ES2365829T3 - METHOD FOR THE DETECTION OF HEPATIC CANCER, OF THE RISK OF HEPATIC CANCER, OF THE RISK OF RECURRENCE OF HEPATIC CANCER, OF THE MALIGNITY OF HEPATIC CANCER AND OF THE PROGRESS IN THE TIME OF THE BASIC-BASED HEPATHIC CANCER CANCER. - Google Patents

Abstract

Method for detecting the presence and / or quantity of liver cancer specific methylated cytosines in a region containing CpG sequences in the BASP1 gene and optionally in the SRD5A2 gene comprising the following steps (a) to (d): (a) isolate the genomic DNA of a patient sample; (b) contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines; (c) amplifying a region containing methylated cytosines of the BASP1 gene and optionally of the SRD5A2 gene in the genomic DNA using a PCR method; and (d) determining the presence and / or amount of liver cancer specific methylated cytosines in a region containing CpG sequences of the BASP1 gene and optionally of the SRD5A2 gene in the sample of said patient, so that the presence and / or amount of Methylated cytosines specific to liver cancer indicate that the patient is affected by liver cancer, has an increased risk of liver cancer, presents a risk of recurrence after liver cancer treatment, has a malignant liver cancer or has a liver cancer that is progressing time.

Description

TECHNICAL SECTOR OF THE INVENTION

The present invention discloses a method, kit and reagent for detecting liver cancer, the risk of liver cancer, the risk of liver cancer recurrence, and liver cancer malignancy, and for monitoring the progression over time of the liver cancer by identifying and / or quantifying the methylation of specific genes and / or their DNA fragments in clinical samples.

BACKGROUND OF THE INVENTION

Liver cancer develops from chronic liver diseases (chronic hepatitis and liver cirrhosis), and in most cases in those patients with persistent hepatitis virus infection. In Japan, more than 95% of patients with liver cancer have an infection maintained by hepatitis B virus (HBV) and hepatitis C (HCV), especially more than 80% of cases are derived from diseases associated with HCV. Generally, patients with hepatitis reach more severe phases 20-30 years after the onset of hepatitis, and develop liver cancer after progression to liver cirrhosis. Especially, the phase of liver pre-cirrhosis (grade F3 fibrosis) and liver cirrhosis (grade F4 fibrosis) are associated with high rates of liver cancer, and it is said that these patients will develop liver cancer over a period of 10 years.

The age of onset is 60 years or more, with more cases in older generations. There are cases that develop in relatively early stages and in younger generations after the onset of hepatitis. But the cause is to date unknown. In the past, screening for liver cancer in high-risk patients was periodically screened using an immunological assay of existing tumor markers, AFP (alpha-fetoprotein) and PIVKA-II (protein induced by the absence of vitamin K-II), and monitoring by ultrasound (ultrasonography).

For patients with suspected liver cancer using these tests, the location, size and number of liver cancer are confirmed by imaging techniques, more detailed ultrasound, CT (computed tomography) or MRI (magnetic resonance imaging). In addition, liver cancer is finally diagnosed using a pathological biopsy test, if necessary. Once definitively diagnosed, liver cancer undergoes treatment: hepatectomy, trans-arterial embolization (APR), puncture treatment (percutaneous ethanol injection therapy (PEIT), radiofrequency ablation (RFA) or microwave coagulation therapy ( MCT) However, liver cancer recurrence rates are extremely high, since the virus has not been eliminated and liver diseases have not been cured, even if they have been treated.

If liver cancer is detected at an early stage (less than 3 cm in diameter, less than 5 cm in diameter and only

or well differentiated tumors) by the immunological tests and ultrasound currently used, and treated properly, the prognosis is relatively good. However, the sensitivity and specificity of the existing tumor markers, AFP and PIVKA-II, used for the immunological assay, are insufficient. Especially, in the case of early stage hepatic carcinomas both markers have a remarkably low sensitivity (see non-patent literature 1, 2, 3 and 4). Therefore, it is difficult to detect liver cancer at an early stage, most cases have already progressed to advanced cancer when they are detected, and the relative 5-year survival rate of patients with liver cancer is very poor in comparison with other cancers (see non-patent literature 5).

On the other hand, recent improvements in ultrasound instruments have allowed us to detect liver cancer in early stages, but it is not suitable for screening patients at high risk for liver cancer due to some problems such as the need for around 30 minutes to proof, the need for technical skills, dependence on the technical qualities of the instruments, and that it still has a low penetration rate in general hospitals. In addition, since it is difficult to examine the entire liver, especially the parts hidden by other organs and completely within the liver, there is a risk of missing liver cancer. Therefore, there are no sufficiently satisfactory methods for the detection of liver cancer, especially in early stages.

Recent advances in molecular biology are clarifying the characteristics of DNA modifications without genetic changes and alterations of nucleotide sequences in human cancer cells. The characteristics of changes, mutations, amplification and gene loss are well known as representative examples. In recent epigenetic studies, it has been reported that specific genes are highly and abnormally methylated in variable cells, especially cancer cells. In higher eukaryotes, genomic DNA is only methylated in cytosine residues in CpG dinucleotides, the CpG region is called the "CpG island" and is known to exist in about half of the total human genome. Generally, the CpG island is in the promoter region, and in that case the methylation is closely related to the regulation of gene expression. Briefly, in unmethylated cases genes are expressed (mRNA is transcribed), but in methylated cases it is, on the contrary, suppressed through some processes of the regulatory system. It is known that this regulation of gene expression by methylation is tissue specific, development, differentiation, disease, sex or age. Especially, since the suppression of gene expression due to elevated anomalous methylation of the CpG island is frequently observed in cancer or transformed cells, it is considered to be related to carcinogenesis.

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Based on this evidence from basic studies, to date clinical studies have been conducted in various cancers on the association between elevated anomalous methylation of the CpG island and carcinogenesis. Similar clinical studies have been performed in liver cancer, and it has been reported that high anomalous methylation of certain genes is observed in tumor tissues of patients with liver cancer (see non-patent literature 6, 7, and 8). Most recent developments, for example, the positive correlation between CpG island methylation phenotype (CIMP) and AFP levels in hepatocellular carcinoma (HCC) suggest that CIMP can serve as a molecular marker of advanced HCC development ( see non-patent literature 16) and that hypermethylation of genes, for example, HAI-2 / PB, occurs frequently in primary HCC tumors (see non-patent literature 17).

