KR102254686B1 - Circulating serum exosomal lncRNA marker composition for diagnosing early liver cancer for noninvasive in vitro diagnosis - Google Patents

Abstract

The present invention relates to a serum exosome long non-coding RNA marker composition for early diagnosing liver cancer for non-invasive in vitro diagnosis. To find a novel serum liver cancer biomarker, lncRNAs, which are specifically overexpressed in liver cancer exosomes, are identified by analyzing lncRNA inside exosomes secreted by liver cancer cells, so as to discover a novel serum biomarker with high diagnostic accuracy in early liver cancer for which there is currently no reliable biomarker. As such, by using exosomes to check a cancer cell genome and proteome without biopsy, the composition is highly likely to be utilized as an effective diagnostic biomarker in a disease group with a high risk of biopsy, such as liver cancer, in the future.

Description

Circulating serum exosomal lncRNA marker composition for diagnosing early liver cancer for noninvasive in vitro diagnosis

The present invention relates to a serum exosome long non-coding RNA (lncRNA) marker composition for diagnosing early stage cancer for non-invasive in vitro diagnosis.

Liver cancer is one of the most common carcinomas of Koreans. In 2015, the incidence rate was 29.5 for men and 8.2 for women, ranking 4th among men and 6th among women. In 2015, liver cancer was male among all cancer deaths in 2015. It is a malignant tumor with a poor prognosis enough to occupy 2nd place in and 3rd place in women.

Most of the liver cancer patients have a high risk of bleeding due to the underlying cirrhosis of the liver, so they have to bear a significant risk.Therefore, it is necessary to develop a liquid biopsy technology that diagnoses liver cancer early and acquires genetic information based on blood. . Biopsy is not invasive and simple because it can be diagnosed with blood, compared to taking a considerable risk. Unlike a biopsy, blood can be collected several times during the treatment process, and is suitable for predicting treatment response or residual cancer during the treatment process.

Exosomes are small nano-vesicles with a size of 30-150nm that are produced/secreted from most nucleated cells including cancer cells. As a leader in liquid biopsy technology, various body fluids such as blood, urine, ascites, and saliva It is detected in, and carries the specific genetic information of the parent cell as an exosomal cargo and delivers it to the target cell. Recently, exosomes are attracting attention as a key material for cell-cell communication, and cancer cell-derived exosomes have been pointed out as a key material for tumor penetration and metastasis. Cancer cell exosomes transfer parental genetic information to other target cells in the tumor microenvironment to malignize normal cells, and are believed to play the role of parental cancer cell avatars.

Meanwhile, in the case of alpha-fetoprotein (AFP), which has been previously used as a diagnostic marker for liver cancer, the sensitivity of liver cancer diagnosis is only about 60%. However, there is an urgent need to discover next-generation serum liver cancer biomarkers for predicting treatment response.

Chinese Patent Publication CN 106967820 A (published on July 21, 2017)

An object of the present invention is to provide a biomarker composition for diagnosis of liver cancer comprising a long untranslated RNA LINC00853 as an active ingredient.

In addition, another object of the present invention is to provide a liver cancer diagnostic composition comprising as an active ingredient an agent capable of measuring the expression level of long untranslated RNA LINC00853, and a liver cancer diagnostic kit comprising the same.

In addition, another object of the present invention is to provide a method of providing information necessary for diagnosis of liver cancer comprising the step of measuring the expression level of long untranslated RNA LINC00853.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing liver cancer comprising a long untranslated RNA LINC00853 as an active ingredient.

In addition, the present invention provides a composition for diagnosing liver cancer comprising, as an active ingredient, an agent capable of measuring the expression level of long untranslated RNA LINC00853.

In addition, the present invention provides a liver cancer diagnostic kit comprising the composition.

In addition, the present invention (1) measuring the expression level of the long untranslated RNA LINC00853 from the sample isolated from the patient; (2) comparing the measured expression level of the long untranslated RNA LINC00853 with a control sample; And (3) it provides a method of providing information necessary for liver cancer diagnosis comprising the step of determining the liver cancer when the measured expression level of the long untranslated RNA LINC00853 is higher than that of the control sample.

