are polymerized via phosphodiester bonds. As used herein, "RNA modification" refers to chemical modification of RNA. As used herein, "modified nucleoside" refers to a nucleoside containing one or more modifications. As used herein, "nucleoside" refers to any compound in which a base and a sugar are bonded, but is preferably a ribonucleoside, such as uridine, cytidine, inosine, adenosine, and guanosine. RNA modifications can be characterized by the ratio of modified nucleosides to unmodified nucleosides in the nucleosides constituting RNA. As used herein, "platinum-resistant-specific modified nucleosides" refers to modified nucleosides whose ratio to unmodified nucleosides in the nucleosides constituting RNA contained in extracellular vesicles differs between platinum-sensitive and platinum-resistant ovarian cancer patients. Examples of platinum-resistant specific modified nucleosides include 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), N2,N2-dimethylguanosine (m2,2G), N4-acetylcytidine (ac4C), N6-threonylcarbamoyladenosine (t6A), 7-methylguanosine (m7G), 5-methylcytidine (m5C), 5-methyluridine (m5U), 3-methylcytidine (m3C), N6-methyladenosine (m6A), N2-methylguanosine (m2G), 1-methylguanosine (m1G), N6-isopentenyladenosine (i6A), N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), and N6,N6-dimethyladenosine (m6,6A), 5-methoxycarbonylmethyluridine (mcm5U), 2-thiouridine (S2U), 5-methylaminomethyl-2-thiouridine (mnm5S2U), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-formylcytidine (f5C), 5-carboxycytosine (ca5C), 5-Hydroxymethylcytosine (5hmC), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 2'-O-methyladenosine (Am), 2'-O-methylguanosine (Gm), 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), N6,2'-O-dimethyladenosine (m6Am), 4-thiouridine (S4U), 5-carbamoylmethyluridine (ncm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5S2U), 5-carbamoylmethyl-2-thiouridine (ncm5S2U), dihydrouridine (D), 2'-O-methylinosine (Im), 5-methyldihydrouridine (m5D), 5-carbamoylhydroxymethyluridine (nchm5U), 5-(carboxyhydroxymethyl)uridinemethyl ester ((R)-mchm5U), 5-(carboxyhydroxymethyl)uridinemethyl ester ((S)-mchm5U), 1-methyladenosine (m1A), pseudouridine (φ), etc. are mentioned, preferably 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,The uridine may be one or more selected from the group consisting of 2'-O-dimethyluridine (m5Um) and dihydrouridine (D). The term "multiple types" is not particularly limited as long as it is two or more types, and two or more types can be freely combined. However, from the viewpoint of further improving diagnostic accuracy, three or more types are preferred, four or more types are more preferred, five or more types are more preferred, six or more types are more preferred, seven or more types are more preferred, and a combination of eight types (chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, and D) is even more preferred.

[0018] As used herein, the term "platinum preparation" refers to an anticancer drug that contains a platinum atom in its structure and exerts its anticancer effect by inhibiting DNA replication and / or transcription through binding of the platinum atom to the DNA of cancer cells. Examples of platinum preparations include, but are not limited to, cisplatin, carboplatin, and oxaliplatin.

[0019] The timing of performing the testing method of the present invention is not particularly limited, and may be, for example, after the administration of a platinum preparation in primary therapy has been completed. However, from the viewpoint of reducing the physical burden on the patient, it is preferable to perform the testing method before the administration of a platinum preparation in primary therapy has been completed, and more preferably before the administration of a platinum preparation in primary therapy has been initiated.

[0020] The sample used in the testing method of the present invention is not particularly limited as long as it is a biological sample containing extracellular vesicles (EVs). For example, the sample may be prepared from tissue (e.g., ovarian tissue, fallopian tube tissue), blood, urine, serum, plasma, saliva, or other specimens collected from a living body (e.g., an ovarian cancer patient or a person suspected of having ovarian cancer). Examples of tissue collected from a living body include tissue resections, including tumor tissue, obtained by surgery or endoscopic examination.