Consequently, it is inferred that the use of the identification of the methylation of specific genes in cancer cells or their DNA fragments as a diagnostic method is very useful for the detection of cancer in early stages. In addition, methylation that is considered one of the factors directly related to carcinogenesis, is supposed to develop before the onset of cancer to alter the progression or malignant alteration of the cancer, and that shows different profiles in different patients or clinical conditions. Therefore, the identification of methylation has possibilities to allow its use for the detection of precancerous lesions, the prediction of recurrence after liver cancer treatment, the detection of liver cancer malignancy and the monitoring of progression in the liver cancer time.

However, the previous results of clinical studies have been obtained using tumor tissue DNA after confirmation of the diagnosis in the sample to be analyzed, and cannot comply with the use as screening for the detection of liver cancer in early stages or of the risk of appearance of liver cancer. An assay that uses tissue is not suitable for routine analysis because it is complex to handle and the low sample processing capacity. In addition, sensitivity and specificity are insufficient due to inadequate selection of the genes to be analyzed and the use of a single gene in the analysis. Therefore, in practice it has been very difficult to use DNA fragment methylation in the diagnosis of cancer.

On the other hand, tiny amounts of DNA circulate in the blood, and it is especially known that the amount of DNA in blood increases in cancer patients (see non-patent literature 9 and 10). It is considered to be due to the fact that old cells are destroyed by programmed death (apoptosis) or necrosis and that large amounts of DNA are released into the blood from them, while the tumor tissue continues to proliferate at an abnormal rate. Since in the blood, in addition to the DNA of the tumor tissue, DNA from different tissues or blood cells is found, it has been difficult to discriminate the DNA derived from a particular tissue by conventional technologies. But, the use of blood DNA for the diagnosis of cancer is very promising as a diagnosis of practical application.

REFERENCES

[Patent literature 1] Tetzner R et al., WO 2004/113567: 24-25, 2004 [Non-patent literature 1] Nomura F et al. Am J Gastroenterol 94: 650-4, 1999 [Non-patent literature 2] Martinez Cerezo FJ and others Rev Esp Enferm Dig 84: 311-4, 1993 [Non-patent literature 3] He YM and others Hepatobiliary Pancreat Dis Int 4: 50-4, 2005 [Non-patent literature 4] Han SL and others Hepatogastroenterology 52: 348-51 2005 [Non-patent literature 5] Osaka Cancer Registry, Annual report (“Annual report of the Osaka cancer registry)

[Non-patent literature 6] Yu J and others Cell Res 13: 319-33, 2003 [Non-patent literature 7] Kanai Y and others Hepatology 29: 703-9, 1999 [Non-patent literature 8] Kondo Y and others Hepatology 32: 970-9, 2000 [Non-patent literature 9] Leon SA and others J Immunol Methods 9: 157-64, 1975 [Non-patent literature 10] Ren N and others World J Gastroenterol 12: 3911-4, 2006 [Non-patent literature 11 ] Herman JG et al., Proc Natl Acad Sci USA 93: 9821-6, 1996. [Non-patent literature 12] Cottrell SE et al., Nucleic Acids Res 32: e10, 2004. [Non-patent literature 13] Eads CA et al., Nucleic Acids Res 28: e32, 2000. [Non-patent literature 14] Iizuka N et al., Lancet 361: 923-929, 2003 [Non-patent literature 15] The General Rules for the Clinical and Pathologic Study of Primary Liver Cancer Liver Cancer Study Group of Japan ("General rules for the clinical and pathological study of primary liver cancer. Study group of liver cancer") 2nd ed. Tokyo: Kanehara & CO., LTD, 2003. [Non-patent literature 16] Zhang C et al., Clin Cancer Res 13 (3): 944-952 (2007) [Non-patent literature 17] Fukai K et al., Cancer Res 63: 8674 -8679 (2003) [Non-patent literature 18] Carpenter B et al., Mol Cell Biol 24 (2): 537-549 (2004)

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SUMMARY OF THE INVENTION

The object of the present invention is to solve the problems of liver cancer screening or monitoring by means of immunological tests with conventional tumor markers and ultrasound in patients at high risk of liver cancer, to discover new detectable tumor markers with high sensitivity and specificity, even in cancer. Hepatic in early stages that is difficult to detect by conventional methods, and to disclose a simple, precise and high performance method using said markers, and a kit and a reagent therewith. The present invention also aims to present a method for the detection of liver cancer that can be easily automated using blood samples as detection targets. In addition, using new tumor markers, it is also an object of the present invention to realize the possibility of detecting precancerous lesions and predicting recurrence after liver cancer treatment in patients at high risk of liver cancer, and the detection of cancer malignancy. Hepatic and monitoring of the progression over time of liver cancer in patients with liver cancer.

Accordingly, the inventors have discovered that it is surprisingly possible to detect liver cancer, especially that of early phases, with high sensitivity and specificity, easily and precisely by identifying and / or quantifying the methylation of two or more specific genes, or their corresponding DNA fragments in clinical samples, that it is possible to easily detect the risk of liver cancer, the risk of recurrence after liver cancer treatment and liver cancer malignancy, that it is possible to easily monitor the progression over time of liver cancer, and have achieved the present invention.

