阅读说明：本技术 入组生酮饮食临床研究患者的首选分子分型的构建方法 (Construction method of first-choice molecular typing of group ketogenic diet clinical research patients ) 是由 周福祥 唐蒙 孙雪花 匡浩 张万芳 于 2020-07-10 设计创作，主要内容包括：本发明提供一种入组生酮饮食临床研究患者的首选分子分型的构建方法及首选分子分型方法。该构建方法包括如下步骤：1)对临床大样本结肠癌转录组数据以及结肠非肿瘤组织基因mRNA的表达信息进行分析；2)筛选出糖代谢和酮体代谢重编程相关的靶点分子；3)获得所述多例结肠癌组织的基因mRNA表达信息；4)通过R语言分析靶点分子表达与预后之间的关系；5)将结肠癌患者癌组织分子分型分为三类：预后最差型、中间型及预后最好型；6)选取预后最差的糖酵解型-酮体代谢缺陷型作为入组生酮饮食临床研究患者的首选分子分型。本发明可筛选出其中预后最差型患者,其肿瘤组织对酮体代谢利用较弱,可作为入组生酮饮食临床研究的首选分子分型。(The invention provides a construction method for first-choice molecular typing of a patient in a clinical study of a ketogenic diet and a first-choice molecular typing method. The construction method comprises the following steps: 1) analyzing the colon cancer transcriptome data of a large clinical sample and the expression information of the colon non-tumor tissue gene mRNA; 2) screening out target molecules related to reprogramming of sugar metabolism and ketone body metabolism; 3) obtaining gene mRNA expression information of the plurality of colon cancer tissues; 4) analyzing the relation between target molecule expression and prognosis through R language; 5) cancer tissue molecules of colon cancer patients are classified into three types: worst, intermediate and best prognosis; 6) the glycolytic-ketone body metabolism defect with the worst prognosis is selected as the first molecular typing of the grouping ketogenic diet clinical research patient. The invention can screen the patients with worst prognosis, the tumor tissues of which have weak utilization on ketone body metabolism, and can be used as the first choice molecular typing for clinical research of group ketogenic diet.)

1. The construction method of the preferred molecular typing of the patient entering the ketogenic diet clinical study is characterized by comprising the following steps:

1) analyzing the colon cancer transcriptome data of a large clinical sample and the expression information of the colon non-tumor tissue gene mRNA;

2) comparing the differences of the transcription levels of molecules related to glycolysis, oxidative phosphorylation and ketone body metabolism of colon cancer tissues and colon non-tumor tissues, and screening target molecules related to reprogramming of carbohydrate metabolism and ketone body metabolism, wherein the target molecules comprise GLUT1, PFKFB3, OXCT1 and ACAT 1;

3) acquiring clinical information of multiple colon cancer patients by using a GEO database, and acquiring gene mRNA expression information of multiple colon cancer tissues;

4) analyzing the relation between target molecule expression and prognosis through R language, and determining that GLUT1 and/or PFKFB3 are highly expressed in glycolysis type and the prognosis is poor; determining that GLUT1 and PFKFB3 are low expressed as non-glycolytic; determining that the low expression of the OXCT1 and/or the ACAT1 is ketone body metabolism defect, and the prognosis is poor; and determining that OXCT1 and ACAT1 are highly expressed as ketone body metabolic types;

5) further combining genes related to glycolysis and ketone body metabolism, and classifying cancer tissue molecules of colon cancer patients into three types through survival analysis: the worst prognosis is glycolytic-ketobody metabolism deficient; the intermediate forms are non-glycolytic-ketone body metabolism deficient and glycolytic-ketone body metabolism deficient; the most prognostic is non-glycolytic-ketobody metabotropic;

6) the glycolytic-ketone body metabolism defect with the worst prognosis is selected as the first molecular typing of the grouping ketogenic diet clinical research patient.

2. The method of claim 1, wherein the clinical information of step 3) includes age, gender, TNM stage, overall survival and relapse-free survival.