[0021] As used herein, "extracellular vesicles (EVs)" refer to tiny vesicles with a membrane structure that are secreted from various types of cells, and examples include exosomes, ectosomes, microvesicles, microvesicles, and apoptotic bodies.

[0022] The biological species that can be the subject (subject) of the testing method of the present invention is not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being the most preferred.

[0023] The testing method of the present invention is characterized by detecting modified nucleosides specific to platinum drug resistance in RNA contained in extracellular vesicles in a sample collected from a subject.

[0024] In the present invention, the recovery of extracellular vesicles (EVs) from a sample and the recovery of RNA from EVs can be performed using known or future methods. For example, when a tissue resection is used as a sample, EVs can be recovered using the method described in Jingushi et al., Int J Cancer. 2018. Briefly, the resected tissue can be cultured in tissue culture medium, and then EVs can be recovered from the tissue by centrifugation. RNA can be recovered from EVs by, for example, extracting and purifying RNA using a commercially available RNA extraction kit.

[0025] In the present invention, modified nucleosides in RNA can be detected by any known method or any method to be developed in the future. For example, modified nucleosides can be detected by analyzing the nucleosides obtained by hydrolyzing RNA using mass spectrometry such as LC-MS / MS, GC / MS, or ESI-MS. Hydrolysis of RNA to nucleosides can be performed, for example, by alkaline phosphatase treatment. Modified nucleosides can also be detected by using antibodies against specific modified nucleosides. Detection by mass spectrometry is advantageous over antibody-based detection in that it can simultaneously quantify many types of modified and unmodified nucleosides and can also detect modified nucleosides for which no antibodies exist.

[0026] The testing method of the present invention can include a step of comparing the abundance ratio of modified nucleosides specific to platinum agent resistance to unmodified nucleosides with a cutoff value.

[0027] The cutoff value is a standard value for distinguishing between platinum-sensitive and platinum-resistant ovarian cancer. The cutoff value can be set based on the abundance ratio of platinum-resistant-specific modified nucleosides to unmodified nucleosides contained in extracellular vesicles (EVs) in tissue-derived samples from ovarian cancer patients determined to be platinum-sensitive and / or platinum-resistant. The abundance ratio of platinum-resistant-specific modified nucleosides to unmodified nucleosides can be the ratio of the detected amount (measured amount) of platinum-resistant-specific modified nucleosides to the detected amount (measured amount) of unmodified nucleosides. The cutoff value can be set, for example, by calculating the average or median abundance ratio of platinum-resistant-specific modified nucleosides to unmodified nucleosides contained in EVs in tissue-derived samples from ovarian cancer patients determined to be platinum-sensitive or platinum-resistant. Another method for setting the cutoff value is to use commercially available statistical analysis software to determine the ratio of platinum-resistant modified nucleosides to unmodified nucleosides in EVs from tissue samples of ovarian cancer patients determined to be platinum-sensitive and platinum-resistant. To create an ROC curve, the area under the curve (AUC) is calculated. The AUC ranges from 0 to 1. An AUC of 0.5 indicates chance and no diagnostic ability. An AUC greater than 0.5 indicates diagnostic ability. An AUC of 0.5–0.7 is generally classified as low accuracy, an AUC of 0.7–0.9 as moderate accuracy, and an AUC of 0.9–1.0 as high accuracy. An AUC of 1 indicates 100% sensitivity and 100% specificity.

[0028] The testing method of the present invention may further include a step of determining that the patient is platinum-resistant or platinum-resistant when the abundance ratio of modified nucleosides specific to platinum resistance to unmodified nucleosides is equal to or greater than (or above) or less than (or below) a cutoff value.

