JP2007057309A - Detection method of uterine cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for finding out a physiological element capable of becoming the specific tumor marker of uterine cancer which is not yet found out and detecting the uterine cancer based on the physiological element. <P>SOLUTION: The mass distribution within a molecular weight range of 1,000-200,000 Da and development intensity of protein in the blood specimen of a subject are measured and the difference found out by comparing the mass distribution and the pattern of relative development intensity with the pattern in a normal person is used as an index to detect the uterine cancer of the subject. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cancer detection means, and more specifically to a specific tumor marker for endometrial cancer.

  Uterine cancer is the second most common cancer in women after stomach cancer and breast cancer, and is broadly divided into cervical cancer and endometrial cancer. Endometrial cancer, which is directly related to the present invention, has been known as a cancer with a relatively low onset frequency among uterine cancers, but in recent years, its frequency has been regarded as a problem.

  Endometrial cancer is a cancer that occurs in the endometrium, and broadly includes both endometrial cancer that occurs in the endometrium and uterine sarcoma that occurs in the myometrium, but in general the endometrium It means cancer. In the present invention, endometrial cancer is defined as endometrial cancer.

  Although early symptoms of endometrial cancer include irregular organ bleeding (especially after menopause), it is relatively slow and is one of the cancers that are difficult to detect early.

  Diagnosis of endometrial cancer includes endometrial cytology, endometrial histology, complete endometrial curettage / hysteroscopy, ultrasound, CT, MRI, and other diagnostic imaging as needed. It has been broken.

  Among cancer diagnostic means, a so-called tumor marker is mentioned as an extremely effective means in recent years. A tumor marker is a biological substance that exhibits a characteristic behavior in blood or urine in response to the presence of a specific tumor. Since the presence of a specific tumor can be detected by quantifying a tumor marker and using that quantitative value as an index, the tumor marker greatly contributes to the early detection of the specific tumor because of its simplicity. It is. For example, SCC and the like are known as tumor markers for cervical cancer and the like. As tumor markers for ovarian cancer, CA125, α-fetoprotein, LDH, CEA, CA72-4, CA19-9 and the like are known.

  However, a specific tumor marker for endometrial cancer is not known at present, and as described above, it is not easy to use an existing test means for endometrial cancer. This is clearly one of the causes that hinder early detection of endometrial cancer.

  Early detection of endometrial cancer not only saves the patient's life, but also connects the hope of childbirth. That is, the principle of treatment of endometrial cancer is surgical treatment, and the principle is total hysterectomy + excision of appendages (ovary and fallopian tubes) + pelvis and para-aortic lymphadenectomy. For women, the desire to give birth is cut off at the same time as treatment. However, in the very early stage (clinical progression stage Ia of atypical endometrial adenocarcinoma (Grade I)), it is possible to select uterine preservation therapy, but in order to find endometrial cancer at this stage Requires the aid of diagnosis with sensitive tumor markers.

  Therefore, an object of the present invention is to find a biological element that can be a specific tumor marker for endometrial cancer that has not yet been found, and to provide means for detecting endometrial cancer based on this.

  As a result of repeated studies aimed at solving this problem, the inventor has surprisingly been able to detect a subject's endometrial cancer with extremely high accuracy by using a specific blood protein as an index. As a result, the present invention was completed.

  That is, the present invention measures mass distribution and expression intensity in the range of molecular weight 1000 to 200000 Da in a protein in a blood sample of a subject, and finds the pattern of the mass distribution and relative expression intensity in comparison with a pattern in a healthy person. The present invention provides a detection method (hereinafter also referred to as the present detection method) for detecting endometrial cancer (in the present invention, endometrial cancer) in the subject using the difference as an index.

  As described above, a specific tumor marker for endometrial cancer has not yet been found, and the present invention is expected to make a great contribution to the early detection of endometrial cancer.

  A blood sample means blood collected from a subject or a processed product thereof, which may be arterial blood or venous blood, but is usually venous blood, particularly peripheral venous blood. Is used. In addition, the blood sample used in the present invention can collect whole blood by a conventional method such as vacuum blood sampling or syringe blood sampling. In this detection method, it is preferable to use serum out of whole blood, plasma and serum. Further, by further pre-fractionating normal serum and using it as a sample, the detection sensitivity can be further increased. Such pre-fractionation is performed, for example, by treating serum with an anion exchange solid phase carrier.

  For the measurement of the mass distribution, any apparatus or system capable of mass spectrometry of proteins can be used without particular limitation. For example, devices such as a quadrupole mass spectrometer, a time-of-flight mass spectrometer, a quadrupole ion trap mass spectrometer, a Fourier transform ion cyclotron mass spectrometer, a protein chip and a time-of-flight mass spectrometer, For example, a protein chip system integrated with a computer in which software used for measurement and analysis is installed can be used. Preferably, a time-of-flight mass spectrometer or a protein chip system can be used, and particularly preferably, a protein chip system manufactured by Ciphergen can be used. The protein chip system manufactured by the company irradiates the blood protein captured by the solid phase carrier with a laser beam to desorb the blood protein from the solid phase carrier in an ionized state. Proteins can be detected with a time-of-flight mass spectrometer.

