KR20130066481A - Method for analyzing cancer-specific glycan and use of it for diagnosing cancer - Google Patents

Abstract

PURPOSE: A method for analyzing a cancer-specific sugar chain is provided to search a sugar chain in blood as a novel prediction factor with excellent specificity and sensitivity. CONSTITUTION: A method for providing information for cancer diagnosis comprises: a step of providing a first plate comprising a plurality of wells arranged in a first array type(S1); a step of isolating one or more sugar chains from a sample by treating the sample containing serum isolated from a patient with cancer using the first plate(S2); a step of providing a second plate containing a matrix material ionized by absorption of energy(S3); a step of mixing one or more sugar chains with the matrix material using a plurality of mixing regions and irradiating the plurality of mixing regions by each laser to ionize one or more sugar chains(S4); a step of moving one or more ionized sugar chains under an electric field and detecting by a detector(S5); a step of generating third mass spectrum using blood collected from a subject person(S6); a step of drawing one or more peaks corresponding to the cancer-specific sugar chain using first and second mass spectra, and acquiring cancer progression information(S7). [Reference numerals] (AA) Start; (BB) End; (S1) Provide a first plate containing a plurality of wells arranged in an array type; (S2) Extract one or more sugar chains by processing the blood of a patient with cancer using the first plate; (S3) Provide a second plate containing a plurality of mixing regions arranged in an array type; (S4) Mix the sugar chains with a matrix material using each mixing region and ionize the sugar chains by irradiating the each mixing region with laser; (S5) Detect using a detector after moving the ionized sugar changes under an electric field; (S6) Calculate mass spectrum by measuring the moving time of one or more sugar chains; (S7) Compare with the calculated mass spectrum using the blood of a person without disease

Description

METHOD FOR ANALYZING CANCER-SPECIFIC GLYCAN AND USE OF IT FOR DIAGNOSING CANCER

The examples relate to methods of analyzing cancer specific sugar chains and applications to cancer diagnosis using the same.

It is known that the biomarker in the blood exhibits a characteristic in which the substance in the tissue is secreted into the circulatory system during the occurrence of the disease or under a specific physiological state and thus is present in excess in the blood. In-depth characterization and analysis of protein structures through in-depth characterization of biopolymers has enabled the development of proteomics technology to enable sensitive and accurate measurement of changes in disease predictors. Although many studies have been conducted on proteins as markers that detect the qualitative and quantitative changes of certain substances in the blood to comprehensively determine the condition of the disease, the therapeutic effect of the drug, and its association with other diseases. The discovery of predictors and their applications in diagnostic medicine are falling short of expectations.

Meanwhile, about 70% of the proteins constituting the human body are glycosylated. As a part of the study of proteomics, a research field called glycomics is a study that targets the structure and properties of glycan. Recently, the development of research and application technology to apply the blood sugar chain as a predictor of disease and to apply it to the diagnostic medicine field for precise analysis is emerging. Previous studies and inventions have shown that glycosylation specifically changes in various malignant tumors, including ovarian cancer, breast cancer, prostate cancer, liver cancer, lung cancer, gastric cancer, and pancreatic cancer. That is, glycoprotein (protein glycation) of the protein is changed in the process of cancerous, and the level of oligosaccharides constituting the serum glycoprotein in each disease is changed. Some of these glycoprotein changes have been suggested as potential biomarkers of disease. For example, U.S. Patent No. 7,651,847 describes the possibility of ovarian cancer and breast cancer through pattern analysis of mass spectrometry on repeated fragments of oligosaccharides separated from the blood of certain cancer patients. Techniques for predicting cancer progression stages are disclosed. In particular, US Pat. No. 7,651,847 focused on mucin in proteins. Mucins are classified as glycoproteins combined with high glycosilation, a large portion of which is known as oligosaccharides. Most epithelial cell carcinomas (ie epithelial tissue cancers) are often accompanied by large amounts of mucin production. For example, CA125 (cancer antigen 16) (mucin 16) is a typical tumor marker of ovarian cancer and has been reported to increase its secretion in the body of ovarian cancer patients. Mucin 1 is also known to be associated with breast cancer.

