CN111996249A

CN111996249A - Cancer diagnosis and disease course monitoring method

Info

Publication number: CN111996249A
Application number: CN202010156417.2A
Authority: CN
Inventors: 王弢; 巴兆粉; 丁凤英
Original assignee: Jiangsu Microdiag Biomedical Technology Co ltd; Suzhou Purui Ahmed Medical Laboratory Ltd
Current assignee: Jiangsu Microdiag Biomedical Technology Co ltd; Suzhou Purui Ahmed Medical Laboratory Ltd
Priority date: 2019-05-27
Filing date: 2020-03-10
Publication date: 2020-11-27

Abstract

The present disclosure provides methods for cancer diagnosis and disease course monitoring. In particular, the present disclosure provides methods for predicting a subject's risk of developing cancer or monitoring the course of disease in a cancer patient by detecting the level of exosome mrnas of one or more tumor microenvironment-associated genes in a body fluid of the subject or cancer patient. The present disclosure also provides kits for carrying out the above methods.

Description

Cancer diagnosis and disease course monitoring method

Technical Field

The present disclosure relates to the field of disease diagnosis and prognosis. More specifically, the disclosure relates to the field of early diagnosis and prognosis of pan-cancer, and monitoring of the course of cancer.

Technical Field

According to the calculation data of the incidence of all cancers in the population of all ages and sexes in the world, in 2018, the number of new cancer cases is estimated to reach 1810 ten thousands and the number of death cases reaches 960 ten thousands globally. In this enormous new case of cancer, asian accounts for up to 50%; in 960 ten thousand cancer deaths, asia accounts for up to 70% more. Among 1810 ten new cases of cancer, the number of new cases in China accounts for 380.4 ten thousands; and of 960 ten thousand cancer death cases, 229.6 death cases account for China.

From the cancer species of onset, lung cancer is the cancer with the highest incidence worldwide, up to 11.6%, followed by breast cancer (11.6% by weight, 5000 fewer than lung cancer), colorectal cancer (10.2%), prostate cancer (7.1%), stomach cancer (5.7%). From the mortality of various cancer types, the highest mortality rate is still lung cancer (18.4%), and the mortality rates of other cancers are respectively: colorectal cancer (9.2%), gastric cancer (8.2%), liver cancer (8.2%), breast cancer (6.6%).

At present, the early diagnosis of cancer in the first-aid patients in hospitals in various places in China is less than 10%, and most patients lose the optimal cancer treatment period. Therefore, it is crucial to improve the early cancer detection rate. WHO also explicitly states: early detection is key to improving cancer cure.

Exosomes refer to small membrane vesicles (30-150nm) containing complex RNAs and proteins, which today refer specifically to discoidal vesicles with diameters between 40-100 nm. In 1983, exosomes were first found in sheep reticulocytes, which were named "exosomes" by Johnstone in 1987. Many cells secrete exosomes under both normal and pathological conditions. It is mainly from the multivesicular body formed by the invagination of intracellular lysosome particles, and is released into extracellular matrix after the fusion of the outer membrane of the multivesicular body and cell membrane.

Exosome is a nano-scale vesicle with phospholipid bilayer membrane structure, which contains various components of parent cells, including various substances such as protein, nucleic acid, lipid, carbohydrate and the like, and is widely distributed in body fluids such as serum, saliva, urine, lavage fluid and the like. The formation of exosomes is closely related to the state of parent cells, the content of exosomes in a tumor microenvironment is particularly rich, and the exosomes are closely related to the occurrence and development of tumors, immune escape and microenvironment establishment. The exosome can protect nucleic acid substances in the exosome and prevent the nucleic acid substances from being rapidly degraded. Therefore, the detection of the exosome content is more specific than the traditional tumor marker, and the tumor diagnosis based on the exosome content can monitor the change of the marker in time in the disease development process, so that the detection is easier to monitor and the sample is easier to collect. The advantages of the exosome enable the exosome to have remarkable advantages in the field of tumor diagnosis, and a new direction is provided for noninvasive diagnosis of tumors.

Species and distribution of RNA in exosomes: miRNA (about 40.4%), piwiRNA (about 40.0%), pseudogene (about 3.7%), lncRNA (about 2.4%), tRNA (about 2.1%), and mRNA (about 2.1%). In the prior art, based on exosome cancer diagnosis and detection, it is common to detect exosome proteins or miRNA in exosomes. Due to the low mRNA content in exosomes, it is difficult to detect or become a clinically significant biomarker in cancer detection.

Disclosure of Invention

The present inventors have surprisingly found that mRNA levels of genes associated with the tumor microenvironment in exosomes can be detected, whether by conventional RT-PCR methods or by more sensitive detection methods such as ddPCR detection, high throughput sequencing based detection such as ngs (next Generation sequencing) detection or gene chip detection. Furthermore, the inventor finds that the mRNA level of the tumor microenvironment-associated gene in the body fluid exosome of the subject or cancer patient has significance in clinical diagnosis and cancer course monitoring.

Accordingly, in one aspect, the present disclosure relates to a method of detecting mRNA levels of one or more tumor microenvironment-associated genes in a body fluid exosome of a subject, the method comprising the steps of:

a. isolating exosome RNA from a bodily fluid of the subject;

b. detecting the mRNA level of the tumor microenvironment-associated gene by a method selected from the group consisting of conventional RT-PCR detection, ddPCR detection, high throughput sequencing-based detection, and gene chip detection.

In some embodiments, isolating exosome RNAs from the bodily fluid of the subject comprises:

a1. isolating exosomes from a bodily fluid of the subject; and

a2. isolating RNA from the exosomes.

Methods known in the art can be used to isolate exosomes from a subject's bodily fluid, as well as to isolate RNA from exosomes.

In other embodiments, isolating exosome RNA from a bodily fluid of the subject comprises:

a1. pre-treating the body fluid; and

a2. directly isolating exosome RNAs from the body fluid.