Briefly, the main point of the present invention is a method for the detection of liver cancer, the detection of liver cancer risk, the detection of recurrence after liver cancer treatment, the detection of liver cancer malignancy and the monitoring of liver cancer. the progression over time of liver cancer. More specifically, the present invention relates to the following processes:

A method for detecting the presence and / or amount of methylated cytosine specific for liver cancer in a region containing CpG sequences in the BASP1 gene or optionally in the SRD5A2 gene comprising steps (a) to

(d) mentioned below, in which the presence and / or amount of methylated cytosine specific for liver cancer in the BASP1 gene and optionally in the SRD5A2 gene in a sample of a patient is indicative that the patient is affected by a cancer Hepatic, presents an increased risk of liver cancer, presents a risk of recurrence after treatment of liver cancer, presents a malignant liver cancer or presents a liver cancer that is progressing over time, with the following steps:

(to)

isolate genomic DNA from a patient sample;

(b)

contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines;

(C)

amplifying a region containing methylated cytosines of the BASP1 gene and optionally of the SRD5A2 gene in the genomic DNA using a PCR method; Y

(d)

to determine the presence and / or quantity of methylated cytosines specific to liver cancer in a region that contains CpG sequences of the BASP1 gene and optionally of the SRD5A2 gene in the patient sample, indicating that the patient is affected by liver cancer, presents a increased risk of liver cancer, presents a risk of recurrence after liver cancer treatment, has a malignant liver cancer or has a liver cancer that is progressing over time.

Another object of the present invention is a method to indicate that a patient is affected by liver cancer, has an increased risk of liver cancer, presents a risk of recurrence after liver cancer treatment, has a malignant liver cancer or has liver cancer that is progressing over time by detecting the presence and / or amount of methylated cytosine specific for liver cancer in regions that contain CpG sequences in at least the BASP1 gene and the SPINT2 gene, optionally in an additional gene of the SRD5A2, CFTR, CCND2 genes, APC and RASSF1 in the patient sample by the method comprising the following steps (a) to (d) and by use in combination with said presence and / or amount of methylated cytosine:

(to)

isolate genomic DNA from a patient sample;

(b)

contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines;

(C)

amplify a region containing methylated cytosines of the BASP1 gene and the SPINT2 gene,

optionally of an additional gene between the SRD5A2, CFTR, CCND2, APC and RASSF1 genes in the genomic DNA using a PCR method; Y

(d)

determine the presence and / or amount of liver cancer specific methylated cytosines in a region containing CpG sequences of the BASP1 gene and the SPINT2 gene, and optionally an additional gene between the SRD5A2, CFTR, CCND2, APC and RASSF1 genes in the sample of the patient,

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indicating that the patient is affected by liver cancer, has an increased risk of liver cancer, presents a risk of recurrence after liver cancer treatment, has a malignant liver cancer or has a liver cancer that is progressing over time.

A more specific object of the present invention is a method according to any of the processes mentioned above, in which at least one nucleotide sequence of a pair of primers is complementary to a nucleotide sequence of the genomic DNA of a region containing residues of cytosine that can be methylated, and that can specifically hybridize to said genomic DNA nucleotide sequence when said cytosines are methylated.

Alternatively, a preferred embodiment of the present invention is a method according to the aforementioned processes, in which at least one nucleotide sequence of a pair of PCR primers can specifically hybridize to a region that does not contain any cytosine residue that can be methylated and it is used simultaneously with oligonucleotides to block amplification by said PCR primers, in which said nucleotide sequences of said oligonucleotides partially overlap with the nucleotide sequences of said PCR primers, are complementary to nucleotide sequences in other parts of the DNA. genomic in a region containing residues that can be methylated and in which said oligonucleotides can specifically hybridize to said nucleotide sequences of genomic DNA when said cytosines are not methylated.

It is a preferred embodiment of any of the processes described, that in which as a chemical treatment in step (b) a reagent for the conversion of unmethylated cytosine into uracil is added to the genomic DNA, so that essentially all cytosines do not methylated of said DNA become uracil. Preferably, the reagent for the conversion of unmethylated cytosines into uracil in said DNA is a reagent containing bisulfite, for example, sodium bisulfite and / or sodium sulphite and / or 6-hydroxy-2,5,7 acid , 8-tetramethylchroman-2-carboxylic acid.

It is another preferred embodiment of any of the processes described, that in which as an enzymatic treatment in step (b) a restriction enzyme is added to the genomic DNA that is unable to digest the restriction sites containing methylated cytosines and can digest the restriction sites that do not contain methylated cytosines.

Blood samples are preferred, to which the concentrations of the conventionally used tumor marker proteins are simultaneously applied, for example AFP and / or PIVKA-II, and / or the amount of free circulating DNA, for example, the amount of GSTP1 gene .

The methods of the present invention are preferred for determining liver cancer in early stages.

The samples to be used, according to the present invention, are derived from serum, plasma, whole blood, tissues, blood cells, excretions or human internal / external secretions.

In addition, preferred embodiments, according to the present invention, are those in which in step (c) an amplification is performed, by real-time PCR using a fluorescent probe.

The invention also relates to a method, in which a pair of PCR primers can specifically hybridize to genomic DNA sequences that do not contain any cytosine residue that can be methylated at at least its 3 'end, and in which it is determined the presence and / or amount of methylated cytosine in products amplified by said PCR primers by sequencing analysis or mass spectrometry in step (d).

According to the present invention, the following pairs of primers comprising two nucleic acid primers are preferred:

1) BASP1_MSF (Sequence Identifier #: 1) AND BASP1_MSR (Sequence Identifier No.: 2), and

optionally at least one pair of primers selected from the group of primer pairs

next:

2) SPINT2_MSF (Sequence Identifier No. 3) and SPINT2_MSR (Sequence Identifier No. 4),

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3) APC_MSF (Sequence Identifier No. 5) and APC_MSR (Sequence Identifier No. 6), 4) CCND2_MSF (Sequence Identifier No. 7) and CCND2_MSR (Sequence Identifier No. 8), 5) CFTR_MSF (Identifier No. sequence #: 9) and CFTR_MSR (sequence identifier #: 10), 6) RASSF1_MSF (sequence identifier #: 11) and RASSF1_MSR (sequence identifier #: 12),

and 7) SRD5A2_MSF (Sequence Identifier No.: 13) and SRD5A2_MSR (Sequence Identifier No.: 14). The following preferred probes according to the invention are selected from the group consisting of: 1) BASP1_TMP (Sequence Identifier No.: 31), and optionally at least one probe selected

from the following group of probes:

2) SPINT2_TMP (Sequence Identifier #: 32),

3) APC_TMP (Sequence Identifier #: 33),

4) CCND2_TMP (Sequence Identifier #: 34),

5) CFTR_TMP (Sequence Identifier #: 35),

6) RASSF1_TMP (Sequence Identifier #: 36), and

7) SRD5A2_TMP (Sequence Identifier No.: 37). Another object of the present invention is a kit, to be used for any of the methods of detecting the presence and / or amount of methylated cytosine bases specific to liver cancer or to indicate that a patient is affected by liver cancer or may suffer from liver cancer in the future, said kit comprising:

(to)

at least one of the primer pairs comprising Sequence Identifier #: 1 and Sequence Identifier No.: 2 and optionally at least one of the other primer pairs specified previously, and / or

(b)

at least one of the probes comprising Sequence Identifier #: 31 and optionally at least one of the other probes specified previously.