The present invention relates to a serum exosome long non-translated RNA marker composition for early stage cancer diagnosis for non-invasive in vitro diagnosis, by analyzing lncRNA inside exosomes secreted by liver cancer cells to find new serum liver cancer biomarkers, liver cancer exosomes By identifying the lncRNA that is specifically overexpressed in, we discovered a new serum biomarker with high diagnostic accuracy in early liver cancer, which does not currently have a reliable biomarker. As described above, since exosomes are used to identify cancer cell genomes and proteins without tissue examination, it is highly likely to be used as a useful diagnostic biomarker in disease groups with a high risk of tissue examination, such as liver cancer in the future.

1 is a schematic diagram for purification and separation of exosomes.
Figure 2 shows the clinical association with the overexpression of LINC00853 in the liver cancer cohort.
3 shows the results of Exosomal LINC00853 overexpression in the blood of a liver cancer patient.
4 shows the expression and AUROC results of LINC00853 in staged liver disease.
5 shows the results of AF and comparative ROC analysis in patients by mUICC stage.
6 shows the evaluation of the positive rate of AFP and LINC00853 in each disease group and the positive rate of LINC00853 according to the presence or absence of AFP in liver cancer patients.

The present invention provides a biomarker composition for diagnosis of liver cancer comprising a long untranslated RNA LINC00853 as an active ingredient.

Preferably, the long untranslated RNA LINC00853 may be derived from serum exosomes, but is not limited thereto.

"Long untranslated RNA LINC00853" of the present invention refers to NCBI accession no. NR_047498.1 may be, but is not limited thereto.

As used herein, the term "diagnosis" refers to determining the susceptibility of an object to a specific disease or disease, determining whether an object currently has a specific disease or disease, or having a specific disease or disorder. Determining the prognosis of an object, or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).

In addition, the present invention provides a composition for diagnosing liver cancer comprising, as an active ingredient, an agent capable of measuring the expression level of long untranslated RNA LINC00853.

Specifically, the agent capable of measuring the expression level of the long untranslated RNA LINC00853 may be a primer or probe that specifically binds to the long untranslated RNA LINC00853, but is not limited thereto.

Preferably, the long untranslated RNA LINC00853 may be derived from serum exosomes, but is not limited thereto.

Preferably, the liver cancer may be early liver cancer, but is not limited thereto.

In addition, the present invention provides a liver cancer diagnostic kit comprising the composition.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3'hydroxyl group, capable of forming a base pair with a complementary template, and acting as a starting point for template strand copying. Refers to the nucleic acid sequence. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. PCR conditions, the length of the sense and antisense primers can be appropriately selected according to techniques known in the art.

In the present specification, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to a few hundred bases capable of specifically binding in addition to mRNA, and is labeled so that the presence or absence of a specific mRNA, expression You can check the amount. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA (DNA) probe, a double strand DNA (DNA) probe, an RNA probe, or the like. Selection of an appropriate probe and conditions for hybridization can be appropriately selected according to techniques known in the art.

In addition, the present invention (1) measuring the expression level of the long untranslated RNA LINC00853 from the sample isolated from the patient; (2) comparing the measured expression level of the long untranslated RNA LINC00853 with a control sample; And (3) it provides a method of providing information necessary for liver cancer diagnosis comprising the step of determining the liver cancer when the measured expression level of the long untranslated RNA LINC00853 is higher than that of the control sample.

Preferably, the liver cancer may be early liver cancer, but is not limited thereto.

Specifically, the method of measuring the expression level of the long untranslated RNA LINC00853 is RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay ( RPA; RNase protection assay), Northern blotting, DNA chip, Western blot, ELISA (enzyme linked immunosorbent asay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchteroni ( Ouchterlony) Immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation assay, Complement Fixation Assay, FACS or protein chip may be used, but are not limited thereto.

In the present specification, the term "sample isolated from a patient" refers to tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid that differ from the control in the expression level of the long untranslated RNA LINC00853, which is a biomarker for diagnosis of liver cancer of the patient. , Or a sample such as urine, but is not limited thereto. In the present invention, preferably, the sample may be blood, and more preferably, the sample may be blood-derived serum exosomes.

