JP5405059B2 - Cancer diagnosis support system, cancer diagnosis support information provision device, and cancer diagnosis support information provision method - Google Patents

Description

The present invention relates to a cancer diagnosis support system , a cancer diagnosis support information providing apparatus, and a cancer diagnosis support information providing method.

  Conventionally, serodiagnosis for examining tumor markers in serum, histological diagnosis by biopsy (biopsy), and cytodiagnosis are known as cancer diagnosis. However, these are not reliable, or there are variations in individual judgments and medical institution judgments. Therefore, in recent years, molecular diagnosis based on genes and proteins expressed in vivo has been examined as a uniform diagnosis method for cancer with little variation by diagnosticians. As molecular diagnosis based on the protein, various methods using, for example, cyclin-dependent kinase (hereinafter also simply referred to as “CDK”) have been proposed (see, for example, Patent Documents 1 and 2).

  In Patent Document 1, a ratio obtained from the activity value and expression level of CDK1 (CDK1 specific activity) and a ratio obtained from the activity value and expression level of CDK2 (CDK2 specific activity) are measured, and CDK2 specific activity / CDK1 specific activity is measured. Is compared with a preset threshold value, and a method for determining the malignancy of cancer based on these results is described. In Patent Document 2, the expression level of a predetermined gene such as p53BP2 gene, cathepsin B gene, cathepsin L gene, Ki67 / MiB1 gene, thymidine kinase gene, p27 gene or the expression product thereof is normalized with respect to the control gene. A method for predicting a patient's clinical outcome compared to the amount found in a reference cancer tissue set is described.

International Publication No. 2005/116241 Pamphlet JP 2005-519624 A

  In the above method, a predetermined threshold value set in advance and a measured value of a parameter are compared, and prediction of a cancer state and prediction of a clinical result are performed based on the comparison result. However, in such a method, even if the measured value of the parameter is very close to the threshold value, the determination is made based on the threshold value. Therefore, for example, even if the clinical specimen is actually at a high risk of recurrence, the measured value of the parameter obtained from the clinical specimen is slightly lower or higher than the threshold value. Results may be obtained. In such a case, it cannot be said that accurate determination is performed by the above method, and when such a determination result is used in cancer diagnosis, accurate diagnosis may not be performed.

The present invention has been made in view of such circumstances, and when diagnosing a cancer patient by a doctor, another cancer patient having a measured value that approximates a measured value of a predetermined item of a malignant tumor of the cancer patient. providing cancer recurrence rate and / or disease-free survival diagnostic support information diagnosis support system for cancer that is as possible out to provide as the, diagnosis support information providing apparatus and diagnostic support information providing method for cancer cancer For the purpose.

Diagnosis support system for cancer of the present invention is directed to the first cancer patient by measuring the predetermined two kinds of measurement items for prepared specimen using a malignant tumor collected from the first cancer patient a measurement unit take measurements, the first of said predetermined two types of measurement items for each of the specimens prepared with malignant tumors taken from different cancer patients and cancer patients a plurality of measurements generated by measuring, and clinical information after malignant tumor removal of each of the plurality of cancer patients, and serial憶部for storing data including a reference area, and Viewing section, Reading the data of the plurality of measurement values and the reference range from the storage unit, with the two types of measurement items as coordinate axes, the plurality of measurement values and the measurement values related to the first cancer patient are plotted, and Includes measurements for the first cancer patient And a control unit for displaying a view distribution in which the reference range is displayed on the display unit, wherein the storage unit, information as the clinical information, as to whether or not the survival of information about the recurrence and / or post-operative cancer And the control unit obtains from information on recurrence of the cancer of the cancer patient corresponding to the plurality of measurement values within the reference range and / or information on presence or absence of postoperative survival. wherein the display before Symbol display unit diagnosis support screen showing the recurrence rate and / or disease-free survival of cancer.

Since the above-described configuration is adopted in the cancer diagnosis support system of the present invention, other cancer patients having measurement values that approximate the measurement values of the predetermined items of the malignant tumor of the first cancer patient may be used. The recurrence rate and / or disease-free survival rate can be provided as diagnostic support information . Malignant tumors having similar measured values are considered to be similar in nature to each other, and other cancers provided by the cancer diagnosis support system of the present invention when diagnosing the first cancer patient. the recurrence rate and / or disease-free survival rate of cancer patients can and use the child as a diagnostic support information.

In the cancer diagnosis support system of the present invention, it is preferable that the measurement value includes a value related to expression of the cell cycle protein and / or activity of the cell cycle protein.

The cell cycle protein is preferably configured to be a cyclin-dependent kinase.
In addition, the measured value includes the ratio between the activity value of the first cyclin-dependent kinase (first CDK) and the expression level (first ratio), and the activity value and expression of the second cyclin-dependent kinase (second CDK). It is preferable to be configured so as to be a ratio to the amount (second ratio).

Moreover, the clinical information further, it is preferably configured to include information about the post-operative treatment.

A method for providing cancer diagnosis support information in a cancer diagnosis support information providing apparatus including a storage unit and a display unit according to the present invention is a specimen prepared using a malignant tumor collected from a first cancer patient a step of accepting an input of a predetermined two first measurements of cancer patients measurement items generated by measuring per were pre Symbol taken from a plurality of cancer patients different from the first cancer patient A plurality of measurement values and reference range data generated by measuring the two predetermined measurement items for each specimen prepared using a malignant tumor are read from the storage unit, and the two measurement items A distribution diagram in which the reference range including the measurement values relating to the first cancer patient is plotted, and the plurality of measurement values and the measurement values relating to the first cancer patient are plotted. It is displayed on the display unit Reading a step, the information as to whether or not the survival of information about the recurrence and / or post-operative cancer cancer patients corresponding to the plurality of measurement values within the reference range from the storage unit, the read the Executing a step of displaying on the display unit a diagnosis support screen showing a cancer recurrence rate and / or disease-free survival rate obtained from information on cancer recurrence and / or information on whether or not there is postoperative survival. It is characterized by that.

The method for providing information for assisting cancer diagnosis of the present invention, it is possible to provide a diagnosis support information adapted by a first cancer patient, if example embodiment, when diagnosing a first cancer patient, the first The cancer recurrence rate and / or disease-free survival rate of other cancer patients having a measurement value close to the measurement value of the predetermined item of the malignant tumor of the cancer patient can be provided as diagnosis support information .

According to the cancer diagnosis support system, the cancer diagnosis support information providing apparatus, and the cancer diagnosis support information providing method of the present invention, when a doctor diagnoses a cancer patient, the predetermined malignant tumor of the subject cancer patient is determined. other cancer recurrence rate of cancer patients and / or disease-free survival with a measurement value approximate to the measurement value of the item as possible out to provide a diagnosis support information.

  Hereinafter, embodiments of a cancer diagnosis support system (hereinafter also simply referred to as “diagnosis support system”) of the present invention will be described in detail with reference to the accompanying drawings. The diagnosis support system of the present embodiment includes a measurement value of a predetermined measurement item related to a malignant tumor of a cancer patient to be diagnosed, a measurement value of a predetermined measurement item related to a malignant tumor of another cancer patient, and the other Information for supporting cancer diagnosis can be provided based on sample data associated with clinical information after malignant tumor removal of cancer patients.

  Malignant tumors are life-threatening tumors that invade or metastasize to other tissues and grow in various parts of the body. Malignant tumors include carcinomas that are malignant tumors derived from epithelial tissues and sarcomas that are malignant tumors derived from non-epithelial tissues. Specific examples of the malignant tumor include malignant tumors that can be located in the breast, lung, liver, stomach, large intestine, pancreas, uterus, testis, ovary, thyroid, parathyroid, lymphatic lineage, and the like. Malignant tumors can be collected from cancer patients suffering from breast cancer, lung cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, prostate cancer and the like.

  In the diagnosis support system of the present embodiment, clinical information of other cancer patients having measurement values that approximate the measurement values of the predetermined measurement items related to the malignant tumor of the cancer patient to be diagnosed is the diagnosis target. It can be provided as information to support diagnosis for cancer patients (hereinafter also referred to as “cancer diagnosis support information”). The clinical information provided by the diagnosis support system includes, for example, information on the presence or absence of recurrence of other cancer patients having measured values within the reference range. In the diagnosis support system, information such as recurrence rate and disease free survival calculated based on information on the presence or absence of recurrence of other cancer patients having measured values within the reference range, It can also be provided as clinical information.

  Furthermore, in the diagnosis support system of the present embodiment, the malignancy of the cancer is determined based on the measurement value of the predetermined measurement item related to the malignant tumor of the cancer patient to be diagnosed, and the determination result is determined based on the determination result. It can also be provided as cancer diagnosis support information together with clinical information of cancer patients. Specific examples of the malignancy of cancer include ease of metastasis, ease of recurrence, and poor prognosis.

  The predetermined measurement item measured in the diagnosis support system of the present embodiment is not particularly limited as long as it is a measurement item related to a gene and / or protein in a malignant tumor, and the type of gene or protein and the type of measurement item are: The type is selected according to the type of diagnosis and diagnosis support information provided.

  Examples of the predetermined measurement items include Japanese Patent Publication No. 2002-504683, Japanese Patent Publication No. 2002-519681, Japanese Patent Application Laid-Open No. 2002-335997, and Patent Document 1 shown above (International Publication No. 2005/116241 pamphlet). ) And the like, the measured values relating to CDK are mentioned. Specific examples of the measurement values related to the CDK include, for example, the expression level of CDK, the activity value of CDK, the ratio of the activity value to the expression level of CDK (for example, specific activity, the reciprocal of the specific activity, etc.) Can be mentioned. In addition to CDK, in order to predict the recurrence risk of cancer in Japanese Patent Application Laid-Open No. 2005-58113, Japanese Patent Application Laid-Open No. 2006-223303, Japanese Patent Application Laid-Open No. 2005-519624 described above, etc. The expression level of the gene used may be a predetermined measurement item. In addition, the expression level of a gene used for predicting the sensitivity of an anticancer agent in International Publication No. 2005/007846, Japanese Patent Application Laid-Open No. 2005-341862, or the like may be used as a predetermined measurement item.

Hereinafter, as the diagnosis support system of the present embodiment, the expression level and activity value of CDK in a malignant tumor collected from a cancer patient are measured, and the malignancy of cancer (ease of recurrence) based on the obtained measurement value. A diagnosis support system that provides information on the above will be described as an example.
Prior to the description of the diagnosis support system, first, [1] the usefulness of CDK in cancer diagnosis will be described.

[1] Usefulness of CDK in the diagnosis of cancer The measurement value regarding cyclin-dependent kinase (CDK) accurately reflects the state of malignant tumor in a patient suffering from cancer. Therefore, the expression level and activity value of two or more cyclin-dependent kinases in tissues including malignant tumors are measured. For example, the ratio between the activity value and expression level of the first CDK and the activity value of the second CDK Based on the ratio between the expression level and the expression level, the nature of the tissue containing the malignant tumor, the ease of recurrence of cancer, and the like can be evaluated. Moreover, the measured value regarding CDK shows a similar measured value, respectively, in a cancer patient having a malignant tumor in a similar state. Therefore, the expression levels and activity values of two or more cyclin-dependent kinases in tissues containing malignant tumors are measured, for example, the ratio between the activity value and expression level of the first cyclin-dependent kinase and the second cyclin Based on the ratio between the activity value and the expression level of the dependent kinase, the nature of the tissue including the malignant tumor, the ease of recurrence of cancer, and the like can be diagnosed.

  Thus, as a measurement value regarding CDK, for example, the ratio (for example, specific activity) of the activity value and expression level of at least one CDK possessed by a certain tissue, and / or the activity value and expression of a plurality of CDKs. A numerical value calculated by the amount [for example, the ratio of the ratio of the activity value of the first CDK to the expression level (A1) and the ratio of the activity value of the second CDK to the expression level (A2) (for example, “A1 / A2 Or “A2 / A1”) etc.] can be used.