A further object of the present invention is a reagent, to be used with any of the methods to detect the presence and / or amount of methylated cytosine bases specific to liver cancer or to indicate that a patient is affected by liver cancer or may suffer from liver cancer in the future, said reagent comprising:

(to)

at least one of the primer pairs comprising Sequence Identifier #: 1 and Sequence Identifier No.: 2 and optionally at least one of the other primer pairs specified previously, and / or

(b)

at least one of the probes comprising Sequence Identifier #: 31 and optionally at least one of the other probes specified previously.

The present invention further relates to an instrumental system, to be used in any of the methods described previously, comprising:

(to)

at least one of the pairs of primers specified previously, and / or

(b)

at least one of the probes specified previously.

The present invention also relates to the use of a previously specified pair of primers or probe, in particular Sequence Identifier No. 1 and Sequence Identifier No. 2 and / or Sequence Identifier No. 31, to detect the presence and / or quantity of methylated cytosine bases specific to liver cancer in a region that contain CpG sequences in a gene related to HCC in a sample of a patient, or to detect in a patient liver cancer, a risk of liver cancer, a risk of recurrence after treatment of liver cancer, a malignant liver cancer or a liver cancer that progresses over time.

It is possible to detect liver cancer, especially that in early stages, in clinical samples easily and accurately by the method of analysis of the present invention, and it is expected to contribute to improving the outcome of the treatment of patients with liver cancer by detection in advance of the risk of liver cancer and recurrence after treatment. As another use, it is expected to provide an indication to decide the treatment regimen for liver cancer by detecting its malignancy and monitoring its progression over time.

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DETAILED DESCRIPTION OF THE INVENTION

(1) Methylated liver cancer specific genes

The inventors selected genes whose level of expression is specifically reduced in tumor tissues, that is, those in which their CpG islands are most likely to be highly methylated, comparing the levels of gene expression in seventy-six cases of tumor tissue and Sixteen cases of non-tumor tissue removed from patients with liver cancer through surgery, in addition, they chose twenty-three specific candidate liver cancer methylated genes that present, in fact, CpG islands in their promoter regions. Next, two methylated candidate genes specific for liver cancer were selected, by performing a methylation analysis for said candidate genes from genomic DNA derived from tumor tissues removed from patients with early-stage liver cancer. In addition, by confirming the detection of liver cancer-derived methylated DNA fragments specific to these two genes in the blood of patients with liver cancer, these two genes have finally been identified (the BASP1 gene (membrane-bound signal protein, abundant in the brain 1) and the SRD5A2 gene (alpha 2 polypeptide of the steroid-2-alphareductase)) as specific methylated liver cancer genes.

Both genes, the BASP1 gene and the SRD5A2 gene, have already been cloned and their cDNA sequences (mRNA) have been registered in GenBank (BASP1: NM_006317, SRD5A2: NM_000348). However, it has never been indicated that the CpG islands of these two genes are highly methylated specifically in cancer. The BASP1 gene, for example, has been associated with the regulation of transcription function during renal development (see non-patent literature 18). The inventors of the present invention are the first who have surprisingly demonstrated experimentally that these two genes are highly methylated in liver cancer tissues but rarely in non-tumor liver tissues. That is, they have found that these two genes are highly methylated specifically in liver cancer and are suitable markers for the detection thereof.

On the other hand, of the twelve genes of which their specific anomalous methylation of liver cancer has been reported in previously published references, nine specific candidate methylated liver carcinoma genes were selected, by performing genomic DNA methylation analysis derived from Tumor tissues removed from patients with early liver cancer. In addition, by confirming the detection of methylated DNA fragments derived from liver cancer specific to five of said nine genes in the blood of patients with liver cancer, these five genes have finally been identified (SPINT2 gene (Serine protease inhibitor gene, kunitz type 2), APC gene (adenomatous polyposis of the colon), CCND2 gene (Cyclin D2), CFTR gene (ATP-binding cassette, regulator of transmembrane conductance of cystic fibrosis) and RASSF1 gene (Family domain 1 of association with Ras ( RaIGDS / AF-6))).

All these five genes, the SPINT, APC, CCND2, CFTR and RASSF1 genes, have already been cloned and their cDNA sequences (mRNA) have been registered in GenBank (SPINT2: NM_021102, APC: NM_000038, CND2: NM_001759, CFTR: NM- 000492, RASSF1: NM_007182). There are some notifications that indicate that the CpG islands of these five genes are highly methylated specifically in liver cancer, but their frequency of methylation has been variable and there are no reports in patients with HCV positive liver cancer. The fact that these five genes are highly methylated in tumor tissues of HCV positive liver cancer but rarely in non-tumor liver tissues, has been first objectified by the inventors. That is, they have found that these five genes are highly methylated specifically in HCV positive liver cancer and are suitable markers for liver cancer detection, especially in HCV positive liver cancer.

The present invention relates in particular to a method for detecting the presence and / or quantity of hepatic carcinoma-specific methylated cytosines in a region containing CpG sequences in the BASP1 gene and optionally in the SRD5A2 gene comprising the following steps (a ) to (d):

(to)

isolate genomic DNA from a patient sample;

(b)

contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines;

(C)

amplifying a region containing methylated cytosines in the BASP1 gene and optionally in the SRD5A2 gene in the genomic DNA using a PCR method; Y

(d)

determine the presence and / or amount of specific methylated cytosines of liver cancer in a

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region containing CpG sequences of the BASP1 gene and optionally of the SRD5A2 gene in the sample of said patient,

so that the presence and / or quantity of liver cancer-specific methylated cytosines in the BASP gene and optionally in the SRD5A2 gene in a sample of said patient is indicative that said patient is affected by liver cancer, presents an increased risk of liver cancer, presents a risk of recurrence after treatment of liver cancer or presents a malignant liver cancer or has a liver cancer that is progressing over time.

