阅读说明：本技术 宠物中的癌的检查方法 (Method for examining cancer in pet ) 是由 广津崇亮 海渕觉 杉本敏美 石井秀始 今野雅允 植田裕司 小林省吾 江口英利 土岐祐 于 2019-08-14 设计创作，主要内容包括：本发明提供宠物等非灵长类哺乳动物中的癌的检查方法。根据本发明,提供例如在非灵长类哺乳动物中对癌进行检查或诊断的方法,其包括评价线虫对该对象的尿样的趋性行为的步骤。(The present invention provides a method for detecting cancer in a non-primate mammal such as a pet. According to the present invention, there is provided a method of detecting or diagnosing cancer, for example in a non-primate mammal, comprising the step of assessing nematode chemotactic behavior for a urine sample from the subject.)

1. A method of detecting or diagnosing cancer in a non-primate mammal comprising: a step of evaluating the chemotactic behavior of nematodes on urine samples of the subject.

2. The method of claim 1, wherein the mammal is a mammal selected from the group consisting of canines, felines, and rodents.

3. The method of claim 1 or 2, wherein the mammal is a dog.

4. A method according to claim 1 or 2 wherein the mammal is a cat.

5. The method of claim 1 or 2, wherein the mammal is a rodent.

Technical Field

The invention provides a method for detecting cancer in a pet.

Background

Various methods have been developed to detect cancer simply and inexpensively. Using the principle that nematodes attract urine of cancer patients and nematodes avoid urine of healthy people, cancer can be diagnosed in people using nematode chemotactic behavior as an indicator (see patent document 1).

Documents of the prior art

Patent document

Patent document 1: WO2015/088039

Disclosure of Invention

The invention provides a method for detecting cancer in a pet.

The present inventors have found that, in the case of pets such as dogs, cats and mice suffering from cancer, nematodes show a chemotactic behavior towards the urine of the pet. The present invention is based on such an insight.

That is, according to the present invention, the following inventions are provided.

(1) A method of detecting or diagnosing cancer in a non-primate mammal comprising: a step of evaluating the chemotactic behavior of nematodes on urine samples of the subject.

(2) The method according to the above (1), wherein the mammal is a mammal selected from the group consisting of canines, felines and rodents.

(3) The method according to (1) or (2) above, wherein the mammal is a dog.

(4) The method according to (1) or (2) above, wherein the mammal is a cat.

(5) The method according to (1) or (2) above, wherein the mammal is a rodent.

Drawings

Figure 1 shows the chemotactic behavior of nematodes on urine of rodents with cancer or rodents without cancer (healthy). One histogram corresponds to each volume.

Figure 2 shows the chemotactic behavior of nematodes on urine of either cancer-bearing dogs or non-cancer-bearing dogs (healthy). One histogram corresponds to each volume.

Figure 3 shows the chemotactic behavior of nematodes on urine of cats with cancer or cats without cancer (healthy). One histogram corresponds to each volume.

Detailed Description

In the present specification, the term "subject" refers to a non-human mammal, for example, a non-primate mammal. In the present specification, the term "subject" is used in a meaning including a healthy subject, a subject suspected of having cancer, and a subject having cancer. In the present specification, the term "subject suspected of having cancer" includes subjects not specifically suspected of having cancer, in addition to subjects specifically suspected of having cancer.

In the present specification, "cancer" refers to malignant tumor. Carcinomas can be broadly classified as hematopoietic tumors, epithelial carcinomas (Carcinoma), and non-epithelial sarcomas (Sarcoma). Examples of hematopoietic tumors include leukemia, malignant lymphoma, and myeloma. Examples of epithelial cancers include lung cancer, breast cancer, stomach cancer, colorectal cancer, liver cancer, uterine cancer, ovarian cancer, head and neck cancer, and tongue cancer. Examples of sarcomas include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma.