3. The method for constructing the preferred molecular typing for the patients in the clinical study of incoming ketogenic diet of claim 1, wherein the transcriptome gene expression data of step 1) is selected from the TCGA database of the GEPIA bioinformatics website.

4. The preferred molecular typing method for grouping ketogenic diet clinical study patients is characterized by comprising the following steps:

1) making pathological tumor tissue excised by a patient operation into a tissue slice or a chip, and carrying out Opal multicolor fluorescence detection;

2) analyzing the fluorescence detection image, and marking each positive cell with a color point;

3) observing the percentage of positive cells in the cells by using a microscope, wherein the percentage is X%, the positive cells are judged to be high expression when the percentage is larger than a defined value, and the positive cells are judged to be low expression when the percentage is smaller than or equal to the defined value;

4) when GLUT1 and/or PFKFB3 in the tissue chip were highly expressed and OXCT1 and/or ACAT1 were low expressed, they were the first to type patients for the clinical study of the invasive ketogenic diet.

5. The method for first-choice molecular typing of patients in a clinical study of incoming ketogenic diet as claimed in claim 4, wherein the high expression is obtained when ACAT1> 5%, OXCT1> 10%, PGC1a > 7%, PFKFB3> 30%, GLUT1> 0.5% and the low expression is obtained when the expression level is lower than or equal to the defined level.

6. The method for the first choice molecular typing of the patients in the clinical study of incoming ketogenic diet as claimed in claim 4, wherein the antibody used in Opal multicolor fluorescence detection in step 1) is ACAT1, OXCT1, PFKFB3 and GLUT 1.

7. The method for the preferred molecular typing of a patient in a clinical study on ketogenic diet as claimed in claim 4, wherein the determination in step 3) is made based on the ratio of the immunohistochemical marker CK and the marker molecule double positive marker to the total cells in the tissue, and the determination of high expression is made when the ratio is greater than a defined value, and the determination of low expression is made when the ratio is less than or equal to a defined value.

Technical Field

The invention relates to disease molecular typing detection, in particular to a preferred molecular typing construction method and a preferred molecular typing method for grouping ketogenic diet clinical research patients.

Background

The abnormal energy metabolism is one of ten major features of tumor cells, and is mainly expressed as the "Warburg effect", and the tumor cells take glucose glycolysis as a main energy acquisition mode, take a large amount of glucose and generate lactic acid even under aerobic conditions, and promote cell proliferation. Many studies suggest that altering the energy metabolism pattern of a tumor may be effective in controlling its growth. The Ketogenic Diet (KD) has been recognized for its safety and effectiveness as a diet formulated with high fat, low carbohydrate, moderate amounts of protein and other nutrients. The long-term ketogenic diet can simulate the hunger state of human body, and the fatty acid is oxidized and decomposed by the liver to generate an intermediate metabolite ketone body: acetoacetate, beta-hydroxybutyrate, acetone for peripheral tissue energy supply. Because the metabolism of the ketone body in the cell depends on the complete inner mitochondrial membrane and the ketone body catabolic enzyme, normal cells are easy to adapt to the ketone body as a high-efficiency metabolite, most tumor cells do not have the adaptability, and the low expression of any ketone body metabolic enzyme can cause the remarkable reduction of the utilization rate of the ketone body. Based on the fact that the sensitivity of the tumor ketogenesis therapy (KD) depends on the low expression of the metabolic enzyme of the ketone body, a molecular typing method related to tumor metabolism needs to be researched urgently, the metabolic heterogeneity of different tumor cells is discussed, and the effect of the ketogenesis therapy can be predicted.