[0029] For example, when detecting one or more modified nucleosides specific to platinum resistance selected from the group consisting of 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um) and dihydrouridine (D), if the value set based on the ROC curve in the Examples described below is used as the cutoff value, if the abundance ratio of chm5U to uridine (U) contained in the EV in the test sample (chm5U / U) is less than the cutoff value, it can be determined that the sample is resistant to platinum agents, and if chm5U / U is equal to or greater than the cutoff value, it can be determined that the sample is sensitive to platinum agents. If the ratio of Cm to cytidine (C) in EVs in the test sample (Cm / C) is equal to or greater than the cutoff value, the patient is determined to be platinum-resistant; if Cm / C is less than the cutoff value, the patient is determined to be platinum-sensitive. If the ratio of m1I to inosine (I) in EVs in the test sample (m1I / I) is equal to or greater than the cutoff value, the patient is determined to be platinum-resistant; if m1I / I is less than the cutoff value, the patient is determined to be platinum-sensitive. If the ratio of m5U to U in EVs in the test sample (m5U / U) is equal to or greater than the cutoff value, the patient is determined to be platinum-resistant; if m5U / U is less than the cutoff value, the patient is determined to be platinum-sensitive. If the ratio of m6A to adenosine (A) in EVs in the test sample (m6A / A) is equal to or greater than the cutoff value, the patient is determined to be platinum-resistant; if m6A / A is less than the cutoff value, the patient is determined to be platinum-sensitive. If the ratio of m6,6A to A contained in the EV in the test sample (m6,6A / A) is equal to or greater than the cutoff value, the sample is judged to be resistant to platinum agents, and if m6,6A / A is less than the cutoff value, the sample is judged to be sensitive to platinum agents.If the abundance ratio of m5Um to U contained in EVs in a test sample (m5Um / U) is equal to or greater than a cutoff value, the sample is determined to be platinum-resistant, whereas if m5Um / U is less than the cutoff value, the sample is determined to be platinum-sensitive. If the abundance ratio of D to U contained in EVs in a test sample (D / U) is equal to or greater than a cutoff value, the sample is determined to be platinum-resistant, whereas if D / U is less than the cutoff value, the sample is determined to be platinum-sensitive. Platinum resistance can also be determined when a set of multiple types of modified nucleosides specific to platinum resistance contains characteristics of platinum resistance. In one embodiment, when measuring the abundance ratios of two or more of chm5U / U, Cm / C, m1I / I, m5U / U, m6A / A, m6,6A / A, m5Um / U, and D / U, platinum resistance can be determined when at least one of them indicates platinum resistance by comparison with the cutoff value. In one aspect, when measuring the abundance ratio of two or more of chm5U / U, Cm / C, m1I / I, m5U / U, m6A / A, m6,6A / A, m5Um / U and D / U, platinum resistance can be determined when an abundance ratio of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more or 100% indicates platinum resistance by comparison with the cutoff value. As cutoff values ​​set based on the ROC curve, for example, the cutoff value of chm5U / U is 1.600 to 3.000, the cutoff value of Cm / C is 0.007 to 0.013, the cutoff value of m1I / I is 0.490 to 0.920, the cutoff value of m5U / U is 0.005 to 0.011, the cutoff value of m6A / A is 0.0007 to 0.0013, the cutoff value of m6,6A / A is 0.0014 to 0.0026, the cutoff value of m5Um / U is 0.0021 to 0.0039, the cutoff value of D / U can be set as a value selected from the range of 0.470 to 0.880, but is not particularly limited.

[0030] Furthermore, for example, if the ratio of modified nucleosides specific to platinum drug resistance to unmodified nucleosides contained in extracellular vesicles (EVs) in a tissue-derived sample from an ovarian cancer patient determined to be platinum drug sensitive is used as a cutoff value, when the ratio of chm5U to uridine (U) contained in EVs in the test sample (chm5U / U) is less than the cutoff value, the patient is determined to be platinum drug resistant, and when chm5U / U is equal to or greater than the cutoff value, the patient is determined to be platinum drug sensitive. If the ratio of Cm to e(C) (Cm / C) exceeds the cutoff value, the patient is determined to be platinum-resistant; if Cm / C is equal to or less than the cutoff value, the patient is determined to be platinum-sensitive; if the ratio of m1I to inosine (I) contained in EVs in the test sample (m1I / I) exceeds the cutoff value, the patient is determined to be platinum-resistant; if m1I / I is equal to or less than the cutoff value, the patient is determined to be platinum-sensitive; and if the ratio of m5U to U contained in EVs in the test sample (m5U / U) exceeds the cutoff value, the patient is determined to be platinum-sensitive. If m5U / U is greater than the cutoff value, the patient is determined to be platinum-resistant; if m5U / U is equal to or less than the cutoff value, the patient is determined to be platinum-sensitive; if the ratio of m6A to A contained in EVs in the test sample (m6A / A) is greater than the cutoff value, the patient is determined to be platinum-resistant; if m6A / A is equal to or less than the cutoff value, the patient is determined to be platinum-sensitive; if the ratio of m6,6A to A contained in EVs in the test sample (m6,6A / A) is greater than the cutoff value, the patient is determined to be platinum-resistant; If the ratio of m5Um to U contained in EVs in the test sample (m5Um / U) is greater than the cutoff value, the test sample is determined to be platinum-resistant; if m5Um / U is less than the cutoff value, the test sample is determined to be platinum-resistant; if the ratio of D to U contained in EVs in the test sample (D / U) is greater than the cutoff value, the test sample is determined to be platinum-resistant; and / or if D / U is less than the cutoff value, the test sample is determined to be platinum-sensitive.