In the present specification, "detection of cancer" may be referred to as "detection of cancer cells", "identification of cancer", "determination of cancer", "a method for assisting diagnosis of cancer", "a method for obtaining preliminary information for diagnosis of cancer", or the like. The method of the present invention is industrially applicable.

As used herein, "nematode" refers to caenorhabditis elegans (C.elegans) ((C.elegans))Caenorhabditis elegans). With respect to nematodes, strains isolated from various environments are registered and published at the nematode genetics center (CGC) of the department of biology, Minnesota university, USA, and can be sold separately. Thus, the skilled person can obtain most strains known from CGC. From the viewpoint of enabling propagation by self-fertilization, hermaphrodite may be preferably used.

In the present specification, "chemotactic behavior" means an attracting behavior or an avoidance behavior. The attraction behavior is a behavior of reducing a physical distance from a certain substance, and the avoidance behavior is a behavior of extending a physical distance from a certain substance. The substance inducing the attracting behavior is referred to as an attracting substance, and the substance inducing the avoiding behavior is referred to as an avoiding substance. The nematode has a property of being attracted by an attracting substance through olfaction and avoiding an avoidance substance. The behavior induced by the inducing substance is referred to as an inducing behavior (sometimes referred to as "positive" in the present specification), and the behavior avoiding the avoiding substance is referred to as an avoiding behavior (sometimes referred to as "negative" in the present specification). In addition, the combination of the attraction behavior and the avoidance behavior is referred to as chemotactic behavior.

In the present specification, the "wild strain" is a wild strain of a nematode, and for example, the N2 Bristol strain is a general wild strain.

According to the present invention, there is provided a method for detecting cancer (or a method for predicting cancer or a method for diagnosing cancer or a method for obtaining basic information on the therapeutic effect) in a subject having cancer or suspected of having cancer, comprising the step of evaluating the chemotactic behavior of nematodes with respect to a urine sample of the subject.

The subject is preferably a non-human mammal or a non-human primate, and preferably rodents (e.g., mice, rats, hamsters, chinchillas, rabbits, guinea pigs, mice, prairie dogs, squirrels, and squirrels), canines (e.g., dogs, foxes, raccoons, and auricular foxes), and felines (e.g., cats, and wild cats) can be used as the subject in the present invention.

As nematodes, wild-type nematodes (e.g., strain N2 Bristol) can be used. Since the nematode strain shows an attractive behavior to urine of a cancer patient, such nematodes showing an attractive behavior to urine of a cancer patient can be used as nematodes in the present invention.

A urine sample can be obtained from a subject. The urine sample can be diluted with a solvent such as water when evaluated by the method of the present invention. In the case of dilution, the dilution ratio may be, for example, 5 to 20000 times, for example, 10 to 10000 times, for example, 10 to 5000 times, 10 to 2000 times, for example, 100 to 1000 times, and nematodes may exhibit a chemotactic behavior with respect to a urine sample after dilution. In one aspect of the present invention, the dilution ratio of urine may be set to, for example, 100 times or 1000 times, or 2 or more dilution ratios including 100 times and 1000 times. For example, the dilution ratio of the urine sample of a dog may be 10-2000 times, for example, 100-1000 times. For example, the dilution ratio of the urine sample of the cat may be 5 to 20000 times, 10 to 10000 times, for example 500 to 5000 times.

The evaluation of the nematode chemotactic behavior can be carried out as follows: the urine sample and the nematodes are placed at a predetermined distance (for example, a distance of about 1cm to about 5 cm) from each other, and whether the urine sample shows attraction behavior or avoidance behavior of the nematodes is observed. The chemotactic behavior can be observed on a solid medium such as an agar medium.