In some embodiments, the conventional RT-PCR assay comprises:

b1. performing reverse transcription by using the exosome RNA as a template and a random primer or specific primers of the tumor microenvironment-associated gene and an internal reference gene; and

b2. performing fluorescent quantitative PCR by using the reverse transcription product as a template and the amplification primers of the tumor microenvironment related gene and the internal reference gene,

thereby obtaining the mRNA level of the tumor microenvironment-associated gene.

In some embodiments of the above methods, steps b1 and b2 are performed in a one-step PCR reaction. In one-step PCR reactions, the reaction system contains reverse transcriptase, polymerase and associated reverse transcription and PCR reaction reagents, and the reverse transcription and PCR reactions are performed in one reaction system.

In some embodiments of the above methods, the fluorescent quantitative PCR may be selected from a high resolution melting curve (HRM) method and a probe method.

In some embodiments, the high-throughput sequencing-based detection comprises:

b1. performing reverse transcription by using the exosome RNA as a template and a random primer or specific primers of the tumor microenvironment-associated gene and an internal reference gene;

b2. performing multiplex PCR using the reverse-transcribed product as a template and amplification primers for the tumor microenvironment-associated gene and the reference gene;

b3. using the products of the multiplex PCR to build a library; and

b4. sequencing the established library to obtain the mRNA level of the genes related to the tumor microenvironment.

In some embodiments of the above methods, steps b1 and b2 are performed in a one-step multiplex PCR reaction. In one-step multiplex PCR reactions, the reaction system contains reverse transcriptase, polymerase and associated reverse transcription reaction and PCR reaction reagents, and the reverse transcription and PCR reactions are completed in one reaction system.

In other embodiments, the high-throughput sequencing-based detection comprises:

b1. (ii) creating a library by transcriptome using the exosome RNA; and

b2. sequencing the obtained whole transcriptome library to obtain the mRNA level of the tumor microenvironment associated gene.

In some embodiments, the gene chip assay comprises:

b2. performing multiplex PCR using the reverse-transcribed product as a template and amplification primers for the tumor microenvironment-associated gene and the reference gene; and

b3. hybridizing the products of the multiplex PCR with a gene chip so as to obtain the mRNA level of the genes related to the tumor microenvironment,

wherein the gene chip comprises specific probes of the tumor microenvironment related gene and the reference gene.

In some embodiments of the above methods, steps b1 and b2 are performed in a one-step multiplex PCR reaction.

In other embodiments, the gene chip assay comprises:

b1. (ii) creating a library by transcriptome using the exosome RNA; and

b2. hybridizing the established complete transcriptome library with a gene chip to obtain the mRNA level of the genes related to the tumor microenvironment,

In any embodiment of the above method, the reference gene is selected from β -Actin (ACTB), GAPDH, β 2-MG, G6PDH, UBC, Tub, PLA, GUSB, HPRT, and the like.

In some embodiments involving methods using conventional RT-PCR or ddPCR, 2 of the mRNA of the marker is calculated^–ΔΔCTValues to obtain exosome mRNA levels of the markers, e.g., as described in detail in example 1. In other embodiments, the exosome mRNA levels of the markers are obtained by quantifying the mRNA of the markers to copy number using a standard curve.

In the RT-PCR method, the level of exosome mRNA of the marker can also be obtained using a semi-quantitative PCR method. The semi-quantitative PCR method uses gel electrophoresis and gel imaging and quantitative analysis to detect mRNA.

In another aspect, the present disclosure relates to a method for the diagnosis of pan-cancer, the method comprising the step of detecting mRNA levels of one or more tumor microenvironment-associated genes in a bodily fluid exosome of the subject.

In some embodiments, the tumor microenvironment-associated gene comprises EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL12, or a combination thereof. For example, the tumor microenvironment-associated genes include at least one, at least two, at least three, at least four, at least five, or all six genes selected from EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL 12.

In some embodiments, the tumor microenvironment-associated gene comprises EGF, VEGFB, or a combination thereof.

In some embodiments of the above diagnostic methods, the tumor microenvironment-associated gene comprises EGF and wherein an increase in the level of mRNA of EGF in the body fluid exosomes of the subject as compared to a control level from an individual not having cancer is indicative of the subject being at risk for developing cancer.

In some embodiments of the above diagnostic methods, the tumor microenvironment-associated gene comprises TGF- β 1, and wherein an increase in the level of mRNA of TGF- β 1 in a bodily fluid exosome of the subject as compared to a control level from an individual without cancer indicates that the subject is at risk for developing cancer.

In some embodiments of the above diagnostic methods, the tumor microenvironment-associated gene comprises VEGFB, and wherein a decrease in the level of mRNA of VEGFB in the subject's humoral exosomes as compared to a control level from an individual without cancer indicates that the subject is at risk for developing cancer.

In some embodiments of the above diagnostic methods, the tumor microenvironment-associated gene comprises MMP9, and wherein an increase in the level of MMP9 mRNA in a bodily fluid exosome of the subject as compared to a control level from an individual not having cancer is indicative of the subject being at risk for developing cancer.

In some embodiments of the above diagnostic methods, the tumor microenvironment-associated gene comprises CXCL8, and wherein an increase in the level of mRNA for CXCL8 in a bodily fluid exosome of the subject as compared to a control level from an individual not having cancer is indicative of the subject being at risk for developing cancer.

In some embodiments of the above diagnostic methods, the tumor microenvironment-associated gene comprises CXCL12, and wherein an increase in the level of mRNA of CXCL12 in a bodily fluid exosome of the subject as compared to a control level from an individual not having cancer is indicative of the subject being at risk for developing cancer.

