The present invention relates to a method for detecting proliferation of cancer in a human cancer patient after cancer treatment.
Cancer is versatile, so that no effective fixed treatment protocol has been established. It seems important to specify a personalized specific treatment protocol. Thus, it is a critical issue in cancer treatment to evaluate therapeutic effects of the cancer treatment. In addition, although cancer of interest appears to be temporarily removed by treatment, cancer that has not been completely excised by surgery remains in some cases. It is important to evaluate these matters from the viewpoints of preventing recurrence or selecting a treatment protocol prepared for recurrence.
Biomarkers have been developed that indicate the presence of cancer. Unfortunately, cancer is versatile, so that there is a maker-specific limit for its detectability and sensitivity. Consequently, other indicators that each indicate the presence of cancer are needed. Besides, another issue is that in diagnostic imaging, cancer cannot be detected unless the cancer grows to a certain size or more.
Summary of Invention
In a first embodiment the present invention provides a method for detecting proliferation of cancer in a human cancer patient after cancer treatment, comprising:
evaluating nematode taxis behavior to a time-series of urine from the cancer patient, wherein
it is determined that the proliferation of cancer is detected when avoidance to the urine from the cancer patient is weakened or attraction thereto is strengthened as the time-series for evaluating results obtained progresses, and wherein the time-series has been scheduled with an interval of from one week to three months.
The method according to the first embodiment, wherein it is determined that the proliferation of cancer is detected when the tactic behavior to the urine from the cancer patient is changed from avoidance to attraction as the time-series for evaluating results obtained progresses.
 The invention further provides the use of a composition comprising a nematode worm in the method according to embodiment 1.
Brief Description of Drawing
[Figure 1] Figure 1 shows the pre-operation and post-operation history and the results of recurrence monitoring in a cancer patient by a urine test using nematode worms.
As used herein, the "subject" means a human cancer patient.
As used herein, the "cancer" means malignant tumor. Cancer can be largely grouped into hematopoietic tumor, carcinoma, and non-epithelial sarcoma (sarcoma). Examples of the hematopoietic tumor include leukemia, malignant lymphoma, and myeloma. Examples of the carcinoma include lung cancer, breast cancer, gastric cancer, colon cancer, uterine cancer, ovarian cancer, head and neck cancer, and tongue cancer. Examples of the sarcoma include osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma. As used herein, the "cancer" encompasses early cancer such as stage 0 early cancer and stage 1 early cancer.
As used herein, the wording "detecting a therapeutic effect of cancer treatment" can be read as the wording "evaluating a therapeutic effect of cancer treatment", "predicting a therapeutic effect of cancer treatment", "diagnosing a therapeutic effect of cancer treatment", or "non-diagnostically helping diagnose a therapeutic effect of cancer treatment".
As used herein, the wording "detecting proliferation of cancer" in the post-operation context can be read as the wording "detecting a possibility of recurrence", "predicting recurrence", "predicting a possibility of recurrence", or "evaluating a possibility of recurrence".
As used herein, the "recurrence" refers to a phenomenon where although a therapeutic effect appears to be exerted, cancer then occurs at the same site or another site. That is, the word "recurrence" is used, in a sense, to include metastasis. The "recurrence" may also be called "relapse".
As used herein, the "nematode" means Caenorhabditis elegans.
In the Caenorhabditis Genetics Center (CGC) of College of Biological Sciences, University of Minnesota, nematode strains isolated from various environments are stored, open to the public, and available on request. Thus, those skilled in the art can acquire almost any known strains from the CGC. From the viewpoint of reproduction through self-fertilization, hermaphrodites may be preferably used.
As used herein, the "taxis behavior" means attraction behavior or avoidance behavior. The attraction behavior means a behavior of making shorter the physical distance to a certain substance. The avoidance behavior means a behavior of making longer the physical distance to a certain substance. An attraction behavior-inducing substance is called an attractant. An avoidance behavior-inducing substance is called a repellent. Nematode has characteristics such as approaching to an attractant and escaping from a repellent based on the olfaction thereof. A behavior of approaching to an attractant is referred to as attraction behavior (as used herein, sometimes referred to as "positive"). A behavior of escaping from a repellent is referred to as avoidance behavior (as used herein, sometimes referred to as "negative"). In addition, the attraction behavior and the avoidance behavior are collectively called as tactic behavior.