  As a premise for the measurement of the mass distribution, it is often necessary to bring a blood sample into contact with a solid phase carrier and capture the blood protein on the solid phase carrier. For example, in each of the above protein chip systems, blood protein is captured by a protein chip whose main component is a solid phase carrier having various properties suitable for protein analysis, and each protein is unique to the captured protein. The desired measurement can be performed by performing mass spectrometry based on the principle of. The solid phase carrier used here includes a hydrophobic solid phase carrier, a cation exchange type solid phase carrier, an anion exchange type solid phase carrier, a metal ion modified solid phase carrier, a normal phase type solid phase carrier, and a reverse phase type solid phase carrier. Examples include carriers suitable for expression analysis of phase carriers and the like. In addition, a specific binding molecule, for example, an affinity carrier on which an activated molecule, an antibody, a receptor protein, a nucleic acid or the like is solid-phased can be used for analyzing specific binding. Among these, a solid phase carrier for expression analysis, that is, a hydrophobic solid phase carrier, a cation exchange type solid phase carrier, an anion exchange type solid phase carrier, a metal ion modified solid phase carrier, a normal phase type solid phase carrier, Alternatively, it is preferable in the present invention to use a reverse phase type solid phase carrier, and among these, a cation exchange type solid phase carrier, a metal ion modified solid phase carrier, a copper ion modified solid phase carrier, or a reverse type A phase-type solid support is particularly suitable.

  In particular, in order to obtain a continuous pattern in the molecular weight range of 1000 to 200000 Da, the captured proteins captured by these immobilized carriers are provided as peak patterns in a state separated from the immobilized carrier by ionization. Is done. As the ionization method, in addition to the above-mentioned ionization method by laser beam irradiation (for example, matrix-assisted laser desorption ionization (MALDI) method), electron impact ionization (EI) method, chemical ionization (CI) method, field desorption (FD) method, fast atom collision (FAB) method, electrospray ionization (ESI) method and the like.

  In this detection method, protein peaks in blood are continuously analyzed in the molecular weight range of 1000 to 200000 Da, and the peak pattern obtained by this continuous analysis is determined as a pattern of endometrial cancer-affected type and a healthy person. If the subject's peak pattern is categorized as an endometrial cancer-affected pattern divided into type patterns (meaning at least not having endometrial cancer), it is detected as positive for endometrial cancer. It can be carried out. In particular, the molecular weight range of the blood protein to be analyzed can be about 1000 to 20000 Da.

  It is possible to analyze the whole peak pattern of blood protein existing in the above molecular weight range of 1000 to 200000 Da, and to detect uterine body cancer of a subject based on the result. Analysis can also be performed by paying attention to the characteristic peak of blood protein. It is preferable to select a blood protein to be focused on in a combination that maintains the detection accuracy of endometrial cancer as much as possible.

  Specifically, performing the following (1) and / or (2) is a preferred embodiment of this detection method.

  (1) In a blood sample of a subject, one or more selected from blood proteins having substantially the center of mass distribution of a group consisting of 6493 Da, 6487 Da, 3244 Da, 6691 Da, 6684 Da, 3343 Da, and 19318 Da, When the amount of the one or more blood proteins is higher than that of a healthy person, this is used as an index of endometrial cancer in the subject.

  For blood proteins having substantially the mass distribution center represented by the group (1), a cation exchange type solid phase carrier is used to capture a blood sample, and mass distribution analysis is performed on the captured protein. It is preferable to measure by.

  (2) One or more blood proteins selected from blood proteins having substantially the mass distribution center of the group consisting of 5625 Da, 9370 Da, and 28165 Da are detected in the blood sample of the subject, and the one or more blood proteins are detected. When the amount of uterine is decreased as compared with a healthy person, this is used as an index of endometrial cancer in the subject.

  The blood protein substantially having the center-of-mass distribution represented in the group (2) is obtained by capturing a blood sample on a metal ion-modified solid phase carrier and performing mass distribution analysis on the captured protein. It is preferable to measure. The modified metal ion in the metal ion modified solid phase carrier is preferably a copper ion.

  The term “substantially” that defines the mass distribution center of the specific blood protein has the meaning of defining in consideration of the maximum measurement error. Specifically, the maximum error is ± 0.2%. In the present specification, when a specific blood protein is indicated by the center of mass distribution, it means a “substantial” numerical value unless otherwise specified.