  Here, recurrence refers to the case where the same malignant tumor is reproduced in the remaining organ after partial excision of the organ to remove the malignant tumor, and the tumor cells are separated from the primary lesion and transported to the remote tissue (remote organ). It refers to the case where it proliferates autonomously (metastasis recurrence). In general, when cancer is likely to recur within 5 years after removal surgery, it is said to be “prone to relapse”. Predicting recurrence within 5 years after excision surgery is clinically significant because patients with recurrence within 5 years have a high mortality rate. For example, in stage classification, stage III has a recurrence rate of 50% and is more likely to recur than stage II (recurrence rate of 20%).

  Cyclin-dependent kinase is a general term for a group of phosphorylating enzymes activated by binding to cyclin, and functions at a predetermined time in the cell cycle depending on the type. The CDK inhibitor is a general term for a group of factors that bind to a cyclin / CDK complex and inhibit the activity of the cyclin / CDK complex.

Here, the cell cycle refers to a cycle from when a cell starts to proliferate, undergoes events such as DNA replication, chromosome distribution, nuclear fission, cytokinesis, and the like to become two daughter cells and return to the starting point. As shown in FIG. 14, the cell cycle is divided into four phases of G ₁ phase, S phase, G ₂ phase and M phase. The S phase is a DNA replication phase, and the M phase is a mitotic phase. G ₁ phase is the period from the completion of mitosis and the start of DNA synthesis, is a preparation period for the cell to cells enter phase S. Beyond (R point in animal cells) critical point in the G ₁ phase, it starts the cell cycle, usually without stopping in the middle, a round. G ₂ phase is a period until the start of mitosis from completion of DNA synthesis, which is a preparation period for the cell to cells enter phase M. The main check points of the cell cycle are immediately before entering the S phase from the G ₁ phase (G ₁ check point) and the transition phase from the G ₂ phase to mitosis (G ₂ / M check point). In particular, the G ₁ checkpoint is important because it relates to the start of the S period. This is because, after passing through a certain point in the G ₁ phase, even if the proliferation signal disappears, the cell advances the cell cycle from S phase to G ₂ phase to M phase to G ₁ phase without stopping proliferation. is there. The cells that have stopped growing enter a resting phase (G ₀ phase) having a DNA content in the G ₁ phase, and are in a state deviated from the cell cycle. By inducing proliferation in the G ₀ phase, it is possible to proceed to the S phase after a slightly longer time than the G ₁ phase in the cell cycle.

  The cyclin-dependent kinase (CDK) used in the determination method is not particularly limited, and examples thereof include CDK1, CDK2, CDK4, and CDK6. The CDK further includes CDK belonging to cyclin A-dependent kinase, CDK belonging to cyclin B-dependent kinase, CDK belonging to cyclin D-dependent kinase, CDK belonging to cyclin E-dependent kinase, and the like. The cyclin A-dependent kinase is not particularly limited as long as it is a CDK that binds to cyclin A and exhibits activity, and examples thereof include CDK1 and CDK2. The cyclin B-dependent kinase is not particularly limited as long as it is a CDK that binds to cyclin B and exhibits activity, and examples thereof include CDK1. The cyclin D-dependent kinase is not particularly limited as long as it is a CDK that binds to cyclin D and exhibits activity, and examples thereof include CDK4 and CDK6. The cyclin E-dependent kinase may be a CDK that binds to cyclin E and exhibits activity, and is not particularly limited, and examples thereof include CDK2.

  As shown in Table 1, each of these CDKs becomes a cyclin / CDK complex (hereinafter also referred to as “active CDK”) bound to the corresponding cyclin, and has a cell cycle as shown in Table 1. A predetermined period is activated. For example, CDK1 binds to cyclin A or B, CDK2 binds to cyclin A or E, and CDK4 and CDK6 bind to cyclin D1, cyclin D2, or cyclin D3 to be activated. On the other hand, the CDK activity may be inhibited by a CDK inhibitor as shown in Table 1. For example, p21 inhibits CDK1 and CDK2. p27 inhibits CDK2, CDK4 and CDK6. p16 inhibits CDK4 and CDK6.

Among the CDKs, the expression level and activity value of each of two or more types of CDKs are measured, and the ratio between the expression level and activity value of each CDK (that is, the CDK specific activity represented by the following formula or its reciprocal) Ask.
CDK specific activity = CDK activity value / CDK expression level

The CDK activity value refers to the level of kinase activity (unit is represented by U (unit)) indicated by the amount of a substrate that binds to a predetermined cyclin and phosphorylates. Examples of the substrate that phosphorylates CDK include histone H1 for active CDK1 and active CDK2, and Rb (retinoblastoma protein) for active CDK4 and active CDK6. It is done. The CDK activity value can be measured by a conventionally known enzyme activity measurement method. Specifically, a sample containing activated CDK is prepared from cell lysate of the measurement sample, the sample and ³² P-labeled ATP with the ^{([γ- 32 P] ATP)} , ^{32 P-in} substrate protein There is a method in which the amount of ³² P-labeled phosphorylated substrate is incorporated and measured, and quantified based on a calibration curve prepared with a standard product. Moreover, as a method not using the label of the radioactive substance, a method disclosed in JP-A-2002-335997 can be mentioned. In this method, a sample containing a target active CDK is prepared from a cell lysate of a measurement sample, adenosine 5′-O- (3-thiotriphosphate) (ATPγS) is reacted with a substrate protein, The substrate protein is labeled by introducing a monothiophosphate group into the serine residue or threonine residue of the substrate protein, and binding a fluorescent labeling substance or labeling enzyme to the sulfur atom of the introduced monothiophosphate group, and labeled thiophosphate In this method, the amount of labeling based on the group (fluorescence amount when a fluorescent labeling substance is used) is measured and quantified based on a calibration curve prepared with a standard product.

  A sample to be used for activity measurement is prepared by specifically collecting a target CDK from a solubilized solution of a tissue containing a malignant tumor to be measured. In this case, it may be prepared using an anti-CDK antibody specific for the target CDK, or a predetermined cyclin-dependent kinase (for example, cyclin A-dependent kinase, cyclin B-dependent kinase, cyclin E-dependent kinase). In the case of activity measurement, it is prepared using an anti-cyclin antibody. In either case, a CDK other than the active CDK is included in the sample. For example, a complex in which a CDK inhibitor is bound to a cyclin / CDK complex is also included. Further, when an anti-CDK antibody is used, CDK alone, a complex of CDK and cyclin and / or CDK inhibitor, a complex of CDK and other compounds, and the like are included. Therefore, the activity value is measured as a unit (U) of a phosphorylated substrate in a state where an active type, an inactive type, and various competitive reactions coexist.

  The CDK expression level is the target CDK level (unit corresponding to the number of molecules) measured from the cell lysate, and can be measured by a conventionally known method for measuring the target protein level from a protein mixture. For example, an ELISA method, a Western blot method, or the like may be used, or measurement may be performed by the method disclosed in Japanese Patent Application Laid-Open No. 2003-130871. The target protein (CDK) may be captured using a specific antibody. For example, by using an anti-CDK1 antibody, it is possible to capture all of CDK1 present in the cell (including CDK alone, a complex of CDK and cyclin and / or CDK inhibitor, a complex of CDK and other compounds).

  Therefore, the specific activity calculated by the above formula corresponds to the ratio of CDK showing activity among the CDKs present in the cells, and can be said to be a CDK activity level based on the proliferation state of the malignant tumor cell to be determined. The CDK specific activity thus determined does not depend on the measurement sample preparation method. In particular, a measurement sample (a cell lysate) prepared from a biopsy material is easily affected by the number of non-cellular tissues contained in the actually collected tissue, for example, extracellular matrix. Therefore, the significance of using the specific activity or the reciprocal thereof excluding such influence is large, and the correlation with the clinical character is high as compared with the conventional simple activity value.

  By knowing two or more kinds of CDK specific activities or their reciprocals, it is possible to know which CDK activity is dominant, and what is the proportion of cells in any stage of the cell cycle, Alternatively, it is possible to know at which time the cell ratio is dominant.

The type of CDK whose specific activity is measured is not particularly limited, and may be selected as appropriate. In general, since cancer cells are actively proliferating beyond normal growth control, it is considered that the proportion of cells in the S phase and G ₂ phase is large. Therefore, it is considered that the tissue is cancerous in such a case. Moreover, it can be said that the said cancer progresses rapidly and is malignant. Furthermore, aneuploidy is considered to occur when the abnormal M phase has passed or the M phase has been advanced to the G ₁ phase and the S phase has been entered. Therefore, it can be said that the low proportion of cells present in the M phase also indicates that the cancer is malignant. Therefore, CDK1 is used as the first cyclin-dependent kinase, CDK2 is used as the second cyclin-dependent kinase, and the CDK1 specific activity is classified into groups according to the magnitude of the CDK1 specific activity. The activity value reflects the cell ratio in S phase. When there are many cells in the S phase, it can be determined that the tissue in which the cells are constituent cells is clinically malignant, that is, malignant cancer with a poor prognosis that easily metastasizes.

  From the above, in the diagnosis support system of the present embodiment, the specific activity of two or more types of CDK is obtained for the cancer patient to be diagnosed, and the CDK specific activity approximates the CDK specific activity of the cancer patient. By providing clinical information of the cancer patient, it is possible to provide information useful for diagnosis of the cancer patient to be diagnosed.

[2] Diagnosis Support System Next, a diagnosis support system according to an embodiment of the present invention will be described. The diagnosis support system according to the present embodiment uses the above-described CDK1 specific activity and CDK2 specific activity as measured values of predetermined measurement items. Specifically, the diagnosis support system according to the present embodiment acquires the expression levels and activity values of CDK1 and CDK2 of the subject cancer patient. CDK1 specific activity and CDK2 specific activity are calculated from the obtained expression levels and activity values of CDK1 and CDK2. Then, the reference range is determined based on the calculated CDK1 specific activity and CDK2 specific activity. The sample data of other cancer patients having CDK1 specific activity and CDK2 specific activity within the reference range are searched and specified from the sample data of other cancer patients stored in advance. Then, the recurrence rate is calculated based on the specified sample data, and the risk of recurrence is determined based on the calculated recurrence rate. Then, a screen including the calculated recurrence rate result and recurrence risk determination result is generated, and the generated screen is displayed on the display device.

FIG. 1 is a perspective explanatory view of a diagnosis support system according to an embodiment of the present invention. The diagnosis support system according to the present embodiment includes a measuring device A and a solubilizing device B. The measuring apparatus A includes a measuring unit 501 and a data processing unit 12. The measurement unit 501 measures the activity value and expression level of CDK1, and the activity value and expression level of CDK2, and includes a detection unit 4, a chipset unit 1 disposed in the front part of the apparatus main body unit 20, Pipette cleaning for cleaning the first reagent setting unit 5 and the second reagent setting unit 6, the activity measuring unit 2 disposed in the rear part of the apparatus main body unit 20, the waste liquid tank 7 for storing the waste liquid, and the pipette. Dispensing mechanism 3 that is disposed above the tank 8 and the apparatus main body 20 and can move the pipette in three directions (X direction, Y direction, and Z direction), and the back of the apparatus main body 20 The fluid part 9 and the main body control part 10 are mainly configured. The data processor 12 is communicably connected to the main body controller 10. Further, the measuring apparatus A is provided with a pure water tank 13, a cleaning liquid tank 14, a waste liquid tank 15, and an air pressure source 11. The pure water tank 13 stores pure water for channel cleaning at the end of measurement, and is connected to the fluid portion 9 by a pipe 21. The cleaning liquid tank 14 stores cleaning liquid for cleaning the pipette. The pipe 22 is connected to the pipette washing tank 8, and the waste liquid tank 15 for containing the waste liquid is connected to the waste liquid tank 7 through the pipe 23. Furthermore, in the diagnosis support system according to the present embodiment, the measurement apparatus A is provided with a solubilization apparatus B for obtaining a specimen that can be processed by the measurement apparatus A from a biological sample.
Hereinafter, the solubilizing apparatus B and the measuring apparatus A will be described in order.