[0031] Furthermore, for example, if the ratio of modified nucleosides specific to platinum drug resistance to unmodified nucleosides contained in extracellular vesicles (EVs) in a tissue-derived sample from an ovarian cancer patient determined to be platinum drug resistant is used as a cutoff value, when the ratio of chm5U to uridine (U) contained in EVs in the test sample (chm5U / U) exceeds the cutoff value, the patient is determined to be platinum drug sensitive, and when chm5U / U is equal to or less than the cutoff value, the patient is determined to be platinum drug resistant. If the ratio of Cm to inosine (C) (Cm / C) is less than the cutoff value, the patient is judged to be platinum-sensitive; if Cm / C is equal to or greater than the cutoff value, the patient is judged to be platinum-resistant; if the ratio of m1I to inosine (I) contained in EVs in the test sample (m1I / I) is less than the cutoff value, the patient is judged to be platinum-sensitive; if m1I / I is equal to or greater than the cutoff value, the patient is judged to be platinum-resistant; and if the ratio of m5U to U contained in EVs in the test sample (m5U / U) is the cutoff value, the patient is judged to be platinum-resistant. If m5U / U is less than the cutoff value, the patient is judged to be susceptible to platinum agents; if m5U / U is equal to or greater than the cutoff value, the patient is judged to be resistant to platinum agents; if the ratio of m6A to A contained in EVs in the test sample (m6A / A) is less than the cutoff value, the patient is judged to be susceptible to platinum agents; if m6A / A is equal to or greater than the cutoff value, the patient is judged to be resistant to platinum agents; if the ratio of m6,6A to A contained in EVs in the test sample (m6,6A / A) is less than the cutoff value, the patient is judged to be susceptible to platinum agents; If the ratio of m5Um to U contained in EVs in the test sample (m5Um / U) is less than the cutoff value, the patient is determined to be platinum-resistant; if m5Um / U is greater than or equal to the cutoff value, the patient is determined to be platinum-resistant; if the ratio of D to U contained in EVs in the test sample (D / U) is less than the cutoff value, the patient is determined to be platinum-resistant; and / or if D / U is greater than or equal to the cutoff value, the patient is determined to be platinum-resistant.

[0032] The testing method of the present invention allows for early determination of the treatment method (e.g., whether or not to use a platinum drug) and treatment schedule for ovarian cancer patients determined to be platinum-sensitive or platinum-resistant using the testing method of the present invention. For example, for ovarian cancer patients determined to be platinum-resistant using the testing method of the present invention before the start of primary therapy, a treatment other than a platinum drug (e.g., a monotherapy not cross-reactive with a platinum drug) can be selected as the primary treatment. For ovarian cancer patients determined to be platinum-sensitive using the testing method of the present invention before the start of primary therapy, a treatment containing a platinum drug can be selected as the primary treatment. For ovarian cancer patients determined to be platinum-resistant using the testing method of the present invention during or after the completion of primary therapy containing a platinum drug, administration of the platinum drug can be discontinued and / or a treatment other than a platinum drug can be selected as the secondary treatment. For ovarian cancer patients determined to be platinum-sensitive using the testing method of the present invention during or after the completion of primary therapy containing a platinum drug, a treatment containing a platinum drug can be selected as the secondary treatment. Furthermore, the testing method of the present invention can predict the prognosis of ovarian cancer patients determined to be platinum-resistant, with a high probability of cancer recurrence within six months after administration of a platinum drug.