  As described above, when analyzing by focusing on the whole blood protein peak pattern in a specific molecular weight range or the above individual peak patterns (single marker analysis), for example, the above-described apparatus (for example, manufactured by Ciphergen) Can be comprehensively grasped as a mass distribution of blood protein in a specific mass range. Similarly, it is possible to grasp the presence, absence, increase or decrease of the individual blood proteins. In particular, by processing the protein mass distribution information in the blood sample of the subject through software having an algorithm that correlates the relationship between the mass distribution and the endometrial cancer, it is very simple and accurate, Detection of endometrial cancer in a subject can be performed. As a data processing method in this case, for example, univariate analysis or multivariate analysis (hierarchical cluster analysis, principal component analysis, classification analysis, or the like) can be performed.

  The present invention provides a method for detecting endometrial cancer that can detect endometrial cancer with high accuracy using a specific blood protein as a tumor marker.

  Hereinafter, although the concrete form of this invention is described as an Example, the scope of the present invention is not limited by this description.

(1) Blood samples Blood samples were collected from 26 cases of endometrial cancer (stages I to IV) and 25 cases of healthy subjects as initial test subject candidates, and visually checked and a protein chip [cation exchange chip (CM10)]. A hemolysis check was performed using the hemoglobin peak as an index (100 mM Sodium Acetate, pH 4).

  For the endometrial cancer group, 20 cases were selected excluding a total of 6 cases, 3 cases in the recurrence / chemochemistry, 2 cases in stage IV, and the oldest case, taking into account the age difference between the normal group. In order to reduce the difference in age between 2 cases in which the influence of hemoglobin was recognized in the 40s and the endometrial cancer group sample, 5 samples were excluded from the younger age group, a total of 5 cases. An example was chosen.

  Blood was collected from the peripheral venous blood vessels of 20 cases each of the endometrial cancer group and the healthy subject group selected in this manner, and used as a serum sample by a conventional method. The serum specimen was once frozen, thawed on ice, and centrifuged at 20000 × g for 10 minutes, and the supernatant was collected. The pre-fractionation was processed on a 96-well format filter plate, and all supernatants were collected using a Biomek2000 Laboratory Work Station (Beckman) equipped with a DPC Micromix 5 shaker. In this way, 20 μL of a denaturation buffer, 30 μL of U9 (9 M urea, 2% CHAPS, 50 mM Tris-HCl, pH 9) was added to the serum sample obtained as a supernatant and shaken at 4 ° C. for 20 minutes. Bio Sepra Q Ceramic HyperDF (anion exchange resin) was then pre-equilibrated with 50 mM Tris-HCl, pH 9, and prepared to 50% slurry. 180 μL of this resin was added to each well of the filter plate, and equilibrated three times with 200 μL of U1 buffer (9-fold diluted U9 buffer with 50 mM Tris-HCl, pH 9). Add the serum sample denatured with U9 to the resin, wash the sample well with 50 μL of ΜL buffer, add to the resin, shake at 30 ° C for 30 minutes, collect the non-adsorbed fraction, 50 mM Tris-HCl, pH 9, 0.1% OGP 100 μL was added to the resin. This washing solution was collected and combined with the non-adsorbed fraction to obtain Fraction 1. Thereafter, the protein was eluted by a stepwise pH gradient of pH 7, 5, 4, 3 (each elution buffer, 100 μL × 2). In this stepwise protein elution process, elution at pH 7 is performed with an elution buffer (50 mM HEPES with 0.1% OGP pH 7), and elution at pH 5 is performed with an elution buffer (100 mM NaAcetate with 0.1% OGP pH 5). Elution at pH 4 was performed with an elution buffer (100 mM NaAcetate with 0.1% OGP pH 4), and elution at pH 3 was performed with an elution buffer (50 mM NaCitrate with 0.1% OGP pH 3). Finally, the protein firmly bound to the resin was eluted with an organic solvent (33.3% isopropanol / 16.7% acetonitrile / 0.1% trifluoracetic acid).

(2) Adsorption of the sample to the protein chip As the solid phase carrier mounted on the protein chip, cation exchange resin (CM10) (binding / washing buffer: 100 mM NaAcetate pH4), the same (binding / washing buffer: 50 mM HEPES pH7) ), A copper ion modified carrier (IMAC30) (binding / washing buffer: 50 mM NaPhosphate, pH 7.0, 0.5M NaCl), and a reverse phase carrier (H50) (binding / washing buffer: 50 mM HEPES pH 7). Under all conditions, sinapinic acid (SPA) and CHCA (α-cyano-4-hydroxycinnamic acid) were used as matrix molecules. In addition, operations such as dispensing were all performed using Biomek2000 (Beckman).