Disclosure of Invention

The invention provides a construction method for the first-choice molecular typing of a patient in clinical study of the grouped ketogenic diet and a first-choice molecular typing method for solving the technical problems.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the construction method of the preferred molecular typing of the group ketogenic diet clinical research patients comprises the following steps:

1) analyzing the colon cancer transcriptome data of a large clinical sample and the expression information of the colon non-tumor tissue gene mRNA;

2) comparing the differences of the transcription levels of molecules related to glycolysis, oxidative phosphorylation and ketone body metabolism of colon cancer tissues and colon non-tumor tissues, and screening target molecules related to reprogramming of carbohydrate metabolism and ketone body metabolism, wherein the target molecules comprise GLUT1, PFKFB3, OXCT1 and ACAT 1;

3) acquiring clinical information of multiple colon cancer patients by using a GEO database, and acquiring gene mRNA expression information of multiple colon cancer tissues;

4) analyzing the relation between target molecule expression and prognosis through R language, and determining that GLUT1 and/or PFKFB3 are highly expressed in glycolysis type and the prognosis is poor; determining that GLUT1 and PFKFB3 are low expressed as non-glycolytic; determining that the low expression of the OXCT1 and/or the ACAT1 is ketone body metabolism defect, and the prognosis is poor; and determining that OXCT1 and ACAT1 are highly expressed as ketone body metabolic types;

5) further combining genes related to glycolysis and ketone body metabolism, and classifying cancer tissue molecules of colon cancer patients into three types through survival analysis: the worst prognosis is glycolytic-ketobody metabolism deficient; the intermediate forms are non-glycolytic-ketone body metabolism deficient and glycolytic-ketone body metabolism deficient; the most prognostic is non-glycolytic-ketobody metabotropic;

6) the glycolytic-ketone body metabolism deficient type with the worst prognosis is selected as the first choice for typing the patients in the clinical study of the ketone-entering diet.

Preferably, the clinical information of step 3) includes age, gender, TNM stage, overall survival and relapse-free survival.

Preferably, the transcriptome gene expression data in step 1) is selected from the TCGA database of the GEPIA bioinformatics website.

The preferred molecular typing method for grouping ketogenic diet clinical study patients comprises the following steps:

1) making pathological tumor tissue excised by a patient operation into a tissue slice or a chip, and carrying out Opal multicolor fluorescence detection;

2) analyzing the fluorescence detection image, and marking each positive cell with a color point;

3) observing the percentage of positive cells in the cells by using a microscope, wherein the percentage is X%, the positive cells are judged to be high expression when the percentage is larger than a defined value, and the positive cells are judged to be low expression when the percentage is smaller than or equal to the defined value;

4) when GLUT1 and/or PFKFB3 in the tissue chip were highly expressed and OXCT1 and/or ACAT1 were low expressed, they were the first to type patients for the clinical study of the invasive ketogenic diet.

Preferably, high expression is obtained when ACAT1> 5%, OXCT1> 10%, PGC1a > 7%, PFKFB3> 30%, GLUT1> 0.5%, and low expression below or equal to a defined value.

Preferably, the antibodies used in Opal multicolor fluorescence detection in step 1) are ACAT1, OXCT1, PFKFB3 and GLUT 1.

Preferably, the determination in step 3) is carried out according to the proportion of the immunohistochemical marker CK and the indicator molecule double positive marker in the total cells of the tissue, the determination is carried out when the ratio is greater than a defined value, the determination is high expression, and the determination is low expression when the ratio is less than or equal to the defined value.

Compared with the prior art, the invention has the following beneficial effects: screening and analyzing clinical large-sample colon cancer transcriptome data, screening target molecules related to glycometabolism and ketone body metabolism reprogramming with expression difference with paracarcinoma, then carrying out combined typing by using the difference molecules, verifying the relation between the metabolic molecule typing and prognosis based on bioinformatics analysis and 180-point tissue chip expression level, and screening out a metabolic typing Glycolysis type ketone body metabolism defective Glycolysis +/Ketolysis-patient with worst prognosis, wherein the tumor tissue of the patient has weak utilization on ketone body metabolism and can be used as the first choice typing for clinical study of ingoing ketodiet.

Drawings

FIG. 1 is a graph showing the relationship between a molecule for regulating ketone body metabolism, OXCT1, and prognosis of colon cancer.