[Solubilizer]
The solubilizer B prepares a liquid specimen that can be processed by the measuring apparatus A from a biological sample such as a tissue extracted from a patient prior to the processing by the measuring apparatus A. An operation unit 31 disposed above the front surface of the body unit 30, a drive unit 32 including a pair of pestle 34 for pressing and crushing the biological sample, and a container 35 for storing the biological sample are set. And the sample setting unit 33.

The drive unit 32 can move the pestle 34 up and down and give the pestle 34 a rotational movement, whereby the biological sample injected into the container 35 is pressed or ground. A control unit (not shown) that controls the operation of the drive unit 32 is built in the housing unit 30.
The operation unit 31 is provided with an operation button 31a, an operation lamp 31b, and a display unit 31c for displaying an apparatus state, an error message, and the like. Further, a cooling means (not shown) is provided in the specimen setting unit 33, and the biological sample in the container set in the recess on the upper surface of the specimen setting unit 33 is kept at a constant temperature.
The supernatant of the biological sample solubilized by the solubilizer B and further centrifuged by a centrifuge (not shown) is collected in a predetermined specimen container and set in the first reagent setting unit 5 of the measuring apparatus A. .

[First reagent setting unit]
A cooling means (not shown) is arranged in the first reagent setting unit 5 similarly to the sample setting unit 33, and a sample in a container set in a recess on the upper surface of the first reagent setting unit 5, CDK1 antigen (calibration 1), CDK2 antigen (calibration 2), fluorescently labeled CDK1 antibody, fluorescently labeled CDK2 antibody, and the like are maintained at a constant temperature. In the present embodiment, a total of 20 recesses are provided in 5 rows and 4 rows, so that a maximum of 20 containers can be set.

[Second reagent set part]
Next to the first reagent setting unit 5, a second reagent setting unit 6 is disposed. In the second reagent setting unit 6, a plurality of recesses are formed in the same manner as the first reagent setting unit 5. In these recesses, a container containing a buffer, a substrate solution, a fluorescence enhancing reagent and the like is set.
Prior to the processing by the measuring apparatus A, the protein solid phase chip is set in the chip setting unit 1 and the column is set in the activity measurement unit 2.

[Chipset part]
The chip set unit 1 is made of an aluminum block. As shown in FIGS. 2 to 3, the chip set unit 1 has a recess 102 for placing the protein solid phase chip 101 on the upper surface and three suctions on the bottom. It has a mouth 103. More specifically, the chip set unit 1 includes a rectangular first concave portion 102 on the upper surface and three rectangular second concave portions 104 on the bottom of the first concave portion 102. The second recesses 104 are made independent from each other by the partition wall 105, and when the protein solid phase chip 101 is placed on the chip set unit 1, the second recesses 104 are not in communication with each other. On the bottom surface of the first recess 102, a rectangular frame-shaped rubber elastic gasket 106 is disposed on the periphery of the second recess 104.

The second recess 104 includes a cross-shaped groove 107 at the bottom and a suction port 103 at the center of the bottom so that the groove bottom of the groove 107 becomes deeper from the periphery of the second recess 104 toward the center. It is inclined. The suction port 103 communicates with a nipple 108 provided for connection to an external suction air pressure source 11. The nipple 108 is connected to the other end of a tube 109 having one end connected to the suction pneumatic pressure source 11 side. The tube 109 is provided with an opening / closing valve 110.
The protein solid phase chip 101 to be described in detail later is horizontally loaded through the bottom gasket 106 of the first recess 102. After the protein-containing sample solution is injected or dropped into each well of the protein solid phase chip 101, the suction pump is activated.

  As a result, the protein solid-phase chip 101 is airtightly adsorbed to the bottom surface of the first recess 102 via the gasket 106, and the sample liquid in each well is sucked through the porous film described later, for the purpose of measurement. Is formed in a solid phase on the porous membrane. 2 and 3, reference numeral 130 denotes a pressing mechanism for pressing and fixing the protein solid phase chip 101 against the bottom surface of the first recess 102. After the protein solid-phase chip 101 is placed on the first recess 102, the pressing mechanism 130 slides in the direction of the arrow in the figure so that the upper portion presses the upper surface of the protein solid-phase chip 101 and 1 It fixes to the recessed part 102.

  As shown in FIGS. 4 to 5, the protein solid phase chip 101 includes a porous membrane 111 and a filter paper 112, and an upper plate 113 and a lower plate 114 for sandwiching the porous membrane 111 and the filter paper 112. It is configured. The protein solid phase chip 101 has a function of bringing an antibody solution containing a cyclin-dependent kinase antibody into contact with a biological sample (analyte).

  4-5, the upper plate 113 is comprised from three mutually independent plates, ie, the 1st upper plate 113a, the 2nd upper plate 113b, and the 3rd upper plate 113c. Each upper plate has a rectangular plate shape, and each of the first upper plate 113a and the second upper plate 113b has twelve oval through holes 115 arranged in a matrix in four rows and three columns. The third upper plate 113c is similarly provided with 16 oval through holes 115 arranged in a matrix of 4 rows and 4 columns. Each upper plate has an independent region for sample processing in which a plurality of through holes are formed. Further, a groove 116 is formed along the short side on the bottom surface of each upper plate.

On the other hand, a rectangular plate-like lower plate 114 is formed with a total of 40 oval through holes 117 arranged in a matrix at positions corresponding to the through holes 115 of the upper plates 113a, 113b, 113c. Has been. The through hole 117 has the same shape and cross-sectional area as the through hole 115. The lower plate 114 has regions in which a plurality of through holes are formed corresponding to the regions of the upper plates 113a, 113b, and 113c.
On the upper surface of the lower plate 114, there are three regions corresponding to the regions of the upper plates 113a, 113b, and 113c on the upper plate 113a, 113b, and 113c. Partition walls 119 are formed. And the three rectangular porous membrane installation area | regions are divided by the said convex part 118 and the partition 119 inside. The upper plate 113 and the lower plate 114 can be made of, for example, vinyl chloride resin.

2-5, the laminated body of the porous membrane 111 and the filter paper (filter) 112 is mounted in the porous membrane installation area of the lower plate 114, and then the grooves of the upper plates 113a, 113b, 113c. The protein solid phase chip 101 can be formed by attaching the upper plates 113a, 113b, and 113c to the lower plate 114 so that 116 is sequentially fitted to the convex portions 118 of the corresponding lower plate 114. Thereby, each through-hole 115 and each through-hole 117 are mutually coaxially arrange | positioned.
In the protein solid phase chip described above, the upper plate is divided into three, and the three regions can be sucked independently. However, the number of the upper plates may be two or four. It may be the above and is not particularly limited in the present invention. The number can be appropriately selected in consideration of the number of measurement items and the number of samples.

[Sample preparation unit for activity measurement]
As shown in FIGS. 6 to 10, the activity measurement sample preparation unit 2 includes a plurality of sample preparation units 211 each having a column 201 and a fluid manifold 213 for measuring the activity value of CDK. Used.

  A column 201 shown in FIG. 6 is formed of a cylindrical body made of a vinyl chloride resin, and includes a carrier holding unit 202 that holds a carrier 206 used for isolating a target substance in a liquid sample. The carrier holding unit 202 has a liquid storage unit 204 for receiving and storing the liquid sample into which the liquid sample is introduced. The column 201 is provided with an opening 205 that can inject or collect a liquid sample from the outside at the upper part of the liquid storage part 204, introduces the liquid sample to the fluid manifold 213 at the lower part of the carrier holding part 202, and supplies liquid from the fluid manifold 213. A connection channel 203 for receiving a sample is provided. The column 201 constitutes means for bringing a substrate solution containing a predetermined substrate into contact with a biological sample (specimen).

  The carrier 206 is made of a cylindrical monolithic silica gel, and unlike the particle carrier, the monolithic silica gel has a structure in which a three-dimensional network skeleton and its voids are integrated. A predetermined CDK antibody is immobilized on the monolithic silica gel. The carrier 206 is inserted into the carrier holding portion 202 from the lower opening of the column 201 and is elastically pressed and supported by the fixing pipe 208 via the O-ring 207. The fixing pipe 208 is press-fitted from the lower opening of the column 201, and the fixing pipe 208 and the hole of the O-ring 207 form the connection flow path 203.

  A loading flange 209 for loading and fixing the column 201 in the sample preparation unit 211 is formed at the lower end of the column 201. The flange 209 is an oblong flange formed by cutting out both sides of a disk-shaped flange having a diameter D in parallel so as to have a width W (W <D).

FIG. 7 is a perspective view of the sample preparation unit 211. As shown in FIG. 7, the sample preparation unit 211 includes an L-shaped support plate 212. The support plate 212 includes a fluid manifold 213 and a syringe pump 214. And a stepping motor 215 with a speed reducer are fixed.
A screw shaft 216 is connected to the output shaft of the stepping motor 215. A drive arm 217 that is screwed onto the screw shaft 216 is connected to the tip of the piston 218 of the syringe pump 214. When the screw shaft 216 is rotated by the stepping motor 215, the piston 218 moves up and down. The syringe pump 214 and the fluid manifold 213 are connected by a liquid feeding tube 250 via connectors 219 and 220. The syringe pump 214 is connected to a chamber 234 (see FIG. 10) in which a liquid (cleaning liquid) for filling the flow path is accommodated by a liquid feeding tube 220b through a connector 220a.

As shown in FIGS. 8 to 9, the fluid manifold 213 includes a column connection part 221 to which the lower opening of the column 201 is connected.
The fluid manifold 213 includes a flow path 223 inside, and a solenoid valve 225 that opens and closes between the flow path 223 and the column connection portion 221 on the lower surface. The fluid manifold 213 has a connector connection screw hole 226 for connecting the connector 220 to the side surface, and the screw hole 226 is connected to the flow path 223.

  FIG. 10 is a fluid circuit diagram of the sample preparation unit 211 and shows a state where the syringe pump 214 is connected to the fluid manifold 213 via the connector 220. A chamber 234 is connected to the syringe pump 214 via an electromagnetic valve 233, and a positive pressure is applied to the chamber 234 from a positive pressure source 235.

Here, a method of loading the column 201 into the fluid manifold 213 will be described.
As shown in FIGS. 8 to 10, a column loading recess 227 for receiving the lower end of the column 201 is formed on the upper surface of the fluid manifold 213, and the center of the bottom of the recess 227 penetrates the column connection portion 221 and the bottom portion. An O-ring 228 is attached to the circumference of the. On the upper surface of the fluid manifold 213, two L-shaped holding plates 229 and 230 are fixed in parallel at an interval wider than the width W and narrower than D around the column loading recess 227.

  In order to prevent the specimen or reagent that has passed through the carrier 206 inside the column 201 fixed to the fluid manifold 213 from coming into contact with the liquid (washing liquid) that fills the flow path 223 inside the fluid manifold 213 and diluting, the column 201 is replaced with the column 201. Before fixing in the loading recess 227, the electromagnetic valve 225 is opened (the electromagnetic valve 233 is closed), and the syringe pump 214 is suctioned by about 16 μL. Thereby, the liquid level of the column connection part 221 falls and an air gap is formed.

  After that, the column 201 is loaded into the column loading recess 227 so that the flange 209 passes between the pressing plates 229 and 230 and rotated by 90 degrees clockwise or counterclockwise. As a result, the diameter D portion of the flange 209 is engaged with the holding plates 229 and 230, and the flange 209 is fixed by the holding plates 229 and 230 due to the elasticity of the O-ring 228. When removing the column 201, the column 201 may be rotated by 90 degrees in either the left or right direction while pressing the column 201.