The above cancer may be any kind of cancer, and specific examples thereof include, but are not limited to: basal cell carcinoma, cholangiocarcinoma; bladder cancer; bone cancer; breast cancer; peritoneal cancer; cervical cancer; bile duct cancer; choriocarcinoma; bowel cancer (e.g., colon and rectal cancer); connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; glioblastoma; liver cancer; kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphomas, including hodgkin lymphoma and non-hodgkin lymphoma; melanoma; a myeloma cell; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; b cell lymphoma; chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia, etc.

In some embodiments, the cancer is selected from lung cancer, gastric cancer, intestinal cancer, and breast cancer.

In any of the embodiments of the diagnostic methods described above, the bodily fluid may be selected from the group consisting of blood, urine, alveolar lavage fluid, cerebrospinal fluid, pleural fluid, and nipple aspirate. For example, in some embodiments, the bodily fluid is blood.

In some embodiments of the above diagnostic methods, the mRNA level of the tumor microenvironment-associated gene is detected by the methods of the present disclosure for detecting mRNA levels of one or more tumor microenvironment-associated genes in a bodily fluid exosome of the subject.

Preferably, the diagnostic method of the invention has a sensitivity of at least 65%, such as 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity.

Preferably, the diagnostic method of the invention has a specificity of at least 65%, e.g. 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity.

In one aspect, the disclosure relates to a method of disease course monitoring of a cancer patient, the method comprising the step of comparing the mRNA levels of one or more tumor microenvironment-associated genes in the patient's bodily fluid exosomes at a first time point and a second time point, or comprising the step of comparing the mRNA levels of one or more tumor microenvironment-associated genes in the patient's bodily fluid exosomes at a first time point and a plurality of other time points, wherein the second time point and the plurality of other time points are subsequent to the first time point.

In some embodiments of the above method of disease course monitoring, the tumor microenvironment-associated gene comprises EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL12, or a combination thereof. For example, the tumor microenvironment-associated genes include at least one, at least two, at least three, at least four, at least five, or all six genes selected from EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL 12.

In some embodiments of the above method of disease progression monitoring, the tumor microenvironment-associated gene comprises EGF and wherein a decreased level of mRNA of EGF in the fluid exosomes of the patient at the second time point as compared to the first time point is indicative of a good prognosis.

In some embodiments of the above method of disease progression monitoring, the tumor microenvironment-associated gene comprises TGF- β 1, and wherein a decrease in the level of mRNA of TGF- β in the fluid exosomes of the patient at the second time point as compared to the first time point is indicative of a good prognosis.

In some embodiments of the above method of disease progression monitoring, the tumor microenvironment-associated gene comprises VEGFB, and wherein an increase in the level of mRNA of VEGFB in the humoral exosomes of the patient at the second time point as compared to the first time point is indicative of a good prognosis.

In some embodiments of the above methods of disease progression monitoring, the tumor microenvironment-associated gene comprises MMP9, and wherein a decrease in the level of mRNA for MMP9 in the patient's humoral exosomes at the second time point as compared to the first time point is indicative of a good prognosis.

In some embodiments of the above methods of disease course monitoring, the tumor microenvironment-associated gene comprises CXCL8, and wherein a decrease in the level of mRNA for CXCL8 in the patient's humoral exosomes at the second time point as compared to the first time point is indicative of a good prognosis.

In some embodiments of the above methods of disease course monitoring, the tumor microenvironment-associated gene comprises CXCL12, and wherein a decrease in the level of mRNA for CXCL12 in the patient's humoral exosomes at the second time point as compared to the first time point is indicative of a good prognosis.

The particular type of cancer for which the course of disease is monitored is not limited, and it may be any of the types of cancer listed above. In some embodiments, the cancer is selected from lung cancer, gastric cancer, intestinal cancer, and breast cancer.

In some embodiments of the above method of disease-course monitoring, the bodily fluid is selected from the group consisting of blood, urine, alveolar lavage, cerebrospinal fluid, pleural fluid, and nipple aspirate. For example, in some embodiments, the bodily fluid is blood.

In some embodiments of the above method of disease course monitoring, the patient receives cancer treatment between the first time point and the second time point. In some embodiments, the cancer treatment is selected from chemotherapy, radiation therapy, immunotherapy, surgical treatment, or a combination thereof. For example, in some embodiments, the cancer treatment comprises a surgical treatment.

In any of the embodiments of the method of disease progression monitoring described above, the mRNA level of the tumor microenvironment-associated gene is detected by the method of detecting mRNA levels of one or more tumor microenvironment-associated genes in a bodily fluid exosome of the subject of the present disclosure.

Preferably, the method of disease course monitoring of the invention has a sensitivity of at least 65%, for example 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sensitivity.

Preferably, the method of disease course monitoring of the invention has a specificity of at least 65%, such as 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% specificity.

In one aspect, the present disclosure relates to a kit for diagnosis of pan-cancer, the kit comprising:

a. exosome RNA extraction reagent; and

b. and (3) a detection reagent for mRNA of one or more tumor microenvironment related genes.

In another aspect, the present disclosure relates to a kit for disease course monitoring of cancer, the kit comprising:

a. exosome RNA extraction reagent; and

The specific type of cancer is not limited, and it may be any of the types of cancer listed above. In some embodiments, the cancer is selected from lung cancer, gastric cancer, intestinal cancer, and breast cancer.

In some embodiments of the above kits, the tumor microenvironment-associated gene comprises EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL12, or a combination thereof. For example, the tumor microenvironment-associated genes include at least one, at least two, at least three, at least four, at least five, or all six genes selected from EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL 12.

In some embodiments of the above kit, the kit further comprises an exosome-isolating reagent for isolating exosomes from a bodily fluid.

In some embodiments, the exosome-separating agent is for separating exosomes from a bodily fluid selected from blood, urine, alveolar lavage fluid, cerebrospinal fluid, pleural fluid, or nipple aspirate. For example, in some embodiments, the exosome-separating agent is used to separate exosomes from blood.