FIG. 2 is a graph showing the relationship between the molecule ACAT1 for regulating ketone body metabolism and prognosis of colon cancer.

FIG. 3 is a graph of the relationship between glycolytic regulator GLUT1 and colon cancer prognosis.

FIG. 4 is a graph of the relationship of the glycolytic regulator PFKFB3 to colon cancer prognosis.

FIG. 5 is a graph of molecular association of ketone body metabolism typing in relation to prognosis of colon cancer patients. The results of Kaplan-Meier survival analysis and Log Rank (Mantel-Cox method) statistical analysis are as follows, OXCT1& ACAT 1: chi Square is 8.871, and p is 0.031.

FIG. 6 is a graph of the relationship between glycolytic metabolic molecule association typing and prognosis for colon cancer patients. The results of Kaplan-Meier survival analysis and Log Rank (Mantel-Cox method) statistical analysis are as follows, GLUT1& PFKFB3: Chi Square 8.562, and p 0.014.

FIG. 7 is a graph of the relationship between glycolysis and molecular association of ketone body metabolism and prognosis of colon cancer patients. Kaplan-Meier survival analysis and Log Rank (Mantel-Cox method) statistical analysis gave the following glycolysis & ketone body metabolism: Chi Square ═ 21.88, p < 0.001.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention is described in detail below by way of examples.

The embodiment of the invention provides a construction method for the first-choice molecular typing of a patient in a clinical study of a grouped ketogenic diet, which comprises the following steps:

1) analyzing the colon cancer transcriptome data of a large clinical sample and the expression information of the colon non-tumor tissue gene mRNA;

in this step, 275 colon Cancer patients selected from the TCGA (the Cancer genome atlas) database were analyzed online by the GEPIA bioinformatics website for transcriptome data, and 41 colon non-tumor tissue gene mRNA expression information.

2) Comparing the differences of the transcription levels of molecules related to glycolysis, oxidative phosphorylation and ketone body metabolism of colon cancer tissues and colon non-tumor tissues, and screening target molecules related to reprogramming of carbohydrate metabolism and ketone body metabolism, wherein the target molecules comprise GLUT1, PFKFB3, OXCT1 and ACAT 1;

in this step, colon cancer tissue and colon nontumor tissue are compared for differences in glycolysis, oxidative phosphorylation and transcription levels of molecules related to ketone body metabolism. Compared with normal colon tissue, glycolysis related molecules GLUT1, PFKFB3, HK2 and PKM2 in colon cancer tissue are highly expressed, and the expression of ketolase BDH1, OXCT1, ACAT1 and oxidative phosphorylation molecule PGC1a is reduced. And the OXCT1 and ACAT1 expression level tumor patients have large span in different individuals, which indicates that the patients have significant difference of ketone body metabolic capability. Therefore, the target molecules related to reprogramming of sugar metabolism and ketone body metabolism with expression difference with paracarcinoma are screened out. These molecules include GLUT1, PFKFB3, OXCT1, ACAT 1.

3) Acquiring clinical information of multiple colon cancer patients by using a GEO database, and acquiring gene mRNA expression information of multiple colon cancer tissues;

in this step, information including 3 data sets GSE17536(172 cases), GSE17537(55 cases), and GSE39582(555 cases) for 782 colon cancer patients is obtained from a geo (gene Expression omnibus) database GPL570 platform. The data also contains relevant clinical data including age, gender, TNM staging, overall survival, recurrence-free survival. The data were normalized using the R language to obtain mRNA expression information of genes of 782 colon cancer tissues in total.