Hereinafter, the present invention will be described in detail according to examples that do not limit the present invention. It goes without saying that the following examples of the present invention are intended to embodi the present invention and do not limit or limit the scope of the present invention. Therefore, what can be easily inferred by experts in the technical field to which the present invention pertains from the detailed description and examples of the present invention is interpreted as belonging to the scope of the present invention.

<Experimental Example>

The following experimental examples are intended to provide experimental examples commonly applied to each of the examples according to the present invention.

One. Exosomes Separation and verification

(1) Purifying/separating each exosome from liver cancer cells, normal hepatocytes, and hepatic stellate cell culture media

Separation of exosomes using ultracentrifugation: Each liver cancer cell line culture was centrifuged at 300 g for 10 minutes to remove cell debris, and then centrifuged at 3000 g for 20 minutes to remove apoptotic bodies. . By ultracentrifugation at 100,000 g for 70 minutes, an exosome fraction was separated. The exosomes suspended in D-PBS medium were concentrated to 250 μl through ultrafiltration using a 0.2 μm pore filter.

Purification of exosomes using Optiprep density gradient centrifugation: To further purify the exosomes obtained above, sucrose gradient was performed. By diluting sucrose buffer and 60% Optiprep gradient media (Sigma-Aldrich), 40%, 20%, 10%, 5% solutions were prepared, layered in order in a centrifuge tube, and the exosomes extracted by ultracentrifugation above were Loaded. Ultracentrifugation was performed at 100,000 g for 18 hours, and exosomes located between 40% and 20% were collected (FIG. 1).

(2) Isolation of exosomes and extraction of RNA from patient serum

Serum of normal and liver cancer patients was extracted using 300ul of Exosome RNA isolation kit (SeraMir™ Exosome RNA Amplification (Cat #RA806A-1)).

72ul of ExoQuick™ was added to 300ul of patient serum, mixed (Vortex), and stored at 4°C for about a day, and then a sample of ExoQuick™ put in 300ul of patient serum was centrifuged at 13,000rpm at 4°C for 2 minutes.

After removing all the supernatant, leaving only the generated pellet, the pellet was dissolved in 100 ul of PBS (1×) (Hyclone™), and 300 ul of Lysis buffer and 200 ul of 100% EtOH were added, followed by mixing (Vortex) for 10 seconds.

Prepare Spin Column and Collection Tube, transfer the entire sample (600ul) to the column, centrifuge at 13,000rpm, 4℃ for 1 minute, remove the solution that has come down to the Collection Tube, and add 400ul Wash Buffer to the column. After centrifugation at 4° C. for 1 minute and removal of the solution that came down to the Collection Tube, the solution was repeated, centrifuged at 13,000 rpm, 4° C. for 2 minutes, and finally dried.

After adding 30ul of elution buffer, centrifugation was performed at 2,000rpm, 4℃, for 2 minutes, followed by final centrifugation at 13,000rpm, 4℃, for 1 minute. The concentration was measured by Nano Drop with the obtained solution.

(3) RNA extraction from patient serum

Serum of normal and liver cancer patients was 300ul, and RNA was extracted with TRIzol™ reagent (Invitrogen).

2. Liver disease by stage whole transcriptome Liver cancer specific from data lncRNA Marker screening

Compared to the normal control group, the expression was not significantly different in hepatitis and cirrhosis, but we tried to select genes whose expression is specifically increased only in early and advanced liver cancer.When only lncRNA was selected in the TCGA_LIHC dataset, a total of 14269 were selected and then liver cancer patients. Of 50 normal peripheral tissues and 371 liver cancer tissues, 3674 lncRNAs were selected that were statistically significant (P<0.05, 1.5 fold changes) between the two groups. Through a heatmap and volcano plot, a more detailed analysis was performed to identify 7 liver cancer-specific lncRNAs.

3. Public Omix Verified using data Exosomes LINC00853 Candidate selection

The association between the expression level and prognosis of the liver cancer sample of lncRNA overexpressed in liver cancer exosomes was analyzed in the TCGA data set, and LINC00853, which was significantly overexpressed in the liver cancer sample and showed a correlation with the prognosis, was selected and verified in an actual liver disease patient cohort. I did.