In addition, the present invention relates to a method for indicating that a patient is affected by liver cancer, has an increased risk of liver cancer, presents a risk of recurrence after treatment of liver cancer or has a malignant liver cancer or has a cancer liver disease that is progressing over time by detecting the presence and / or quantity of methylated cytosines specific to liver cancer in regions that contain CpG sequences of at least the BASP1 and SPINT2 gene, optionally in an additional gene between the SRD5A2, CFTR, CCND2 genes, APC and RASSF1 in the sample of said patient, said method comprising the following steps (a) to (d):

(to)

isolate genomic DNA from the patient's sample;

(b)

contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines;

(C)

amplifying a region containing methylated cytosines of the BASP1 gene and the SPINT2 gene, optionally of an additional gene between the SRD5A2, CFTR, CCND2, APC and RASSF1 genes in the genomic DNA using a PCR method; Y

(d)

determine the presence and / or amount of liver cancer specific methylated cytosines in a region containing CpG sequences of the BASP1 gene and the SPINT2 gene, and optionally an additional gene between the SRD5A2, CFTR, CCND2, APC and RASSF1 genes in the sample of said patient.

A cancer detectable by the present invention is preferably a liver cancer, more preferably a hepatocellular carcinoma (HCC) and more preferably a HCV positive HCC. In the present invention, the detection of liver cancer means judging whether or not an individual is suspected of having liver cancer. When an individual is suspected of having liver cancer, it is definitively diagnosed by imaging techniques and pathological tests.

According to the present invention, the detection of the risk of liver cancer means judging whether it is highly possible or not for an individual to develop liver cancer in the near future.

According to the present invention, the detection of liver cancer recurrence means judging whether or not there is a probability of liver cancer recurrence in an individual after treatment thereof.

According to the present invention, the detection of the malignancy of a liver cancer means judging whether the liver cancer that is developing in an individual is highly malignant or not.

According to the present invention, monitoring the progression of liver cancer over time means judging whether the liver cancer that is developing in an individual is progressing over time or not.

In addition, the present invention relates to a method that uses clinical samples and detects liver cancer by identifying and / or quantifying the methylation of the BASP1 gene and optionally the SRD5A2 gene, or its DNA fragments in said samples, or the BASP1 gene and the SRD5A2 gene and at least one of the following genes: SPINT2 gene, APC gene, CCND2 gene, CFTR gene and RASSF1 gene, or their DNA fragments in said samples. Combinations comprising three or more genes are preferably used, the use of the BASP1, SRD5A2 and SPINT2 genes being more preferred according to the present invention.

According to the present invention, it is possible to use the combination of blood concentrations of the conventionally used tumor marker proteins and / or the amount of free circulating DNA in blood with the results identified and / or the quantified values of methylated cytosine in said seven genes . The tumor marker proteins conventionally used are preferably AFP and / or PIVKA-II, in particular AFP. The amount of free circulating DNA is preferably determined for the GSTP1 gene. To determine DNA, DNA can be used before or after bisulfite treatment (BIS), with the use of DNA after bisulfite treatment being preferred.

In principle, clinical samples do not have any limitations, provided they are obtained from individuals. For example, according to the present invention, serum, plasma, whole blood, blood cells, excretions or internal / external secretions are applicable. Serum, plasma and whole blood are preferred, and the use of serum is particularly preferred. Biopsies and surgically removed tissue are suitable as tissues, excretions, feces and urine are suitable, and bile and pancreatic fluid are suitable as samples of internal / external secretions.

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Neither do individuals or patients present limitations, as long as they are human. The present invention preferably applies to patients at high risk for liver cancer (chronic hepatitis or liver cirrhosis), and in particular to patients at high risk for liver cancer positive for HCV or patients with liver cancer. Patients who have had liver cancer and who have received appropriate treatments can be included as patients at high risk for liver cancer,

(2) identification and / or quantification of methylation

According to the present invention, "identification and / or quantification of the methylation of specific genes" means to identify the methylation of the cytosine residues contained in CpG dinucleotides or CpG island in regions promoting specific genes in clinical samples and / or quantifying the level of said methylation

Methods for the identification and / or quantification of methylation are preferably methylation-specific PCR (MSP) (see non-patent literature 11) and / or PCR-HM (HeavyMethyl PCR) (see non-patent literature 12) and TaqMan-MSP and / or TaqMan-PCR-HM (see non-patent literature 13) with TaqMan probes being particularly preferred according to the present invention. As amplification reagents, a reagent of its own can be used according to public methods, preferably using the LightCycler-TaqMan Master Kit (Roche Diagnostics GmbH).

MSP means a method according to the present invention, in which at least one of the sequences of a pair of PCR primers is complementary to a genomic DNA sequence in a region containing cytosine residues that can be methylated, and can specifically hybridize to said nucleotide sequence of genomic DNA when said cytosines are methylated. In particular, PCR amplification occurs with PCR primers that can specifically hybridize to the nucleotide sequence containing said methylated cytosines, when the cytosines that can be methylated are methylated. PCR-HM means a method according to the present invention, in which at least one of a pair of primers can specifically hybridize to a region that does not contain any cytosine residue that can be methylated and used simultaneously with oligonucleotides to block amplification by said primers. PCR, in which said nucleotide sequences of said oligonucleotides partially overlap with the nucleotide sequences of said PCR primers, are complementary to nucleotide sequences of other parts of the genomic DNA in a region containing residues that can be methylated and wherein said oligonucleotides can specifically hybridize to said genomic DNA nucleotide sequences when said cytosines are not methylated. In particular, PCR primers are used simultaneously can specifically hybridize to nucleotide sequences that do not contain cytosines that can be methylated, that is, non-mutilation-specific PCR primers, independent of cytosine methylation, and oligonucleotides (blocking probes) suitable for blocking amplification by said primers.

