JP6593787B2 - Risk assessment program and apparatus for lymph node metastasis - Google Patents

Description

The present invention relates to a program, a storage medium, and an apparatus based on a method for evaluating a risk of metastasis in a lymph node.

The number of breast cancer patients in Japan is the highest among female carcinomas, and both the number of sufferers and deaths from breast cancer are increasing year by year. The incidence of breast cancer in 2011 is 1 in 18 and the mortality rate has increased to 20%. Breast cancer is more likely to metastasize than other organs, and recent studies have reported that lymph node metastasis occurs at a relatively early stage. Since the presence or absence of this lymph node metastasis greatly affects the survival rate of the affected person, early diagnosis and early treatment of lymph node metastasis of breast cancer are desired.

Lymph node metastasis is an important prognostic factor for survival in patients with malignant tumors such as breast cancer. As a method for treating lymph node metastasis in breast cancer, axillary lymph node dissection that removes the axillary lymph nodes around the breast has been performed. Recently, however, it has been reported that there is no significant difference in survival with or without axillary lymph node dissection when used in combination with local radiation therapy or chemotherapy, unless the lymph nodes have clearly large metastases. The clinical significance has been reviewed. Preoperative diagnosis of axillary lymph nodes is effective for minimal lymph node dissection. As a preoperative diagnosis of an axillary lymph node, a needle biopsy is known in which a needle is inserted into a lymph node and the tissue is removed while checking with an ultrasonic device (see Non-Patent Document 1).

Lymphatic system, section in the pressure of the lymph nodes: plays an important role in order to keep the (Int ra nodal pressure INP) constant. Lymphatic vessels that function as recovery systems can be a route for cancer metastasis. When tumor cells invade surrounding lymph vessels, they move to nearby lymph nodes. The lymph node that tumor cells first invade is called the sentinel lymph node. It has been reported that the sentinel lymph node having a tumor has a statistically significantly higher intranodal pressure than the sentinel lymph node having no tumor (see Non-Patent Document 2). Non-Patent Document 2 reports that INP in the axilla where tumor metastasis was observed was statistically significantly higher than INP in the axilla where no tumor was found.

In solid tumors and around tumors, it has been reported that IFP (interstitial fluid pressure) shows a higher value than normal tissues (see Non-Patent Documents 3 to 7). In particular, it has been reported that the normal tissue IFP shows a value close to 0 mmHg, whereas the value in the tumor is about 10 to 40 mmHg (see Non-Patent Documents 5 to 7).

Nathanson SD, Burke M, Slater R, Kapke A. Ann Surg Oncol. 2007; 14: 3102-10 Nathanson SD, Mahan M. Ann Surg Oncol. 2011; 18: 3791-6. Heldin CH, Rubin K, Pietras K, Ostman A. Nature reviews Cancer. 2004; 4: 806-13 Jain RK. Science. 1996; 271: 1079-80 Curti BD, Urba WJ, Alvord WG, Janik JE, Smith JW, 2nd, Madara K, et al. Cancer research. 1993; 53: 2204-7. MiIosevic MF, Fyles AW, Wong R, Pintilie M, Kavanagh MC, Levin, W et al. Cancer. 1998; 82: 2418-26 Swartz MA, Lund AW. Nature reviews Cancer. 2012; 12: 210-9.

In the conventional method for identifying a metastatic lymph node using ultrasound, CT, MRI, PET or the like as described in Non-Patent Document 1, a micrometastasis or lymph node metastasis route of a lymph node having a maximum minor axis of 10 mm or less is used. There is a problem that it is difficult to identify.

Therefore, a method capable of solving the problems found in the conventional methods is desired. In particular, since the fatality rate after the discovery of metastasis is very high, a method that can predict metastatic lymph nodes in a state where metastasis has not progressed to the extent that it can be detected as a tumor is desired.

Therefore, it is conceivable to identify a metastatic lymph node by measuring the pressure of INP in the lymph node. Certainly, Non-Patent Documents 5 to 7 describe that INP and the like increase in a lymph node-related tissue in which a tumor has already been formed or metastasis has already been found. However, these documents do not describe that it is possible to predict metastatic lymph nodes by measuring INP.

Therefore, the present invention can identify a micrometastasis and a lymph node metastasis path of a lymph node having a maximum minor axis of 10 mm or less, which is difficult with the conventional method for identifying a metastatic lymph node, and is detected as a tumor. The invention provides a method for assessing the risk of metastasis in a lymph node and a means for achieving the method, which can predict a metastatic lymph node in a state where metastasis has not progressed as much as possible. It was a problem to be solved.

As a result of intensive studies, the present inventors have inoculated tumor cells into the subiliac lymph nodes using MXH10 / Mo- lpr / lpr (abbreviation: MXH10 / Mo / lpr) mice, and this subiliac Metastasis was formed in the axillary lymph node, which is the lymph node of the lymph node, and the INP values of both the sentinel node and the secondary lymph node were successfully measured. Based on these measurement results, it was found that the INP value in the secondary lymph node was correlated with the degree of tumor growth, and surprisingly, the INP value increased before the secondary lymph node volume change occurred. . The fact that the lymph node metastasis path could be identified from the measurement of the INP values of both the sentinel lymph node and the secondary metastatic lymph node was first made by the present inventors.

Based on what we have been able to do, we have succeeded in identifying and determining lymph nodes where tumor cell metastasis can occur by measuring changes in pressure in secondary lymph nodes. Moreover, the knowledge that the antitumor effect by chemotherapy, radiotherapy, etc. can be determined by measuring the pressure change of a secondary lymph node was acquired. The present invention is a completed invention based on such successful examples and findings.

Therefore, according to the present invention,
(1) measuring the intranodal pressure of the regional lymph node of the primary lesion in the subject;
(2) including a step of evaluating that there is a risk of metastasis in regional lymph nodes when the intra-nodal pressure tends to increase,
A method is provided for assessing the risk of metastasis in lymph nodes.

According to another aspect of the invention,
(A) obtaining a measurement of the intranodal pressure of the regional lymph node of the primary lesion in the subject;
(B) evaluating the risk of metastasis in regional lymph nodes by confirming an increasing tendency of intranodal pressure from the measured values;
A program for assessing the risk of metastasis in lymph nodes is provided.

According to another aspect of the present invention, a computer-readable storage medium recording the program of the present invention is provided.