4. Liver disease for verification Cohort Collection of blood and clinical information

After passing the IRB screening at Ajou University Hospital, the study was conducted by pre-sale of serum and clinical information of 183 liver disease cohorts (29 normal, 28 chronic hepatitis, 35 cirrhosis, 91 liver cancer) through the Human Resource Bank. .

5. qPCR Liver disease through analysis Cohort Patient Serum and Serum In exosomes LINC00853 expression level measurement

To measure LINC00853 expression, 300ul of patient serum was used, TRIzol reagent (Cat #15596018) (Ivitrogen), Exosome RNA isolation kit (SeraMir™ Exosome RNA Amplification (Cat #RA806A-1)) (SBI, System Biosciences) was used. , PrimeScript™ RT Master Mix (Perfect Real Time) (Cat #RR036A) (TaKaRa), miScript RT II kit (Cat #218161) (QIAGEN) was used to synthesize cDNA.

Serum RNA and RNA Free Water (RFW) were combined and adjusted to 8ul, and 2ul of 5X PrimeScript RT Master Mix was added to synthesize a total of 10ul.

Add serum exosome RNA and RNA Free Water (RFW) to 12ul, add 8ul of miScript RT Ⅱ Buffer (4ul for 5× miScript Hiflex buffer, 2ul for 10× miScript Nucleics Mix, 2ul for miScript Reverse TranScriptase Mix). CDNA was synthesized with 20ul.

The cDNA conditions were set as follows.

(When using PrimeScript RT master mix)

A. Stage 1: 37℃, 15 minutes

B. Stage 2: 85℃, 10℃ after 5 seconds, -ing

(When using miScript RT Ⅱ kit)

A. Stage 1: 37℃, 60 minutes

B. Stage 2: 95℃, 10℃ after 5 minutes, -ing

The synthesized cDNA was diluted 1/20 and used.

Gene Accession No. Forward sequence Reverse sequence LINC00853 NR_047498.1 5'-AAAGGCTAGGCGATCCCACA-3' 5'-ACTCCCTAGCTTGGCTCTCCT-3' HMBS NM_001024382.2 5'-GGAGGGCAGAAGGAAGAAAACAG-3' 5'-CACTGTCCGTCTGTATGCGAG-3'

PCR was performed using the primers having the sequence of Table 1. At this time, the necessary primer was purchased and used from M.biotech (Hanam, Korea). qPCR MasterMix (2X, High ROX) (Gendepot, Cat #Q5602) was used for qRT-PCR in a total of 10ul using 5ul.

qRT-PCR conditions were set as follows.

A. Stage 1: 2 minutes at 95℃

B. Stage 2: 95℃ for 15 seconds, 60℃ for 34 seconds, 72℃ for 30 seconds

C. Repeat Stage 2 45 cycles

6. LINC00853 Statistical analysis of the results

After calculating the relative concentration of serum using the Microsoft Office Excel program, statistical analysis was performed using MedCalc statistical software and SPSS v22 analysis program.

< Example 1> Early liver cancer detection serum Exosomes LINC00853 to be selected For integrated genome analysis

To identify long untranslated RNA (lncRNA) that plays a role in liver cancer incidence, when only lncRNA was first selected from the TCGA_LIHC dataset, a total of 14269 were selected (Genecode v22, biotype), and then normal peripheral tissues of liver cancer patients 50 Statistically significant (P<0.05, 1.5 fold changes) between the two groups from 371 samples of dog and liver cancer tissues, 3674 lncRNAs were selected.

When 3674 lncRNAs were expressed as a heatmap, it was found that 3140 genes were overexpressed in liver cancer and 534 genes were underexpressed. It was found that relatively many lncRNAs were overexpressed.

Through the volcano plot, when more detailed analysis was performed, 7 liver cancer driver genes could be identified. Of these, only LINC00853 was selected as a novel liver cancer-specific lncRNA unknown in the research results to date.

In order to verify the expression of LINC00853, it was confirmed that the three data sets (GSE94660, GSE124535) including TCGA_LIHC were statistically significant overexpression in all three data in liver cancer.

As a result of checking the expression level of LINC00853 by stage in GSE114564, which is the sequencing data for each stage of liver cancer, it was confirmed that the expression gradually increased as the liver cancer progressed.