In some embodiments of the above kits, the detection reagent is used to detect the mRNA level of the tumor microenvironment-associated gene by a method selected from the group consisting of high throughput sequencing-based assay, gene chip assay, RT-PCR assay, and ddPCR assay.

In some embodiments, the detection reagents comprise specific amplification primers and/or probes for the tumor microenvironment-associated genes. In some embodiments, the kit further comprises amplification primers and/or probes specific for an internal reference gene. For example, the reference gene may be selected from β -Actin (ACTB), GAPDH, β 2-MG, G6PDH, UBC, Tub, PLA, GUSB, HPRT, etc.

In some embodiments, the kit further comprises one or more reagents for a PCR reaction. In some embodiments, the reagents include polymerase, dntps, buffer, and deionized water, among others. In other embodiments, the reagents include polymerase, UNG enzyme, dNTPs, dUTP, buffer, deionized water, and the like.

In some embodiments, the kit further comprises one or more reagents for a reverse transcription reaction, such as reverse transcriptase, reverse transcription primers, dntps, buffer, deionized water, and the like.

In some embodiments, the kit further comprises one or more reagents for reverse transcription and PCR reactions. In some embodiments, the reagents include reverse transcriptase, polymerase, dntps, buffer, and deionized water, among others. In other embodiments, the reagents include reverse transcriptase, polymerase, UNG enzyme, dNTP, dUTP, buffer, deionized water, and the like.

In some embodiments of the above kits, the kit is used to carry out the methods of the present disclosure for the diagnosis of pan-cancer or for the monitoring of the course of disease in a cancer patient.

In one aspect, the present disclosure relates to the use of exosome RNA extraction reagents and detection reagents for mRNA of one or more tumor microenvironment-associated genes in the preparation of a kit for the diagnosis of pan-cancer.

In another aspect, the disclosure relates to the use of an exosome RNA extraction reagent and a detection reagent for mRNA of one or more tumor microenvironment-associated genes in the preparation of a kit for disease course monitoring of cancer.

In one aspect, the present disclosure relates to the use of exosome-isolating reagents, exosome RNA-extracting reagents and detection reagents for mRNA of one or more tumor microenvironment-associated genes in the preparation of a kit for the diagnosis of pan-cancer.

In another aspect, the disclosure relates to the use of exosome-isolating reagents, exosome RNA-extracting reagents and detection reagents for mRNA of one or more tumor microenvironment-associated genes in the preparation of a kit for disease course monitoring of cancer.

In some embodiments of the above uses, the exosome-separating agent is used to separate exosomes from a body fluid selected from blood, urine, alveolar lavage fluid, cerebrospinal fluid, pleural fluid, or nipple aspirate. For example, in some embodiments, the exosome-separating agent is used to separate exosomes from blood.

In some embodiments of the above uses, the tumor microenvironment-associated gene comprises EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL12, or a combination thereof. For example, the tumor microenvironment-associated genes include at least one, at least two, at least three, at least four, at least five, or all six genes selected from EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL 12.

In some embodiments of the above uses, the detection reagent is used to detect the mRNA level of the tumor microenvironment-associated gene by a method selected from the group consisting of high throughput sequencing-based detection, gene chip detection, RT-PCR detection, and ddPCR detection.

In some embodiments of the above use, the detection reagent comprises an amplification primer and/or a probe specific for the tumor microenvironment-associated gene. In some embodiments, the kit further comprises amplification primers and/or probes specific for an internal reference gene.

For example, the reference gene may be selected from β -Actin (ACTB), GAPDH, β 2-MG, G6PDH, UBC, Tub, PLA, GUSB, HPRT, etc.

In some embodiments of the above uses, the kit is for performing the methods of the present disclosure for diagnosis of pan-cancer or for monitoring the course of disease in a cancer patient.

Drawings

Figure 1 shows the differences in exosome mRNA levels in plasma samples from healthy humans and cancer patients, for each marker detected using the HRM method. The left panel shows the results of ROC curve analysis of each marker, and the right panel shows a scatter plot of exosome mRNA levels.

Figure 2 shows scatter plots of exosome mRNA levels in plasma samples from healthy people, different kinds of cancer patients and inflammatory patients using individual markers detected by HRM.

Figure 3 shows the difference in exosome mRNA levels of each marker detected using the probe method in plasma samples from healthy people and cancer patients. The left panel shows the results of ROC curve analysis of each marker, and the right panel shows a scatter plot of exosome mRNA levels.

Fig. 4 shows a scatter plot of exosome mRNA levels in plasma samples from healthy populations, breast cancer patients and breast benign hyperplasias detected using the probe method for each marker.

Figure 5 shows changes in exosome mRNA levels in plasma samples from paired pre-and post-operative cancer patients using probe methods for each marker.

Examples

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and should not be taken as limiting the scope of the invention.

Example 1 exosome mRNA detection method

In the following examples, listed tumor microenvironment-associated markers were analyzed by mRNA assays designed to run on a fluorescent quantitative PCR detection platform and a ddPCR platform, respectively. The performance of markers is tested independently/in combination in non-cancerous and cancerous plasma exosomes to provide diagnostic performance in the detection of markers for healthy human diversion, high risk population monitoring, prediction and early diagnosis of pan-cancer, and disease course monitoring in cancer patients.

Analyzing the performance of each marker using the Lightcycle480 platform, the markers comprising:

EGF、TGF-β1、VEGFB、MMP9、CXCL8(IL-8)、CXCL12(SDF-1)。

1. plasma treatment, plasma exosome separation and exosome mRNA extraction

Blood was collected in an EDTA anticoagulant tube (5 mL) and plasma was separated as soon as possible after blood collection (plasma separation was performed within half an hour). Plasma separation step: centrifuging at 800g for 10 min to remove red blood cells and white blood cells; and (3) second centrifugation: 16000g, centrifuge for 10 min. The plasma obtained was used for immediate detection or stored at-80 ℃. When retesting, the plasma is thawed.