According to another aspect of the invention,
(A) a lymph node pressure measuring means for obtaining a measured value of the intranodal pressure of the regional lymph node of the primary lesion in the subject;
(B) computing means for analyzing the intranodal pressure obtained by the lymph node pressure measuring means and calculating a tendency of the intranodal pressure;
(C) an evaluation means for evaluating that there is a risk of metastasis in a regional lymph node by confirming an increasing tendency of the intranodal pressure from the tendency of intranodal pressure calculated by the computing means; An apparatus for evaluating lymph node metastasis for evaluating the risk of metastasis is provided.

In the method, program, medium and apparatus of the present invention, preferably, the regional lymph node is a sentinel lymph node.

According to the present invention, it is possible to identify a micrometastasis of a lymph node having a maximum minor axis of 10 mm or less, which has been difficult by a conventional method using CT, MRI, PET, ultrasonic images and the like. In addition, by measuring the intranodal pressure of the lymph nodes, it was difficult to do with the previous method, such as sentinel lymph nodes and downstream lymph nodes such as downstream lymph nodes such as secondary lymph nodes and tertiary lymph nodes located downstream thereof Can be identified. As a result, secondary and tertiary lymph nodes can be confirmed, and development of a minimally invasive treatment for lymph node metastasis and reduction of the dissection range can be expected.

According to the present invention, it is possible to obtain a particularly remarkable effect that a metastatic lymph node can be identified in a predictable manner in a state where metastasis has not progressed so as to be detected as a tumor. In addition, according to the present invention, it can be expected to determine a therapeutic effect by chemotherapy, radiation therapy, or the like. In particular, it can be expected that the determination of the therapeutic effect is performed in real time without using an uneconomical apparatus such as CT, MRI, and PET by using it together with an ultrasound image.

According to the present invention, it is expected that the above-described identification and determination can be performed at a low cost and in real time, and further, an excellent effect can be obtained that it can be automatically processed on a personal computer.

After performing PCR with 6 types of primers corresponding to each gene, GE-Luc / GFP cells express VEGF family and bFGF known to be involved in angiogenesis by performing gel electrophoresis. It is the photograph figure which confirmed doing. It is the photograph which confirmed that FM3A-Luc cell expressed VEGF-A by implementing gel electrophoresis after implementing PCR using the primer corresponding to VEGF-A. It is the figure which showed the ultrasonic image in the subiliac lymph node by the ultrasonic high-resolution imaging system for small animals (VEVO) acquired just before tumor cell inoculation. It is the figure which showed the ultrasonic image in the subiliac lymph node by VEVO acquired immediately after tumor cell inoculation. It is the figure which showed the three-dimensional ultrasonic image of the axillary lymph node created with VEVO. It is the schematic of an INP pressure measurement system. SiLN: subiliac lymph node, PALN: axillary lymph node It is a photograph figure in the middle of measuring INP using the INP pressure measurement system. It is a simplified diagram of the INP measurement plan of control group mice. In the figure, “IVIS” represents the measurement date of luciferase activity measured with a biological imaging observation apparatus (IVIS), and “VEVO” represents the volume measurement date of the axillary lymph node. It is a simplified diagram of the INP measurement plan of KM-Luc / GFP cell inoculated group mice. In the figure, “IVIS” represents the measurement date of luciferase activity, and “VEVO” represents the volume measurement date of the axillary lymph node. It is a simplified diagram of the INP measurement plan of FM3A-Luc cell inoculated group mice. In the figure, “IVIS” represents the measurement date of luciferase activity, and “VEVO” represents the volume measurement date of the axillary lymph node. It is the image figure which visualized light emission of the luciferase acquired by IVIS of the mouse | mouth inoculated with KM-Luc / GFP cell. It is the graph obtained by calculating the amount of luciferase luminescence of the subiliac and axillary lymph nodes by IVIS of mice inoculated with KM-Luc / GFP cells. It is the image figure which visualized the light emission of the luciferase acquired by IVIS about the mouse | mouth inoculated with FM3A-Luc cell. It is the graph obtained by calculating the amount of luciferase luminescence of the subiliac and axillary lymph nodes by IVIS for mice inoculated with FM3A-Luc cells. It is a B-mode ultrasonic image figure of the maximum smooth surface of the axillary lymph node acquired by VEVO about the mouse | mouth which inoculated KM-Luc / GFP cell. It is the graph obtained by calculating the measurement result of the volume of an axillary lymph node from the ultrasonic image acquired by VEVO about the mouse | mouth inoculated with KM-Luc / GFP cell. It is a B-mode ultrasonic image figure of the maximum smooth surface of the axillary lymph node acquired by VEVO about the mouse | mouth inoculated with FM3A-Luc cell. It is the graph obtained by calculating the measurement result of the volume of an axillary lymph node from the ultrasonic image acquired by VEVO about the mouse | mouth inoculated with FM3A-Luc cell. It is the graph showing the INP measured value in a subiliac lymph node about the mouse | mouth which inoculated KM-Luc / GFP cell. It is the graph showing the INP measured value in an axillary lymph node about the mouse | mouth which inoculated KM-Luc / GFP cell. It is the graph showing the INP measurement value in a subiliac lymph node about the mouse | mouth inoculated with FM3A-Luc cell. It is the graph showing the INP measured value in an axillary lymph node about the mouse | mouth inoculated with FM3A-Luc cell. It is the scatter diagram which showed the relationship between the luciferase activity measured value and INP measured value in a subiliac lymph node about the mouse | mouth inoculated with KM-Luc / GFP cell. It is the scatter diagram which showed the relationship between the luciferase activity measured value and INP measured value in an axillary lymph node about the mouse | mouth inoculated with KM-Luc / GFP cell. It is the scatter diagram which showed the relationship between the luciferase activity measured value and INP measured value in a subiliac lymph node about the mouse | mouth inoculated with FM3A-Luc cell. It is the scatter diagram which showed the relationship between the luciferase activity measured value and INP measured value in an axillary lymph node about the mouse | mouth inoculated with FM3A-Luc cell. It is a graph showing the INP measurement value of the secondary lymph node after sentinel lymph node resection. FIG. 4 is a graph showing changes in luciferase activity over time in cancer-bearing lymph nodes described in Example 3. It is the image figure which visualized light emission of the luciferase acquired by IVIS of the model mouse in Example 3. FIG. 6 is a graph showing changes over time in cancer-bearing lymph nodes INP in Example 3. It is the graph which showed the change over time of the volume of the lymph node measured using VEVO in Example 3. It is the graph which showed the change over time of the blood vessel density of the lymph node measured using VEVO in Example 3. It is a B-mode ultrasonic image figure of the maximum smooth surface which concerns on the blood vessel density of the lymph node measured using VEVO in Example 3. FIG.