However, measuring the concentration of mucin, such as mucin 1 or mucin 16 (CA 125), is complicated for several reasons. Mucins have a molecular structure of heterogeneous glycoproteins of polymers having a molecular weight of about 2.5 × 10 ⁶ to about 2.9 × 10 ⁶ Daltons (Da), and antigen-antibody immunospecific reactions currently used for mucin determination Inaccurate and not quantitative

In addition, mucin, which is commonly measured such as CA125, tends to show different numerical values depending on the type of tumor of ovarian cancer. In addition, when the CA125 concentration is higher than normal, only about 50% of patients with stage 1 ovarian cancer have a problem of showing a false positive response that increases the CA125 concentration even by pregnancy or other cancers. Moreover, in women with mild endometriosis or pelvic inflammatory disease, a high number of high levels of CA125 levels occur. As such, the marker CA125 is particularly unsuitable for screening for early ovarian cancer.

US Patent No. 7,651,847

According to an aspect of the present invention, there is provided a method for analyzing cancer-specific sugar chains that can detect blood sugar chains as a new predictor having better specificity and sensitivity than conventionally used ovarian cancer diagnostic predictors. Can be. The cancer-specific sugar chain analysis method is performed in a short time by high-density separation and fractionation of a large amount of serum by Matrix Assisted Laser Desorption / Ionization Time of Flight (MALDI-TOF) mass spectrometry. High throughput can be achieved.

Furthermore, according to another aspect of the present invention, by using the method for analyzing cancer-specific sugar chains, it is possible to provide an application method for cancer diagnosis that can determine whether cancer has developed and the degree of cancer progression.

According to one or more exemplary embodiments, a method of analyzing cancer-specific sugar chains may include providing a first plate including a plurality of wells arranged in a first array; Processing a sample comprising serum isolated from blood of a cancer patient using the first plate to extract one or more sugar chains from the sample; Providing a second plate comprising a plurality of mixing regions arranged in a second array form; Mixing the at least one sugar chain with a matrix material using the plurality of mixed regions, and ionizing the at least one sugar chain by irradiating a laser to each of the plurality of mixed regions; Moving the at least one sugar chain ionized under an electric field and detecting it by a detector; Deriving a first mass spectrum of the at least one sugar chain by measuring the time of arrival of the detector of each of the at least one sugar chain; And comparing the derived first mass spectrum with a second mass spectrum derived using blood of a normal person to determine cancer specific sugar chains.

Screening method for diagnosing cancer according to an embodiment may be performed using the above-described method for analyzing cancer specific sugar chains. The screening method for diagnosing cancer may include generating a third mass spectrum using blood of a diagnosis subject; Deriving, from the third mass spectrum, one or more peaks corresponding to the cancer specific sugar chains determined using the first mass spectrum and the second mass spectrum; And comparing the one or more peaks with the peaks of the first mass spectrum and the second mass spectrum, respectively, to obtain cancer progress information of the diagnosis subject.

Using a method for analyzing cancer specific sugar chains according to an aspect of the present invention, blood sugar chains as a new predictor having better specificity and sensitivity than cancer predictors such as CA125 (cancer antigen 125) used in the past Can be excavated. In addition, the pretreatment process of the sample is performed using a first plate arranged in a plurality of wells, and matrix-assisted laser desorption / ionization time analysis using a second plate arranged in an array form in a plurality of mixing regions. By performing the Mass Assisted Laser Desorption / Ionization Time of Flight (MALDI-TOF) mass spectrometry process, the entire process from blood to mass spectrometry can be highly integrated and integrated to achieve high efficiency throughput.

In addition, the cancer-specific sugar chain analysis method according to an aspect of the present invention, by using the MALDI-TOF mass spectrometry to reduce the occurrence of fragmentation (fragmentation) during the ionization process of sugar chains to determine the mass value for the intact form It can be measured and has a very large detection range, so it is easy to analyze materials with large mass values, has high resolution and accurate analysis compared to other types of mass spectrometry, and achieves high sensitivity. Analyzing the sample also has the advantage that the amount of sample consumed is very small.

Furthermore, the data measured using the MALDI-TOF mass spectrometry is easy to interpret because it appears as a simple spectrum without multiple charged ions, and has the advantage of small size and fast processing speed. In addition, the effect of foreign substances such as buffer solution and salt contained in the sample is relatively small, and the time from preparation of the sample to the measurement is very short, which is suitable for analyzing a large amount of samples, and the use and maintenance of the device It is easy and inexpensive.