When the survival analysis according to the expression of LINC00853 was performed on the TCGA_LIHC dataset, the prognosis was poor in the patient group with high expression of LINC00853 in the overall survival rate and disease-free survival rate (Logrank P=0.002, P=0.006, respectively) (Fig. 2).

< Example 2> Of LINC00853 serum Exosomes Liver cancer diagnosis As a marker Specificity( Diagnostic power ) evaluation

In order to evaluate LINC00853 as a non-invasive diagnostic marker, when the expression was measured using qRT-PCR in the serum of 10 normal patients and 10 liver cancer patients, there was no difference in expression between normal patients and liver cancer patients.

Exosome, a type of small extracellular vesicles produced by various cells, is a biomarker for various diseases including cancer, and has been reported in various research results as an important research subject in basic research and pharmaceutical fields. Therefore, it was decided to measure the expression of LINC00853 in RNA isolated from serum exosomes.

In order to confirm that the serum exosomes were well separated, when proteins were confirmed through western blot in the exosome markers CD63 and CD81, it was confirmed that the expression of CD63 and CD81 appeared in the serum exosome, and ER It was confirmed that the marker Grp78 was detected only in cell lysate.

Pictures of exosomes extracted from the blood of PKH26-stained liver cancer patients could be observed.

As a result of confirming the expression of LINC00853 in the extracted serum exosome RNA, it was confirmed that the normal group was almost not expressed, whereas all of the liver cancer patient samples were overexpressed (FIG. 3).

< Example 3> Of LINC00853 Liver disease In the cohort Diagnostic power evaluation

To evaluate the diagnosis of liver cancer of LINC00853 in patients who visited the Department of Gastroenterology at Ajou University Hospital, normal liver (n=29), chronic hepatitis (n=28), cirrhosis (n=35), mUICC I (n=32), mUICC II (n=14), mUICC III/IV (n=45) LINC00853 expression was confirmed in patient serum exosomes by quantitative real-time PCR. It was confirmed that the expression of LINC00853 was statistically significantly increased in serum exosomes of liver cancer patients compared to non-liver cancer (NL, CH, LC) (Welch's t-test, compare to normal liver; *P<0.05, **P<0.01 , ***P<0.001, compare to CH; #P<0.05, ##P<0.01, ###P<0.001, compare to LC; §P<0.05, §§P<0.01, §§§P< 0.001).

As a result of ROC analysis in diagnosing liver cancer, serum exosomes LINC00853 had a very high AUC of 0.934 (95% CI: 0.887-0.966) for liver cancer diagnosis. In addition, the cut-off value was found to be 14-fold (Fig. 4).

< Example 4> Exosomes Of LINC00853 Early liver cancer As a marker Verification result

In order to determine whether to detect early liver cancer, the AUC was measured by dividing the mUICC stage.

It was compared with the existing liver cancer serum marker, AFP, to confirm statistically significant superiority.

As shown in FIG. 5, in the diagnosis of liver cancer in the entire liver cancer cohort, AFP showed an AUC of 0.713, LINC00853 was AUC of 0.935, and when compared in CH and LC, the high-risk groups of liver cancer excluding normal, the AFP of AUC was 0.601. , LINC00853 showed a significant difference of 0.908 (P<0.0001).

In the diagnosis of liver cancer in the early liver cancer cohort including mUICC I and II, AFP had an AUC of 0.604, LINC00853 had an AUC of 0.965, and when compared in CH and LC, the high-risk groups of liver cancer excluding normal, AFP had an AUC of 0.541. LINC00853 also showed a significant difference at 0.950 (P<0.0001).

In the diagnosis of liver cancer in the early liver cancer cohort of mUICC stage I with a tumor size of 2 cm or less, AFP showed an AUC of 0.548, LINC00853 was AUC 0.969, and when compared in CH and LC, high-risk liver cancer groups excluding normal, the AUC of AFP was 0.604 and LINC00853 showed a significant difference as 0.956 (P<0.0001).