Details of the present invention will be described below.
The present invention relates to a program for evaluating a risk of metastasis in a lymph node, and is a program for causing a computer to realize at least the following steps (A) and (B).
(A) A step of obtaining a measured value of intranodal pressure of the regional lymph node of the primary lesion in the subject (B) By confirming an increasing tendency of intranodal pressure from the measured value, there is a risk of metastasis in the regional lymph node. Step to evaluate that there is

The program of the present invention is based on a method for evaluating the risk of metastasis in lymph nodes, and the method includes at least the following steps (1) and (2).
(1) The step of measuring the intranodal pressure of the regional lymph node of the primary lesion in the subject (2) The step of evaluating that there is a risk of metastasis in the regional lymph node when the intranodal pressure tends to increase

In the present invention, “assessment of the risk of metastasis in lymph nodes” means, for example, sentinel lymph nodes called lymph nodes where tumor cells first metastasize from the primary lesion, and regional lymph nodes downstream from the sentinel lymph nodes. It can be understood as assessing the risk of metastasis caused by the migration of tumor cells to the node. In addition, assessing the risk of metastasis means both predicting metastasis that metastasis may occur in the future even if no metastasis can be found, and detecting metastasis that confirms that metastasis has actually occurred. Include.

The reason why IFP is high in and around solid tumors has not yet been clarified, and the technical scope of the present invention is not limited by any reasoning. It may be due to the inflow of interstitial fluid from the resulting tumor neovascularization and microvasculature associated with abnormal lymphatic structure and function into the stroma. On the other hand, there is a probability that leakage from tumor neovascularization does not contribute to the increase in INP. Thus, for example, the increase in INP may have been caused by the growth of a tumor in a lymph node, an encapsulated region with a capsule. However, there is a probability that the increase in INP correlates with the growth of the tumor in the lymph node itself.

In step (1), the intranodal pressure (INP) of the regional lymph node of the primary lesion in the subject is measured.

The subject is not particularly limited as long as it is an organism having a lymph node, and examples thereof include mammals such as humans, rats, mice, rabbits, dogs, cats, and the like, but preferably humans.

The regional lymph node is not particularly limited as long as it is highly likely that tumor cells migrate from the primary lesion and develop metastasis.For example, in addition to the lymph node directly connected to the primary lesion, the downstream lymph node in the vicinity of the primary lesion The lymph nodes in are included.

In the present invention, the regional lymph node of the primary lesion in the subject is preferably a regional lymph node downstream of the primary lesion. The regional lymph node can be appropriately selected by those skilled in the art, and examples thereof include an axillary lymph node, a cervical lymph node, a thoracic lymph node, and an abdominal lymph node.

The method for measuring INP of the lymph node is not particularly limited. For example, as described in the examples described later, a pressure transducer is connected to the first mouth and physiological saline is contained in the second mouth. There is a method using a device equipped with a three-way cock that connects a syringe and a syringe needle to the third mouth. When using this device, the syringe needle is pushed in to fill the needle and pressure transducer with saline, and the needle is inserted into the lymph node to measure the pressure. The pressure measured in this way is the intranode pressure (INP). The pressure detected by the pressure transducer is preferably obtained as a value amplified by a transducer amplifier connected to the pressure transducer. Further, it is more preferable to connect a transducer amplifier and a personal computer equipped with analysis software, and collect and analyze the pressure measured continuously or intermittently on the personal computer.

INP is, for example, an intra-node INP value that is a difference value before and after insertion into a lymph node. When measuring INP, the skin of the subject may be incised and an injection needle may be inserted into the exposed lymph node, or the site of the lymph node located above the subject's skin. You may stab an injection needle. During the measurement of INP, it is preferable to fix the subject, and it is more preferable to fix the subject under anesthesia.

In the step (2), when the INP measured in the step (1) tends to increase, it is evaluated that there is a risk of metastasis in the regional lymph node where the INP is measured or in another regional lymph node.

A method for determining whether or not the INP has an upward tendency is not particularly limited. For example, when the INP increases with time by measuring the INP, it is determined that the INP has an upward tendency. be able to. In this case, the period for measuring INP is not particularly limited, and examples thereof include several hours to several tens of days, preferably 1 day or more, more preferably 3 days or more, and further preferably 6 days or more. The interval and the number of times of measuring INP are not particularly limited. For example, it can be measured several times to several tens of times at regular intervals or unequal intervals within the measurement period, and can be 6 hours or 12 hours within the measurement period. It is preferable to measure every 24 hours or 48 hours.

For example, as soon as the primary lesion is confirmed, the INP of the regional lymph node is measured, and the INP value, for example, the INP value at the lower limit of detection, preferably the INP value of 0.1 Pa or more, more preferably the INP value of 1 Pa or more, more preferably When an INP value of 10 Pa or higher, more preferably 20 Pa or higher is detected, it can be evaluated that the measured lymph node has a risk of metastasis, assuming that the INP tends to increase.

The evaluation result may be expressed in any way, for example, predicting that the measured lymph node is a secondary metastatic lymph node, and determining that the measured lymph node is a secondary metastatic lymph node, etc. Can be expressed as

By applying the method of the present invention, the prognosis and effects of treatment of the primary lesion can be confirmed. Examples of the method according to another aspect of the present invention include a method for evaluating the prognosis of a treated primary lesion, which includes the following steps (1 ′) and (2 ′).
(1 ′) the step of measuring the intranodal pressure of the regional lymph node of the treated primary lesion in the subject (2 ′) when the intranodal pressure tends to increase, the treatment at the primary lesion is insufficient Process to evaluate

Moreover, as a method of another aspect of this invention, the method of evaluating the chemical | medical agent for treating a primary focus including the following processes (1 '') and (2 '') is mentioned, for example.
(1 ″) measuring the intranodal pressure of the regional lymph node of the treated primary lesion in the subject (2 ″) when the intranodal pressure tends to be maintained, the treatment is performed on the primary lesion The process of evaluating that it has an effect

In the method according to another aspect of the present invention, the treatment of the primary lesion is not particularly limited. For example, dissection treatment for excising part or all of the primary lesion, chemical treatment for administering a drug, radiation treatment for irradiating radiation, etc. Is mentioned. Even if such treatment is applied to the primary lesion, metastasis may occur if the regional lymph node INP tends to increase, and it can be evaluated that treatment of the primary lesion is insufficient. On the other hand, if the regional lymph node INP tends to be maintained, the possibility of metastasis is reduced, and it can be said that a certain therapeutic effect on the primary lesion was obtained. According to these methods of another aspect of the present invention, minimally invasive dissection and reduction of dissection frequency; determination of drug screening usage and dose; determination of irradiation time and interval Can be expected.