When the frozen plasma is detected, the plasma is thawed in a water bath at 37 ℃. After centrifugation at 12000rpm for 15min, filtration was carried out with a 0.8 μm filter. Plasma exosome RNA was extracted using QIAGEN Kit (exo-RNeasy Serum/Plasma Midi Kit (50) cat.no.77044), and the specific procedures were performed according to the Kit instructions.

2. Reverse transcription reaction

14 μ l of ddH was used₂And O, after eluting the total RNA of the exosome, adding all the RNA into a reverse transcription reaction system. The reverse transcription reaction was performed using 1. mu.l of random primers according to the reverse transcription reaction system shown in Table 1 below. The reverse transcription Kit was purchased from Thermo Scientific (RevertAID First Strand cDNA Synthesis Kit # K1621).

TABLE 1 reverse transcription reaction System

3. Fluorescent quantitative PCR

3.1HRM method

The levels of each marker mRNA in plasma exosomes were detected using ACTB as an internal reference gene using a dye method. RT-PCR was performed to detect the relative levels of biomarker mRNA according to the protocol shown in Table 2 below.

TABLE 2 RT-PCR (HRM method) assay protocol

3.2 Probe method

The cDNA was detected by MGB type fluorescently labeled probe using ACTB as an internal reference gene, and the level of mRNA of each marker in plasma exosomes was detected by a two-step probe method. RT-PCR was performed to detect the relative expression levels of biomarker mRNA according to the protocol shown in Table 3 below.

TABLE 3 RT-PCR (Probe method) assay protocol

3.3ddPCR method

The reaction was performed in a QX200 Droplet Digital PCR system using ddPCR supermix for probes. ddPCR was performed to detect the relative expression levels of biomarker mRNA according to the protocol shown in Table 4 below.

TABLE 4.ddPCR detection protocol

The determination of each marker was repeated twice in plasma exosome samples of non-cancer/cancer control groups, while control groups were set up, including positive quality control (total RNA extracted from marker-positive cell lines, positive cell lines: H3122/H2228) and negative quality control (DNA/non-reverse transcribed RNA). And analyzing the sample results.

4. Statistical analysis

mRNA expression level of sample 2^-ΔΔCTAnd (3) calculating by using the ACTB as an internal reference, wherein the Ct value means that: the number of cycles that the fluorescence signal in each reaction tube reached the set threshold, the ct value for each mRNA was linear with concentration, and the highest number of detection cycles was calculated for the undetected samples as 50.

Reference gene normalization sample differences:

Δ CT treatment sample ═ CT (target gene) -CT (reference gene)

Δ CT Yang-control ═ CT (Yang-control) -CT (Netherlands gene)

Δ Δ CT ═ Δ CT treatment samples- Δ C positive control controls

Final calculation 2^–ΔΔCT

The differential expression of mRNA of genes associated with the tumor microenvironment between plasma samples of cancer/healthy human patients was analyzed. Different plasma samples were compared using GraphPad Prism software and displayed as a scatter plot.

The ROC curve is used to judge the specificity and sensitivity of individual markers in cancer diagnosis.

Example 2 differentiating Effect of Gene markers associated with tumor microenvironment on cancer and healthy population

Detecting mRNA of gene markers related to a tumor microenvironment in plasma exosomes of clinical samples (including healthy people, patients with gastric cancer, intestinal cancer, lung cancer and breast cancer) to obtain corresponding experimental data. And (3) analyzing the experimental data, and selecting a marker which is suitable for shunting cancer and healthy people or has clinical significance for early diagnosis and prediction of pan-cancer.

Grouping samples:

plasma samples were collected for 200 total cases, with healthy human samples from the department of medical inspection at proemmad, suzhou and the second national hospital of cupling, anhui, and cancer samples from the department of the chest hospital, shanghai, and the first subsidiary hospital of the university of technology, hannan. Hemolysis, lipemia and other abnormal plasma were excluded.

The final study group of plasma samples was 140, including plasma samples of 62 normal controls and 78 cancer patients.

And (3) detection:

sample processing and RNA extraction procedures are described in example 1.

The detection method comprises the following steps: HRM method. The number of samples analyzed for each marker is shown in table 5 below.

The statistical method comprises the following steps: calculation 2^–ΔΔCTSee example 1 for details.

TABLE 5 number of samples analyzed for each marker

Marker substance	Total number of samples	Healthy human sample	Cancer patient samples
				EGF	140	62	78
TGF-β1	98	38	60
				VEGFB	111	36	75
MMP9	98	42	56
				CXCL8(IL-8)	83	36	47
CXCL12(SDF-1)	57	18	39

As a result:

exosome marker 2 for cancer and healthy groups^–ΔΔCTThe value comparison shows that: EGF, TGF-beta 1, VEGFB, MMP9, CXCL8(IL-8),The mRNA level of CXCL12(SDF-1) is clinically diagnostic by ROC analysis, and statistical by t-test (P)<0.0001), the sensitivity/specificity (%) of detection was 80.77/87.10, 65.00/71.05, 82.67/83.33, 76.79/80.95, 87.23/80.56 and 84.62/61.11, respectively. Specific results are shown in fig. 1 and table 6 below.

TABLE 6 test results

Example 3 differentiating Effect of markers on different cancer populations

Based on the experimental results of example 2, this example was further tested against a panel of superior markers as a shunt between cancer and healthy people, including EGF, VEGFB, MMP9, CXCL12(SDF-1), and CXCL8 (IL-8). The difference in the mRNA level of the above marker genes in plasma exosomes of patients of different cancer species was examined.