When the column 201 is loaded into the fluid manifold 213 of the sample preparation unit 211, the concave portion 227 of the fluid manifold 213 is filled with a fluid manually or automatically dispensed to prevent bubbles from being mixed. When the tip of the is inserted into the recess 227, the fluid overflows due to its volume. In order to prevent this liquid from flowing out to the periphery, an overflow liquid storage recess 231 is provided around the column mounting recess 227, and the overflow liquid is sucked and discharged to a part of the overflow liquid storage recess 231 by a pipette. An overflow liquid discharge recess 232 is provided.
Various specimens and reagents are injected or aspirated into or from a predetermined location by the dispensing mechanism unit 3 equipped with a pipette.

  Here, the operation when the sample or reagent in the upper opening 205 of the column 201 is injected will be described. When a sample or reagent is injected into the opening 205, the electromagnetic valve 225 is first opened (the electromagnetic valve 233 is closed), and the syringe pump performs a suction operation. As a result, the air gap and the specimen or reagent pass through the electromagnetic valve 225 and are sucked to the syringe pump side. Next, the syringe pump performs a discharge operation. As a result, the specimen or reagent passes through the electromagnetic valve 225 and is fed into the column 201.

[Dispensing mechanism]
As shown in FIG. 1, the dispensing mechanism unit 3 includes a frame 352 for moving the pipette X direction, a frame 353 for moving the pipette Y direction, and a plate 354 for moving the pipette Z direction.
The frame 352 includes a screw shaft 355 for moving the plate 354 in the arrow X direction, a guide bar 356 for supporting and sliding the plate 354, and a stepping motor 357 for rotating the screw shaft 355.

The frame 353 includes a screw shaft 358 for moving the frame 352 in the arrow Y direction, a guide bar 359 for supporting and sliding the frame 352, and a stepping motor 361 for rotating the screw shaft 358. .
The plate 354 includes a screw shaft 367 for moving the arm 368 supporting the pipette 362 in the arrow Z direction, a guide bar for supporting and sliding the arm 368, and a stepping motor 370 for rotating the screw shaft 367. And.
In this embodiment, since the dispensing mechanism unit 3 includes a pair of pipettes 362, a reagent or the like is injected into two sample containers at the same time, or the contents are aspirated from the two sample containers at the same time. And the measurement process can be performed efficiently.

[Fluid part]
As shown in FIG. 1, a fluid unit 9 for manipulating fluid connected to the pipette washing tank 8 for washing the pipette 362 and each sample preparation unit 211 is disposed on the back of the apparatus main body 20. Has been. As shown in FIG. 10, the fluid section 9 includes an electromagnetic valve 225 of each sample preparation section 211, an electromagnetic valve 233 that controls fluid when the syringe 214 is filled with liquid from the cleaning liquid chamber, and liquid suction by the pipette 362. An electromagnetic valve that controls the fluid when discharging, an electromagnetic valve that controls the fluid when sucking the liquid discarded from the pipette 362 in the waste liquid tank 7, and a fluid when cleaning the pipette 362 in the pipette cleaning tank 8. It has an electromagnetic valve to control.

[Detection unit]
The detection unit 4 is based on a fluorescent labeling substance that reflects the amount of protein captured by the porous membrane 111 of the protein solid phase chip 101 and based on the bound fluorescent labeling substance and the amount of phosphate groups. Measures the amount of fluorescence, irradiates the protein solid phase chip 101 with excitation light, detects the generated fluorescence, and outputs an electrical signal having a magnitude corresponding to the detected fluorescence intensity to the main body control unit 10 To do. As the detection unit 4, a commonly used light source unit, illumination system, and light receiving system can be appropriately employed.

[Data processing section]
FIG. 11 is a block diagram illustrating a partial configuration (a control system that controls the diagnosis support system) of the diagnosis support system according to the present embodiment. As shown in FIG. 11, the data processing unit 12, which is a personal computer, includes a control unit 77, an input unit 78, and a display unit 79.

  The control unit 77 has a function for transmitting an operation start signal of the apparatus to a main body control unit 10 to be described later. When an operation start command is transmitted from the control unit 77, the main body control unit 10 adjusts the temperature of the drive signal for driving the stepping motor 215 of each sample preparation unit 211 and the temperature of the first reagent setting unit 5. A drive signal, a drive signal for driving the stepping motors 357, 361, and 370, and a drive signal for driving the electromagnetic valve in the fluid section 9 are output. Further, the control unit 77 has a function for analyzing the detection result obtained by the detection unit 4. The detection result obtained by the detection unit 4 is transmitted to the main body control unit 10. The main body control unit 10 transmits the detection result obtained by the detection unit 4 to the control unit 77.

  The display unit 79 is provided for displaying the analysis result and the like obtained by the control unit 77.

  Next, the configuration of the personal computer used as the data processing unit 12 will be described in detail. As shown in FIG. 12, the control unit 77 mainly includes a CPU 91a, a ROM 91b, a RAM 91c, an input / output interface 91d, an image output interface 91e, a communication interface 91f, and a hard disk 91g. The CPU 91a, ROM 91b, RAM 91c, input / output interface 91d, image output interface 91e, communication interface 91f, and hard disk 91g are connected by an electric signal line (bus) so that electric signals can be communicated.

The CPU 91a can execute the computer program stored in the ROM 91b and the computer program loaded in the RAM 91c. Then, the CPU 91a executes an application program 91h as described later and executes an operation described later, thereby allowing the personal computer to function as the data processing unit 12.
The ROM 91b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and stores a computer program executed by the CPU 91a, data used for the same, and the like.

  The RAM 91c is configured by SRAM, DRAM, or the like. The RAM 91c is used for reading out computer programs recorded in the ROM 91b and the hard disk 91g. Further, when these computer programs are executed, they are used as a work area of the CPU 91a.

  The hard disk 91g is installed with various computer programs to be executed by the CPU 91a, such as an operating system and application programs, and data used for executing the computer programs. Furthermore, the expression level and activity value of CDK1 and CDK2 are acquired, and the CDK1 specific activity and CDK2 specific activity are calculated from the acquired expression level and activity value of CDK1 and CDK2, and the calculated CDK1 specific activity and CDK2 specific activity are calculated. Based on the reference range, determine the sample data of other cancer patients with CDK1 specific activity and CDK2 specific activity within the determined reference range, and calculate the recurrence rate based on the specified sample data An application for determining the risk of recurrence based on the calculated recurrence rate, generating a screen including the calculated recurrence rate result and recurrence risk determination result, and causing the display unit 79 to display the generated screen The program 91h is also installed on the hard disk 91g.

  Further, in order to acquire the expression level and activity value, the hard disk 91g includes a first database 91i that stores a calibration curve that is conversion data for converting the fluorescence intensity into the expression level or activity value. In addition, you may make it obtain | require a calibration curve for every measurement of an expression level or an activity value. The first database 91i of the hard disk 91g contains data for use in the calculation for determining the reference range, default value data for the reference range, and reference range set value data that has been input and used in the past. It is remembered. Further, the first database 91i of the hard disk 91g stores a reference value for determining a recurrence risk by comparing with the calculated recurrence rate.

  The hard disk 91g is sample data in which measured values such as the activity values and expression levels of cancer patients are associated with clinical information such as the presence / absence of recurrence of the patient, information on postoperative treatment, and information on survival. Is stored in the second database 91j.

  The hard disk 91g is installed with an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation. In the following description, the application program 91h according to the present embodiment is assumed to operate on the operating system.

  The input / output interface 91d includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, an analog interface including a D / A converter, an A / D converter, and the like. Has been. An input unit 78 is connected to the input / output interface 91d, and the user can input data to the data processing unit 12 by using the input unit 78.

The communication interface 91f is, for example, an Ethernet (registered trademark) interface. The data processing unit 12 can transmit and receive data to and from the main body control unit 10 using a predetermined communication protocol through the communication interface 91f.
The image output interface 91e is connected to a display unit 79 constituted by an LCD, a CRT, or the like, and outputs a video signal corresponding to the image data given from the CPU 91a to the display unit 79. The display unit 79 displays an image (screen) according to the input video signal.

[Main unit control unit]
On the back of the apparatus main body 20, a main body control unit 10 is connected to each sample preparation unit 211, the detection unit 4, the stepping motors 357, 361, 370, the fluid unit 9, etc. Yes.

As shown in FIG. 13, the main body control unit 10 includes a CPU 301a, a ROM 301b, a RAM 301c, a communication interface 301d, and a circuit unit 301e.
The CPU 301a can execute computer programs stored in the ROM 301b and computer programs read out to the RAM 301c.
The ROM 301b stores a computer program to be executed by the CPU 301a, data used for executing the computer program, and the like.

The RAM 301c is used for reading a computer program stored in the ROM 301b. Further, when these computer programs are executed, they are used as a work area of the CPU 301a.
The communication interface 301d is, for example, an Ethernet (registered trademark) interface. The main body control unit 10 can transmit and receive data to and from the data processing unit 12 using a predetermined communication protocol through the communication interface 301d.

  The circuit unit 301e includes a plurality of drive circuits and a signal processing circuit (not shown). The drive circuits are provided corresponding to the sample preparation unit 211, the first reagent setting unit 5, the detection unit 4, the stepping motors 357, 361, and 370, and the fluid unit 9, respectively. Each drive circuit generates a control signal (drive signal) for controlling the corresponding unit (or the sample preparation unit 211 if the drive circuit corresponds to the sample preparation unit 211) in accordance with the instruction data given from the CPU 301a. Then, a control signal is transmitted to the unit. The output signal of the sensor provided in the unit is given to the drive circuit, and the drive circuit converts this output signal into a digital signal and gives it to the CPU 301a. The CPU 301a generates the instruction data described above based on the output signal of the given sensor.

  The signal processing circuit is connected to the detection unit 4. The detection unit 4 outputs a detection signal indicating the fluorescence intensity, and this detection signal is given to the signal processing circuit. The signal processing circuit performs signal processing such as noise removal processing, amplification processing, and A / D conversion processing on the detection signal. Data of detection results obtained as a result of signal processing is given to the CPU 301a.

[3] Cancer Diagnosis Support Next, the operation of the diagnosis support system according to the present embodiment will be described.

(1) Pretreatment with solubilizer B Prior to the treatment with the measuring device A, a liquid specimen is collected from the tissue containing the malignant tumor extracted from the cancer patient using the solubilizer B. As the procedure, first, the tissue is put into a container using tweezers. Next, when this container is set in the sample setting section 33 of the solubilizer B shown in FIG. 1 and the start button of the operation section 31 is pressed, the pestle 34 is lowered to a predetermined position, and the tissue in the container is moved to the container. Press against the bottom of the.

  In this state, a solubilizing solution (such as a buffer containing a surfactant and a protease inhibitor) is automatically or manually injected into the container. Thereafter, the tissue is ground by the rotation of the pestle 34. After the predetermined time has elapsed, the driving of the pestle 34 is stopped, and the pestle 34 is further moved upward, and then the container is taken out from the sample setting unit 33. Next, the solubilized contents in the container are centrifuged and the supernatant is manually collected as a specimen.

(2) Setting of sample or the like in measuring apparatus A The supernatant liquid is put into two sample containers and diluted at different dilution ratios, and then the sample container is set at a predetermined position of the first reagent setting unit 5. Of the two samples, one is a sample for expression level measurement and the other is a sample for activity value measurement.
In addition, the protein solid phase chip 101 is set in the chip setting unit 1, and eight columns 201 are set in the sample preparation unit 211 of the activity measurement unit 2.

(3) Overall Flow of Processing by Diagnosis Support System FIGS. 15 to 20 show an overall flow of processing by the diagnosis support system. In addition, in the determination in the following flowchart, when “Yes” and “No” are not illustrated, the bottom is Yes and the right (left) is No. The processes described below are processes controlled by the control unit 77 and the main body control unit 10.