In the method of the present invention, as long as the object of the present invention can be achieved, various processes and operations can be added before or after the above-described process or in the middle of the process. As such a step, for example, there is a step of confirming the presence of other metastasis or tumor other than the INP measurement of the regional lymph node. For example, a step of obtaining lymph node images such as CT, MRI, PET, and ultrasound; a step of measuring interstitial fluid volume (IFV) of regional lymph nodes; a step of measuring volume of regional lymph nodes Can be mentioned. The step of measuring the INP of the regional lymph node may be performed with reference to Non-Patent Document 2, for example. In addition, the step of measuring the volume of the regional lymph node refers to the description of the examples described later, and creates a three-dimensional image by joining two-dimensional ultrasound images of the regional lymph nodes taken continuously by VEVO. Then, the volume of this three-dimensional image can be calculated on the VEVO software, and the volume of the entire regional lymph node can be measured.

Specific embodiments of the method of the present invention are as follows, for example, but the present invention is not limited thereto.
[Specific embodiment]
A state where the primary lesion has been found or the subject has been treated under anesthesia, the skin where the regional lymph node of the primary lesion is located is incised, and the skin tissue is exfoliated on the muscle tissue fascia Secure with. Next, connect the pressure transducer to the first mouth, connect the syringe containing physiological saline to the second mouth, and connect the syringe needle to the third mouth. The INP is measured by filling the needle and pressure transducer with saline by pushing in and inserting the needle into the regional lymph node. INP may be measured by inserting a needle near the center of the lymph node. The obtained INP measurements are collected and analyzed by a transducer amplifier and a personal computer connected to the pressure transducer. When INP increases over time from the start of measurement, INP tends to increase, and it is determined that there is a possibility of metastasis in the measured regional lymph nodes or that there is already metastasis.

In the method of the present invention, when it is evaluated that there is a risk of metastasis in a lymph node, it is preferable to perform antitumor treatment on the regional lymph node where INP was measured or other regional lymph nodes. As described above, the method of the present invention enables early treatment or preventive treatment of metastasis of regional lymph nodes existing downstream of the primary lesion.

The program of the present invention is basically a program for evaluating the risk of metastasis in lymph nodes for causing a computer to implement the above-described method of the present invention.

According to another aspect of the present invention, a computer-readable storage medium recording the program of the present invention is provided. Examples of the medium type include, but are not limited to, a magnetic or electric medium. The program of the present invention is recorded in accordance with the type of the medium, and the recording method can be used without limiting methods known in the art.

According to another aspect of the present invention, there is provided a lymph node metastasis evaluation apparatus for evaluating the risk of metastasis in a lymph node based on the method, program or medium of the present invention, and such an apparatus has the following configuration. Can take:
(A) a lymph node pressure measuring means for obtaining a measured value of the intranodal pressure of the regional lymph node of the primary lesion in the subject;
(B) computing means for analyzing the intranodal pressure obtained by the lymph node pressure measuring means and calculating a tendency of the intranodal pressure;
(C) Evaluation means for evaluating that there is a risk of metastasis in the regional lymph node by confirming the increasing tendency of the intranodal pressure from the tendency of intranodal pressure calculated by the computing means.

The lymph node metastasis evaluation apparatus of the present invention is an apparatus that enables the program of the present invention, and belongs to the technical scope of the present invention. The lymph node metastasis evaluation apparatus of the present invention includes, as shown in FIG. 5, for example, a T-type cock stopper provided with an injection needle, a syringe and a pressure transducer, and a pressure transducer amplifying apparatus ((a) pressure measuring means in lymph node) ) And a computer ((b) calculation means and (c) evaluation means) installed with the program of the present invention can be configured to realize the program of the present invention.

The operation method of the lymph node metastasis evaluation apparatus of the present invention includes, for example, the following steps (A) and (B):
(A) A step of obtaining a measured value of intranodal pressure of the regional lymph node of the primary lesion in the subject (B) By confirming an increasing tendency of intranodal pressure from the measured value, there is a risk of metastasis in the regional lymph node. Step to evaluate that there is

The operation method of the lymph node metastasis evaluation apparatus of the present invention can be easily carried out using a computer by creating an appropriate computer program (or software) that can process data according to the above procedure. Such a computer program can itself be useful as it can be used to implement the method of operating the lymph node metastasis assessment apparatus. An example of such a computer program is the program of the present invention.

The program and apparatus of the present invention can be used not only in clinical situations but also in situations such as testing and research. Further, the program and apparatus of the present invention can be handled without particular limitation such as not only clinical specialists such as doctors but also specialist assistants and device manufacturers. When used in a clinical setting, it can be used at any stage before, during, or after the diagnosis of a doctor. Therefore, the program and apparatus of the present invention can be used as a means for early detection of regional lymph node metastasis, or as a preliminary determination means for the purpose of preventing regional lymph node metastasis. For the purpose of preventing metastasis after tumor treatment, it can also be used as a retrospective determination means. If the determination result using the program and apparatus of the present invention and the diagnosis result of the doctor are used together, the risk of metastasis of the regional lymph node can be evaluated with very high accuracy. You may use the program and apparatus of this invention for the screening of an anticancer agent, or the performance evaluation of an anticancer agent.

In addition, the program or apparatus of the present invention measures the increase in regional lymph node INP induced by cancer metastasis from the primary lesion, thereby determining the presence or absence of lymph node micrometastasis and the direction of lymph flow between lymph nodes. It makes detection possible. Therefore, in combination with an ultrasound diagnostic imaging system, in addition to enabling detection of micrometastasis in preoperative, intraoperative and postoperative regional lymph nodes in real time, it is possible to grasp the flow of the lymph network Therefore, development of minimally invasive lymph node dissection and a new lymphatic drug administration system can be expected.