In addition, according to another application method for diagnosing cancer according to another aspect of the present invention, cancer markers determined by using the above-described method for analyzing cancer-specific sugar chains may be used to determine whether cancer develops and / or the progress of cancer. can do.

1 is a flowchart illustrating a method of analyzing cancer specific sugar chains according to an embodiment.
2 is a flowchart illustrating a process of separating one or more sugar chains from blood in the method for analyzing cancer specific sugar chains according to an embodiment.
Figure 3a is a photograph showing the operation of the first plate in the reactor using a microwave (microwave) in the method for analyzing cancer-specific sugar chains according to an embodiment.
3B is a schematic diagram of a first plate consisting of 96 wells.
4 is a photograph showing the use of the second plate in the method for analyzing cancer specific sugar chains according to one embodiment.
5A and 5B are graphs showing exemplary mass spectra obtained from a human serum of Sigma by the method for analyzing cancer specific sugar chains according to one embodiment.
6A and 6B are graphs showing mass spectra derived from blood of a cancer patient and mass spectra derived from blood of a normal person, respectively, by the method for analyzing cancer specific sugar chains according to an embodiment.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

According to one embodiment, a method for analyzing cancer-specific sugar chains may include a matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass of a sugar chain corresponding to a cancer marker. It may be configured to analyze by an assay. After separating serum from the blood of a cancer patient and digesting it back to the repeating unit molecular group, the molecular weight distribution and relative intensities of the digested fragments can be obtained in the mass spectrum. In addition, cancer-specific sugar chains can be determined by comparing the mass spectra obtained in this way with mass spectra obtained from serum of normal persons, not cancer patients, through pattern analysis.

Furthermore, the screening method for diagnosing cancer according to one embodiment diagnoses the progress and / or extent of cancer of a diagnosis subject by using a cancer specific sugar chain determined by the method for analyzing cancer specific sugar chains described above. It can be configured to. That is, the mass spectrum may be derived from the blood of the diagnosis subject by the above-described process, and the derived mass spectrum may be used to determine whether cancer has developed in the body of the diagnosis subject and to what stage the cancer has progressed.

In the embodiments described herein, methods for analyzing cancer specific sugar chains and screening methods for cancer diagnosis have been described based on ovarian cancer. However, cancer to which the embodiments of the present invention are applicable is not limited to ovarian cancer, and breast cancer, prostate cancer, liver cancer, lung cancer, gastric cancer, pancreatic cancer, or any other type of tumor not described herein may be used. It may be the subject of an embodiment.

1 is a flowchart illustrating a method of analyzing cancer specific sugar chains according to an embodiment.

Referring to FIG. 1, a first plate including a plurality of wells arranged in a first array form may be provided (S1). The first plate is a means for performing a process of separating one or more sugar chains from a sample as a pretreatment process for performing MALDI-TOF mass spectrometry. Each of the plurality of wells may be equipped with a container such as a tube into which a sample is injected, or a hole or a groove into which a sample may be directly injected, and the like. A plurality of wells may be arranged in an array form on the first plate. By using a plurality of wells arranged in an array form, a large amount of samples may be processed at one time, thereby rapidly processing a sample.

When the first plate is provided, one or more sugar chains may be separated from the sample by treating the sample including the serum separated from the blood of the cancer patient using the first plate (S2).

2 is a flowchart illustrating a process of separating one or more sugar chains from blood in a method for analyzing cancer specific sugar chains according to an embodiment.

Referring to FIG. 2, first, serum may be separated from blood of a cancer patient to prepare a sample (S21). To this end, blood collected from cancer patients may be kept at low temperature for a time for coagulation, and then only serum may be separated from blood by centrifugation. For example, blood may be left on ice for 1 hour to allow clot formation, followed by centrifugation at an acceleration of about 3500 g at a temperature of about 4 ° C. for about 10 minutes to separate serum. If storage of the separated serum is necessary, the serum may be transferred to a sterile cryogenic tube and stored at a temperature of about 80 ° C below zero.

Next, a sample containing the separated serum may be injected into each of the plurality of wells of the first plate (S22). In the present specification, the injection may mean directly injecting a sample into a corresponding region, or when the sample is injected into another container such as a tube, it may mean to mount a container into which the sample is injected. During sample injection for each well, the sample may be mixed with the buffer solution and injected. Ammonium bicarbonate may be used as the buffer solution, but is not limited thereto.