  First, when the apparatus main body 20 is powered on, the main body control unit 10 is initialized (step S1). In this initialization operation, initialization of the program, return to the origin position of the drive part of the apparatus main body 20 and the like are performed.

  When the data processing unit 12, which is a personal computer, is turned on, the control unit 77 is initialized (step S201). In this initialization operation, initialization of the program and the like are performed. When the initialization is completed, a menu screen (not shown) including an input screen display button for instructing display of the input screen is displayed on the display unit 79. The user can select an input screen button for instructing display of an input screen of the menu screen by operating the input unit 78.

Next, in step S202, the control unit 77 of the data processing unit 12 determines whether or not the input screen is being displayed. If the control unit 77 determines that the input screen is being displayed (Yes), it proceeds to step S205. If it determines that the input screen is not being displayed (No), it proceeds to step S203. Proceed.
In step S203, the control unit 77 of the data processing unit 12 selects whether or not an input screen display instruction has been issued (that is, an input screen button for instructing display of the menu screen input screen is selected). Whether or not) is determined. When it is determined that an input screen display instruction has been made (Yes), the control unit 77 proceeds to step S204, and when it is determined that an input screen display instruction has not been made (No), step S204 is performed. The process proceeds to S301.

  In step S <b> 204, the input screen is displayed on the display unit 79 by the control unit 77 of the data processing unit 12.

  Next, in step S205, when the user operates the input unit 78, sample information such as the ID number and age of the subject cancer patient is input. Thereafter, in step S206, the information input by the input unit 78 is stored in the hard disk 91g. Then, when the user operates the input unit 78 of the personal computer 12 and selects the start button displayed on the input screen, the measurement start instruction is performed.

In step S207, the control unit 77 determines whether an instruction to start measurement has been issued. If the control unit 77 determines that an instruction to start measurement has been given (Yes), it proceeds to step S208. If it determines that an instruction to start measurement has not been given (No), it proceeds to step S301. To proceed. In step S <b> 208, a measurement start signal is transmitted from the control unit 77 to the main body control unit 10.
Next, in step S2, it is determined whether or not the measurement control signal has been received by the main body control unit 10. If the main body control unit 10 determines that the measurement start signal has been received (Yes), the process proceeds to step S3. If the main body control unit 10 determines that the measurement start signal has not been received (No), the process proceeds to step S8. Proceed to the process.

Next, in step S3, a preparation process for the expression level measurement sample is performed. In step S3, the sample is aspirated from the sample container set in the first reagent setting unit 5. Then, the aspirated specimen is subjected to a predetermined process to prepare an expression level measurement sample.
Furthermore, in step S4, a preparation process for the activity value measurement sample is performed. Here, the sample is aspirated from the sample container set in the first reagent setting unit 5. Then, the aspirated specimen is subjected to a predetermined process to prepare an activity value measurement sample.

Next, in step S5, the chip set unit 1 in which the protein solid phase chip 101 including the expression level measurement sample and the activity value measurement sample is set moves from the position shown in FIG. .
In step S6, each well of the protein solid phase chip 101 is irradiated with excitation light, and fluorescence emitted from each sample is detected.
In step S <b> 7, the detected detection result is transmitted from the main body control unit 10 to the control unit 77 of the personal computer 12.

  Next, in step S209, the control unit 77 determines whether or not a detection result has been received. If the control unit 77 determines that the detection result has been received (Yes), the control unit 77 proceeds to step S210. On the other hand, when the detection result is not received, the control unit 77 executes the process of step S209 again.

  In step S210, the control unit 77 executes analysis processing from the acquired detection result.

  Thereafter, in step S211, the control unit 77 outputs an analysis result such as the result of the recurrence rate calculated in step S210 and the result of the determined recurrence risk, and displays the result on the display unit 79.

FIG. 21 and FIG. 22 show examples of display screens for diagnosis support information.
In the display screen illustrated in FIG. 21, the display unit 401 displays the ID number, age, and the like of a cancer patient to be diagnosed. In addition, the information display unit 402 displays a CDK1 specific activity value and a CDK2 specific activity value as measured values of predetermined measurement items related to a malignant tumor of a cancer patient to be diagnosed. The display button icon 403 is a display button icon for shifting to a display screen (FIG. 22) of information for each treatment group after malignant tumor extraction such as no treatment, hormone therapy, and chemotherapy. The information display screen (FIG. 22) for each treatment group after malignant tumor removal is displayed as a window different from the window in which the screen in FIG. 21 is displayed when the user presses the display button icon 403. Instead of being displayed as a separate window, the screen shown in FIG. 21 may be switched to the screen shown in FIG. The distribution map display unit 404 displays a distribution map of CDK1 specific activity values and CDK2 specific activity values as measured values of predetermined measurement items related to malignant tumors of other cancer patients. In this distribution chart, points corresponding to the CDK1 specific activity value and the CDK2 specific activity value of the malignant tumor of the cancer patient to be diagnosed are drawn. Further, in the distribution chart, a reference range 602 determined based on the measured value 601 (CDK1 specific activity value and CDK2 specific activity value of a malignant tumor) of the test cancer patient is drawn.
In the display screen illustrated in FIG. 21, the distribution chart display unit 404 may display a three-dimensional distribution chart instead of the two-dimensional distribution chart of the CDK1 specific activity value and the CDK2 specific activity value. In this case, in addition to the CDK1 specific activity value and the CDK2 specific activity value, other parameters are coordinate axes.
FIG. 22 shows an example of a display screen for information on hormone treatment groups after malignant tumor removal. The information display unit 405 displays information such as a recurrence rate and a recurrence risk.
According to the diagnosis support system of the present embodiment, the recurrence rate is calculated based on information on the presence or absence of recurrence of other cancer patients exhibiting similar specific activities. The recurrence rate calculated here can be shown as an expected recurrence rate of a cancer patient to be diagnosed. Therefore, the user can obtain more accurate diagnosis support information by the diagnosis support system of the present embodiment. The information display unit 406 displays information such as Disease Free Survival.

  In step S301, the control unit 77 determines whether or not an input screen for setting values such as a reference value for determining a recurrence risk and a value (radius) for determining a reference range is being displayed. Is done. When it is determined that the setting value input screen is being displayed (Yes), the control unit 77 proceeds to step S305, and when it is determined that the setting value input screen is not being displayed (No), the control unit 77 proceeds to step S305. The process proceeds to S302.

In step S302, the control unit 77 determines whether an instruction to display a setting value input screen has been issued. When it is determined that the setting value input screen display instruction has been performed (Yes), the control unit 77 proceeds to step S303, and when it is determined that the setting value input screen display instruction has not been performed ( In No), the process proceeds to step S307.
In step S303, the RAM 91g of the control unit 77 stores data such as a reference value for determining a recurrence risk and a value (radius) for determining a reference range stored in the first database 91i of the hard disk 91g. read out.

  In step S <b> 304, the control unit 77 displays a setting value input screen on the display unit 79. Here, when the user operates the input unit 78, a new value is input as a setting value such as a value (radius) for determining a reference value or a reference range.

In step S305, the control unit 77 determines whether a setting value has been input. When it is determined that the set value has been input (Yes), the control unit 77 proceeds to step S306, and when it is determined that the set value has not been input (No), the process proceeds to step S307. Proceed.
In step S306, the input new setting value is stored in the first database 91i of the hard disk 91g.

  In step S307, the control unit 77 determines whether an instruction to shut down is received. When it is determined that an instruction to shut down is accepted (Yes), the control unit 77 proceeds to step S308, and when it is determined that an instruction to shut down is not accepted (No), the control unit 77 returns to step S202. In step S <b> 308, a shutdown signal is transmitted from the control unit 77 to the main body control unit 10. In step S309, the control unit 77 performs a shutdown process of the personal computer 12, and the process is completed.

  In step S8, the main body control unit 10 determines whether or not a shutdown signal has been received. When determining that the shutdown signal has been received (Yes), the main body control unit 10 proceeds to step S9. When determining that the shutdown signal has not been received (No), the main body control unit 10 proceeds to step S2. Return. In step S9, the main body control unit 10 shuts down the apparatus main body 20, and the process ends.

(4) Expression Level Measurement Sample Preparation Processing FIG. 17 shows a flow of an example of expression level measurement sample preparation processing in Step S3.
First, in step S21, the preserving solution stored in advance in each well of the protein solid phase chip is discharged, and the inside of each well is washed. The washing is performed by injecting the washing solution into each well from above through the pipette of the dispensing mechanism unit 3, and then sucking the washing solution injected under the negative pressure from below the protein solid phase chip through the porous membrane. Is called. The same is true for the following cleaning steps.

Next, a sample for expression level measurement is aspirated from the sample container set in the first reagent setting unit 5 with a pipette, and this sample is injected into a plurality of predetermined wells. Is sucked by negative pressure. As a result, the protein is immobilized on the porous membrane of the protein solid phase chip (step S22).
Next, the inside of the predetermined well is cleaned with a cleaning liquid in the same manner as in step S21. Thereby, components other than protein are removed from the porous membrane of the protein solid phase chip (step S23).

  Thereafter, a blocking solution is injected into the predetermined well, and the blocking solution remaining in the well after being left for 15 minutes or longer (for example, 30 minutes) is discharged (step S24). As a result, the fluorescently labeled CDK1 antibody (fluorescently labeled CDK1 antibody) and the fluorescently labeled CDK2 antibody (fluorescently labeled CDK2 antibody) are immobilized on the portion of the porous membrane where the protein is not immobilized. Can be prevented. Commercially available products can be used as the fluorescently labeled CDK1 antibody and the fluorescently labeled CDK2 antibody.

Next, a fluorescently labeled CDK1 antibody and a fluorescently labeled CDK2 antibody are respectively injected into predetermined wells. At that time, each fluorescently labeled antibody is injected into two wells. After 20 to 30 minutes, the injected fluorescent label is discharged after the reaction between the fluorescently labeled antibody and the protein (CDK1 or CDK2) immobilized on the porous membrane is completed (step S25).
Finally, in the same manner as in step S23, the inside of the predetermined well is cleaned with a cleaning solution (step S26). Then, the main body control unit 10 returns the process to step S4 of the main routine shown in FIG.

(5) Activity Value Measurement Sample Preparation Process FIG. 18 shows a flow of an example of the activity value measurement sample preparation process in step S4. In the activity value measurement sample preparation process, the activity measurement unit 2 shown in FIG. 1 includes four sample preparation units 211 on the front side in the figure, and four sample preparation units on the back side in the figure. What is provided with 211 is used. Each sample preparation unit 211 of the activity measurement unit 2 is divided into a first sample preparation unit (Ac1), a second sample preparation unit (Ac2), a third sample preparation unit (Ac3), and a fourth sample from the left in the figure. The preparation unit (Ac4), and from the left in the figure, the fifth sample preparation unit (Ac5), the sixth sample preparation unit (Ac6), the seventh sample preparation unit (Ac7), and the eighth sample preparation unit (Ac8) And

  First, for each of the first to eighth sample preparation sections (Ac1 to Ac8), a buffer that is a cleaning reagent is injected into the opening 205 with a pipette of the dispensing mechanism section 3. For each of the first to eighth sample preparation units (Ac1 to Ac8), the syringe pump 214 and the electromagnetic valve 225 operate as described above, so that the buffer of the liquid storage unit 204 flows through the carrier 206. After being drawn into the path 223, it passes through the carrier 206 again and is returned to the liquid storage unit 204. The buffer returned to the liquid storage unit 204 in all the columns 201 is sucked and discarded by the pipette of the dispensing mechanism unit 3 (step S31).