In addition, it is known that an increase in IFP in the tumor reduces the amount of drug delivery and causes hypoxia to reduce the radiotherapy effect. Therefore, if the risk of metastasis of regional lymph nodes can be detected at an early stage, efficient chemotherapy and radiotherapy can be expected. Therefore, the present invention contributes not only to assessing the risk of metastasis in regional lymph nodes, but also to achieving efficient treatment of metastasis in regional lymph nodes.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various modes as long as the problems of the present invention can be solved. .

By measuring the intranodal pressure (INP) of both sentinel and secondary lymph nodes, it is possible to evaluate lymph nodes where tumor cell metastasis is expected, and chemotherapy, radiation therapy, etc. It was demonstrated as follows that the anti-tumor effect can be determined by.

[1. Experimental Materials and Methods]
1-1. Animal model The mouse model MXH10 / Mo- lpr / lpr (hereinafter also referred to as MXH10 / Mo / lpr) independently developed by the present inventors was used (see References 1 and 2 described later). MXH10 / Mo / lpr is a recombinant inbred line obtained by mating over 20 generations of siblings based on MRL / Mp- lpr / lpr (MRL / lpr) and C3H / HeJ- lpr / lpr (C3H / lpr) mice. (RI) A group of mice. In this lineage group, lines that randomly develop glomerulonephritis, vasculitis, arthritis, etc. in various combinations are recognized. Among the group strains, MXH10 / Mo / lpr mice are the tenth strains that are particularly stable in the onset of lymphadenopathy. In particular, the subiliac lymph nodes (SiLN) and axillary lymph nodes (PALN) of MXH10 / Mo / lpr mice are swollen and visualized to the same size as human lymph nodes (about 10 mm in major axis) at about 12 weeks of age. Excellent in accessibility and accessibility. MXH10 / Mo / lpr mice lack the Fas gene for apoptosis, but maintain basic lymph node anatomy such as medulla, cortex, paracortex. Furthermore, the fatal glomerulonephritis seen in MRL / MpJ- lpr / lpr which is the founder of this mouse and is a disease model mouse of collagen disease does not develop.

In this example, a total of 72 female MXH10 / Mo / lpr mice aged 14 to 16 weeks were used. The body weight was 27-37 g, and the major diameters of the subiliac and medial axillary lymph nodes measured with calipers were 10.8 ± 1.9 mm (mean ± SD; n = 12) and 10.4 ± 1.6 mm, respectively. (N = 12).

1-2. As tumor cell-inoculated cells, mouse fibroblast-derived MRL / KM-Luc / GFP cells (see References 3 to 5; hereinafter also referred to as KM-Luc / GFP cells) and mouse breast cancer cell-derived FM3A- Luc cells were used. KM-Luc / GFP cells are obtained by transfecting MRL / N1 cells, which are tumor cells derived from MRL / MpTn-gld / gld mouse fibroblasts, with pEGFPPLuc plasmid constitutively expressing the luciferase gene. (See references 4 and 5). 10% (V / V) FBS (fetal bovine serum; Thermo Fisher Scientific), 1% (V / V) L-glutamine-P. S. Dulbecco's modified Eagle's medium (Sigma-Aldrich) containing the solution (Sigma-Aldrich) was used as a medium and cultured at 37 ° C., 5% CO ₂ + 95% air. Further, FM3A-Luc cells are obtained by introducing pGL4.51 (Invitrogen) by electroporation into FM3A cells, which are mouse breast cancer cells, using Gene Pulser Xcell (BioRad). Yes, the luciferase gene is constitutively expressed. 10% (V / V) FBS (fetal bovine serum), 1% (V / V) L-glutamine-P. S. RPMI-1640 (Sigma-Aldrich) containing the solution was used as a medium, and cultured at 37 ° C. in 5% CO ₂ + 95% air. KM-Luc / GFP cells express VEGF-A, VEGF-B and VEGF-D, and FM3A-Luc cells express VEGF-A (see FIGS. 1 and 2).

1-3. Cell-inoculated tumor cells were prepared using phosphate buffered saline (PBS; Sigma-Aldrich) and Matrigel (Collaborative Biomedical Products) without Ca ²⁺ and Mg ²⁺ in suspension. After adjusting the concentration to 3 × 10 ⁵ cells / mL, the mice were inoculated into the unilateral subiliac lymph node. At that time, under inhalation anesthesia with 1 to 2% (V / V) isoflurane (Abbott Japan), the needle of the injection needle into the lymph node with a high frequency ultrasonic imaging device for small animals (VEVO) (Visual Sonic) This was done while confirming the point (see FIGS. 3A and 3B). It can be confirmed that the tip of the injection needle is located at the center of the subiliac lymph node (FIG. 3A), and it can be confirmed that a mass of tumor cells is formed immediately after inoculation (FIG. 3B). The inoculated state was kept for about 1 minute and allowed to stand so that the tumor cells did not exude after tumor cell inoculation. A transducer having a center frequency of 25 MHz, a distance resolution of 70 μm, and an azimuth resolution of 140 μm was used. The inoculum is 60 μL. During the measurement, the mouse is placed under inhalation anesthesia. As a control group, mice inoculated only with a turbid solution of PBS and Matrigel were prepared (n = 12).

1-4. Measurement of luciferase activity KM-Luc / GFP and FM3A-Luc used in this example are luciferase-expressing cells, and the number of cells can be confirmed by luciferin-luciferase reaction. Since cells that have caused a luciferin-luciferase reaction generate luminescence, the amount of luminescence was measured using a bioluminescence imaging system (IVIS; Lumina Imaging System) (Xenogen). In this example, luciferase activity was measured for subiliac lymph nodes directly inoculated with tumor cells and axillary lymph nodes that were assumed to be metastatic lesions. 200 μl of luciferin (D-luciferin potassium salt) adjusted to a concentration of 15 mg / ml was injected intraperitoneally, and measured 10 minutes later using IVIS. During the measurement, the mouse was placed under inhalation anesthesia.