Next, the serum in the sample can be digested (S23). For example, dithiothreitol may be injected into a sample and heated to a temperature of about 95 ° C. to denature the protein. In addition, one or more sugar chains may be separated from the protein by mixing a sample in which the protein is denatured (S24). For example, peptide N-glycosidase F (PNGase F) enzyme may be injected into a sample to cut sugar chains by mixing enzymes. In one embodiment, the sample may be irradiated with microwaves when the enzyme is mixed. For example, the microwave may be irradiated for about 10 minutes at an intensity of about 400 W at a temperature of about 37 ° C., but is not limited thereto.

3A is a photograph illustrating a process of manipulating a first plate in a reactor using microwaves in a method for analyzing cancer specific sugar chains according to an embodiment, and FIG. 3B is a schematic diagram of a first plate including 96 wells.

3A and 3B, the first plate may be a 96 well plate including 96 wells arranged in an array. As shown in FIG. 3A, a 96 well plate may be placed in a chamber of a microwave mixer and the mixer may be operated to allow for enzymatic mixing in a sample located in the well. However, this is exemplary and the form of the first plate and the equipment for performing the enzyme mixing using the same is not limited to that shown in the figure.

Next, the protein may be removed from the sample using alcohol precipitation (S25). For this purpose, the sample may be precipitated by cooling the sample after mixing with absolute ethanol. For example, the sample and ethanol may be mixed at a ratio of about 1: 4, and then the protein may be precipitated at a low temperature of about 80 ° C. Once the protein has precipitated, only the supernatant except the protein can be transferred to a new tube by centrifugation. Centrifugation may be performed using an acceleration of about 3500 g at a temperature of about 4 ° C, but is not limited thereto. The sample extracted as described above may be dried using a high speed vacuum dryer or the like.

Next, one or more sugar chains may be eluted from the dried sample by a solid phase extraction process (S26). For example, distilled water, an acetonitrile (ACN) solution at a concentration of about 80%, and distilled water may be sequentially added to a graphite carbon column. The dried sample may be dissolved in distilled water and then put in a column. Next, distilled water may be flowed to the column to remove salt, and then ACN solution may be injected to elute sugar chains. The ACN solution injected for elution can be about 10% ACN solution, about 20% ACN solution, or about 40% ACN solution with about 0.05% trifluoacetic acid solution. have. The above solution may be introduced into the column in an amount of about 4 mL to about 10 mL, and as a result, one or more sugar chains may elute.

In one embodiment, the material is dispensed into a plurality of wells of the first plate at a time in performing one or a plurality of steps of the above-described processing of the sample using the first plate (S22 to S26); It is also possible to use a multi pipette system that can extract material from multiple wells at once. For example, a liquid pipette 96 manufactured by Mettler Toledo may be used as the multi pipette system, but is not limited thereto. By using a multi-pipette system and a first plate containing a plurality of wells, a large amount of samples can be processed at once and standardization and automation of the process can provide rapid and clinically reproducible results.

Referring back to FIG. 1, a second plate including a plurality of mixing regions arranged in a second array shape may be provided (S3). Each mixing region may consist of holes or grooves into which a sample and a matrix material are injected, or a container such as a tube into which the materials are injected, may be mounted, and is not limited to a specific shape. Since a plurality of mixing regions are arranged in the form of a second array, a large amount of samples can be processed at one time. The number of mixing regions may be the same as or different from the number of wells on the first plate described above.

In one embodiment, the first plate may be a 96 well plate comprising 96 wells, and the second plate may be a 384 well plate comprising 384 wells. Figure 4 shows a 384 well plate as a photograph showing the use form of the second plate in the method for analyzing cancer specific sugar chains according to one embodiment. However, this is exemplary and the number of mixing regions on the second plate may be different from that described above and may also be the same or less than the number of wells on the first plate.

In one embodiment, one or more sugar chains may be mixed with the matrix material in the liquid state and injected into each of the plurality of mixing regions. For example, they may be injected onto a solvent capable of dissolving both one or more sugar chains and the matrix material, and a liquid mixed with one or more sugar chains and the matrix material simultaneously may be injected into each mixing region on the second plate. As the injected liquid dries on the mixing zone, the sugar chains and the matrix material may crystallize.