Said blocking probes partially overlap with said PCR primers. Therefore, when said blocking probes hybridize to the target nucleotide sequences, amplification is blocked because said PCR primers are not capable of hybridizing. Said blocking probes are complementary to sequences containing cytosines that can be methylated from elsewhere and can specifically hybridize to nucleotide sequences containing said unmethylated cytosines when said cytosines are not methylated. Accordingly, amplification is performed with said primers and the absence or presence of methylation can be detected depending on the absence or presence of said amplification with said primers, only when the cytosines are methylated.

Furthermore, it is possible to apply other methods for the identification and / or quantification of the methylation according to the invention, for example, sequencing analysis or mass spectrophotometry, sequencing analysis using Pyrosequencer (Biotage AG) being preferred.

According to the present invention, it is necessary to extract the DNA from the clinical samples for their treatment prior to the identification and / or quantification of the methylation of specific genes. For example, commercialized kits such as MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics GmbH) or QIAamp DNA Blood Midi Kit (QIAGEN GmbH) or reagents prepared according to known methods can be used for DNA extraction. DNA extraction is preferably used using the SP Kit for Serum and Plasma Extractor kit (Wako Pure Chemical Industries, Ltd.).

The extracted DNA is treated with a bisulfite reagent converting the unmethylated cytosines of the DNA into uracils (BIS treatment). For BIS treatment, commercialized kits such as the EpiTect Bisulfite Kit (QIAGEN GmbH), the EZ DNA Methylation-Gold Kit (Zymo Research Corporation) or the DNA Modification Kit (Chemicon • International, Inc.), or reagents prepared according to known methods can be used .

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DNA whose unmethylated cytosines have been converted to uracils by said BIS treatment is amplified in regions that include CpG islands by a method of nucleic acid amplification such as PCR, and it is confirmed that unmethylated cytosines have been regularly converted to uracils using said amplified products.

5 Specifically, it is performed by nucleic acid amplification using primers to amplify nucleotide sequences of methylated cytosines that are not converted to uracils by the BIS treatment and of non-methylated cytosines that are converted to uracils, and confirming said products amplified by electrophoresis and / or sequencing analysis of said amplified products.

The length of said amplified products may normally be less than 1,000 bp, preferably it is less than 180 bp and particularly preferably it is around 100 bp.

Table 1 shows the groups of preferred primers and the length of the products amplified by said groups of primers of the present invention.

Table 1: Groups of primers for MSP amplification of liver cancer specific methylated genes

Primer group

Direct primer Reverse primer Products Length (bp)

one

BASP1_MSF (Sequence Identifier # 1) BASP1_MSR (Sequence Identifier # 2) 96

2

SPINT2_MSF (Sequence Identifier # 3) SPINT2_MSR (Sequence Identifier No. 4) 118

3

APC_MSF (Sequence Identifier No. 5) APC_MSR (Sequence Identifier # 6) 127

4

CCND2_MSF (Sequence Identifier No. 7) CCND2_MSR (Sequence Identifier No. 8) 85

5

CFTR_MSF (Sequence Identifier No. 9) CFTR_MSR (Sequence Identifier No. 10) 139

6

RASSF1_MSF (Sequence Identifier No. 11) RASSF1_MSR (Sequence Identifier No. 12) 75

7

SRD5A2_MSF (Sequence Identifier # 13) SRD5A2_MSR (Sequence Identifier # 14) 93

8

BASP1_F (Sequence Identifier No. 15) BASP1_R (Sequence Identifier No. 16) 458

9

SPINT2_F (Sequence Identifier No. 17) SPINT2_R (Sequence Identifier No. 18) 361

10

APC_F (Sequence Identifier No. 19) APC_R (Sequence Identifier No. 20) 479

eleven

CCND2_F (Sequence Identifier No. 21) CCND2_R (Sequence Identifier No. 22) 427

12

CFTR_F (Sequence Identifier No. 23) CFTR_R (Sequence Identifier No. 24) 445

13

RASSF1_F (Sequence Identifier No. 25) RASSF1_R (Sequence Identifier No. 26) 410

14

SRD5A2_F (Sequence Identifier No. 27) SRD5A2_R (Sequence Identifier No. 28) 465

fifteen

SPINT2_HMF (Sequence Identifier No. 29) SPINT2_HMR (Sequence Identifier No. 30) 162

20 From the above nucleic acid amplification, TaqMan probes are shown in Table 2 which allow specific identification and / or quantification of methylated cytosines by real-time PCR with the simultaneous use of primer groups 1 to 7 described in the table. 1. These probes are marked with two different fluorescent substances at their 5 'and 3' ends. The use of fluorescent substances is not limited, as long as they are two different fluorescent substances. The 5 ’end is preferably marked by

25 example, with FAM and the end 3 ’particularly with TAMRA.

image9

Table 2: TaqMan probes to identify MSP amplified products of liver cancer specific methylated genes

Primer group

TaqMan probe 5 ’end fluorescent marker 3 ’fluorescent archer

one

BASP1_TMP (Sequence Identifier No. 31) FAM TAMRA

2

SPINT2_TMP (Sequence Identifier No. 32) FAM TAMRA

3

APC_TMP (Sequence Identifier No. 33) FAM TAMRA

4

CCND2_TMP (Sequence Identifier No. 34) FAM TAMRA

5

CFTR_TMP (Sequence Identifier No. 35) FAM TAMRA

6

RASSF1_TMP (Sequence Identifier No. 36) FAM TAMRA

7

SRD5A2_TMP (Sequence Identifier No. 37) FAM TAMRA

fifteen

 SPINT2_TMP (Sequence Identifier No. 32) FAM TAMRA

5 From the above nucleic acid amplification, the blocking probes that allow specifically identifying and / or quantifying the methylated cytosines by means of a PCR-HM reaction with the simultaneous use of the primer group 15 described in Table 1 are shown in Table 3. These blocking probes are protected with a marked substance. The substance labeled in principle is not limited, as long as the extension reaction can be blocked from said blocking probe, and is preferably phosphate labeled. In addition, said

10 blocking probes can be used as a single reagent and preferably both probes are used at the same time.