1-5. Axillary lymph node volume measurement Mice inoculated with tumor cells were fixed under inhalation anesthesia, and axillary lymph node volume was measured over time with VEVO. The ultrasonic images were integrated on the VEVO software, and the two-dimensional images taken continuously were connected to produce a three-dimensional image (see FIG. 4). The volume of this three-dimensional image was calculated on VEVO software, and the volume change of the entire axillary lymph node was measured. At this time, a transducer having a center frequency of 25 MHz, a distance resolution of 70 μm, and an azimuth resolution of 140 μm was used.

1-6. INP measurement in the lymph node The INP value in the subiliac lymph node and the axillary lymph node was measured for the control group and the mouse inoculated with the tumor cells. As a measuring instrument, a pressure transducer (BLPR2; World Precision Instruments) and a transducer amplifier (Bridge-8; World Precision Instruments) were used. A personal computer equipped with analysis software (LabScribe2; iWork) was connected to an amplifier and a pressure transducer. A Terumo syringe (Terumo) containing physiological saline and a 21G Terumo injection needle (Terumo) were connected to the transducer via a three-way stopcock (Terumo). The syringe needle and the pressure transducer were filled with physiological saline by pushing in the syringe, and the pressure was measured by inserting the syringe needle into the node. The difference between before and after insertion into the lymph node was taken as the intra-node INP value. During the measurement, the mouse was anesthetized with 1-2% (V / V) isoflurane using a small animal tabletop anesthesia apparatus (Shinano). Under anesthesia, a skin incision from the lower iliac to the axilla was added to the abdomen of the mouse, and the skin tissue was fixed in a state of peeling on the abdominal muscle tissue fascia, and INP was measured (see FIGS. 5 and 6). . After the measurement, the mice were euthanized by cervical dislocation, and then the lymph nodes were removed and stored overnight in 10% (V / V) formalin. The INP pressure was measured by distributing the mice on each day of pressure measurement. The day of tumor cell inoculation was defined as day 0 (Day 0), and KM-Luc / GFP cells were divided into 12 mice each on Day 3 and Day 6 (see FIG. 7B). For the Day 6 group, luciferase activity and axillary lymph node volume were measured over time. FM3A-Luc cells were divided into groups of 12 each on Day 6, Day 10 and Day 14 (see FIG. 7C). For the Day 14 group, luciferase activity and axillary lymph node volume were measured over time. Moreover, 12 animals were used for pressure measurement also in the control group inoculated with cells (see FIG. 7A).

1-7. About the measurement result of the luciferase activity by statistical processing IVIS, it shows by the average value +/- standard deviation (mean +/- SD), and other measured values are shown by average value +/- standard error (mean +/- SEM). The multi-group test used one-way analysis of variance (ANOVA), and the multiple comparison test performed the Tukey-Kramer test. When P <0.01 (**) and P <0.05 (*), it was evaluated as statistically significant. The software used was JMP 11.0 Academic Edition (SAS Institute Japan).

[2. result]
2-1. Measurement results of luciferase activity by IVIS measuring luciferase activity in subiliac and axillary lymph nodes are shown in FIGS. FIG. 8 shows the measurement results of mice inoculated with KM-Luc / GFP cells, and FIG. 9 shows the results of mice inoculated with FM3A-Luc cells. From FIG. 8A and FIG. 9A, it was possible to confirm tumor engraftment in the subiliac and axillary lymph nodes. In particular, luminescence of axillary lymph nodes by IVIS could be confirmed at Day 6 in the KM-Luc / GFP cell inoculated group and at Day 14 in the FM3A-Luc cell inoculated group. 8B and 9B show changes over time in the amount of luciferase luminescence by calculating and graphing the amount of luciferase luminescence in the subiliac and axillary lymph nodes by IVIS. In FIG. 8B, the value of luciferase activity in Day 6 was statistically significant compared to Day 0 in both the subiliac and axillary lymph nodes. Similarly, in FIG. 9B, the value of luciferase activity in Day 14 was statistically significant compared to Day 0 in both the subiliac and axillary lymph nodes. From FIG. 8B and FIG. 9B, it was confirmed that in both types of cells, the tumor first grew in the subiliac lymph node, and the tumor engrafted and grew later in the axillary lymph node.

2-2. Changes in Volume of Axillary Lymph Nodes Changes in volume of axillary lymph nodes due to VEVO are shown in FIGS. FIG. 10 shows the measurement results of mice inoculated with KM-Luc / GFP cells, and FIG. 11 shows the results of mice inoculated with FM3A-Luc cells. 10A and 11A show ultrasonic images of the maximum smooth surface of the axillary lymph node, and FIGS. 10B and 11B are graphs showing changes in the lymph node volume calculated by the VEVO software. Although both KM-Luc / GFP and FM3A-Luc showed a slight increase in volume, the amount of change was slightly different.

2-3. INP measurement results in the subiliac and axillary lymph nodes INP measurement results in the lymph nodes are shown in FIGS. In addition, scatter diagrams showing the relationship between the INP value and the luciferase activity are shown in FIGS. FIGS. 12 and 14 show the measurement results of mice inoculated with KM-Luc / GFP cells, and FIGS. 13 and 15 show the results of mice inoculated with FM3A-Luc cells. Further, FIGS. 12A to 15A are measurement results of subiliac lymph nodes, and FIGS. 12B to 15B are measurement results of axillary lymph nodes. 12 and 13, in both types of cells, the INP values in the subiliac and axillary lymph nodes tend to increase, and the tendency is so significant that a statistically significant difference is obtained under the iliac. confirmed. 14 and 15, it can be considered that the value of luciferase activity increases as the INP value increases, that is, the tumor size increases in both the subiliac and axillary lymph nodes.

[3. Evaluation]
By making a metastatic lesion in the axillary lymph node using MXH10 / Mo / lpr mice, changes in INP values in the sentinel lymph node and the secondary metastatic lymph node during the metastasis process were grasped. As a result, it was suggested that there is a correlation between the degree of tumor growth and the INP value in the lymph node. Thus, it was shown that tumor cell metastasis to the lymph node can be assumed by measuring the pressure change of the lymph node. It was also suggested that the measurement of lymph node INP values may be effective in determining the antitumor effect by chemotherapy or radiotherapy.