Grouping samples:

a total of 170 plasma samples of different patients and healthy persons were collected, 80 cancer samples, 62 normal control samples and 28 inflammation samples. The normal control samples were from the department of medical inspection at proruimed, suzhou and the second hospital, cupling, anhui, the cancer samples were from the first subsidiary hospital, the thoracic hospital, shanghai, and the university of technology, and the inflammation samples (enteritis, gastritis) were from the hospital, cupling, anhui. Hemolysis, lipemia and other abnormal plasma were excluded.

And (3) detection:

sample processing and RNA extraction procedures are described in example 1.

The detection method comprises the following steps: HRM method. The number of samples analyzed for each marker is shown in table 7 below.

TABLE 7 number of samples tested for each marker

Marker substance	Healthy person	Lung cancer	Stomach cancer	Intestinal cancer	Breast cancer	Gastritis (gastritis)	Enteritis (enteritis)
								EGF	62	28	26	9	17	9	18
VEGFB	36	11	37	15	14	9	19
								MMP9	42	19	29	9	0	0	0
CXCL8	36	19	20	9	8	0	0
								CXCL12	18	0	20	9	8	0	0

As a result:

the specificity of detecting four kinds of cancers by EGF reaches more than 87%, and the sensitivity is highest by breast cancer (93.75%) and gastric cancer (80.00%); the sensitivity of VEGFB for detecting four cancers reaches over 78 percent, wherein the sensitivity of intestinal cancer and gastric cancer is the highest and reaches 100 percent; the specificity of MMP9 for detecting three cancers is over 80 percent, the sensitivity is the highest for lung cancer (89.47 percent), and the sensitivity is lower for intestinal cancer (44.41 percent); the specificity of the CXCL8 detection of four cancers is above 75%, the sensitivity is highest with lung cancer (89.47%), and the specific results of breast cancer (87.75%) and stomach cancer (85%) are shown in the following tables 8-12.

TABLE 8 analysis of the sensitivity and specificity of EGF in different cancer species

TABLE 9 analysis of sensitivity and specificity of VEGFB in different cancer species

TABLE 10 sensitivity and specificity analysis of CXCL8 in different cancer species

TABLE 11 sensitivity and specificity analysis of MMP9 in different cancer species

TABLE 12 sensitivity and specificity analysis of CXCL12 in different cancer species

Furthermore, by the scatter plot analysis shown in fig. 2, it can be seen that there is a clear distinction between plasma exosome samples, gastroenteritis patient sample samples and healthy control samples from different kinds of cancer patients. The mRNA of the tumor microenvironment related gene can be used as an effective index for different cancer populations, inflammatory populations and healthy populations. The results are integrated to show that the mRNA of EGF, VEGFB, MMP9 and CXCL8(IL-8) in the plasma exosome has different expression amounts in different cancer species, can be used as a good index for cancer screening and auxiliary diagnosis, and has important clinical significance and medical development value.

Example 4 diagnostic and prognostic Effect of combinations of markers on cancer

The markers described above were examined individually and compared for different combinations of sensitivity and specificity in 2 and more using the method of example 1. The combination of EGF and VEGFB is illustrated below only, and the present disclosure is not limited to this two marker combination.

Grouping samples:

the samples analyzed included 20 healthy control plasma exosome samples from the second national hospital, holy, anli; 58 cancer plasma exosome samples from the department of technology hospital, shanghai city and the first subsidiary hospital of the university of science and technology, he henan.

And (4) judging a result:

negative and positive readings were performed according to the cutoff values listed in example 2: an EGF cutoff value > 0.224; the VEGFB cutoff value ≦ 0.0526. And if one of the two markers is positive, the result is judged to be positive, and if both markers are negative, the result is judged to be negative.

As a result:

the detection specificity of single EGF mRNA and VEGFB mRNA is respectively 90.00 percent and 100.00 percent, the sensitivity is respectively 79.31 percent and 81.03 percent, and after the two items are jointly detected, the sensitivity is obviously improved and reaches 93.10 percent. In addition, the negative predictive value after the two joint tests is improved to 81.82 percent, and the total coincidence rate is as high as 92.31 percent, and the specific results are shown in the following tables 13-15.

TABLE 13 EGF individual assay results

TABLE 14 Single assay results for VEGFB

TABLE 15 results of two-item Combined test (EGF + VEGFB)

By combining the results, the combined detection of the two markers can obviously improve the sensitivity and the accuracy without reducing the specificity after comparing the combined detection of the plasma exosome markers in the cancer patients with the single detection result.

Example 5 evaluation of the diagnostic and prognostic Effect of markers on cancer based on Probe detection methods

The method used the method described in example 1 for plasma exosome RNA extraction and reverse transcription. The RT-PCR method adopts a probe method to detect the mRNA of plasma exosomes of a large number of clinical samples (including healthy people and patients with gastric cancer, intestinal cancer, lung cancer and breast cancer). Based on the advantages of the probe method detection, the following analysis is carried out on the experimental data so as to accurately detect the expression quantity of the mRNA of the plasma exosomes of the cancer group and the non-cancer group.

Grouping samples:

plasma samples of 39 different cancer species were collected, as well as 20 normal controls. The normal control samples were from the department of medical inspection at proemmad, suzhou and the second national hospital, cupling, anhui, and the cancer samples were from the department of medicine, the Shanghai city, and the first subsidiary national hospital, the university of technology, Henan. Hemolysis, lipemia and other abnormal plasma were excluded.

As a result:

the detection by a probe method shows that the plasma exosome mRNA of EGF, VEGFB, MMP9, CXCL8 and CXCL12 can obviously distinguish a cancer group from a healthy group. In addition, the mRNA detection results of the markers EGF, VEGFB, MMP9, CXCL8 and CXCL12 detected by the probe method are similar to the results of the HRM method, the ROC analysis has clinical diagnosis significance, and the t test has statistical significance (P < 0.0001); the sensitivity/specificity (%) of the detection was 76.92/90.00, 94.87/100.00, 97.44/58.97, 89.74/100.00 and 87.18/85.00, respectively. Specific results are shown in fig. 3 and table 16 below.