Meanwhile, in another embodiment, the matrix material may be previously injected into each of the plurality of mixing regions of the second plate. That is, the second plate is uniformly applied to the plurality of mixing regions in advance of the matrix material to prepare a pre-spot plate, and a solution in which one or more sugar chains are dissolved is added to each mixing region to add the matrix material and the crystalline state. Can be mixed. For example, the second plate is placed on a u- Focus plate which is treated with a hydrophobic / hydrophilic substance to induce high integration of the sample on the surface of the plate, and the dihydroxybenzoic acid for analyzing the obtained sugar chain. The plate may be applied in advance to each of the mixing regions. The microfocus plate is described in US Patent No. 7,619,215 and Korean Patent Nos. 10-0544860 and 10-0883908.

In one embodiment, the sugar chains present in the fraction eluted with an ACN solution of about 10% and 20% concentrations are neutral sugar chains, the neutral sugar chains being sodium chloride when the matrix is applied to the second plate because they are not readily ionized. Can be added together to induce ionization in the bipolar mode when obtaining the MALDI-TOF spectrum of the neutral sugar chain. In this case, each of the mixing zones was about 1700 to about 2000 μm in diameter and about 1.5 to about 2.0 μg of matrix was uniformly applied to each of the mixing zones. The acidic sugar chains are obtained from the eluted aliquots using an ACN solution of about 40% concentration. In this case, sodium chloride is removed from the second plate in the same way, and only the dihydroxybenzene acid is applied before crystallization, followed by injection / mixing of the samples. Can be analyzed in the cathode mode.

The matrix material is a material that easily absorbs energy from the laser and ionizes, and MALDI-TOF mass spectrometry is configured to indirectly ionize the sample by an ion transfer process using the matrix. For example, the matrix material may be an organic compound having a structure that is easily excited by a laser, or may be an aromatic organic compound. In addition, a mixture of two or more substances may be used as the matrix material in order to dissolve the organic compound well and to dissolve the sample well.

In obtaining the mass spectrum using MALDI-TOF mass spectrometry, in order to maximize the analysis sensitivity of the sample, a sample mounting preparation method of mixing the sample and the matrix material and then drying and crystallizing the mixed material is very important. Various factors affect the analysis, such as the relative mixing ratio of the sample and the matrix material and the drying rate, and errors in the sample pretreatment may reduce the reproducibility of the analysis. In one embodiment, in order to minimize the effects of sample pretreatment and to obtain crystals that are simple and have high uniformity, a prespot that has been mechanically and accurately applied with a matrix material in advance to the mixing region into which one or more sugar chains to be analyzed is injected spot plate may be used as the second plate.

When the second plate is prepared, one or more sugar chains extracted from the sample may be injected into each of the plurality of mixed regions, and the sugar chains may be ionized by irradiating a laser to the mixed regions (S4). First, as shown in FIG. 4, one or more sugar chains extracted from a sample may be mixed with a matrix material previously applied to each mixing region in a crystallized state. Alternatively, the sugar chains and the matrix material may be mixed in a solution phase and then injected into the mixed region to be dried to crystallize. Next, the sugar chains can be ionized by irradiating a laser to each mixed region. For example, the laser may be a solid type yag laser, but is not limited thereto. Since the matrix material is made of a material that easily absorbs energy from the laser and is ionized, one or more sugar chains may be indirectly ionized through an ion transfer process by the matrix material.

Next, the ionized sugar chains may be moved under an electric field and then detected by a detector (S5). The detector may be placed in a spatially spaced position from the second plate, and ions generated from the sugar chain may be accelerated by the electric field and moved in the direction of the detector. In this case, the space between the detector and the second plate to which the ions move may be maintained in a vacuum for accurate measurement. According to the solvent of the sample, ions having different polarities may be detected, and the electric field applied to the ions depending on the solvent may be changed into a positive mode or a negative mode. For example, a solvent containing an ACN solution at a concentration of about 10% or about 20% can be detected in the positive electrode mode, and a solvent containing an ACN at a concentration of about 40% can be detected in the negative electrode mode.

However, the numerical values described above are exemplary, and the amount of each of the sample and the matrix, the type and amount of the matrix material, the polarity of the applied electric field, and the like can be appropriately determined by applying a known MALDI-TOF mass spectrometry technique.