[4. References]
1. Shao L, Mori S, Yagishita Y, Okuno T, Hatakeyama Y, Sato T, et al. Lymphatic mapping of mice with systemic lymphoproliferative disorder: Usefulness as an inter-lymph node metastasis model of cancer. J Immunol Methods. 2013; 389: 69-78.
2. Tanaka Y, Komori H, Mori S, Soga Y, Tsubaki T, Terada M, et al. Evaluating the role of rheumatoid factors for the development of rheumatoid arthritis in a mouse model with a newly established ELISA system.The Tohoku journal of experimental medicine. 2010; 220: 199-206.
3. Li L, Mori S, Kodama M, Sakamoto M, Takahashi S, Kodama T. Enhanced Sonographic Imaging to Diagnose Lymph Node Metastasis: Importance of Blood Vessel Volume and Density. Cancer research. 2013; 73: 2082-92.
4. Ito MR, Terasaki S, Itoh J, Katoh H, Yonehara S, Nose M. Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand. Arthritis Rheum. 1997; 40: 1054-63.
5. Furukawa H, Kitazawa H, Kaneko I, Kikuchi K, Tohma S, Nose M, et al. Mast cells inhibit CD8 + T cell-mediated rejection of a malignant fibrous histiocytoma-like tumor: Involvement of Fas-Fas ligand axis. Am J Immunol. 2009; 5: 89-97.
6. Rofstad EK, Tunheim SH, Mathiesen B, Graff BA, Halsor EF, Nilsen K, et al. Pulmonary and lymph node metastasis is associated with primary tumor interstitial fluid pressure in human melanoma xenografts. Cancer research. 2002; 62: 661- Four.
7. Ellingsen C, Hompland T, Mathiesen B, Rofstad EK. Microenvironment-associated lymph node metastasis of human cervical carcinoma xenografts. Acta Oncol. 2012; 51: 465-72.
8. Hompland T, Ellingsen C, Ovrebo KM, Rofstad EK. Interstitial Fluid Pressure and Associated Lymph Node Metastasis Revealed in Tumors by Dynamic Contrast-Enhanced MRI. Cancer research. 2012; 72: 4899-908.

Even after removal of the sentinel lymph node, it was demonstrated as follows that the INP value of the secondary metastatic lymph node increases when metastasis occurs.

[1. Experimental Materials and Methods]
1-1. Animal model Sixteen female MXH10 / Mo / lpr mice aged 13 to 15 weeks were used. The body weight was 27 to 37 g, and the major diameters of the subiliac and medial axillary lymph nodes measured with calipers were 10.8 ± 1.9 mm (mean ± SD; n = 16) and 10.4 ± 1.6 mm, respectively. (N = 16).

1-2. Cell culture mouse-derived fibroblasts KM / Luc were used. 10% (V / V) FBS (Thermo Fisher Scientific), 1% (V / V) L-glutamine-P. S. DMEM (Sigma-Aldrich) containing the solution (Sigma-Aldrich) was used as a medium, and cultured at 37 ° C., 5% CO ₂ + 95% air.

1-3. A cell suspension diluted in PBS as a cell-inoculated cell solution and Matrigel (Basement membrane matrix; Becton Dickinson) were mixed at a ratio of 2: 1 to prepare a final concentration of 3.3 × 10 ⁵ cells / mL. did. Anesthesia was performed with 2% (V / V) isoflurane (Abbott Japan Co., Ltd.), and the cell solution was inoculated locally into the subiliac lymph nodes of mice under 60 μL under the guidance of a high-frequency ultrasonic imaging apparatus.

1-4. In vivo, 6 hours after inoculation of sentinel lymph node excised tumor cells, on days 3 and 9, the subintestinal lymph node, which is the sentinel lymph node, was exposed to expose a high-frequency coagulation incision device (Erbotom VI050C; ERBE Elektromedizin) It was excised with. After excision, the exposed site was sutured and ligated. On day 9, intranodal pressure (INP) values of axillary lymph nodes were measured.

[2. Results and Evaluation]
When the subiliac lymph nodes inoculated with tumor cells were removed 6 hours, 3 days and 6 days after inoculation, if the sentinel lymph nodes were removed 6 hours after inoculation, the secondary lymph nodes had an INP value. No increase was observed, and a marked increase in the INP value was observed 3 days and 6 days after the inoculation (see FIG. 16). From the results of FIGS. 8A and 8B shown in Example 1, metastasis does not occur when the sentinel lymph node is excised 6 hours after tumor cell inoculation, but metastasis can occur after 3 days after inoculation. Therefore, FIG. 16 shows that a significant increase in the INP value was observed 3 and 6 days after the inoculation due to the occurrence of metastasis in the secondary lymph node. Indicates that the error occurred.

We conducted an experiment to treat tumors with ultra-early lymph node metastases, which can be detected by measuring intranodal pressure (INP), which is extremely difficult to detect with conventional imaging modalities. It was demonstrated as follows that the therapeutic effect of a cancer drug can be evaluated by INP.

[1. Experimental Materials and Methods]
1-1. Cell culture MRL / Tn- gld / gld KM-Luc / GFP cell line transformed with spleen-derived MRL / N1 cells was used. The medium contains 10% FBS (Sigma-Aldrich), 1% L-glutamine-penicillin-streptomycin solution (Sigma-Aldrich) and 0.5% Geneticin G418 (Wako Pure Chemical Industries). Sigma-Aldrich). KM-Luc / GFP cells were confirmed to be free from mycoplasma contamination in cell culture using a mycoplasma detection kit (R & D Systems) before inoculating mice.

1-2. Preparation of Lymph Node Carcinoma Model MXH10 / Mo / lpr mice (n = 51), which is a lymph node metastasis mouse model with constant lymphatic swelling, were used. Inoculate 60 μL of a cell solution in which 20 μL of MRL / KM-Luc cell suspension diluted with PBS to 1.0 × 10 ⁵ cells / mL and 40 μL of Matrigel is inoculated into the subiliac lymph nodes (SiLN) of mice. A lymph node cancer model was prepared. The day of inoculation was defined as day 0, 200 μL of luciferin (Promega) adjusted to a concentration of 15 mg / mL on days 3 and 6 was injected intraperitoneally into the mouse, and 10 minutes later, luciferase activity using IVIS (Xenogen) Was measured.

1-3. Measurement of INP The mouse was opened in 2% isoflurane anesthesia, SiLN and PALN were exposed, a pressure transducer (World Precision Instruments) was connected, and a 21G needle filled with physiological saline to the tip was lymphatic Inserted into the center of the knot. The measurement was performed for 5 minutes, and the average value was set to INP. After the measurement, the mouse was immediately euthanized in the anesthesiology department.