Method for analyzing urine using nematode

The assay using nematode urine can be performed as follows: a test sample (for example, urine) obtained from a subject is placed at a distance from nematodes, and whether the nematodes show an attracting behavior or an avoiding behavior to the test sample is observed. Then, when the attracting behavior is exhibited, the subject may be evaluated as suffering from cancer or having such a possibility. When the avoidance behavior is exhibited, the subject may be evaluated as not having cancer or as having the possibility of having cancer. An example of an analytical method using a human sample is disclosed in WO2015/088039, the entire content of which is incorporated by reference in the present specification.

In more detail, the analysis method using urine of nematodes may include, for example:

disposing a sample to be tested (e.g., a urine sample) obtained from a subject in a petri dish (e.g., a petri dish into which a solid medium is introduced);

disposing nematodes in a culture dish in which a sample to be tested is disposed, at a position spaced apart from the sample to be tested by a predetermined distance;

after deployment, the nematode is allowed to behave.

Analytical methods using nematode urine, for example, may include: in the case where the nematode exhibits chemotactic behavior for the test sample, the subject is determined to have cancer, or to have a likelihood of having cancer.

The chemotactic behavior of nematodes can be assessed by the difference or ratio of the number of nematodes close to the sample being tested to the number of nematodes further away from the sample being tested. When the evaluation is performed based on the difference in the number of nematodes, the test sample may induce an attracting behavior as a whole and evaluate the test sample as a sample from a cancer patient when the difference is positive, and/or induce an avoidance behavior as a whole and evaluate the test sample as a sample from a subject not suffering from cancer when the difference is negative. In the case of evaluation based on the ratio of the number of nematodes, the test sample may induce an attracting behavior and be evaluated as a sample derived from a cancer subject when the ratio is 1 or more, and/or induce an avoidance behavior and be evaluated as a sample derived from a subject not suffering from cancer when the ratio is less than 1. In addition, the nematode chemotaxis behavior can be evaluated using the chemotaxis index as an index, for example, as described below.

{ formula, A is the number of nematodes showing attractive behavior to the test sample, and B is the number of nematodes showing avoidance behavior to the test sample. }

When the tropism index is positive, the test sample induces attraction behavior as a whole and can be evaluated as a sample from a subject suffering from cancer, and when the tropism index is negative, the test sample induces avoidance behavior as a whole and can be evaluated as a sample from a subject not suffering from cancer.

A closer to 1 of the tropism index means that the ratio of the nematodes showing attraction behavior is larger, and a closer to-1 of the tropism index means that the ratio of the nematodes showing avoidance behavior is larger, and a closer to 0 of the nematodes showing neither attraction behavior nor avoidance behavior. The larger the absolute value of the tendency index is, the more clear the behavior evaluation result is. If the nematode showing the convergent behavior and the nematode showing the avoidance behavior are of the same degree, the test may be positive (if the test is positive, a further precision test may be performed), or may be excluded from the evaluation target. In the present invention, the individuals from which the positive urine sample is obtained can be further subjected to a close examination to determine whether the individual has cancer.

When urine at two or more different dilution ratios is examined, from the viewpoint of improving sensitivity, it can be determined as cancer if either of the urine is positive, and from the viewpoint of improving specificity, it can be determined as cancer if both of the urine is positive.

A subject assessed as having cancer by the methods of the invention may thereafter receive treatment for the cancer (e.g., chemotherapy, radiotherapy, and surgical resection, combinations thereof, and the like). Subjects evaluated as having cancer by the method of the present invention can be further subjected to a precision examination to make a definitive diagnosis of cancer.

Examples

Example 1: cancer examination using rodent urine

In this example, nematode chemotactic behavior was evaluated on rodent urine samples.

As the rodent, a mouse was used. Urine samples were collected from healthy mice and from mice with cancer. As healthy mice, mice that did not develop cancer with C-Met deletion (C57Bl/6 system) were used, and as mice with cancer, mice having the KrasG12D mutation were used (see Noguchi K.et al, Oncology Letters,16:1892-1898, 2018). Urine samples were diluted 100-fold and nematode chemotactic behavior of urine samples was evaluated using the method described in WO 2015/088039. As the nematode, a wild-type nematode (Bristol N2 strain) was used. The trending behavior was evaluated using a trending index. The measurement was repeated 12 times (hereinafter, the same applies) to obtain an average value of the tropism index of each individual. The results of the chemotactic behavior of urine samples are shown in FIG. 1.