As shown in Fig. 6, when looking at the positive rate in the normal liver, AFP was measured equal to 0% and LINC00853 was 0%. In the hepatitis group, AFP was measured at 30% and LINC00853 was measured at 11%. In the cirrhosis group, AFP was 50% and LINC00853 was 19%. In the mUICC stage I liver cancer patient group, AFP was 9% and LINC00853 was 94%, which was significantly higher than that of AFP, a marker for measuring liver cancer. In mUICC II, AFP was 29% and LINC00853 was 86%. In mUICC III/IV liver cancer patients, AFP was 60% and LINC00853 was 73%. Overall, the positive rate of LINC00853 was significantly lower than that of AFP in the non-liver cancer group, and the positive rate of LINC00853 was significantly higher than that of AFP in the early liver cancer group.

When comparing the positive rate of LINC00853 according to the presence or absence of AFP in all liver cancer patients, LINC00853 was found to be 86% positive in liver cancer patients with negative AFP.

When comparing the positive rate of LINC00853 according to the presence or absence of AFP in the UICC stage I/II liver cancer patient group, LINC00853 showed a very high diagnostic power as 92% positive in liver cancer patients with a negative AFP.

When comparing the positive rate of LINC00853 according to the presence or absence of AFP in the mUICC stage I liver cancer patient group, LINC00853 was 97% positive in liver cancer patients with a negative AFP. Showed possibility.

In conclusion, the present invention relates to the diagnostic ability of liver cancer using LINC00853 overexpressed in liver cancer exosomes, and exhibits quite excellent sensitivity and specificity with only a small amount of blood, enabling accurate diagnosis of early liver cancer, accuracy, simplicity, and economy of diagnosis. It can be said that it is effective.

The present invention discovered lncRNA overexpressed in hepatic cancer cell exosomes, and firstly verified it in public ohmic data to select LINC00853, and the clinical effectiveness of the selected LINC00853 was verified in an independent liver disease cohort, resulting in excellent diagnostic accuracy. Was confirmed.

The detection rate of early liver cancer using ultrasound is 60-80%, which is less than expected, and AFP, known as a serum biomarker, also plays a minor role in early liver cancer, and the present invention shows remarkable liver cancer diagnosis accuracy using a small amount of blood. Therefore, the present invention is expected to play an innovative role in screening for high-risk liver cancer in the future.

Claims (11)

A biomarker composition for diagnosing liver cancer comprising a long untranslated RNA LINC00853 as an active ingredient. The biomarker composition for diagnosing liver cancer according to claim 1, wherein the long untranslated RNA LINC00853 is derived from serum exosomes. ◈ Claim 3 was abandoned upon payment of the set registration fee.◈ A composition for diagnosing liver cancer comprising an agent capable of measuring the expression level of long untranslated RNA LINC00853 as an active ingredient. ◈ Claim 4 was abandoned upon payment of the set registration fee. The composition for diagnosing liver cancer according to claim 3, wherein the agent capable of measuring the expression level of the long untranslated RNA LINC00853 is a primer or probe that specifically binds to the long untranslated RNA LINC00853. ◈Claim 5 was abandoned upon payment of the set registration fee.◈ The composition for diagnosing liver cancer according to claim 3, wherein the long untranslated RNA LINC00853 is derived from serum exosomes. ◈Claim 6 was abandoned upon payment of the set registration fee.◈ The composition for diagnosing liver cancer according to claim 3, wherein the liver cancer is early liver cancer. ◈ Claim 7 was abandoned upon payment of the set registration fee. A kit for diagnosing liver cancer comprising the composition of any one of claims 3 to 6. ◈ Claim 8 was abandoned upon payment of the set registration fee. (1) measuring the expression level of long untranslated RNA LINC00853 from the sample isolated from the patient;
(2) comparing the measured expression level of the long untranslated RNA LINC00853 with a control sample; And
(3) A method of providing information necessary for diagnosis of liver cancer, comprising determining that the measured expression level of the long untranslated RNA LINC00853 is higher than that of a control sample. ◈ Claim 9 was abandoned upon payment of the set registration fee.◈ The method of claim 8, wherein the sample is blood. ◈ Claim 10 was abandoned upon payment of the set registration fee. The method of claim 9, wherein the sample is a blood-derived serum exosome. ◈ Claim 11 was abandoned upon payment of the set registration fee. The method of claim 8, wherein the liver cancer is early liver cancer.

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