1-4. Treatment intervention with anticancer agent Cisplatin (CDDP), an anticancer agent, was diluted to 3 mg / kg body weight of mice with physiological saline, and 60 μL of CDDP solution was inoculated on the first and fourth days after tumor inoculation. Injection into the subiliac lymph node. Treatment evaluation was quantitatively analyzed from bioluminescence imaging with a bioluminescence imaging device (IVIS) 3 days and 6 days after tumor inoculation.

1-5. Measurement of blood vessel density with ultrasonic contrast agent Sonazoid (16 μl for sonazoid injection; Daiichi Sankyo Co., Ltd.) which is a contrast agent for ultrasonic diagnosis was used. 16 μl of perflubutane microbubbles were suspended in 2 ml of water for injection, and 100 μl per mouse was injected into the tail vein, and SiLN was photographed using VEVO (Vevo770; Visualsonics). During image acquisition, the mouse was left under anesthesia on a heat stage set at around 37 ° C. In the contrast mode, 300 frames were photographed every 0.5 mm in the longitudinal direction of the SiLN, and blood vessels were imaged using the contrast agent detection software developed uniquely.

1-6. Statistical analysis The luciferase activity and INP measurement results were subjected to statistical analysis using GraphPad Prism 6 (GraphPad Software). The value was expressed as mean ± SD, and statistically significant when P <0.05.

[2. Results and Evaluation]
2-1. Measurement of Luciferase Activity FIGS. 17A and 17B show changes in luciferase activity over time after tumor inoculation to lymph nodes. As shown in FIGS. 17A and B, tumors were inoculated into lymph nodes and treatment intervention was not performed (□: Tumor + PBS) showed an increase over time (P <0.001). On the other hand, the treatment intervention group (◯: Tumor + CDDP) showed no significant increase in activity 6 days after tumor inoculation. In addition, the untreated group was significantly larger than the treated group and non-tumor-inoculated group (P <0.001).

2-2. Tumor growth and INP in lymph nodes
FIG. 18 shows how the INP changes over time. In the untreated group, INP increased with tumor progression (P <0.001). Moreover, it was confirmed that INP of the treatment group was smaller than that of the non-treatment group (P <0.001). In the group administered with CDDP alone, no change was observed in INP, which was smaller than that in the untreated group (P <0.001).

2-3. Measurement of Lymph Node Volume FIG. 19 shows changes in lymph node volume over time. There was no change in the untreated group by 6 days after tumor inoculation. In addition, it was confirmed that the volume of the treatment group and the CDDP-inoculated group decreased over time, and the volume after 6 days after tumor inoculation was smaller than that of the untreated group (P <0.001).

The group inoculated with CDDP showed a decrease in volume regardless of the presence or absence of a tumor (P <0.001).

2-4. Measurement of blood vessel density FIGS. 20A and 20B show changes in blood vessel density of lymph nodes 3 days and 6 days after tumor inoculation. Tumor growth and CDDP inoculation did not change blood vessel density. That is, the blood vessel density on the maximum smooth surface did not change over time in any group, and there was no difference between the groups.

2-5. Evaluation From the above results, it was shown that INDP is suppressed to a low value in cancer-bearing lymph nodes where administration of CDDP should originally increase. CDDP binds to DNA at the base atom site to inhibit DNA synthesis and exerts an antitumor effect, and thus does not inhibit tumor-induced angiogenesis. In addition, the blood vessel density at 6 days after tumor inoculation when INP became high did not change from that at day 3 of tumor inoculation, and there was no change in blood vessel density in the group administered CDDP regardless of the presence or absence of cancer. Therefore, it is suggested that leakage from tumor neovascularization does not contribute to the increase of INP. The increase in INP may be caused by the growth of the tumor in the lymph node, an encapsulated area with a capsule. Therefore, the administration of CDDP inhibited tumor growth in the lymph nodes, and therefore it was confirmed that INP could be reduced, and that measurement of INP could evaluate tumor growth in cancer-bearing lymph nodes. It was also shown that INP measurement can be used for diagnosis of early stage metastatic lymph nodes. The increase in INP correlates with the growth of tumor in the lymph node itself, and tumor neovascularization in the lymph node may not affect the increase in INP.

According to the method and program of the present invention, since the risk of metastasis in lymph nodes can be efficiently evaluated as compared with the conventional method using CT, MRI, PET, ultrasound images, etc., tumor metastasis Probability can be reduced, and it can be expected to increase the survival rate of cancer patients. Thus, the method and program of the present invention contribute to human health and welfare.

Claims (6)

(A) obtaining a measured value of intranodal pressure of a regional lymph node downstream of the sentinel lymph node of the primary lesion in the subject over time ;
(B) Evaluating the risk of metastasis in the lymph nodes to make the computer realize the step of evaluating that there is a risk of metastasis in the regional lymph nodes by confirming the increasing tendency of the intranodal pressure from the measured values Program to do. The program according to claim 1, wherein the regional lymph node is a regional lymph node selected from the group consisting of an axillary lymph node, a cervical lymph node, a thoracic lymph node, and an abdominal lymph node . A computer-readable storage medium in which the program according to claim 1 or 2 is recorded. (A) an intranodal pressure measuring means for obtaining a measured value of the intranodal pressure of the regional lymph node downstream of the sentinel lymph node of the primary lesion in the subject;
(B) a computing means for analyzing the intranodal pressure obtained over time by the lymph node pressure measuring means and calculating a tendency of the intranodal pressure;
(C) an evaluation means for evaluating that there is a risk of metastasis in a regional lymph node by confirming an increasing tendency of the intranodal pressure from the tendency of intranodal pressure calculated by the computing means; A lymph node metastasis evaluation device that evaluates the risk of metastasis. The device according to claim 4, wherein the regional lymph node is a regional lymph node selected from the group consisting of an axillary lymph node, a cervical lymph node, a thoracic lymph node, and an abdominal lymph node . (1) measuring the intranodal pressure of the regional lymph node downstream of the sentinel lymph node of the primary lesion in the subject over time ;
(2) A method for evaluating a risk of metastasis in a lymph node, comprising a step of evaluating that there is a risk of metastasis in a regional lymph node when the intra-nodal pressure tends to increase.