Next, the mass spectrum of one or more sugar chains can be derived by measuring the detector arrival time of each sugar chain (S6). The ions produced from the sugar chains are separated by the mass-to-charge ratio during the acceleration and movement by the electric field. Thus, the time to which each ion reaches the detector can be used to specify the mass-to-charge ratio of the ion. By performing the above procedure on the ions of one or more sugar chains, the mass-to-charge ratio and the intensity of one or more sugar chains can be derived in the form of a mass spectrum.

5A and 5B are graphs showing mass spectra derived from a human serum sold by Sigma, by a method for analyzing cancer specific sugar chains according to one embodiment. 5A shows the mass spectrum in the positive mode of one or more sugar chains eluted using an ACN solution at about 10% concentration, and FIG. 5B is in the positive mode of one or more sugar chains eluted using an ACN solution at about 20% concentration. The mass spectrum of is shown.

As shown, in the molecular weight range of about 1000 Daltons (Da) to about 3000 Da, the positional distribution of the mass-to-charge ratio (m / z) of each sugar chain and their relative intensities can be identified and Mass spectra can be obtained to determine relative heights. In addition, the molecular structure is shown below each spectrum and the numbers below each structure are theoretically calculated mass values of the corresponding sugar chains, and the mass value of each sugar chain measured (the number shown in the red box at the top of each peak). ) And the theoretical calculated value showed a small difference of less than about 20 × 10 ⁻³ to 50 × 10 ⁻³ Daltons (about 20 to 50 ppm (parts per million)) on average.

In addition, to examine the reproducibility of the data obtained from each of the 10% and 20% fractions injected into each of the plurality of mixing zones of the second plate, the mass spectra were analyzed by analyzing the samples injected into 10 different mixing zones. The relative intensities of each peak of were compared. As a result, the minimum deviation of the peak intensity was about 0.49%, the maximum deviation was about 13.7%, and the average deviation was about 7.5%.

By the above-described processes (S1 to S6), the mass spectrum of blood sugar chains was derived from the blood of cancer patients. Next, cancer specific sugar chains, that is, cancer markers, may be determined by comparing the derived mass spectrum with the mass spectrum derived from blood of a normal person (S7). Normal person as used herein refers to any subject who has been found to have not developed the cancer as a control for the cancer patient described above. The mass spectrum of the blood of a normal person can be obtained by the same procedure as that of obtaining the mass spectrum of blood of the cancer patient described in this example (S1 to S6), or by other different methods not described herein. It may be.

By comparing the mass spectra obtained from cancer patients and respective normal blood with respect to one or more sugar chains by pattern analysis, cancer specific sugar chains that are indicative of the onset and / or progression of a particular cancer can be determined. Pattern analysis of the mass spectrum can be done by comparing the intensity of the peak corresponding to each sugar chain in the mass spectrum of a cancer patient with the intensity of the peak corresponding to each sugar chain in the mass spectrum of a normal person, or other known pattern analysis It may be made by a method.

FIG. 6A shows a mass spectrum of 20% aliquot derived from blood of a cancer patient by the method of analyzing cancer specific sugar chains according to one embodiment, and FIG. 6B shows a mass spectrum of 20% aliquot derived from blood of a normal person . As shown, it can be seen that the intensity peaks of the mass-to-charge ratios of about 1257.42, about 1419.47, about 1485.53, about 1647.59, about 1809.64, and about 1850.67 molecules show a relatively large difference between cancer patients and normal subjects. Sugar chains with a high charge ratio can be determined as cancer markers.

Table 1 below shows the mass values and structures of the cancer markers determined by the method for analyzing cancer specific sugar chains according to one embodiment.

Mass versus Charge ratio  (m / z) ^(※) rescue 1257.42

1419.47

1485.53

1647.59

1809.64

1850.67

Man,

Hex,

GlcNAc,

HexNAc,

NeuAc,

Fuc ^※

Man,

Hex,

GlcNAc,

HexNAc,

NeuAc,

Fuc

As shown in Table 1, each sugar chain determined as a cancer marker is mannose (Man, Mass to charge ratio 162.05), hexose (Hex, mass to charge ratio 162.05), N-acetylglucosamine ( N-acetylglucosamine; GlcNAc, mass-to-charge ratio 203.08), N-acetylhexosamine; HexNAc, mass-to-charge ratio 203.08, N-acetylneuraminic acid (NeAcAc, mass charge One or more of several sugars, such as Harvey 291.09) and fucose (Fuc, mass-to-charge ratio 146.08).