Table 3: Blocking probes for PCR-HM of methylated genes specific to liver cancer

Primer group

TaqMan probe Marker at the 3 ’end

fifteen

 SPINT2_HMBF (Sequence Identifier No. 38) Phosphate

fifteen

 SPINT2_HMBR Phosphate

(Sequence Identifier No. 39)

The present invention relates to the primers and probes above, as well as to a method for the detection of liver cancer, the detection of liver cancer risk, the detection of recurrence after liver cancer treatment, the detection of cancer malignancy Hepatic and monitoring of the progression over time of liver cancer. In addition, the present invention includes a kit and a reagent for the detection of liver cancer, the reagent may include primers and probes and the kit may contain reagents for DNA extraction,

20 BIS treatment and nucleic acid amplification. This invention includes an instrumental system for the detection of liver cancer, the detection of liver cancer risk, the detection of recurrence after liver cancer treatment, the detection of liver cancer malignancy and the monitoring of the progression over time of the liver cancer using a method, a kit, a reagent, primers and probes for the detection of liver cancer, the detection of liver cancer risk, the detection of recurrence after

25 treatment of liver cancer, detection of liver cancer malignancy and monitoring the progression over time of liver cancer.

As those skilled in the art will readily understand, some bases can be removed or added to the 5 ′ end of sequence identifiers # 1 to 14 of said primers on condition that they can amplify said seven genes with specific cytosine methylation. Accordingly, the primers of the present invention include primers in which some bases have been removed or added to sequence identifiers # 1 to 30, as long as they meet said condition. Some bases may be removed or added from the ends of said probes with the proviso that they can hybridize with nucleotide sequences that include cytosines

image10

methylated in amplified products using sequence identifiers # 1 to 14 in said primers, that is, that retain the specificity specific hybridization specificity of said probes.

Accordingly, the probes of the present invention include probes in which some bases have been removed or added to sequence identifiers No. 31 to 37, as long as they meet said condition. In addition, they comprise complementary chains of said probes. Some bases may be removed or added to the ends of said blocking probes with the proviso that they can block amplification using primer group 15. Accordingly, the blocking probes of the present invention include blocking probes in which they have been removed or added. some bases to sequence identifiers No. 38 and 39, as long as they meet that condition.

Next, the present invention will be explained in greater detail by showing examples that will be performed in practice using known samples. However, the scope to which this invention extends is not limited to these examples completely.

Example 1. Identification of methylated genes specific to liver cancer

Using "Affymetrix Human Genome U95A DNA Chips®", one hundred genes were selected whose expression level was specific and significantly reduced twice or more in tumor tissues, that is, in which it was more likely that their CpG islands were highly methylated , comparing the expression levels between seventy-six cases of tumor tissues and sixteen cases of non-tumor tissues removed from patients with liver cancer through surgery. In addition, from these genes twenty-three methylated candidate genes specific for liver cancer were selected that actually possess CpG islands in their promoter regions.

Next, 1 µg of tumor tissue genomic DNA obtained from twenty cases of patients with early-stage liver cancer was treated with BIS (liver cancer whose stage of classification is stage I or II, and the tumor size is less than 1.3 and 3.6 cm), regions that include CpG islands of the previous twenty-three candidate genes were amplified by PCR using said BIS treated DNA as a template and the state of methylation was examined by direct sequencing. The methylation status of genomic DNAs from normal liver tissues and leukocytes were also analyzed simultaneously as control samples. Specifically, as a result of the methylation analysis, four genes that were highly methylated in tumor tissues but were not selected in normal liver tissues and leukocytes were selected.

In addition, the analysis of methylation with the same genomic DNA procedure of pairs of tumor tissue and non-tumor tissue extracted from the liver of twenty new cases of liver cancer patients different from those of previous liver cancer, two candidate methylated genes were determined specific to liver cancer that were highly methylated in these tumor tissues but not in non-tumor tissues. In addition, upon confirming the blood detection of liver cancer patients from liver cancer-derived methylated DNA fragments specific to these two genes, these two genes (BASP1 and SRD5A2 genes) were finally identified as specific methylated liver cancer genes.

On the other hand, twelve genes were selected, whose specific methylation of liver cancer had been notified in previously published references. Next, 1 µg of tumor tissue genomic DNA obtained from twenty cases of patients with early-stage liver cancer (liver cancer whose classification stage is stage I or II, and the tumor size is less than between 1, was treated with BIS) 3 and 3.6 cm), regions including CpG islands of said twelve candidate genes were amplified by PCR using said BIS treated DNA as a template, and the methylation status was examined by direct sequencing. The methylation status of genomic DNAs from normal liver tissues and leukocytes as control samples was also analyzed simultaneously. As a result of the mutilation analysis, nine genes were selected that were highly methylated in tumor tissues but were not in normal liver tissues and leukocytes.

In addition, the analysis of the methylation of these nine genes with the same procedure in the genomic DNA of pairs of tumor and non-tumor tissues extracted from the liver of twenty new cases of patients with liver cancer different from those of previous liver cancer, were determined nine methylated candidate genes specific for liver cancer that were highly methylated in these tumor tissues but not in non-tumor tissues. In addition, upon confirming the blood detection of liver cancer patients from liver cancer-derived methylated DNA fragments specific to five of these nine genes, these five genes (SPINT2, APC, CCND2, CFTR and RASSF1 genes) were finally identified as genes specific methylates of liver cancer.

Example 2. Evaluation of the diagnostic performance of specific methylated liver cancer genes in a small number of cases

Total DNA of 1 mL of serum obtained from nine cases of liver cancer positive for HCV in early stages (well differentiated liver cancer) and nineteen cases of patients at high risk of liver cancer (chronic hepatitis and liver cirrhosis) positive for HCV using the SP Kit for Serum and Plasma Extractor kit (Wako Pure Chemical Industries, Ltd.) and 50 µL of DNA solution was prepared.

image11

Said solution was treated with BIS using a self-prepared reagent preparing 50 µL of DNA solution

5 treated with BIS. Next, the specific methylation PCR (MSP) of each of the seven methylated liver cancer specific genes (BASP1, SPINT2, APC, CCND2, CFTR, RASSF1 and SRD5A2) genes was performed with 5 µL of said DNA solution treated with BIS. In said MSP, quantification of methylated DNA was performed by real-time PCR by concomitantly adding TaqMan probes specific to each gene. DNA quantification was actually performed by the following procedure.