Cation exchange tip (CM10)
150μL / spot of binding / washing buffer (using 100mM Sodium Acetate, pH4 and 100mM Sodium Acetate, pH7) is added to CM10 chip (Cyphergen Biosystems) with cation exchange group (Carboxymethyl group). And then shaken at room temperature for 5 minutes to remove the buffer. This operation was repeated twice to equilibrate the chip surface. Next, 10 μL of each fraction was added to 90 μL of the binding / washing buffer (diluted 10-fold), and the protein in the sample was adsorbed on the chip surface while shaking at room temperature for 30 minutes. A binding / washing buffer was added at 150 μL / spot, and the chip surface was washed by shaking for 5 minutes at room temperature. This was repeated three times to remove non-adsorbed components, and then desalted twice with 200 μL / spot of MilliQ water. The chip was air-dried and then air-dried after adding 1 μL / spot of 50% saturated sinapinic acid (SPA) or 50% saturated CHCA (α-cyano-4-hydroxycinnamic acid). This addition / air drying operation was repeated twice.

Copper ion modified chip (IMAC30)
In order to immobilize copper ions on the chip surface, 50 μL / spot of 100 mM CuSO ₄ was added to an IMAC30 chip having a nitrilotriacetic acid group (Ciphergen Biosystems), shaken at room temperature for 10 minutes, and then 200 μL / Wash with spot MilliQ water for 2 minutes 2 times, add 0.1 M Sodium Acetate, pH4 50 μL / spot, shake for 5 minutes at room temperature, and then wash with 200 μL / spot MilliQ water for 2 minutes.

  Next, 150 μL / spot of binding / washing buffer was added and shaken at room temperature for 5 minutes to remove the buffer. This operation was repeated twice to equilibrate the chip surface. Next, 10 μL of each fraction was added to 90 μL of the binding / washing buffer (diluted 10-fold), and the protein in the sample was adsorbed on the chip surface while shaking at room temperature for 30 minutes.

  A binding / washing buffer was added at 150 μL / spot, and the chip surface was washed by shaking for 5 minutes at room temperature. This was repeated three times to remove non-adsorbed components, and then desalted with 200 μL / spot of MilliQ water. After the chip was air-dried, 50% saturated sinapinic acid (SPA) or 50% saturated CHCA (α-cyano-4-hydroxycinnamic acid) was added at 1 μL / spot and then air-dried. This addition / air drying operation was repeated twice.

Reversed phase chip (H50)
150 μL / spot of binding / washing buffer was added to an H50 chip (Ciphergen Biosystems) having an alkyl group having 6 to 12 carbon atoms, and the buffer was removed by shaking at room temperature for 5 minutes. This was repeated twice to equilibrate the chip surface. Next, 10 μL of each fraction was added to 90 μL of the binding / washing buffer (diluted 10-fold), and the protein in the sample was adsorbed on the chip surface while shaking at room temperature for 30 minutes. A binding / washing buffer was added at 150 μL / spot to wash the chip surface and remove non-adsorbed components, followed by desalting twice with 200 μL / spot of MilliQ water. The chip was air-dried and then air-dried after adding lμL / spot of 50% saturated sinapinic acid (SPA) or 50% saturated CHCA (α-cyano-4-hydroxycinnamic acid). This addition / air drying operation was repeated twice.

(3) Measurement / data analysis The protein chip was measured with a protein chip reader ModelPBSIIC of Cyphergen. Data acquisition was performed using ProteinChip Software version 3.2 and CiphergenExpress ^™ Data Manager version 3.0. The aiming measurement range is 3000-10000 m / z for low molecular region data, 10000-30000 m / z for high molecular region data, and the maximum measured molecular weight is 100,000 m / z for low molecular region data and 200,000 m / z for high molecular region data. did. A two-point assay was performed per sample, and analysis was performed using the average value of peak intensities.

Data analysis was performed from CiphergenExpress ^™ DataManager version 3.0. After performing baseline correction from the measured spectrum, molecular weight calibration was performed. The proteins used for the molecular weight calibration are shown in Table 1.

  Next, normalization processing was performed using Total Ion Current (TIC) Normalizatio. The analysis target was a peak with Signal / Noise (S / N) larger than 2, and a peak pattern comparison between samples and a u-test (analysis based on p value) using a peak intensity value between groups were performed.

As multimarker analysis, hierarchical cluster analysis, principal component analysis (CiphergenExpress ^™ DataManager version 3.0), and Classification analysis using Biomarker Patterns Software version 5.0 were performed.

(4) Results (A) Single marker analysis The flow of data analysis is shown in FIG. First, as described above, univariate analysis (Mann-Whitney u-test) was performed on the 9175 peak showing S / N> 2, and the 1436 peak with a p value <0.05 was selected. Next, from these, peaks due to noise and individual differences were removed and used as marker candidates.

The peak of the endometrial cancer group sample has an increased intensity The peak of the endometrial cancer group sample can be captured by the cation exchange chip as a peak having an increased intensity compared to the healthy person group sample, and the center of mass is 6493 Da. (6493 Da # 1: captured by a cation exchange chip equilibrated with a pH 7 buffer solution. 6493 Da # 2: captured by a cation exchange chip equilibrated with a pH 4 buffer solution. 1 and # 2 are used in the same meaning.) 6487 Da (# 1, # 2), 3244 Da, 6691 Da (# 1, # 2), 6684 Da (# 1, # 2), 3343 Da (# 1, # 2), In addition, an increase in intensity corresponding to the peak of 19318 Da was observed.