[0033] EXAMPLES In the following, the present invention will be described in more detail with reference to examples in order to deepen understanding of the present invention, but it goes without saying that the present invention is not limited to the descriptions of the examples.

[0034] Example 1 Analysis of RNA Modification in Extracellular Vesicles (EVs) Derived from Cancer Tissues after Ovarian Cancer Surgery In this example, modified nucleosides in extracellular vesicles derived from cancer tissues of ovarian cancer patients were analyzed. A conceptual diagram is shown in Figure 1.

[0035] (Collection of ovarian cancer tissue EVs) This study was approved by the ethics committee of Osaka Medical and Pharmaceutical University, and involved patients who consented. Postoperative ovarian cancer tissues surgically removed before the completion of platinum-based therapy were collected from 35 patients with stage I-IV ovarian cancer (treated with platinum-based drugs) and normal ovarian tissues obtained from 41 patients with early-stage cervical cancer, early-stage endometrial cancer, and early-stage ovarian cancer (with localized cancer and no metastatic lesions in the ovarian tissue or abdominal cavity) who were not receiving platinum-based drugs. The culture medium was then immersed in serum-free DMEM medium (WAKO) and cultured at 37°C for 2 hours. The medium was then centrifuged at 2,000 g for 30 minutes to recover the supernatant. The supernatant was then further centrifuged at 10,000 g for 1 hour to recover ovarian cancer tissue EVs and normal ovarian tissue EVs. Among the ovarian cancer patients receiving the above platinum-based drugs, seven patients who subsequently experienced a recurrence within six months of completing platinum-based therapy were diagnosed as having platinum-resistant disease, while 21 patients who experienced a recurrence more than six months after completing platinum-based therapy were diagnosed as having platinum-sensitive disease.

[0036] (Recovery of RNA from EVs) Each EV solution (0.5 × 10 8 QIAzol (QIAGEN) was added to the particles to make the total volume 700 μL, and the solution was mixed by vortexing for 1 minute. RNA was then extracted and purified according to the protocol of the miRNeasy Mini Kit (QIAGEN).

[0037] (Degradation of RNA nucleotides to nucleosides) dG15N5 was added as an internal standard to 30 μL of RNA solution at 1 / 100 the amount of RNA. A solution of 0.5 U / μL nuclease P1 (Fujifilm Wako Pure Chemical Industries, Ltd.) diluted 5-fold with DNase / RNase-free HO and an equal volume of 0.1 M ammonium acetate (pH 5.3) was mixed. 10 μL of this solution was added to the RNA solution and incubated at 45°C for 2 hours. 0.5 U / μL alkaline phosphatase (BAP) solution (TaKaRa) was then diluted 50-fold with DNase / RNase-free HO, and 5 μL of this solution was added to each solution. The mixture was incubated at 37°C for 2 hours. 60 μL of DNase / RNase-free HO and 20 μL of chloroform (Fujifilm Wako Pure Chemical Industries, Ltd.) were added, vortexed, and centrifuged at 5,000 g for 5 minutes. After centrifugation, the supernatant was transferred to a new 1.5 mL tube and concentrated to dryness. After concentration to dryness, it was redissolved in HPLC-grade ultrapure water (Fujifilm Wako Pure Chemical Industries) to a concentration of 10 ng / μL.

[0038] (Analysis of modified nucleosides using UHPLC-MS / MS) Measurements were carried out under the following conditions.