TABLE 16 test results (Probe method)

Example 6 differentiating Effect of markers on benign diseases and cancer

This example investigates the difference in expression of plasma exosome mrnas of markers between benign adenomas and cancers as useful for benign and malignant identification. In the following, breast cancer and benign hyperplasia of the breast are taken as exemplary cancers and benign tumors.

Grouping samples:

a total of 60 breast cancer, fibroadenoma of the breast (benign hyperplasia) and healthy control plasma samples were collected, including 20 breast cancer samples, 20 benign hyperplasia samples and 20 healthy control samples, all from the subsidiary tumor hospital of the university of counterdenier. Hemolysis, lipemia and other abnormal plasma were excluded.

And (3) detection:

sample processing and RNA extraction procedures are described in example 1.

The detection method comprises the following steps: a probe method;

statistical method 2^–ΔΔCTSee example 1 for details.

As a result:

the result of differential diagnosis analysis on benign and malignant breast diseases shows that EGF, VEGFB, MMP9, CXCL12 and CXCL8(IL-8) can better distinguish benign hyperplasia of mammary glands from breast cancer. As shown in the scatter plot in fig. 4, it can be seen that the exosome mRNA expression level of several markers in the breast benign hyperplasia sample was similar to that of the healthy control group. The EGF, MMP9, CXCL8 and CXCL12 have obvious difference in the expression level of exosome mRNA in breast cancer and breast benign hyperplasia samples, which shows that the expression level of exosome mRNA of EGF, MMP9, CXCL8 and CXCL12 in breast cancer patients is abnormally increased and is obviously higher than that of breast benign hyperplasia patients; VEGFB has reduced exosome mRNA expression level in breast cancer, and is significantly lower than that of healthy control group and benign hyperplasia group. The sensitivity and specificity results for each marker distinguishing breast cancer from benign hyperplasia of the breast are shown in table 17. From the results, the sensitivity of EGF and CXCL8 is as high as more than 90%, and the specificity of EGF, VEGFB, MMP9 and CXCL12 is more than 80%. Therefore, the level of the exosome mRNA of the marker has significant meaning and value for differential diagnosis.

TABLE 17 sensitivity and specificity analysis (Probe method) of markers in breast cancer and benign hyperplasia of mammary glands samples

Example 7 mRNA expression differences of Each marker in plasma exosomes before and after treatment

The method adopts the method described in example 1, detects plasma exosome mRNA before and after operation by RT-PCR by a probe method, analyzes the relative expression quantity of mRNA before and after operation by markers EGF, VEGFB, MMP9, CXCL8(IL-8) and CXCL12(SDF-1), and discusses the difference of the expression of the plasma exosome mRNA of a patient before and after operation of the cancer patient for monitoring relapse prognosis.

Grouping samples:

the sample is collected from 45 pairs of preoperative and postoperative matched samples (gastric cancer, intestinal cancer, breast cancer and the like) of the first subsidiary hospital of Henan university of science and technology, wherein the sample comprises 23 pairs of preoperative and postoperative matched samples of gastric cancer, 9 pairs of preoperative and postoperative matched samples of intestinal cancer and 13 pairs of preoperative and postoperative matched samples of breast cancer.

As a result:

the positive detection rate of markers EGF, MMP9, CXCL8 and CXCL12 in preoperative samples is about 99%, and the expression level of marker mRNA is reduced in more than 50% of samples after operation: wherein total detection of EGF preoperative and postoperative samples is 45 pairs, the positive rate of preoperative samples is 100%, 53.33% of sample mRNA relative expression quantity after operation is reduced, wherein 2^–ΔΔCTThe value is reduced by 25 percent and is 10 pairs, 2^–ΔΔCTThere were 7 pairs with a 50% reduction in value; MMP9 joint detection 39 preoperative and postoperative plasma samples, the preoperative sample positive rate is 98%, and postoperative 61.54% of sample 2^–ΔΔCTA reduction in value of 10 pairs with a 25% reduction; the CXCL8 is used for detecting 30 preoperative and postoperative plasma samples, the positive rate of the preoperative samples is 99.7%, and the mRNA relative expression of the samples is reduced by 56.67% postoperatively; CXCL12 detect 29 pairs altogetherPreoperative and postoperative plasma samples, 100% preoperative positivity and 68.97% postoperative sample 2^–ΔΔCTThe value decreased, with 7 pairs for 25% decrease. In addition, VEGFB in 38 pre-and post-operative sample tests 57.89% of patients were post-operative 2^–ΔΔCTThe values were elevated and 50% of patients were upregulated in 31.58%. The sample number information for each marker analysis is shown in table 18 below, and the detection results are shown in fig. 5 and table 19.

TABLE 18 number of paired samples for each marker analysis

TABLE 19 paired sample assay results for each marker

As can be seen from the above results, the overall trend of the expression levels of the plasma exosome mRNAs of the markers EGF, MMP9, CXCL8(IL-8) and CXCL12(SDF-1) after the operation is reduced, indicating that the surgical treatment is effective; VEGFB in postoperative sample detection in plasma exosome mRNA expression level is increased, proving that the operation has effect. Therefore, monitoring the change of the markers has great significance for judging the effect of tumor surgery and monitoring the disease condition, and the combined detection of the markers has greater clinical value.

Claims

1. Use of one or more tumor microenvironment-associated genes as detection targets for the preparation of a kit for the diagnosis or course monitoring of pan-cancer, said kit detecting the mRNA levels of one or more tumor microenvironment-associated genes in the body fluid exosomes of the subject, characterized in that: the tumor microenvironment-associated genes comprise EGF, TGF-beta 1, VEGFB, MMP9, CXCL8, CXCL12 or a combination thereof, and preferably comprise the combination of EGF/VEGFB.