Next, immunoprecipitation (immune reaction between antibody and CDK) is performed (step S32). First, from one sample container set in the first reagent setting unit 5, the sample 1 for activity value measurement is aspirated by one pipette, and the sample 2 for activity value measurement is aspirated by the other pipette.
Then, the specimen 1 for activity value measurement sucked from the specimen container is first injected into the liquid storage section 204 of the first sample preparation section (Ac1) as shown in FIG. Then, the specimen 1 is sent to the carrier 206 of the first sample preparation unit (Ac1) by the syringe pump 214 and the electromagnetic valve 225 operating as described above. At that time, the specimen 1 reciprocates the carrier 206 of the column 201 once by reciprocating the piston 218 up and down once (aspiration → discharge).

On the other hand, the sample 2 for activity value measurement sucked from the sample container is first injected into the liquid storage unit 204 of the fifth sample preparation unit (Ac5). Then, the specimen 2 is sent to the carrier 206 of the fifth sample preparation section (Ac5) as described above.
Neither CDK1 antibody nor CDK2 antibody is immobilized on the carrier 206 of the column 201 of the first sample preparation section (Ac1) and the fifth sample preparation section (Ac5). Therefore, in the first sample preparation section (Ac1) and the fifth sample preparation section (Ac5), CDK1 and CDK2 are not solid-phased, and the column 201 of the first sample preparation section (Ac1) contains CDK1 and CDK2. The sample 1 is stored, and the sample 2 including CDK1 and CDK2 is stored in the column 201 of the fifth sample preparation unit (Ac5).

Next, the specimen 1 stored in the column 201 of the first sample preparation unit (Ac1) is aspirated by a pipette and injected into the liquid storage unit 204 of the third sample preparation unit (Ac3). Then, the specimen 1 is sent to the carrier 206 of the third sample preparation unit (Ac3) as described above.
On the other hand, the specimen 2 stored in the column 201 of the fifth sample preparation unit (Ac5) is aspirated by a pipette and injected into the liquid storage unit 204 of the fourth sample preparation unit (Ac4). Then, the specimen 2 is sent to the carrier 206 of the fourth sample preparation unit (Ac4) in the same manner as described above.

  CDK1 antibody is immobilized on the carrier 206 of the column 201 of the third sample preparation section (Ac3) and the fourth sample preparation section (Ac4). Therefore, in the third sample preparation unit (Ac3) and the fourth sample preparation unit (Ac4), CDK1 is immobilized, but CDK2 is not immobilized, and the column 201 of the third sample preparation unit (Ac3) The specimen 1 that does not contain CDK1 and contains CDK2 is stored, and the specimen 2 that does not contain CDK1 and contains CDK2 is stored in the column 201 of the fourth sample preparation section (Ac4).

  Next, the specimen 1 stored in the column 201 of the third sample preparation unit (Ac3) is aspirated by a pipette and injected into the liquid storage unit 204 of the seventh sample preparation unit (Ac7). Then, the specimen 1 is sent to the carrier 206 of the seventh sample preparation section (Ac7) as described above.

On the other hand, the specimen 2 stored in the column 201 of the fourth sample preparation unit (Ac4) is aspirated by a pipette and injected into the liquid storage unit 204 of the eighth sample preparation unit (Ac8). Then, the specimen 2 is sent to the carrier 206 of the eighth sample preparation unit (Ac8) as described above.
A CDK2 antibody is immobilized on the carrier 206 of the column 201 of the seventh sample preparation section (Ac7) and the eighth sample preparation section (Ac8). Therefore, in the seventh sample preparation section (Ac7) and the eighth sample preparation section (Ac8), CDK2 is solid-phased, so that the column 201 of the seventh sample preparation section (Ac7) does not contain CDK1 or CDK2. The sample 1 is stored, and the sample 2 containing neither CDK1 nor CDK2 is stored in the column 201 of the eighth sample preparation unit (Ac8).

The sample 1 and the sample 2 stored in the column 201 of the seventh sample preparation unit (Ac7) and the eighth sample preparation unit (Ac8) are each sucked by a pipette and discarded in the waste liquid tank 7.
The first sample preparation unit (Ac1) and the fifth sample preparation unit (Ac5) are used for background activity measurement, and the third sample preparation unit (Ac3) and the fourth sample preparation unit (Ac4) are used for CDK1. For the activity measurement, the seventh sample preparation unit (Ac7) and the eighth sample preparation unit (Ac8) are used for the activity measurement of CDK2.

Thus, by injecting the sample remaining in the column into another column, the background activity measurement, the CDK1 activity measurement and the CDK2 activity measurement can be performed with a small sample amount.
Next, in order to wash away unnecessary components in the specimen, the buffer 1 is fed to the column 201 (step S33).

Thereafter, since the buffer 1 affects the enzyme reaction executed in step S25, the buffer 2 is fed to the column 201 mainly for the purpose of creating a condition for the enzyme reaction. Are washed away (step S34).
Next, a substrate reaction solution containing the substrate Histon H1 and ATPγS is injected into the column 201, and the piston 219 is reciprocated once (step S35). The liquid pushed out from the lower side of the column 201 into the column 201 is stored as it is. By this step, a phosphate group is introduced into Histon H1 using CDK1 or CDK2 as an enzyme. Since the amount of the phosphate group depends on the strength (that is, the activity value) of the CDK1 or CDK2 enzyme, the activity value of CDK1 or CDK2 is determined by measuring the amount of the phosphate group. Can be requested. In addition, the background activity value calculated | required using the 1st sample preparation part (Ac1) and 5th sample preparation part (Ac5) shown by FIG. 23 is used in order to carry out background correction | amendment so that it may mention later. .

Next, the fluorescent labeling reagent is dispensed directly into the column 201 from above the column 201 using a pipette, and the fluorescent labeling substance is bound to the phosphate group introduced into the Histon H1 (step S36). At this time, the liquid in the column 201 is agitated by the pipette repeatedly sucking and discharging the liquid in the column for a predetermined time.
After a predetermined time (for example, 20 minutes) has elapsed from the start of step S26, the reaction stop solution is directly dispensed into the column 201 in the same manner as the fluorescent labeling reagent. Then, the liquid in the column 201 is agitated by repeating the suction and discharge of the liquid in the column for a predetermined time as in step S26 (step S37). This stops the binding of the fluorescent label.

  Next, the first sample preparation unit (Ac1), the third sample preparation unit (Ac3), the fourth sample preparation unit (Ac4), the fifth sample preparation unit (Ac5), the seventh sample preparation unit (Ac7), and the After the liquid in the column 201 of the 8-sample preparation unit (Ac8) has been dispensed into the six wells of the protein solid-phase chip 101, the protein solid-phase chip 101 is aspirated from below (step S38). . As a result, Histon H1 having a phosphate group to which a fluorescent labeling substance is bound is immobilized on the porous membrane of the protein solid-phase chip 101.

Next, the wells are washed in the same manner as in step S21 in the expression level measurement sample preparation process (step S39).
Finally, the quenching reagent for quenching the fluorescence (background quenching) based on the fluorescent labeling substance that has not been bound to the phosphate group introduced into the Histon H1 is dispensed into the well, and the discharge operation is repeated six times. (Step S40). Then, the main body control unit 10 returns the process to step S5 of the main routine shown in FIG.

(6) Analysis Process In the analysis process step (step S210), as shown in FIGS. 19 and 20, an analysis is performed from the fluorescence intensity obtained by the detection unit, and the analysis result is output to the display unit 79. .
First, in step S401, the control unit 77 sends the CDK1 activity, CDK1 expression, CDK2 activity, CDK2 expression, background activity, and background from the light receiving system of the detection unit 4 via the main body control unit 10. Fluorescence intensity is acquired twice for each of the expression.

Next, in step S402, the control unit 77 calculates the average value of the fluorescence intensities obtained by two for each item.
Next, in step S403, the background activity (average value) is subtracted from the fluorescence intensity (average value) of the CDK1 activity. Furthermore, the background activity (average value) is subtracted from the fluorescence intensity (average value) of CDK2 activity. This provides background correction for CDK1 activity and CDK2 activity. Background correction is similarly performed for CDK1 expression and CDK2 expression.

Next, in step S404, the control unit 77 acquires an expression level and an activity value for each item using a calibration curve. The calibration curve is data for converting the fluorescence intensity into the expression level or the activity value. This calibration curve is created in advance using two or more types of samples whose expression levels or activity values are known when the reagent lot is changed, and is stored in the hard disk 91g of the control unit 77.
Next, in step S405, the control unit 77 performs the following equation:
CDK1 specific activity = CDK1 activity value / CDK1 expression level CDK1 specific activity and CDK2 specific activity are calculated according to CDK2 specific activity = CDK2 activity value / CDK2 expression level.

In step S405, the control unit 77 replaces the CDK1 specific activity and the CDK2 specific activity with the following formula:
The reciprocal number of CDK1 specific activity = CDK1 expression level / CDK1 activity value The inverse number of CDK2 specific activity = CDK2 expression level / CDK2 activity value may be calculated as the step of calculating the reciprocal number of CDK1 specific activity and the reciprocal number of CDK2 specific activity.

  Thereafter, in step S406, the control unit 77 determines a reference range. This reference range is sample data having measurement values (CDK1 specific activity and CDK2 specific activity) approximate to the measurement values (CDK1 specific activity and CDK2 specific activity) of predetermined measurement items regarding malignant tumors of cancer patients to be diagnosed. It is an area including Here, the reference range is determined as a circle having a preset radius with the measured value as the center.

  Next, in step S407, the control unit 77 associates the measured values such as the activity values and expression levels of other cancer patients with the clinical information of the other patients from the second database 91j of the hard disk 91g. Read the sample data.

Next, in step S408, the control unit 77 searches and specifies sample data having measured values (CDK1 specific activity and CDK2 specific activity) within the reference range determined in step S406.
In other words, in step S408, a process is performed for identifying the sample values in the second database 91j whose measured values (CDK1 specific activity and CDK2 specific activity) are within the reference range.

  Next, in step S409, the control unit 77 calculates a recurrence rate based on the sample data specified in step S408. Specifically, based on the sample data of other cancer patients identified in step S408, the total number of other cancer patients having measured values within the reference range is counted, and cancer patients who have recurred are included. The recurrence rate can be calculated by calculating the proportion of the recurrence. Here, the recurrence rate is expressed as a percentage (%) when the total number of other cancer patients having measured values within the reference range is 100.

Thereafter, in step S410 and step S411, the recurrence risk is determined by comparing the calculated recurrence rate with a preset reference value.
First, in step S410, the control unit 77 determines whether or not the recurrence rate is less than the first reference value. If Yes, the process proceeds to step S411, whereas if No, there is a recurrence risk. Determined to be “high”. Here, the first reference value was 14%.

  In step S411, the control unit 77 determines whether or not the recurrence rate is less than the second reference value. If Yes, it is determined that the recurrence risk is “low”, while if No, It is determined to stop the determination. Here, the second reference value was 5%.

  The reference value may be one, or may be three or more. In addition, as described above, the risk of recurrence may be determined in two stages, “high” and “low”. Further, the risk of recurrence may be further divided into, for example, a recurrence risk of “high”, “medium”, and “low”. As shown in FIG.

  Thereafter, in step S211, the control unit 77 distributes the CDK1 specific activity and the CDK2 specific activity on the display screen of the display unit, as shown in FIG. 21 and FIG. Plotted and displayed on the figure, and the reference range is displayed on this distribution map, individual information of cancer patients to be diagnosed such as individual number and age, measured values of predetermined items of malignant tumor, recurrence The rate, recurrence risk determination result, etc. are displayed (step S211). In the distribution diagram of the display screen illustrated in FIG. 21, CDK1 specific activity is displayed in logarithm (log) on the horizontal axis, and CDK2 specific activity is displayed in logarithm (log) on the vertical axis. In addition, the display screen illustrated in FIG. 22 displays a decision free survival rate, a recurrence rate, a recurrence risk determination result (“risk determination” in the figure), etc., when hormone therapy is performed. The And the control part 77 returns a process to step S211 of the main routine shown by FIG.