[0039] (UHPLC-MS / MS Conditions) UHPLC-UniSpray / ESI-MS / MS was performed using an ACQUITY UPLC system (Waters) and a Xevo TQ-XS triple quadrupole mass spectrometer (Waters). The column used was an ACQUITY UPLC CSH C18 (1.7 μm, 2.1 × 100 mm) (Waters). The column temperature was set at 42 °C, and the solvent flow rate was 0.4 ml / min. Gradient analysis was performed using two mobile phases: mobile phase A (5 mM CH3COONH4 in H2O) and mobile phase B (5 mM CH3COONH4 in MeOH). The gradient conditions started with 99% mobile phase A / 1% mobile phase B, then changed the ratio of mobile phase B from 1% to 32% over 6 min, and then from 32% to 99% over 0.5 min. The mobile phase B was then maintained at 99% for 4 minutes to wash away any remaining sample from the column. The mobile phase B was then changed to 1% and the column was equilibrated for 5.5 minutes. The measurement time per sample was 15 minutes. Modified nucleosides were detected using a UniSpray ion source in positive ion mode. The UniSpray conditions were: source temperature 125°C; desolvation temperature 600°C; desolvation gas flow 1000 L / h; and cone gas flow 150 L / h. All nucleosides were detected using multiple reaction monitoring (MRM). The detection conditions for the 42 modified nucleosides and 4 unmodified nucleosides used in the experiment, as well as dG15N5 used as an internal standard, are shown in Table 1.

[0040] The compounds in Table 1 were obtained from the following sources: A, C, G, I (Fujifilm Wako Pure Chemical Industries), S4U (Abcam), hm5C (Berry & Associates), t6A (BIOLOG), m1A, m6A, Im, m1G, ca5C, ac4C, f5C, mcm5U, m5Um, Y (Carbosynth), i6A, m22Gm, ho5U, m3Um, D, m5D (Granlen), m7G, mcm5S2U (Santa Cruz Biotechnology), m2G, U (Sigma-Aldrich), Am, Gm, m5C, Cm, m5U (Tokyo Chemical Industry), m6,6A, m6Am, m1I, m22G, m3C, S2U (Toronto Research Chemicals), dG15N5 (Cambridge Isotope Laboratories). m6t6A, cm5U, ncm5U, nchm5U, mchm5U, mnm5S2U, mcm5Um, ncm5Um, and chm5U were synthesized at the Graduate School of Pharmaceutical Sciences, Osaka University. Acetonitrile (CH3CN), ammonium acetate (CH3COONH4), and methanol (MeOH) for LCMS (Fujifilm Wako Pure Chemical Industries, Ltd.) were used.

[0041] Mann-Whitney tests showed that the median amounts of three RNA modifications, 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), and 1-methylinosine (m1I), varied significantly in EVs from ovarian cancer tissues compared with those from normal ovarian cancer tissues (Fig. 2).Furthermore, the median amounts of these three RNA modifications varied significantly in EVs from ovarian cancer tissues from patients diagnosed with platinum-resistant ovarian cancer compared with those from patients diagnosed with platinum-sensitive ovarian cancer (Fig. 3). More specifically, the median ratio of chm5U to uridine (U) (chm5U / U) in the platinum-resistant group was lower than that in the platinum-sensitive group, the median ratio of Cm to cytidine (C) (Cm / C) in the platinum-resistant group was higher than that in the platinum-sensitive group, and the median ratio of m1I to inosine (I) (m1I / IU) in the platinum-resistant group was higher than that in the platinum-sensitive group.