(A) 6493 Da protein (FIGS. 2-3)
Fig. 2 shows the case where the Fr1 fraction prepared using the above pH 9 buffer was treated with a cation exchange chip [CM10: matrix molecule is SPA (LOW)] and pH 7 binding / washing buffer. Shows the results of comparing the distribution of the amount of protein (6493 Da # 1) whose mass distribution center is 6493 Da between the control (healthy person) group and the endometrial cancer group (Group box and whiskers plot and ROC plot) It is. FIG. 3 is a drawing (Group box and whiskers plot) showing the results for the protein (6493 Da # 2) captured in the system shown in FIG. 2 by replacing the binding / washing buffer with a binding / washing buffer of pH 4. And ROC plot). In both systems, it is clearly shown in these drawings that the 6493 Da protein is significantly increased in the endometrial cancer group.

  Therefore, it became clear that 6493 Da protein that can be captured by a cation exchange chip equilibrated with a pH 7 or pH 4 buffer can be used as a marker for endometrial cancer in this detection method.

(B) 6487 Da protein (FIGS. 4-5)
FIG. 4 shows the mass distribution when the Fr1 fraction prepared using the above-mentioned pH 9 buffer was measured with a cation exchange chip [CM10: matrix molecule is CHCA] and a pH 7 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result of having compared the distribution of the amount of protein (6487Da # 1) whose center is 6487Da between a control (normal person) group and a uterine body cancer group. FIG. 5 is a drawing (Group box and whiskers plot) showing the results for a protein (6487 Da # 2) captured in the system shown in FIG. 4 by replacing the binding / washing buffer with a binding / washing buffer of pH 4. And ROC plot). In both systems, it is clearly shown in these figures that the 6487 Da protein is significantly increased in the endometrial cancer group.

  Therefore, it became clear that the 6487 Da protein that can be captured on a cation exchange chip equilibrated with a pH 7 or pH 4 buffer can be used as a marker for endometrial cancer in this detection method.

(C) 3244 Da protein (FIG. 6)
FIG. 6 shows the mass distribution obtained by measuring the Fr1 fraction prepared using the above-mentioned pH 9 buffer with a cation exchange chip [CM10: matrix molecule is CHCA] and a pH 7 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result of having compared the distribution of the protein amount whose center is 3244 Da between a control (healthy person) group and the endometrial cancer group. It is clearly shown in the drawing that the 3244 Da protein is significantly increased in the endometrial cancer group.

Therefore, it was revealed that 3244 Da protein that can be captured on a cation exchange chip equilibrated with a pH 7 buffer can be used as a marker for endometrial cancer in this detection method.
(D) 6691 Da protein (FIGS. 7-8)
FIG. 7 shows the case where the Fr1 fraction prepared using the above pH 9 buffer was treated with a cation exchange chip [CM10: matrix molecule is SPA (LOW)] and a pH 7 binding / washing buffer. Shows the results of comparing the distribution of the amount of protein (6691 Da # 1) whose mass distribution center is 6691 Da between the control (healthy person) group and the endometrial cancer group (Group box and whiskers plot and ROC plot) It is. FIG. 8 is a drawing (Group box and whiskers plot) showing the results for the protein (6691 Da # 2) captured in the system shown in FIG. 7 by replacing the binding / washing buffer with the binding / washing buffer of pH 4. And ROC plot). In both systems, it is clearly shown in these figures that the 6691 Da protein is significantly increased in the endometrial cancer group.

  Therefore, it has been clarified that the 6691 Da protein that can be captured by a cation exchange chip equilibrated with a pH 7 or pH 4 buffer can be used as a marker for endometrial cancer in this detection method.

(E) 6684 Da protein (FIGS. 9-10)
FIG. 9 shows the mass distribution when the Fr1 fraction prepared using the above pH 9 buffer was measured with a cation exchange chip [CM10: matrix molecule is CHCA] and a pH 7 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result of having compared the distribution of the amount of protein 6684Da # 1) whose center is 6684Da between the control (normal person) group and the endometrial cancer group. FIG. 10 is a drawing (Group box and whiskers plot) showing the results for a protein (6684 Da # 2) captured in the system shown in FIG. 9 by replacing the binding / washing buffer with a binding / washing buffer of pH 4. And ROC plot). In both systems, it is clearly shown in these figures that the 6684 Da protein is significantly increased in the endometrial cancer group.

  Thus, it has been clarified that the 6684 Da protein that can be captured by a cation exchange chip equilibrated with a pH 7 or pH 4 buffer can be used as a marker for endometrial cancer in this detection method.