4) Analyzing the relation between target molecule expression and prognosis through R language, and determining that the high expression of GLUT1 and/or PFKFB3 is classified as glycolytic type, and the prognosis is poor; determining that GLUT1 and PFKFB3 are simultaneously low-expressed as non-glycolytic type; determining that the low expression of the OXCT1 and/or the ACAT1 is ketone body metabolism defect, and the prognosis is poor; determining that OXCT1 and ACAT1 are simultaneously highly expressed as ketone body metabolic types;

in this step, the relation between the expression of the metabolic molecules and the prognosis is analyzed by the R language. As a result, the expression of the ketometabolism enzymes OXCT1 and ACAT1 is found to be positively correlated with the prognosis, such as shown in FIG. 1 and FIG. 2, and only GLUT1 and PFKFB3 in the glycolysis-related molecules are significantly negatively correlated with the prognosis, such as shown in FIG. 3 and FIG. 4. Further multi-factor analysis showed that OXCT1, ACAT1 and PGC1 alphaHigh expression of mRNA is a protective factor for prognosis of colon cancer patients, while high expression levels of molecular mRNA represented by GLUT1 and PFKFB3 in glycolytic pathway are prognostic related risk factors. Then we carried out further combinatorial analysis on the same function genes, firstly we calculated the optimal cutoff value of mRNA expression of the four molecules of OXCT1, ACAT1, GLUT1 and PFKFB3 in 807 colon cancer tissues by using X-tile software, then divided the mRNA expression into high and low expression groups, and then carried out combined typing on the molecules to show low expression of either OXCT1 or ACAT1 (OXCT 1)^lowor ACAT1^high、OXCT1^highor ACAT1^low) Simultaneous low expression (OXCT 1)^lowand ACAT1^low) The prognosis has no obvious difference, and the three types of patients are classified as 'ketone body metabolic defects' (Ketolysis-type) according to the biological functions, and the relative two genes are simultaneously highly expressed: OXCT1^high/ACAT1^highRepresentative "ketone body metabolic type" (Ketolysis + type), the former had a poorer prognosis, as shown in FIG. 5. It is suggested that in colon cancer patients with poor prognosis, the ketone body metabolism of cancer tissues is mostly a subtype of 'ketone body metabolism deficiency'. The joint analysis of GLUT1 and PFKFB3 shows that the expression is high simultaneously (GLUT 1)^highand PFKFB3^high) And any high expression (GLUT 1)^highor PFKFB3^low、GLUT1^lowor PFKFB3^high) Has no significant difference in prognosis among patients, and the three types of patients are classified as 'Glycolysis type' (Glycolysis type) according to the molecular metabolism characteristics, and the relative two genes are simultaneously and lowly expressed (GLUT 1)^lowand PFKFB3^low) The "non-glycolytic" type of (1), the former had significantly poorer prognosis, as shown in fig. 6. It is suggested that most of the cancer tissues are of the "glycolytic" subtype in colon cancer patients with poor prognosis.

5) Glycolysis and ketone body metabolism related genes are further combined through survival analysis, and cancer tissue molecules of colon cancer patients are classified into three types through survival analysis: the worst prognosis is glycolytic-ketone body metabolism deficiency (Glycolysis +/Ketolysis-: GLUT 1)^highor PFKFB3^high、OXCT1^lowor ACAT1^low) (ii) a The intermediate form is non-glycolytic-ketone body metabolism defective (Glyc)olysis-/Ketolysis-：GLUT1^lowand PFKFB3^low、OXCT1^loworACAT1^low) And glycolytic-ketone body metabotropic (Glycolysis +/Ketolysis +: GLUT1^highor PFKFB3^high、OXCT1^highand ACAT1^high) (ii) a The most favorable prognosis is the non-glycolytic-ketone body metabotropic (Glycolysis-/Ketolysis +: GLUT1^lowand PFKFB3^low，OXCT1^highand ACAT1^high) As in fig. 7.

6) The glycolytic-ketone body metabolism defect with the worst prognosis is selected as the first molecular typing of the grouping ketogenic diet clinical research patient.