[0042] Furthermore, receiver operating characteristic (ROC) curves were constructed. The AUCs for chm5U, Cm, and m1I in ovarian cancer tissue EVs were 0.79, 0.73, and 0.76, respectively. These curves were deemed to have practical diagnostic capabilities for diagnosing platinum resistance (Figure 4). The best cutoff values ​​(the points on the ROC curve with the shortest distance from the coordinate (0,100)) were calculated as follows: chm5U / U: 2.283, Cm / C: 0.010, and m1I / I: 0.707. Patients with a chm5U / U ratio of 2.283 or greater were classified as chm5U high (chm5U high), those with a chm5U / U ratio less than 2.283 as chm5U low (chm5U low), those with a Cm / C ratio of 0.010 or greater as Cm high (Cm high), those with a Cm / C ratio less than 0.010 as Cm low (Cm low), those with an m1I / I ratio of 0.707 or greater as m1I high (m1I high), and those with an m1I / I ratio less than 0.707 as m1I low (m1I low). The rates of platinum-sensitive and platinum-resistant disease in the high and low inclusion groups for the three RNA modifications were compared using Fisher's exact test. The results showed that the chm5U low group (chm5U low) had a higher rate of platinum drug resistance than the chm5U high group (chm5U high), the Cm high group (Cm high) had a higher rate of platinum drug resistance than the Cm low group (Cm low), and the m1I high group (m1I high) had a higher rate of platinum drug resistance than the m1I low group (Fig. 5).Furthermore, the chm5U high group (chm5U high) had a higher rate of platinum drug sensitivity than the chm5U low group (chm5U low), the Cm low group (Cm low) had a higher rate of platinum drug sensitivity than the Cm high group (Cm high), and the m1I low group (m1I low) had a higher rate of platinum drug sensitivity than the m1I high group (m1I high). Furthermore, a receiver operating characteristic (ROC) curve was created by multiple logistic regression analysis using the measured values ​​of chm5U, Cm, and m1I, and the AUC in ovarian cancer tissue EVs was 0.84 (Figure 6).ROC analysis and Fisher's exact test revealed that the above three RNA modifications could be diagnostic markers for platinum-resistant ovarian cancer.

[0043] (Example 2) Analysis of RNA modification in EVs derived from cancer tissue after ovarian cancer surgery 2 In this example, to further explore potential diagnostic markers for platinum drug resistance, the number of measurement samples was increased to 43, improving analytical accuracy, and modified nucleosides in EVs derived from cancer tissue of ovarian cancer patients were analyzed.

[0044] (Preparation of nucleosides derived from ovarian cancer tissue EVs) Approval was obtained from the ethics committee of Osaka Medical and Pharmaceutical University, and patients who consented were included. As in Example 1, ovarian cancer tissue EVs were collected from postoperative ovarian cancer tissue surgically resected before the completion of platinum administration from patients with stage I to IV ovarian cancer receiving platinum administration. RNA was extracted and purified from each EV solution, and RNA nucleotides were degraded to nucleosides to obtain a 10 ng / μL nucleoside solution. Of the ovarian cancer patients receiving platinum administration, 8 patients who subsequently relapsed within 6 months of the end of platinum administration were diagnosed as platinum-resistant, while 35 patients who relapsed more than 6 months after the end of platinum administration were diagnosed as platinum-sensitive.

[0045] (Analysis of modified nucleosides using UHPLC-MS / MS) Measurements were performed under the same UHPLC-MS / MS conditions as in Example 1. All nucleosides were detected by the MRM method. As in Example 1, 42 types of modified nucleosides and 4 types of unmodified nucleosides, as well as the internal standard dG15N5, were used in the experiment under the detection conditions shown in Table 1.

[0046] Mann-Whitney tests showed that the median amounts of eight RNA modifications, 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um), and dihydrouridine (D), varied characteristically in EVs from ovarian cancer tissues derived from patients diagnosed with platinum-resistant ovarian cancer compared with those from patients diagnosed with platinum-sensitive ovarian cancer (Fig. 7). More specifically, the median ratio of chm5U to uridine (U) (chm5U / U) was lower in the platinum-resistant group than in the platinum-sensitive group, whereas the median ratios of Cm to cytidine (C) (Cm / C), m1I to inosine (I) (m1I / IU), m5U to U (m5U / U), m6A to adenosine (A) (m6A / A), m6,6A to A (m6,6A / A), m5Um to U (m5Um / U), and D to U (D / U) were higher in the platinum-resistant group than in the platinum-sensitive group. These results demonstrate that EVs released by platinum-resistant ovarian cancer tissues characteristically contain chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, and D.