  In the above-described embodiment, in step S401, the control unit 77 performs the CDK1 activity, CDK1 expression, CDK2 activity, CDK2 expression, background activity, and background expression twice each. The fluorescence intensity is obtained, and the average value of the fluorescence intensity obtained for each item is calculated two times. However, the present invention is not limited to this, and is not limited to this. CDK1 activity, CDK1 expression, CDK2 activity, CDK2 For each of the expression, background activity, and background expression, three or more fluorescence intensities may be acquired, and the average value of the fluorescence intensities of each item may be calculated. Alternatively, the fluorescence intensity may be acquired once for each of CDK1 activity, CDK1 expression, CDK2 activity, CDK2 expression, background activity, and background expression. In this case, in step S403, CDK1 activity and expression and CDK2 activity and expression background correction are performed using the fluorescence intensity of each item acquired one by one, not the average value of each item. .

  In the present embodiment, a user such as a doctor sets the radius of the reference range as appropriate, and the reference range is determined as a circle with the set radius. It is desirable that the reference range is determined to have a size that can secure a minimum necessary number of samples that can ensure statistical reliability. Therefore, from the viewpoint of ensuring reliability, information on the number of samples included in this reference range is displayed at the same time as the display of the reference range on the display screen so that the minimum required number of samples can be secured within the reference range. You may let them. Accordingly, the user can easily reset the radius of the reference range to a value that can ensure an appropriate number of samples by referring to the information on the number of samples displayed on the screen.

Further, the diagnosis support system may automatically determine the radius of the reference range. When the diagnosis support system automatically sets the radius, for example,
(I) A predetermined size that includes data on a number of cancer patients that are medically statistically significant, centered on the measurement value of a predetermined measurement item related to a malignant tumor of a cancer patient to be diagnosed Setting an area having
(II) setting a region having a predetermined size including a measurement error / standard deviation by a system, including a measurement value of a predetermined measurement item regarding a malignant tumor of a cancer patient to be diagnosed; or
(III) Measured values obtained by measuring at least once for a given measurement item for one sample, including measured values for a given measurement item for malignant tumors of cancer patients to be diagnosed It is preferable to set a radius capable of setting an area having a predetermined size including the measurement error and standard deviation.

  By determining the reference range as described in (I) above, the control unit 77 can provide more accurate diagnostic support information that is more medically meaningful. Further, the control unit 77 can determine the reference range as described in (II) above, thereby preventing a decrease in accuracy due to a measurement error caused by the system. Further, the control unit 77 can determine the reference range as shown in (III), thereby preventing a decrease in accuracy due to the variation in the measurement value due to the measurement method. In step S406, since the reference range is determined in this way, highly accurate diagnosis support information can be provided.

  In the present embodiment, the reference range is a circle centered on the measured value, but may be another shape such as a rectangle or an ellipse centered on the measured value.

  Below, the result obtained by analyzing the malignant tumor extract | collected from five cancer patients (cancer patients 1-5) used as a diagnostic object using the diagnostic system mentioned above was shown.

(Reference example)
The activity values and expression levels of CDK1 and CDK2 in malignant tumors of breast cancer patients were measured. From the obtained activity value and expression level, the specific activity of CDK1, the specific activity of CDK2, and the ratio of the specific activity of CDK1 and the specific activity of CDK2 were calculated. Next, the vertical axis is the log value of the specific activity of CDK2, and the horizontal axis is the log value of the specific activity of CDK1, and a distribution diagram is prepared.

The following reference values were set as reference values used for risk assessment.
1. ) Ratio of specific activity of CDK1 and CDK2 (specific activity ratio) is 5.0
2. ) Low standard value: CDK1 specific activity is 6
3. ) Intermediate standard value: CDK1 specific activity is 20
4). ) High reference value: CDK1 specific activity is 90

  The eight areas classified by the four reference values are set to any one of recurrence risk “high” (H), recurrence risk “medium” (I), and recurrence risk “low” (L). Specifically, a region that is larger than the reference value related to the specific activity ratio and larger than the middle reference value is defined as a “high” (H) region. Further, a region that is larger than the reference value related to the specific activity ratio, smaller than the middle reference value, and larger than the lower reference value is defined as a “medium” (I) region. Further, a region that is larger than the reference value related to the specific activity ratio and smaller than the lower reference value is defined as a “low” (L) region. On the other hand, for a region smaller than the reference value related to the specific activity ratio, a region larger than the high reference value is set as a “high” (H) region, and a region smaller than the high reference value is set as a “low” (L) region. It was.

Example 1
The activity values and expression levels of CDK1 and CDK2 of the malignant tumor of cancer patient 1 were measured, and the logarithm (log) values of the specific activities of CDK1 and CDK2 were calculated. As a result, the logarithm (log) value of the specific activity of CDK1 was 1.7, and the logarithm (log) value of the specific activity of CDK2 was 1.8. In addition, the results of a large-scale clinical trial of samples from the untreated group + hormone therapy group + anthracycline chemotherapy group were used as a reference data set, and the recurrence risk of cancer patient 1 was determined according to a reference example. It was found to belong to the “low” (L) region.

  Next, based on the logarithm (log) value of the calculated specific activity, the results of a large-scale clinical trial of samples in the untreated group + hormonal therapy group + anthracycline chemotherapy group were used as a reference data set. Using the diagnosis support system, a distribution map was prepared and the recurrence rate was calculated as in the reference example.

  The size (radius) of the reference range for the measured value of cancer patient 1 was 0.3. 24 to 27 show examples of diagnosis support information by the diagnosis support system of the present invention. FIG. 24 shows a distribution diagram of measured values of CDK1 and CDK2 of malignant tumor. In FIG. 24, based on the threshold value 701 according to the reference example, a “high” (H) region, a “medium” (I) region, and a “low” (L) region are displayed together. Moreover, in FIGS. 25-27, the result of the survival curve estimated when each treatment is performed is shown. In the graphs of the respective survival curves shown in FIGS. 25 to 27, the vertical axis represents the disease-free survival rate, and the horizontal axis represents the observation period (days). The criteria for determining the risk of recurrence were “High Risk” when the recurrence rate was 14% or higher, and “Low Risk” when the recurrence rate was less than 5%.

  In the distribution chart shown in FIG. 24, the measurement value 601 of the cancer patient 1 is displayed, and the reference range 602 is further displayed. As shown in FIG. 24, the measured value 601 of this cancer patient 1 belongs to the “low” (L) region according to the recurrence risk determination of the reference example. According to this, information on other cancer patients belonging to the vicinity of the threshold having approximate measurement values or to the “high” (H) region is provided as diagnosis support information. Therefore, a more appropriate judgment can be made. From the information of other cancer patients having measured values approximate to those of the cancer patient 1 provided by the diagnosis support system of the present embodiment, when the cancer patient 1 is not treated, the recurrence rate is 9.1. % Was predicted. Similarly, when hormone therapy was given to cancer patient 1, the recurrence rate was predicted to be 3.8%. Furthermore, when chemotherapy with anthracycline is given to cancer patient 1, the recurrence rate is predicted to be 75%. For malignant tumors of cancer patient 1, chemotherapy with anthracycline is expected. It was predicted that the therapy might not be effective.

  As shown in FIG. 25 and FIG. 26, respectively, in the case of no treatment for cancer patient 1, it is expected that the disease-free survival rate will decrease in about 800 days, but hormone treatment is given. In some cases, disease-free survival is expected to be maintained until at least about 2200 days. Thus, according to the diagnosis support system of the present embodiment, it is possible to provide the cancer patient 1 with information serving as an index for determining that hormonal treatment is one of effective means. On the other hand, as shown in FIG. 25 and FIG. 27, in the case of no treatment for cancer patient 1, it is expected that the disease-free survival rate will decrease in about 800 days, and chemotherapy with anthracycline is When given, it was expected that disease free survival would decrease in about 100 days. Thus, according to the diagnosis support system of the present embodiment, information serving as an index for determining that the anthracycline treatment may not be effective can be provided to the cancer patient 1.

  From these results, according to the diagnosis support system of the present embodiment, it is possible to provide diagnosis support information corresponding to each patient, for example, it is possible to predict the recurrence rate due to a treatment method to be adopted in the future with high accuracy, It suggests that the patient can be provided with information to select a more appropriate treatment.

  The survival curve may be a survival curve created from survival / death information among clinical information of other cancer patients.

(Example 2)
The activity values and expression levels of CDK1 and CDK2 of the malignant tumor of cancer patient 2 were measured, and the logarithm (log) values of the specific activities of CDK1 and CDK2 were calculated. As a result, the logarithm (log) value of the specific activity of CDK1 was 1.5, and the logarithm (log) value of the specific activity of CDK2 was 2.5. In addition, a large-scale clinical test result (332 samples) of samples in the group of no treatment + hormone treatment was used as a reference data set, and the recurrence risk of cancer patient 2 was determined according to a reference example. It was found to belong to.

  Next, based on the logarithm (log) value of the calculated specific activity, a large-scale clinical test result (332 samples) of samples in the group of no treatment + hormone treatment is used as a reference data set, and the diagnosis support system of the present embodiment A distribution map was prepared and the recurrence rate was calculated. The size (radius) of the reference range for the measured value of cancer patient 2 was set to 0.15.

  FIG. 28 shows an example of diagnosis support information by the diagnosis support system of this embodiment. According to the diagnosis support system of the present embodiment, as shown in FIG. 28, information on other cancer patients having measured values approximate to those of the cancer patient 2 provided by the diagnosis support system of the present embodiment, It is shown within the reference range and can be provided as information useful for making a more appropriate diagnosis of the cancer patient 2.

  Moreover, according to the information of other cancer patients having a measured value approximate to the cancer patient 2 provided by the diagnosis support system of the present embodiment for the cancer patient 2, there is a recurrence within the reference range. Includes 2 patients and 9 no recurrence. Based on this, the recurrence rate was predicted to be 18.2%.

(Example 3)
In the same manner as in Example 2, the activity values and expression levels of CDK1 and CDK2 of the malignant tumor of cancer patient 3 were measured. As a result, the logarithm (log) value of the specific activity of CDK1 was 1.5, and the logarithm (log) value of the specific activity of CDK2 was 2. Moreover, when the recurrence risk of the subject cancer patient was determined according to the reference example, it was in the “high” (H) region.

  FIG. 29 shows an example of diagnosis support information by the diagnosis support system of the present embodiment. According to the diagnosis support system of the present embodiment, as shown in FIG. 29, information of other cancer patients having measured values approximate to those of the cancer patient 3 provided by the diagnosis support system of the present embodiment, It is shown within the reference range and can be provided as information useful for making a more appropriate diagnosis of the cancer patient 3.

  Furthermore, the recurrence rate was predicted from information on other cancer patients having measured values approximate to those of the cancer patient 3 provided by the diagnosis support system of the present embodiment. As a result, the reference range includes 4 patients with recurrence and 21 patients without recurrence. Based on this, the recurrence rate was predicted to be 16.0%. As described above, when determined according to the reference example, the cancer patient 3 was determined to belong to the “high” (H) region in the same manner as the cancer patient 2 of Example 2 described above. According to the diagnosis support system, the predicted recurrence rates are different between the cancer patient 3 of the present embodiment and the cancer patient 2 of the second embodiment. More specifically, the measured values (specific activity of CDK1 and specific activity of CDK2) of the cancer patient 2 of Example 2 are the “low” (L) region and “ The measurement value of the cancer patient 3 of this example is “low” (L) among the “high” (H) regions according to the reference example. It exists near the boundary. And the recurrence rate obtained by the diagnosis support system according to the above embodiment for the cancer patient 2 of Example 2 is 18.2%, while the cancer patient 3 of the present embodiment is related to the above embodiment. The recurrence rate obtained by the diagnostic support system is 16%. From this, the diagnosis support system according to the embodiment can obtain a recurrence rate corresponding to the measurement value of the cancer patient, that is, predict the recurrence rate accurately reflecting the state of the malignant tumor of the cancer patient. It turns out that it is possible.