However, the cancer markers described above are merely exemplary and the cancer specific sugar chains that can be analyzed by the embodiments of the present invention are not limited to those described in Table 1 above. For example, various oligosaccharide sugar chains used for morphological analysis of mass spectrometry in US Pat. No. 7,651,847 may be determined as cancer markers by the method for analyzing cancer specific sugar chains according to embodiments of the present invention.

In embodiments, pretreatment of a sample is performed by using a first plate in which wells are arranged in an array form in order to increase mass spectrometry efficiency, and also using a second plate in a prespot form in which mixing regions are arranged in an array form. By performing the MALDI-TOF mass spectrometry procedure, the entire process from blood to mass spectrometry is highly integrated and integrated. As a result, standardization and automation of the process is possible, resulting in rapid and clinically reproducible results. For example, the inventors of the present invention were able to quickly obtain a diagnosis result by analyzing samples obtained from 100 subjects within 2 days.

In one embodiment, a screening method for cancer diagnosis may be provided using a cancer specific sugar chain determined by the method for analyzing cancer specific sugar chains described above.

That is, the first mass spectrum and the second mass spectrum may be generated using the blood of the cancer patient and the normal person, respectively, and the third mass spectrum may be generated using the blood of the diagnosis subject. One or more of the first to third mass spectra may be derived in the same or corresponding manner as described in steps (S1 to S6) of the method for analyzing cancer specific sugar chains. Each of the first to third mass spectra does not necessarily refer to one mass spectrum, and may include a plurality of spectra depending on the number of cancer patients, normal persons, and diagnosis subjects.

Next, the first spectrum and the second spectrum can be compared to identify important cancer markers and establish diagnostic criteria. This may be carried out by the same or corresponding process as in step S7 of the method for analyzing cancer specific sugar chains, for example, through the process of determining and databaseting cancer markers and the relative intensities of the peaks corresponding to the cancer markers. It may also include the step of setting a criterion for distinguishing and determining a cancer patient and a normal person. Next, the incidence of cancer and / or the degree of cancer progression may be diagnosed by comparing the intensity of the peaks corresponding to the cancer specific sugar chains in the third mass spectrum with the obtained criteria.

As described above with reference to FIGS. 6A and 6B, mass spectra obtained from serum of cancer patients and those obtained from serum of normal subjects have mass to charge ratios of about 1257.42, about 1419.47, about 1485.53, about 1647.59, about 1809.64, and about 1850.67 molecular weight. It can be seen that there is a difference between the intensity of the peak and the like. Therefore, the peak differences of cancer-specific sugar chains in the mass spectra of cancer patients and normal subjects are analyzed according to the type of cancer and the degree of cancer, and the samples are analyzed and databased, and then the judgment criteria are derived from the blood of the diagnosis subject. The mass spectrum can then be compared with this criterion to diagnose whether the diagnosis target cancer develops and / or the degree of progression.

Real Patient Serum Numbers (41) Normal Serum Water (30) All (71) A positive judgment
(Judged as a patient) Jinyangseong (31)
CA125 (-) ¹ : 8, (+) ² : 23 False positives (4) Positive predictive rate = 89%
True positive / (positive positive + false positive) Voice judgment
(Normally judged) False negative (10)
CA125 (-): 3, (+): 7 True Voice (26) Speech prediction rate = 72%
True voice / (false voice + true voice) Sensitivity
(31/41) = 76%
CA125 series (-): 11,
(+): 30 Specificity
(26/30) = 87% Hit Rate = 80%
(True positive + true negative)

1. CA125 (-) means that the concentration is less than 35 international units (mL) / mL.

2. CA125 (+) means the concentration is greater than or equal to 35 IU / mL.

Table 2 above is a result obtained by comparing the results of the determination using the method according to an embodiment by receiving 71 different human serum without prior information, compared with the actual information of each received serum obtained after the determination.

First of all, to set the criteria, 52 cancer marker peaks were set, including the peaks mentioned in Table 1, in 10% and 20% aliquots, with serum from 30 ovarian cancer patients and 22 normal patients. Then, by comparing the relative intensities of the cancer patients and normal people of these peaks, a criterion for determining and discriminating between patients and normal people was set.