[0047] Furthermore, receiver operating characteristic curves (ROC) were constructed using ovarian cancer tissue EVs. The AUCs for chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, and D were 0.73, 0.75, and 0.76, respectively. These curves demonstrated practical diagnostic capabilities for platinum resistance (Figure 8, left). Furthermore, ROC curves were constructed using multiple logistic regression analysis of the measured values ​​of chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, and D. The AUC for ovarian cancer tissue EVs was 0.97, demonstrating that the combination of these eight modified nucleosides can diagnose platinum resistance and sensitivity with extremely high accuracy (Figure 8, right).

[0048] The best cutoff values ​​(the point on the ROC curve that is the shortest distance from the coordinate (0,100)) were determined from the ROC curve, and were found to be chm5U / U 2.283, Cm / C 0.01, m1I / I 0.621, m5U / U 0.008, m6A / A 0.001, m6,6A / A 0.002, m5Um / U 0.003, and D / U 0.676. For chm5U / U, Cm / C, m1I / I, m5U / U, m6A / A, m6,6A / A, m5Um / U, and D / U, patients with values ​​equal to or greater than the best cutoff values ​​were classified as follows: chm5U high inclusion group (chm5U high), Cm high inclusion group (Cm high), m1I high inclusion group (m1I high), m5U high inclusion group (m5U high), m6A high inclusion group (m6A high), m6,6A high inclusion group (m6,6A high), m5Um high inclusion group (m5Um high), and D high inclusion group (D high). Patients with values ​​less than the best cutoff values ​​were classified as: chm5U low inclusion group (chm5U low), Cm low inclusion group (Cm low), m1I low inclusion group (m1I low), m5U low inclusion group (m5U low), and m6A low inclusion group (m6A high). The chm5U-low group was classified into the m6,6A-low group (m6,6A low), the m5Um-low group (m5Um low), and the D-low group (D low). The rates of platinum-sensitive and platinum-resistant patients in the high and low chm5U-high and chm5U-low groups were compared using Fisher's exact test. The results showed that the chm5U-low group had a higher rate of platinum resistance than the chm5U-high group, while the Cm, m1I, m5U, m6A, m6,6A, m5Um, and D-high groups had a higher rate of platinum resistance than the Cm, m1I, m5U, m6A, m6,6A, m5Um, and D-low groups, respectively (Figure 9). Furthermore, the chm5U-high group had a higher rate of platinum-sensitivity compared to the chm5U-low group, and the Cm, m1I, m5U, m6A, m6,6A, m5Um, and D-low groups had a higher rate of platinum-sensitivity compared to the Cm, m1I, m5U, m6A, m6,6A, m5Um, and D-high groups, respectively. These findings suggest that chm5U, Cm, m1I, m5U, m6A, m6,6A, m5Um, and D encapsulated in EVs released by ovarian cancer tissue are useful diagnostic markers for platinum-resistant ovarian cancer.

[0049] The testing method of the present invention is expected to provide early information on whether or not ovarian cancer patients are resistant to platinum agents, and if it is possible to determine whether or not a patient is resistant to platinum agents before administering them, it is expected to reduce the burden on patients and significantly reduce cancer medical costs.

Claims

1. A testing method for assisting in identifying platinum agent resistance, comprising detecting modified nucleosides specific to platinum agent resistance in RNA contained in extracellular vesicles in a sample collected from a subject.

2. The method of claim 1, further comprising a step of comparing the abundance ratio of platinum agent resistance-specific modified nucleosides relative to unmodified nucleosides with a cutoff value.

3. The method according to claim 1 or 2, wherein the platinum-resistance specific modified nucleoside is one or more selected from the group consisting of 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um) and dihydrouridine (D).

4. The method according to any one of claims 1 to 3, wherein the sample is derived from tissue of an ovarian cancer patient.

5. The method according to any one of claims 2 to 4, wherein the cutoff value is a value set based on an ROC curve based on the abundance ratio of modified nucleosides specific to platinum drug resistance relative to unmodified nucleosides.

6. The method according to any one of claims 1 to 5, wherein the platinum resistance-specific modified nucleosides are a combination of 5-carboxyhydroxymethyluridine (chm5U), 2'-O-methylcytidine (Cm), 1-methylinosine (m1I), 5-methyluridine (m5U), N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), 5,2'-O-dimethyluridine (m5Um) and dihydrouridine (D).

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