As used herein, the "wild-type strain" is a nematode wild-type strain. Examples include a common wild-type N2 Bristol strain. The wild-type strain as used herein may be a strain by which attraction to urine obtained from each subject with cancer is exhibited and avoidance to urine obtained from each subject without cancer is exhibited.
Method of Analyzing Urine by Using System for Evaluating Nematode Taxis Behavior
The method of analyzing urine by using a system for measuring nematode taxis behavior basically can be put into practice by placing nematode worms at a certain distance from a test sample (e.g., urine) obtained from a subject and observing whether the worms exhibit attraction behavior to the test sample or exhibit avoidance behavior thereto. Then, when the attraction behavior is exhibited, the subject can be evaluated as suffering from cancer or as having a possibility of suffering from cancer. Accordingly, WO 2015/088039
discloses that in a medium-scale study of 242 test subjects, cancer patients were successfully detected with 100% sensitivity and 95% specificity.
More specifically, the method for analyzing urine by using a system for measuring nematode taxis behavior may comprise: for instance,
placing, on a dish (e.g., a dish on which a solid medium is introduced), a test sample (urine) obtained from a human subject;
placing, on the dish on which the test sample is placed, nematode worms positioned at a certain distance from the test sample;
letting the worms behave after the placement; and
evaluating that the subject suffers from cancer or may suffer from cancer when the worms exhibit attraction behavior to the test sample.
Nematode taxis behavior can be evaluated by determining the difference and/or the ratio between the number of worms attracted to the test sample and the number of worms repelled from the test sample. The difference in the number of worms may be used for assessment. When the difference is a positive value, the test sample as a whole induces attraction behavior and can be assessed that the test sample is derived from a cancer patient. When the difference is a negative value, the test sample as a whole induces avoidance behavior and can be assessed that the test sample is derived from a healthy individual. In addition, the nematode taxis behavior can be measured by using, for instance, the following tactic behavior index as an indicator.
wherein, A represents the number of worms exhibiting attraction to a test sample; and B represents the number of worms exhibiting avoidance to the test sample.
If the taxis index is a positive value, the test sample as a whole induces attraction behavior and can be assessed that the test sample is derived from a cancer patient. If the tactic index is a negative value, the test sample as a whole induces avoidance behavior and can be assessed that the test sample is derived from a healthy individual.
As the taxis index is closer to 1, the percentage of worms exhibiting attraction behavior is indicated to be larger. As the taxis index is closer to -1, the percentage of worms exhibiting avoidance behavior is indicated to be larger. If the taxis index is closer to 0, it is interpreted that the worms exhibit neither attraction behavior nor avoidance behavior. The larger the absolute value for the taxis index, the clearer the behavior assessment results.
It has been found that there is a sharp change in nematode taxis behavior to urine from each cancer patient, who has received treatment, between before and after the treatment of cancer. Accordingly, the present inventor has found that, based on the nematode taxis behavior to urine from each cancer patient before and after treatment, therapeutic effects of the cancer treatment can be evaluated.
According to the present inventor, provided is a method for detecting proliferation of cancer (or volume of cancer) in a human cancer patient after cancer treatment, wherein the proliferation of cancer (or volume of cancer) is evaluated based on nematode taxis behavior to a time-series of urine from the cancer patient. In the above method, a time-series of urine collected from a post-treatment cancer patient is prepared and how the nematode tactic behavior to each urine is changed can be observed.
The observation results are used to determine that the cancer has proliferated (or the cancer volume has increased) when avoidance behavior to the urine from the cancer patient is weakened or attraction behavior thereto is strengthened as the time-series for the above urine samples progresses. In a preferred embodiment according to the above method, the observation results are used to determinethat the cancer has proliferated (or the cancer volume has increased) when the tactic behavior to the urine from the cancer patient has been changed from avoidance behavior to attraction behavior as the above time-series progresses.
From the viewpoints of cancer proliferation monitoring the time-series for urine samples is a periodically scheduled time-series. For instance, the time-series contains at least two urine samples and has been scheduled with an interval of from one week to three months. For example,the urine samples may be collected with an interval of from once every 1 week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 1 month. Urine may be collected at the time of periodical urine collection as well as when cancer proliferation and/or recurrence is suspected. As used herein, the "time-series" is used for a plurality of samples collected at two or more different time-points. Accordingly, two or more, three or more, or four or more urine samples may be included in the time-series. If the results of measuring taxis behavior to three or more urine samples are compared, an advantage is that the trend of change (e.g., a change) in taxis behavior is easy to understand.