(F) 3343 Da protein (FIGS. 11-12)
FIG. 11 shows the mass distribution when the Fr1 fraction prepared using the above pH 9 buffer was measured by treatment with a cation exchange chip [CM10: matrix molecule is CHCA] and a pH 7 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result of having compared the distribution of the amount of protein (3343 # 1) whose center is 3343 Da between the control (normal person) group and the endometrial cancer group. FIG. 12 is a drawing (Group box and whiskers plot) showing the results for a protein (3343 Da # 2) captured in the system shown in FIG. 11 by replacing the binding / washing buffer with a binding / washing buffer of pH 4. And ROC plot). In both systems, it is clearly shown in these figures that the 3343 Da protein is significantly increased in the endometrial cancer group.

  Therefore, it has been clarified that the 3343 Da protein that can be captured by a cation exchange chip equilibrated with a pH 7 or pH 4 buffer can be used as a marker for endometrial cancer in this detection method.

(G) 19318 Da protein (FIG. 13)
FIG. 13 shows the case where the fraction prepared using the above pH 7 buffer was measured by treating with a cation exchange chip [CM10: matrix molecule is SPA (HIGH)] and pH 5 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result of having compared the distribution of the protein amount whose mass distribution center is 19318 Da between a control (normal person) group and a uterine body cancer group. It is clearly shown in the figure that the 19318 Da protein is significantly increased in the endometrial cancer group.

  Therefore, it was revealed that 19318 Da protein that can be captured by a cation exchange chip equilibrated with a pH 5 buffer can be used as a marker for endometrial cancer in this detection method.

  As described above, as shown in FIGS. 2 to 13, the mass center that can be captured by the cation exchange chip in the endometrial cancer group is 6493 Da (# 1, # 2), 6487 Da (# 1, # 2), The peaks of 3244 Da, 6691 Da (# 1, # 2), 6684 Da (# 1, # 2), 3343 Da (# 1, # 2), and 19318 Da have significant differences and are recognized as stronger peaks than the control group. It was. From these results, one or more proteins in the blood corresponding to these peaks were selected, and the increase in the peak (blood abundance) was compared with the detection index in comparison with the standard value (peak value in healthy individuals). As a result, it has been found that endometrial cancer can be detected extremely sensitively. Among these, the 3343 Da protein, the 6684 Da protein, and the 6691 Da protein both showed extremely high significant differences in P value <0.000001 for both # 1 and # 2.

In the endometrial cancer group sample, the peak in the endometrial cancer group sample, the peak in which the intensity is reduced compared to the healthy person group sample, the center of mass is the peak of 5625 Da, 9370 Da, and 28165 Da, A corresponding decrease in strength was observed.

(A) 5625 Da protein (FIG. 14)
FIG. 14 shows the control of the distribution of the amount of protein having a mass distribution center of 5625 Da when measured with a copper ion modified chip [IMAC30: matrix molecule is CHCA] and a pH 4 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result compared between the group and the endometrial cancer group. It is clearly shown in the drawing that the 5625 Da protein is significantly decreased in the endometrial cancer group.

  Therefore, it has been clarified that the 5625 Da protein that can be captured by the copper ion modified chip can be used as a marker for endometrial cancer in this detection method.

(B) 9370 Da protein (FIG. 15)
FIG. 15 shows a control of the protein distribution with a mass distribution center of 9370 Da when measured by treatment with a copper ion-modified chip [IMAC30: matrix molecule is CHCA] and pH 4 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result compared between the group and the endometrial cancer group. It is clearly shown in the drawing that the 9370 Da protein is significantly reduced in the endometrial cancer group.

  Therefore, it was revealed that the 9370 Da protein that can be captured by the copper ion modified chip can be used as a marker for endometrial cancer in this detection method.

(C) 28165 Da protein (FIG. 16)
FIG. 16 shows the control of the distribution of the protein amount having a mass distribution center of 28165 Da when measured with a copper ion modified chip [IMAC30: matrix molecule is CHCA] and a pH 4 binding / washing buffer. It is drawing (Group box and whiskers plot and ROC plot) which showed the result compared between the group and the endometrial cancer group. It is clearly shown in the drawing that the 28165 Da protein is significantly decreased in the endometrial cancer group.

  Therefore, it was revealed that 28165 Da protein that can be captured by the copper ion-modified chip can be used as a marker for endometrial cancer in this detection method.

  As shown in FIGS. 14 to 16 above, the peaks of mass centers 5625 Da, 9370 Da, and 28165 Da that can be captured by the copper ion-modified chip in the endometrial cancer group are significantly different from the control group. Was also observed as a weak peak. From these results, one or more of the blood proteins corresponding to these peaks are selected, and the decrease in the peak (blood abundance) is compared with the detection index in comparison with the standard value (peak value in healthy individuals). As a result, it has been found that endometrial cancer can be detected extremely sensitively.