Finally, validation was performed in a colon cancer 180-spot tissue chip (HCola180Su11) purchased from Shanghai core Biotechnology Ltd. The tissue chip has operation time of 2009.11-2010.5, and is followed from discharge 1 month after operation to 2016.6. The clinical data included were age, sex, TNM stage, pathology type, tumor site, overall survival (90 cases), P53, MSH2, MSH6, MLH1, Ki67 immunohistochemical assays. In the chip, 8 spots of the detached piece are removed, and 90 cancer tissue samples are totally obtained, wherein 82 cancer tissue samples have paracancerous controls, metabolic-related molecular subtypes constructed on the basis of mRNA expression conditions of prophase metabolic target molecules in colon cancer tissues are verified in a tissue chip by using Opal multicolor fluorescence detection. Cancer tissue metabolic molecular typing of colon cancer patients was found to be significantly different 3 types in prognosis: glycolytic-ketone body metabolism deficient (Glycolyssi +/Ketolysis-: GLUT1high or PFKPB3high, OXCT1low or ACAT1low) has the worst prognosis with a median survival (mOS) of 22 months; the intermediate type is non-glycolytic-ketone body metabolism deficient (Glycolyssi-/Ketolysis-: GLUT1low and PFKPB3low, OXCT1low or ACAT1low), mOS is 30 months; glycolytic-ketobody metabotropic (Glycolyisi +/Ketolysis +: GLUT1high or PFKPB3high, OXCT1high and ACAT1high), mOS 47.5 months; whereas the non-glycolytic-ketoplast (glycolysii-/ketolisis +: GLUT1low and PFKPB3low, OXCT1high and ACAT1high) prognosis is best, to the point of observation of less than 50% of patients with checkpoint death, mOS is to be further extended for follow-up. This conclusion is consistent with the conclusion drawn from pre-mRNA levels.

This example also provides a preferred method for molecular typing of patients in a clinical study involving a ketogenic diet, comprising the steps of:

firstly, after a primary focus of a clinical tumor patient is removed by an operation, tumor tissues are made into tissue chips, and Opal multicolor fluorescence detection of the tissue chips is carried out; the method comprises the following specific steps:

a) dewaxing: the sections were heated in an oven (60-65 ℃) for at least one hour and held in place so that the melted paraffin evaporated. The sections were washed with xylene for 10 minutes and repeated three times. The hydration reaction was carried out by an ethanol gradient and finally washed with distilled water.

b) Fixing the slices: the tissue was fixed in NBF for 20 minutes and subsequently washed with distilled water.

c) Antigen retrieval: preparing AR6 or AR9 working solution, boiling with high fire, placing into slide, placing with low fire for 15-20 min, and cooling the section to room temperature (at least 15 min) on the test bed after repairing.

d) And (3) sealing: sections were washed with clear water and then treated with TBST. The tissue area to be stained is circled with a composing pen. Tissues were given 10 minutes at room temperature using antibody dilution/blocking solution.

e) Primary antibody incubation: the blocking solution is removed and then a primary antibody solution is applied to the tissue. Antibody concentrations and incubation times must be optimized for Opal detection.

f) And (3) secondary antibody incubation: a secondary antibody solution was applied to the tissue. Opal polymer HRP should be incubated at room temperature for 10 minutes. The sections were washed three times with TBST for two minutes each.

g) Opal fluorophore incubation: opal fluorophore solution was applied to the tissue and then given for 10 min at room temperature. The sections were washed three times with TBST for two minutes each.

h) Removing the antibody: AR buffer performs MWT and the sections are allowed to cool on the bench for at least 15 minutes.

i) Spectral DAPI staining: sections were rinsed with distilled water and then with TBST. Sections were incubated in DAPI solution to room temperature for 5 minutes.

j) Sealing: and (5) dropping an anti-fluorescence quenching mounting agent on the glass slide for mounting.