Secondly, the third mass spectra were obtained in 10% aliquots and 20% aliquots using sera from the subjects (71 patients in total, normal with cancer patients), from which relative intensities at the 52 peaks mentioned above. After the value was obtained, each of them was substituted into the criterion for determination. That is, the relative intensities of the 52 peaks were compared with those of the cancer patient and the normal person, respectively, and compared to which of the cancer patients or the normal person, and the determination results were derived by combining the comparison results of all 52 peaks.

At this time, one serum may be analyzed using a plurality of wells on a plurality of aliquots and / or well plates. The inventors of the present invention obtained 10% aliquots and 20% aliquots from one serum, respectively, and injected 10% aliquots and 20% aliquots into three wells on a well plate, respectively, so that one serum was analyzed in a total of six wells. . Since each aliquot is injected into three wells, a total of up to 156 peaks corresponding to 52 cancer markers are obtained, and the mass spectrum is obtained by deriving a determination result using the values of each of the peaks corresponding to the same cancer marker. We attempted to minimize the deviation in the process. In addition, a determination result may be derived by performing the above-described process a plurality of times for one serum. However, this is merely exemplary, and the type or number of aliquots, the number of wells into which each aliquot is injected, and the number of repetitions of the determination may be appropriately determined in consideration of the purpose or characteristics of the determination, and are limited to those described herein. It doesn't happen.

The result of the judgment by the above process is represented by a number and named as patient proximity rate (%). Thus, if the patient proximity rate derived from each human serum is 50.0% or more, it is determined as positive, that is, a patient, and less than 50.0% as normal. In the case of patients, the CA125 concentration of each serum obtained after the determination was expressed by counting the number of serums of 35 IU / mL or more and the case of less than 35 IU / mL. In the case of normal sera, all CA125 concentrations were less than 35 IU / mL.

In reviewing the above results, the result of the determination according to the embodiment showed that the positive predictive rate was 89%, the specificity was 87%, the hit rate was 80%, and the sensitivity 76% was determined as the CA125 concentration, 73% [ = (23 + 7) / 41]. And considering that the results were based on a database of only 52 sera, further breakthroughs are expected in the future.

In the present specification, a method for analyzing cancer specific sugar chains according to the embodiments has been described with reference to the flowchart shown in the drawings. While the above method has been shown and described as a series of blocks for purposes of simplicity, it is to be understood that the invention is not limited to the order of the blocks, and that some blocks may be present in different orders and in different orders from that shown and described herein And various other branches, flow paths, and sequences of blocks that achieve the same or similar results may be implemented. Also, not all illustrated blocks may be required for implementation of the methods described herein.

Furthermore, some aspects of the method for analyzing cancer specific sugar chains and screening methods for cancer diagnosis using the same may be implemented in the form of a computer program for performing a series of processes, wherein the computer program is a magnetic recording medium. Or it may be recorded on a computer-readable recording medium such as an optical recording medium.

Although the present invention described above has been described with reference to the embodiments illustrated in the drawings, this is merely exemplary, and it will be understood by those skilled in the art that various modifications and variations may be made therefrom. However, such modifications should be considered to be within the technical protection scope of the present invention. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

Claims (2)

Providing a first plate comprising a plurality of wells arranged in a first array form;
Processing a sample comprising serum isolated from blood collected from a cancer patient using the first plate to extract one or more sugar chains from the sample;
Providing a second plate comprising a plurality of mixed regions arranged in a second array form, and a matrix material applied to each of the plurality of mixed regions in a crystallized state and absorbing energy from the laser and ionizing them;
Mixing the one or more sugar chains with the matrix material using the plurality of mixed regions and ionizing the one or more sugar chains by irradiating a laser to each of the plurality of mixed regions;
Moving the at least one sugar chain ionized under an electric field and detecting it by a detector;
Deriving a first mass spectrum of the at least one sugar chain by measuring the detector arrival time of the at least one sugar chain; And
Determining the cancer specific sugar chain by comparing the derived first mass spectrum with a second mass spectrum derived using blood collected from a normal person;
Generating a third mass spectrum using blood collected from the diagnosis subject;
Deriving, from the third mass spectrum, one or more peaks corresponding to the cancer specific sugar chains determined using the first mass spectrum and the second mass spectrum; And
And comparing the one or more peaks with the peaks of the first mass spectrum and the second mass spectrum to obtain cancer progress information of the diagnosis subject.
The method of claim 1,
The cancer progress information, the method of providing information for diagnosing cancer, characterized in that it comprises one or more of cancer development stage and cancer progression stage information.

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