(B) Multi-marker analysis
Hierarchical cluster analysis Hierarchical cluster analysis is a method of classifying objects that have not yet been classified according to a specific standard, and is usually classified according to the degree of similarity between the objects. In this example, the control group and endometrial cancer were determined based on the height of the 701 peak (the amount of protein corresponding to the peak) of all test conditions excluding the noise peak among the peaks having a P value of 0.05 or less. Two groups of the above-mentioned 40 samples consisting of groups were analyzed. As a result, the control group and the endometrial cancer group were clearly classified. Therefore, it was shown that endometrial cancer can be detected by performing hierarchical cluster analysis on the mass distribution pattern of proteins between specific molecular weights in blood samples.

Principal component analysis Principal component analysis is a method of finding a small number of synthesized new variables (principal components) from the variation tendency of data of multiple variables, and classifying objects that have not yet been classified using these principal components. is there. In the case of the present Example, the Percent variance shown in Table 2 is high from the height of the 701 peak (the amount of protein corresponding to the peak) of all test conditions excluding the noise peak among the peaks having a P value of 0.05 or less. The top three principal components 1 (PCAcomp1), principal component 2 (PCAcomp2), and principal component 3 (PCAcomp3) were newly synthesized and analyzed by combining them. The top 10 peaks with the highest contribution rate (Importance) in each main component are listed (Table 2). As a result, the control group and the endometrial cancer group could be classified by the combination of PCAcomp1 and 3, PCAcomp2 and 3, and PCAcomp1,2,3. Therefore, it was shown that endometrial cancer can be detected by performing principal component analysis on the mass distribution pattern of proteins between specific molecular weights in blood samples.

  In addition, the 15 types of peak groups [6493 Da (# 1, 2), 6487 Da (# 1, 2), 3244 Da, 6691 Da (# 1, 2), 6684 Da (# 1, 2), 3343 Da (# 1, 2) ), 19318 Da, 5625 Da, 9370 Da, and 28165 Da, the peak group showing the center of molecular weight distribution] was used, and the same principal component analysis as described above was performed. As a result, even in this case, the control group and the endometrial cancer group could be classified.

Classification analysis
Classification analysis is a method of classifying objects that have already been classified using new criteria. Generally, a rule is constructed based on classified objects, and unclassified objects are matched against the rules. Divided into steps to be classified. In the case of this example, Classification analysis was performed using Biomarker Patterns Software version 5.0. Specifically, the control group and endometrial cancer group on the basis of the height of the 701 peak (the amount of protein corresponding to the peak) of all test conditions excluding the noise peak among the peaks having a P value of 0.05 or less The rule which classifies and was built.

  1) Classification analysis was performed using only the CHCA condition in which the noise peak was removed from the peak having a P value of 0.05 or less in the two-group analysis of the control group and endometrial cancer group. The peak used for the initial classification was MW3343, which was selected as the Candidate Peak during the single marker analysis. As a result of classification using this peak, it was found that diagnosis was possible with an accuracy of 90% Specificity and 95% Sensitivity.

  2) Classification analysis was performed using the 701 peaks of all test conditions except the noise peak at the P value of 0.05 or less in the two-group analysis of the control group and endometrial cancer group. The peak used for the initial classification was MW3343, which was selected as the Candidate Peak during the single marker analysis. As a result of classification using this peak, it was found that diagnosis was possible with an accuracy of 90% Specificity and 95% Sensitivity.

  3) Classification analysis was performed using only the above 15 types of Candidate Peak. The peak used for the initial classification was MW3343. As a result of classification using this peak, it was found that diagnosis was possible with an accuracy of 90% Specificity and 95% Sensitivity.

Summary It was shown that the endometrial cancer group and the control group can be classified by performing the single marker analysis and the multimarker analysis described above. Through these analyses, the mass distribution and expression intensity of the protein in the blood sample in the molecular weight range of 1000 to 30000 Da are measured, and the difference found by comparing the pattern of the mass distribution and the relative expression intensity with the pattern in the healthy person is used as an index. As a result, it was revealed that endometrial cancer in the subject can be detected.