Secondly, analyzing the fluorescence detection image; the method comprises the following specific steps:

a) preparing an image: scanning and analyzing the chip by using an Inform software, firstly performing signal extraction and spectrum splitting, removing autofluorescence, and adjusting fluorescence intensity and pseudo-color.

b) Tissue segmentation: the tumor region and the interstitial region may be segmented for analysis of the tumor, interstitial region, respectively.

c) Cell segmentation: cell segmentation, identification of cells based on nucleus, cytoplasmic identification: distance from the nucleus.

d) Cell typing: cell phenotype identification, each positive cell is marked with a color dot. The fluorescence was set as follows: opal 520-GLUT 1-red; opal 540-PGC-1 a-yellow; opal 570-OXCT 1-cyan; opal 620-CK-magenta; opal 650-PFKFB 3-orange; opal 690-ACAT 1-green.

e) Quantitative analysis: the tumor region and the interstitial region are analyzed independently, and the single positive rate, the double positive rate and the positive intensity score can be analyzed.

Thirdly, observing the percentage of the positive cells in the cells by using a microscope, wherein the percentage is X percent and is judged to be high expression when the percentage is larger than a defined value, and the percentage is low expression when the percentage is smaller than or equal to the defined value; the method comprises the following specific steps:

to avoid errors between the observers' visual fields, a skilled pathology teacher may be asked to read the immunohistochemical experiment results.

The interpretation items and standards are respectively:

a) and (3) determination of positioning: cytoplasm, cell membrane and nucleus

b) Qualitative judgment: negative and positive

c) Quantitative determination:

1. and (3) judging the dyeing intensity: the whole visual field of the tissue site was observed under a low power microscope and classified into weak positive, medium positive and strong positive. Weak positives were pale yellow (+ or 1), medium positives were tan (+ + or 2), and strong positives were tan (+ + + or 3). (Note: if there is both a positive and a strong positive in the tissue, the score is generally 2-3, and similar results are as above)

2. And (3) judging the dyeing positive rate: firstly, observing a tissue point under a low power lens in a whole visual field, then selecting 3 visual fields with different staining intensities, judging under a high power lens, if a target molecule is positioned in a nucleus, randomly recording 100 cells in each visual field, then recording the percentage X1% of positive cells in 100 cells, similarly, after the percentage X2% and X3% of positive cells in another 2 visual fields are viewed on the principle, and finally, taking the average of X1%, X2% and X3% of the staining positive rate of the tissue point; if located in the cytoplasm or the cell membrane, we also selected 3 different fields of staining intensity, and evaluated the positive rate and averaged the values.

3. And (3) judging a result: judging according to CK (cytokeratin, epithelial origin marker, judging and identifying whether the tumor is epithelial origin) and the proportion of the indicator molecule double-positive marker in the total cells of the tissue, and judging that the expression is high when the marker is greater than a threshold value: ACAT1> 5% was high expression, less than or equal to 5% was low expression, and the expression of the remaining target molecules was similarly determined, where OXCT1> 10%, PGC1a > 7%, PFKFB3> 30%, GLUT1> 0.5% was high expression, less than or equal to the defined value was low expression (the defined values were determined only according to our recommended antibodies and experimental conditions, with antibody dilutions such as in table 1 below).

TABLE 1 target molecule immunohistochemical procedure Table

And fourthly, when GLUT1 and/or PFKFB3 in the tissue chip is highly expressed and OXCT1 and/or ACAT1 is low expressed, the molecular typing is the first choice for the clinical research patient of the group-entering ketogenic diet.

Specific application examples are as follows: liu certain is a female of 57 years old, diagnosed as colon hypo-differentiated mucus adenocarcinoma and subjected to colorectal cancer radical treatment, and the postoperative pathological stage is T3N2bM0 and IIIc stage.

The colon cancer tumor sample after the operation is detected according to the scheme, the expression of 4 protein molecules (GLUT1, PFKFB3, ACAT1 and OXCT1) in the tissue sample is detected, and after the pathological physician reads the samples, GLUT1high expression, PFKFB 3high expression, OXCT1high expression and ACAT1low expression of the patient belong to Glycolysis type-ketone body metabolism defect type Glycolysis +/Ketolysis-patients with the worst prognosis, the tumor tissue of the patient has weak utilization on ketone body metabolism and can be used as the first-choice type of clinical study of entering group ketonic diet.