2. Use of a detection reagent for the mRNA of exosomes of one or more tumor microenvironment-associated genes for the preparation of a kit for the diagnosis or course monitoring of pan-cancer, said detection reagent detecting the mRNA level of one or more tumor microenvironment-associated genes in the exosomes of a body fluid of a subject, characterized in that: the tumor microenvironment-associated genes comprise EGF, TGF-beta 1, VEGFB, MMP9, CXCL8, CXCL12 or a combination thereof, and preferably comprise the combination of EGF/VEGFB.

3. The use of claim 1 or 2, wherein an increase in the level of mRNA of EGF, TGF- β 1, MMP9, CXCL8, or CXCL12 in a body fluid exosome of the subject as compared to a control level from an individual not having cancer is indicative of the subject having a risk of developing cancer; or a decrease in the level of mRNA for VEGFB in a body fluid exosome of the subject indicates that the subject is at risk for cancer.

4. The use of claim 1 or 2, wherein the kit or detection reagent detects the mRNA level of the gene associated with the tumor microenvironment by a method selected from the group consisting of conventional RT-PCR detection, ddPCR detection, high throughput sequencing-based detection, and gene chip detection.

5. The use according to claim 4, wherein in the RT-PCR detection, ddPCR detection, high throughput sequencing based detection and gene chip detection methods, reverse transcription and PCR can be performed separately or in a one-step PCR reaction or a one-step multiplex PCR reaction;

wherein the high-throughput sequencing-based detection comprises:

a1. (ii) creating a library by transcriptome using the exosome RNA; and

a2. sequencing the obtained whole transcriptome library to obtain the mRNA level of the tumor microenvironment-associated gene;

or wherein the high throughput sequencing-based detection comprises:

b3. using the products of the multiplex PCR to build a library; and

b4. sequencing the established library to obtain the mRNA level of the tumor microenvironment-associated gene;

wherein the gene chip detection comprises:

c1. performing reverse transcription by using the exosome RNA as a template and a random primer or specific primers of the tumor microenvironment-associated gene and an internal reference gene;

c2. performing multiplex PCR using the reverse-transcribed product as a template and amplification primers for the tumor microenvironment-associated gene and the reference gene; and

c3. hybridizing the products of the multiplex PCR with a gene chip so as to obtain the mRNA level of the genes related to the tumor microenvironment,

wherein the gene chip comprises specific probes of the tumor microenvironment related gene and the reference gene;

or wherein the gene chip detection comprises:

d1. (ii) creating a library by transcriptome using the exosome RNA; and

d2. hybridizing the established complete transcriptome library with a gene chip to obtain the mRNA level of the genes related to the tumor microenvironment,

6. The use of claim 1 or 2, wherein disease course monitoring comprises the step of comparing the mRNA levels of one or more tumor microenvironment-associated genes in the patient's humoral exosomes at a first time point and a second time point or a plurality of other time points, wherein the second time point or plurality of other time points are subsequent to the first time point; further preferably: a decrease in the level of mRNA of EGF, TGF- β, MMP9, CXCL8, CXCL12 in the body fluid exosomes of the patient at the second time point as compared to the first time point is indicative of a good prognosis, or an increase in the level of mRNA of VEGFB in the body fluid exosomes of the patient at the second time point is indicative of a good prognosis.

7. The use of claim 6, wherein the patient receives a cancer treatment between the first and second time points, the cancer treatment selected from chemotherapy, radiation therapy, immunotherapy, surgical treatment, or a combination thereof.

8. The use of any one of claims 1-7, wherein the cancer is selected from lung cancer, gastric cancer, intestinal cancer, and breast cancer, and the bodily fluid is selected from blood, urine, alveolar lavage fluid, cerebrospinal fluid, pleural fluid, and nipple aspirate; preferably, the body fluid is selected from plasma.

9. A kit for diagnosis of pan-cancer or monitoring of the course of cancer, the kit comprising:

a. exosome RNA extraction reagent; and

b. a detection reagent for mRNA of one or more tumor microenvironment-associated genes and an internal reference gene, the detection reagent comprising specific amplification primers and/or probes for the tumor microenvironment-associated genes and the internal reference gene, wherein the tumor microenvironment-associated genes comprise EGF, TGF- β 1, VEGFB, MMP9, CXCL8, CXCL12, or a combination thereof, preferably a combination of EGF/VEGFB; the internal reference gene is selected from beta-Actin (ACTB), GAPDH, beta 2-MG, G6PDH, UBC, Tub, PLA, GUSB, HPRT and the like;

c. optionally further comprising an exosome-isolating agent for isolating exosomes from a bodily fluid.

10. The kit of claim 9, wherein the cancer is selected from lung cancer, gastric cancer, intestinal cancer, and breast cancer; the body fluid comprises blood, urine, alveolar lavage fluid, cerebrospinal fluid, pleural fluid or nipple aspirate, etc., preferably the body fluid is selected from plasma.

11. The kit of claim 9 or 10, wherein the detection reagents are used to detect mRNA levels of the tumor microenvironment-associated gene by a method selected from the group consisting of conventional RT-PCR detection, ddPCR detection, high throughput sequencing-based detection, and gene chip detection.

12. The kit of claim 11, wherein in the RT-PCR detection, ddPCR detection, high throughput sequencing-based detection and gene chip detection methods, reverse transcription and PCR can be performed separately or in a one-step PCR reaction or a one-step multiplex PCR reaction;

wherein the high-throughput sequencing-based detection comprises:

a1. (ii) creating a library by transcriptome using the exosome RNA; and

or wherein the high throughput sequencing-based detection comprises:

b3. using the products of the multiplex PCR to build a library; and

wherein the gene chip detection comprises:

or wherein the gene chip detection comprises:

d1. (ii) creating a library by transcriptome using the exosome RNA; and