It is drawing which showed the flow of the data analysis in a single marker analysis as a diagram. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6493 Da at the time of processing by processing with the binding and washing | cleaning buffer of cation exchange chip [CM10: matrix molecule is SPA (LOW)] and pH7. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6493 Da at the time of processing by processing with the binding / washing buffer of cation exchange chip [CM10: matrix molecule is SPA (LOW)] and pH4. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6487 Da at the time of processing by processing with a cation exchange chip [CM10: matrix molecule is CHCA] and pH7 binding and washing buffer. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6487 Da at the time of processing by processing with the binding / washing buffer of cation exchange chip [CM10: matrix molecule is CHCA] and pH4. It is drawing which shows the result of having analyzed about the peak which shows the protein of 3244 Da at the time of processing by cation exchange chip [CM10: Matrix molecule is CHCA] and pH 7 binding / washing buffer. It is a figure which shows the result of having analyzed about the peak which shows the protein of 6691 Da at the time of processing by processing with the coupling | bonding and washing | cleaning buffer of cation exchange chip [CM10: matrix molecule is SPA (LOW)] and pH7. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6691 Da at the time of processing by processing with the binding / washing buffer of cation exchange chip [CM10: matrix molecule is SPA (LOW)] and pH4. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6684 Da at the time of processing by processing with the coupling | bonding and washing buffer of cation exchange chip [CM10: matrix molecule is CHCA] and pH7. It is drawing which shows the result of having analyzed about the peak which shows the protein of 6684 Da at the time of processing by processing with the binding / washing buffer of cation exchange chip [CM10: matrix molecule is CHCA] and pH4. It is drawing which shows the result of having analyzed about the peak which shows the protein of 3343 Da at the time of processing by cation-exchange chip | tip [CM10: matrix molecule is CHCA] and the pH 7 binding / washing buffer. It is drawing which shows the result of having analyzed about the peak which shows the protein of 3343 Da at the time of processing by cation exchange chip | tip [CM10: a matrix molecule is CHCA] and the coupling | bonding and washing buffer of pH4, and measuring. It is a figure which shows the result of having analyzed about the peak which shows the protein of 19318 Da at the time of processing by processing with a cation exchange chip [CM10: matrix molecule is SPA (HIGH)] and pH5 binding and washing buffer. It is drawing which shows the result of having analyzed about the peak which shows the protein of 5625 Da at the time of processing by processing with the binding / washing buffer of copper ion modification chip [IMAC30: matrix molecule is CHCA] and pH4. It is drawing which shows the result of having analyzed about the peak which shows the protein of 9370 Da at the time of processing by processing with a copper ion modification chip [IMAC30: matrix molecule is CHCA] and pH4 binding and washing buffer. It is drawing which shows the result of having analyzed about the peak which shows the protein of 28165 Da at the time of processing by processing with the binding / washing buffer of copper ion modification chip [IMAC30: matrix molecule is CHCA] and pH4.

Claims (12)

Measuring the mass distribution and expression intensity of the protein in the blood sample of the subject in the range of molecular weight 1000 to 200000 Da, and using the difference found by comparing the pattern of the mass distribution and the relative expression intensity with the pattern in the healthy person as an index, A detection method for detecting endometrial cancer in the subject. The detection method according to claim 1, wherein the measurement range of the molecular weight of the protein in the blood sample of the subject is 1000 to 20000 Da. In the detection method, univariate analysis or one or more multivariate analysis selected from hierarchical cluster analysis, principal component analysis, and classification analysis is used as a means for detecting a difference in pattern of the mass distribution. The detection method according to 1 or 2. The detection method according to claim 1, wherein the following (1) and / or (2) is performed in the detection method.
(1) In a blood sample of a subject, one or more selected from blood proteins having substantially the center of mass distribution of a group consisting of 6493 Da, 6487 Da, 3244 Da, 6691 Da, 6684 Da, 3343 Da, and 19318 Da, When the amount of the one or more blood proteins is higher than that of a healthy person, this is used as an index of endometrial cancer in the subject.
(2) One or more blood proteins selected from blood proteins having substantially the mass distribution center of the group consisting of 5625 Da, 9370 Da, and 28165 Da are detected in the blood sample of the subject, and the one or more blood proteins are detected. When the amount of uterine is decreased as compared with a healthy person, this is used as an index of endometrial cancer in the subject. In the detection method, one or more selected from blood proteins having substantially the center of mass distribution of the group consisting of 6493 Da, 6487 Da, 3244 Da, 6691 Da, 6684 Da, 3343 Da, and 19318 Da according to item (1), The detection method according to claim 4, which is a protein having a property of being captured by a cation exchange type solid phase carrier. In the detection method, at least one selected from blood proteins having substantially the mass distribution center of the group consisting of 6493 Da, 6487 Da, 6691 Da, 6684 Da, and 3343 Da is equilibrated with a pH 4 or pH 7 buffer. 6. The detection method according to claim 5, which is a protein having a property of being captured by a cation exchange type solid phase carrier. In the said detection method, the blood protein concerning item (1) is 1 or more types chosen from the protein which has a mass distribution center of the group which consists of 3343 Da, 6684 Da, and 6691 Da substantially. The detection method in any one of. In the detection method, one or more selected from blood proteins substantially having a mass distribution center of the group consisting of 5625 Da, 9370 Da, and 28165 Da related to item (2) are captured by the metal ion-modified carrier. The detection method according to any one of claims 4 to 7, which is a protein having properties. The detection method according to claim 8, wherein the metal ion of the metal ion modified carrier is a copper ion. In the detection method, the blood protein captured by the solid phase carrier is irradiated with a laser beam to desorb the blood protein from the solid phase carrier in an ionized state. The detection method according to claim 1, wherein the detection method is performed by detecting with a time-type mass spectrometer. The detection method according to claim 1, wherein the blood sample is a serum sample. 12. The detection method according to claim 11, wherein in the detection method, the serum sample is a serum sample that has been subjected to contact treatment with an anion exchange solid phase carrier.