{ formula, A is the number of nematodes showing attractive behavior to the test sample, and B is the number of nematodes showing avoidance behavior to the test sample. }

As shown in fig. 1, the urine sample of healthy mice had a negative tendency index or a tendency index of 0.05 or less in almost all cases, whereas the urine sample of cancer-suffering mice had a tendency index of 3 systems of urine of more than 0.05. This indicates that mice with cancer can be evaluated by assessing nematode chemotactic behavior in urine samples. It should be noted that, when the urine sample of PK-60+98 was diluted 1000 times, nematodes showed an attracting action to the urine sample (at this time, the chemotaxis index was about 0.15). In addition, urine samples from 3 mice with cancer were also shown to attract behavior at 1000-fold dilution. This indicates that the nematodes showed luring behavior in the urine of the cancer model mouse at either of the 100-fold dilution and the 1000-fold dilution. When the tendency index was positive and negative, the sensitivity was 28.6% and the specificity was 100% at 100-fold dilution, and the sensitivity was 42.8% and the specificity was 83.3% at 1000-fold dilution. This indicates that the presence or absence of cancer can be evaluated by evaluating the chemotactic behavior of nematodes for rodent urine.

When the positive trend index was evaluated as positive in any of the 100-fold dilution and the 1000-fold dilution and the negative trend index was evaluated as negative in both the 100-fold dilution and the 1000-fold dilution, the sensitivity was 71.4% (5 out of 7) and the specificity was 83.3% (10 out of 12). From the results, it was revealed that examination of cancer using the nematode chemotactic behavior as an index was effective for rodent mice.

Subsequently, urine samples were collected from healthy dogs and cancer-suffering dogs, and the nematode chemotactic behavior was evaluated using 1000-fold diluted urine in the same manner as in example 1.

As dogs, healthy doll, healthy jackscrew stems, retriever virginiana with angiosarcoma, and scotland stems with liver tumors were used. The results are shown in FIG. 2. As shown in fig. 2, the urine tropism index for healthy dogs was negative, while the urine tropism index for cancer dogs was positive. For dogs, both sensitivity and specificity were 100%. From the results, it was revealed that the examination of cancer using the nematode chemotactic behavior as an index was effective for dogs.

Further, urine samples were collected from healthy cats and cats with cancer, respectively, and the nematode chemotactic behavior was evaluated using 1000-fold dilutions of urine.

As cats, healthy exotic short-haired cats (2), healthy Mix, american short-haired cats with lung tumors, Mix with breast tumors, Mix with adenocarcinoma in the nasal cavity, and bose cats with lymphoma were used. The results are shown in FIG. 3. As shown in fig. 3, the urine tropism index for healthy cats is significantly lower than for cats with cancer. When the positive trend index and the negative trend index were evaluated as positive and negative, the sensitivity was 100% (4 out of 4) and the specificity was 66.7% (2 out of 3). From the results, it was revealed that the examination of cancer using the nematode chemotactic behavior as an index was effective for cats.

From the above results, it was revealed that examination of cancer using nematode chemotactic behavior as an index was effective for dogs and cats, and mice as rodents. Thus, in pets, cancer can be diagnosed inexpensively and quickly by urine examination using nematode chemotactic behavior as an index.

For the above urine samples of dogs and cats, nematode chemotactic behavior was similarly examined by varying the dilution rate of the urine. In dogs, urine samples from dogs with liver tumors and angiosarcoma show a positive chemotaxis index at least at 10-fold to 1000-fold dilutions. In addition, in cats, urine samples from cats with tumors also showed positive chemotaxis indices with at least 10-fold to 10000-fold dilutions.