The present invention further encompasses the use of a composition comprising a nematode worm in the above method.
The following aspects of the disclosed method do not form part of claimed invention.
The above method for evaluating (or method for predicting, method for detecting, or method for diagnosing, or method for acquiring basic information about) an therapeutic effect of cancer treatment may be implemented further in combination with the method for evaluating (or method for determining, method for predicting, method for detecting, or method for diagnosing, or method for acquiring basic information about a therapeutic effect on) proliferation of cancer (or the volume of cancer) in vivo
in a cancer patient after treatment. Specifically, a pre-treatment urine sample from a cancer patient and a post-treatment urine sample from the cancer patient may be used to evaluate therapeutic effects of the cancer treatment, and then, the proliferation of cancer and/or the volume of cancer in vivo
in the cancer patient may be evaluated from a time-series of urine samples (see, for example, Figure 1). Examples
Example 1: Surgical Removal and Recurrence of Tumor
The present inventor has since revealed that nematode exhibits attraction behavior to urine from cancer patients. In this Example, further investigated was how the nematode attraction behavior was associated with removal and recurrence of tumor in a particular cancer patient.
The cancer patient with a very low level of each tumor marker, which was negative in a tumor marker test, was monitored.
The above cancer patient, whose tumor was removed and then recurred, had the following history.
||Sigmoid colon cancer was found to be metastasized into the lung (right middle lobe) (stage 1a colon cancer)|
||Surgical removal of primary lesion and metastatic lesion|
||No matters of concern were found in a blood test or a CT test.|
||No matters of concern were found in a blood test or a CT test.|
|End of February 2016
||Recurrence in the lung (right lower lobe) was found by a CT test.|
Urine was sampled from the above patient once every about 1 month from nearly the date of operation in April 2015 and was frozen and stored.
Next, how nematode tactic behavior was associated with each urine derived from the above patient was examined. The tactic behavior was measured as described in WO 2015/088039
. Figure 1 shows the results.
As shown in Figure 1, after the surgical removal of tumor, attraction behavior to urine from the cancer patient disappeared and avoidance behavior appeared. Immediately after the operation, in particular, a big change in the nematode tactic behavior to urine was already detected. Meanwhile, the day when the recurrence of cancer was detected by CT is February 28, 2016. The nematode worms exhibited, by the end of September 2015, about 5 months before the day, attraction behavior to urine from the above patient. Further, it was also observed that avoidance behavior was weakened over from May to August 2015 before the recurrence.
This result indicates that the concentration of nematode attractant in urine from the cancer patient was very rapidly changed after the removal of cancer. This has revealed that the degree of surgical removal of cancer can be accurately checked by the urine test using nematode worms.
In addition, the nematode worms detected an in-urine attractant at several months before the recurrence was found by CT, which made it possible to predict the recurrence in the patient. This has demonstrated that the cancer patient urine test using nematode worms is useful in evaluating recurrence and/or outcomes of cancer.
Example 2: Urine Test Using Nematode Worms Before and After Operation and Chemotherapy
In this Example, the results of Example 1 were reproduced by increasing the number of test subjects markedly.
Urine before and after operation and chemotherapy was collected from each patient with colon cancer, gastric cancer, pancreatic cancer, esophageal cancer, bile duct cancer, or gallbladder cancer, and was frozen and stored. One month after the treatment, urine was collected from 45 test subjects. Three months after the treatment, urine was collected from 15 test subjects.
nematode tactic behavior to each urine collected was evaluated like Example 1.
As a result, it was observed that in 31 of 45 test subjects (68.9%), the index changed to negative at 1 month after the treatment (i.e., changed from attraction behavior to avoidance behavior). It was also observed that in 10 of 15 test subjects (66.7%), the index changed to negative at 3 months after the treatment.
In this way, the nematode worms changed their tactic behavior from attraction behavior to avoidance behavior between before and after the treatment of each patient with different cancer. This change in tactic behavior seems to result from the phenomenon where nematode sharply detects and responds to a change, between before and after the treatment, in the level of an attractant contained in urine from each cancer patient.
Cases of the above patients include an instance where a cancerous tissue cannot be completely removed by treatment and a case where cancer has already metastasized.
The above results have demonstrated that the cancer patient urine test using nematode worms is useful in evaluating treatment.