Example 4
In the same manner as in Example 2, the activity values and the expression levels of CDK1 and CDK2 of the malignant tumor of cancer patient 4 were measured. As a result, the logarithm (log) value of the specific activity of CDK1 was 0.5, and the logarithm (log) value of the specific activity of CDK2 was 1. Moreover, when the recurrence risk of the cancer patient 4 was determined according to the reference example, it was found to belong to the “low” (L) region.

  FIG. 30 shows an example of diagnosis support information by the diagnosis support system of this embodiment. As shown in FIG. 30, information of other cancer patients having measured values approximate to the cancer patient 4 provided by the diagnosis support system of the present embodiment is shown in the reference range, and the cancer patient 4 Can be provided as information useful for making a more appropriate diagnosis.

  Furthermore, the recurrence rate was predicted from information of other cancer patients having measured values approximate to those of the cancer patient 4 provided by the diagnosis support system of the present embodiment. As a result, the reference range includes 0 patients with recurrence and 8 patients without recurrence. Therefore, the recurrence rate was predicted to be 0%.

(Example 5)
In the same manner as in Example 2, the activity values and expression levels of CDK1 and CDK2 of the malignant tumor of cancer patient 5 were measured. As a result, the logarithm (log) value of the specific activity of CDK1 was 1.2, and the logarithm (log) value of the specific activity of CDK2 was 1.5. Moreover, when the recurrence risk of the cancer patient 5 was determined according to the reference example, it was found to belong to the “low” (L) region.

  FIG. 31 shows an example of diagnosis support information by the diagnosis support system of this embodiment. As shown in FIG. 31, information of other cancer patients having measured values approximate to the cancer patient 5 provided by the diagnosis support system of the present embodiment is shown in the reference range, and the subject is It can be provided as useful information for better diagnosis of cancer patients.

  Next, the recurrence rate was predicted from information on other cancer patients having measured values approximate to those of the cancer patient 5 provided by the diagnosis support system of the present embodiment. As a result, the reference range includes one patient with recurrence and 10 patients without recurrence. Based on this, the recurrence rate was predicted to be 9.1%. Thus, when judged according to the reference example, the test cancer patient of this Example 5 was judged to belong to “low” (L), similarly to the test cancer patient of Example 4 above. However, according to the diagnosis support system of the present embodiment, the predicted recurrence rates are different between the cancer patient 5 of this example and the cancer patient 4 of Example 4. More specifically, the measured value of the cancer patient 4 of Example 4 is far from the boundary between the “high” (H) region and the “medium” (I) region in the “low” (L) region according to the reference example. The measured values of the cancer patient 5 of this example are present in the vicinity of the boundary with the “medium” (I) region in the “low” (L) region according to the reference example. The recurrence rate obtained by the diagnosis support system according to the above embodiment for the cancer patient 4 of Example 4 is 0%, while the diagnosis support according to the above embodiment is performed for the cancer patient 5 of this example. The recurrence rate obtained by the system is 9.1%. From this, the diagnosis support system according to the embodiment can obtain a recurrence rate corresponding to the measurement value of the cancer patient, that is, predict the recurrence rate accurately reflecting the state of the malignant tumor of the cancer patient. It turns out that it is possible.

It is a perspective explanatory view of one embodiment of a diagnosis support system of the present invention. It is a perspective explanatory view of the chip set part and protein solid phase chip in the diagnosis support system shown in FIG. FIG. 2 is a cross-sectional explanatory view of a chip set unit and a protein solid phase chip in the diagnosis support system shown in FIG. It is decomposition | disassembly explanatory drawing of the upper plate and lower plate of a protein solid-phase chip | tip. It is a perspective explanatory view of the protein solid phase chip in a state where the upper plate is mounted on the lower plate. It is a cross-sectional explanatory drawing of the column of the sample preparation part of the activity measurement unit in the diagnosis support system shown in FIG. It is a perspective view of the sample preparation part of the activity measurement unit in the diagnosis assistance system shown by FIG. It is a top view of the fluid manifold of the sample preparation part shown by FIG. It is DD sectional view taken on the line of FIG. FIG. 8 is a fluid circuit diagram of the sample preparation unit shown in FIG. 7. It is a block diagram which shows the partial structure (control system which controls a diagnostic assistance system) of a diagnostic assistance system. It is a block diagram which shows the hardware constitutions of a data processing part. It is a block diagram which shows the hardware constitutions of a main body control part. It is a schematic explanatory drawing of a cell cycle. It is a figure which shows the whole flow of an example of the process by a diagnosis assistance system. It is a figure which shows the whole flow of an example of the process by a diagnosis assistance system. It is a figure which shows the flow of an example of the preparation process of the sample for expression level measurement. It is a figure which shows the flow of an example of the preparation process of the sample for activity measurement. It is a figure which shows the whole flow of an example of the analysis process by a diagnostic assistance system. It is a figure which shows the whole flow of an example of the analysis process by a diagnostic assistance system. It is a figure which shows the example of the display screen of diagnostic assistance information. It is a figure which shows the example of the display screen of diagnostic assistance information. It is explanatory drawing which shows the utilization procedure of the sample etc. in a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system. It is a figure which shows the example of the diagnostic assistance information by a diagnostic assistance system.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Chip set part 2 Activity measurement unit 3 Dispensing mechanism part 4 Detection part 5 1st reagent set part 6 2nd reagent set part 7 Waste liquid tank 8 Pipette washing tank 9 Fluid part 10 Main body control part 11 Air pressure source 12 Personal computer ( Data processing department)
DESCRIPTION OF SYMBOLS 13 Pure water tank 14 Cleaning liquid tank 15 Waste liquid tank 20 Apparatus main body part 30 Case part 31 Operation part 32 Drive part 33 Specimen setting part 34 Pestle 77 Control part 78 Input part 79 Display part 91a CPU
91b ROM
91c RAM
91d Input output interface 91e Image output interface 91f Communication interface 91g Hard disk 101 Protein solid phase chip 111 Porous membrane 112 Filter paper 113 Upper plate 114 Lower plate 115 Through hole 117 Through hole 201 Column 202 Carrier holding part 206 Carrier 211 Sample preparation part 213 Fluid manifold 301a CPU
301b ROM
301c RAM
301d Communication Interface 301e Circuit Unit A Measuring Device B Solubilizing Device 401 Display Unit 402 Information Display Unit 403 Display Button Icon 404 Distribution Map Display Unit 405 Information Display Unit 406 Information Display Unit 501 Measuring Unit 601 Patient Measured Value 602 Reference Range 701 Threshold

Claims (11)

A cancer diagnosis support system,
A measurement unit take measurements relating to the first cancer patient by measuring the predetermined two kinds of measurement items for prepared specimen using a malignant tumor collected from the first cancer patient,
A plurality of measurement values generated by measuring the two predetermined measurement items for each specimen prepared using a malignant tumor collected from a plurality of cancer patients different from the first cancer patient When a serial憶部for storing data including the clinical information after malignant tumor removal of each of the plurality of cancer patients, and the reference range, and
And Viewing section,
Reading the data of the plurality of measurement values and the reference range from the storage unit, with the two types of measurement items as coordinate axes, the plurality of measurement values and the measurement values related to the first cancer patient are plotted, and and a control unit for displaying the reference range is displayed distribution graph includes measurements of the first cancer patient on the display unit,
The storage unit stores information on the recurrence of cancer and / or information on the presence or absence of postoperative survival as the clinical information,
The control unit has a cancer recurrence rate obtained from information on the recurrence of the cancer and / or information on the presence or absence of postoperative survival of the cancer patient corresponding to the plurality of measurement values within the reference range. and / or is displayed before Symbol display unit diagnosis support screen showing the disease-free survival, cancer diagnosis support system. Before SL control section displays the distribution graph on the diagnosis support screen, the diagnosis support system for cancer according to claim 1. The storage unit stores a reference value for recurrence risk determination, and the control unit displays a cancer recurrence risk determination result obtained from the cancer recurrence rate and the reference value on the diagnosis support screen. The cancer diagnosis support system according to claim 1 or 2 , which is displayed . An input unit is further provided, the storage unit stores postoperative treatment information of the plurality of cancer patients as the clinical information, and the control unit is an input for selecting a predetermined postoperative treatment by the input unit The diagnosis support for the cancer recurrence rate and / or disease-free survival rate of the cancer patient who has received the selected post-operative treatment and corresponds to the plurality of measured values within the reference range. The cancer diagnosis support system according to claim 1 or 2 , which is displayed on a screen . The cancer diagnosis support system according to claim 4 , wherein the postoperative treatment includes no treatment, hormone therapy, and chemotherapy . The controller further includes an input unit, and when the control unit receives an input of a range change of the reference range displayed in the distribution chart by the input unit, the control unit corresponds to the plurality of measurement values in the changed reference range. The cancer diagnosis support system according to claim 1 or 2 , wherein a cancer recurrence rate and / or disease-free survival rate is displayed on the diagnosis support screen .   The cancer diagnosis support system according to claim 1, wherein the measurement value includes a value related to expression of a cell cycle protein and / or activity of the cell cycle protein.   The cancer diagnosis support system according to claim 7, wherein the cell cycle protein is a cyclin-dependent kinase.   The measured values are the ratio of the activity value and expression level of the first cyclin-dependent kinase (first CDK) (first ratio), the activity value and expression level of the second cyclin-dependent kinase (second CDK), The cancer diagnosis support system according to claim 8, wherein the ratio is the second ratio. A cancer diagnosis support information providing device,
An input receiving unit that receives input of measurement values relating to a first cancer patient generated by measuring two predetermined measurement items for a sample prepared using a malignant tumor collected from a first cancer patient When,
A plurality of measurement values generated by measuring the two predetermined measurement items for each specimen prepared using a malignant tumor collected from a plurality of cancer patients different from the first cancer patient A storage unit for storing data including clinical information after removal of each malignant tumor of each of the plurality of cancer patients, and a reference range;
A display unit;
Reading the data of the plurality of measurement values and the reference range from the storage unit, with the two types of measurement items as coordinate axes, the plurality of measurement values and the measurement values related to the first cancer patient are plotted, and and a control unit for displaying the reference range is displayed distribution graph includes measurements of the first cancer patient on the display unit,
The storage unit stores information on the recurrence of cancer and / or information on the presence or absence of postoperative survival as the clinical information,
The control unit has a cancer recurrence rate obtained from information on the recurrence of the cancer and / or information on the presence or absence of postoperative survival of the cancer patient corresponding to the plurality of measurement values within the reference range. And / or a cancer diagnosis support information providing apparatus for displaying a diagnosis support screen indicating a disease-free survival rate on the display unit. A method for providing cancer diagnosis support information in a cancer diagnosis support information providing apparatus comprising a storage unit and a display unit ,
Receiving input of measurement values relating to a first cancer patient generated by measuring two predetermined measurement items for a specimen prepared using a malignant tumor collected from a first cancer patient;
Before SL plurality of measurements generated by measuring the predetermined two kinds of measurement items per first respective analyte malignant tumors taken from different cancer patients cancer patient were prepared using the And reference range data from the storage unit, the two types of measurement items as coordinate axes, the plurality of measurement values and the measurement values related to the first cancer patient are plotted, and the first Displaying a distribution map on which the reference range including measurement values related to cancer patients is displayed on the display unit ;
The information on the recurrence of cancer of the cancer patient corresponding to the plurality of measurement values within the reference range and / or the information on the presence or absence of postoperative survival is read from the storage unit, and the read cancer information Displaying on the display unit a diagnosis support screen showing a recurrence rate and / or disease-free survival rate of cancer obtained from information on recurrence and / or information on the presence or absence of postoperative survival;
The execution, provides a method of diagnosis support information for cancer.

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