10 Genes BASP1, SPINT2, CCND2, CFTR and RASSF1

Real-time PCR was performed using the LightCycler • TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler • II (Roche Diagnostics GmbH) as an acid amplification instrument

15 nuclei For each gene, 5 µL of BIS-treated DNA solution was added to a PCR reaction mixture containing each group of primers and each TaqMan probe at the concentrations shown in Table 4 in a 1x Master mix, the PCR being performed in a total volume of 20 L.

For the BASP1, SPINT2, CCND2 and RASSF1 genes, PCR amplification was performed using a

20 initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 63 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds. For the CFTR gene, PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 64 ° C for 60 seconds followed by heating at 40 ° C for 30 seconds. The fluorescent signal was detected after the extension reaction at 72 ° C of each cycle. Be

25 monitored the amplification of the target gene using the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 1,000, 200, 40 and 4 pg / µl for the BASP1, SPINT2, CCND2 and RASSF1 genes and at 200, 50 and 20 pg / µl for the CFTR gene). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of DNA.

30 methylated by 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

Table 4: Composition of the MSP reaction mixture for liver cancer specific methylated genes

Gen

Primer group TaqMan probe

Conc. (ΜM)

Conc. (ΜM)

BASP1

one 0.25 BASP1_TMP (Sequence Identifier No. 31) 0.1

SPINT2

 2 0.25 SPINT2_TMP (Sequence Identifier No. 32) 0.1

CCND2

 4 0.25 CCND2_TMP (Sequence Identifier No. 34) 0.1

CFTR

 5 0.6 CFTR_TMP (Sequence Identifier No. 35) 0.1

RASSF1

6 0.25 RASSF1_TMP (Sequence Identifier No. 36) 0.1

35

APC and SRD5A2 genes

Real-time PCR was performed using a proprietary reagent as an amplification reagent and the

LightCycler • 2.0 (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. For each gene, 40 µl of BIS-treated DNA solution was added after adding the primer group, the TaqMan probe, the

potassium acetate (pH 7.5) and Aptamer48 at the concentrations or amounts shown in table 5 in the mixture of

PCR reaction composed of Tricine (pH 8.3) 50 mM, 3 mM magnesium acetate, 375 µM dNTPs, glycerol al

2.5% and 0.15 units of ZO5 (thermostable DNA polymerase), the PCR being performed in total volume of 20 µL. For

The APC gene amplification was performed by an initial denaturation at 95 ° C for 2 minutes followed by 45 50 cycles of 95 ° C for 10 seconds, 64 ° C for 60 seconds and 72 ° C for 5 seconds followed by a

heating at 40 ° C for 30 seconds.

For the SRD5A2 gene, amplification was performed by initial denaturation at 95 ° C for 2 min.

followed by 50 cycles of 95 ° C for 15 seconds, 66 ° C for 45 seconds and 72 ° C for 5 seconds 50 followed by heating at 40 ° C for 30 seconds. The amplification of the target gene was monitored by

F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and methylated DNA was quantified

in 50 µl of BIS treated DNA using the standard curve performed with simultaneously measured standards

(serial dilution of artificially methylated DNA at 200, 50, 20 and 4 pg / µl for the APC gene, and at 200, 40, 10 and 4 pg / µl

for the SRD5A2 gene). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. Said amount of serum methylated DNA was used for analysis.

Statistical and clinical evaluations.

image12

Table 5: Composition of the MSP reaction mixture for liver cancer specific methylated genes

Gen

Primer group TaqMan probe 2M potassium acetate Aptamer48

Conc. (ΜM)

Conc. (ΜM)

Quantity (µL) Quantity (µL)

APC

3 0.5 APC_TMP (Sequence Identifier No. 33) 0.25 one 0

SRD5A2

7 0.4 SRDSA2_TMP (Sequence Identifier No. 37) 0.05 0.2 0.04

The Fisher coefficient for each gene was calculated using the method reported by lizuka and others (see literature no

5 patent 14) using the amount of methylated DNA of said seven genes in the previous nine cases of patients with liver cancer positive for HCV in early stages and nineteen cases of patients with high risk of liver cancer positive for HCV. The results are shown in Table 6. The higher the Fisher coefficient, the more promising is the gene of the analyzed result. Therefore, it was shown that particularly the BASP1 gene was a promising gene.

10 Table 6: Classification of the ability to discriminate between patients with liver cancer in early stages and patients with HCV high risk of liver cancer by Fisher's coefficient

Classification

Gen Fisher coefficient

one

BASP1 1.10

2

SPINT2 0.58

3

CCND2 0.44

4

SRD5A2 0.37

5

APC 0.31

6

RASSF1 0.26

7

CFTR 0.12

15 In the four best genes of the classification, according to Fisher's coefficient by means of ROC analysis (operative characteristic of the recipient), the diagnostic performance was evaluated by examining the ability of said genes to detect liver cancer in early stages. The diagnostic performance of the combination of the two best genes, BASP1 and SPINT2 genes, and the combination of three genes with the best two and the fourth gene, BASP1, SPINT2 and SRD5A2 genes, were also evaluated. Specifically, the ROC analysis of each individual gene was performed directly

20 using the amount of methylated DNA of each gene (the amount of methylated DNA of the BASP1 gene is called the BASP1 value, naming the rest of the genes in the same way), and was performed using respectively the values that were calculated by entering said amount of methylated DNA in the formula: BASP1 value + SPINT2 value for the combination of two genes, and in the formula: 0.1 X ABSP1 value + SPINT2 value + SRD5A2 value for the combination of three genes.

25 The results are shown in Figure 1. As a result of the previous ROC analysis, while there was no gene with sufficient diagnostic performance to detect early-stage liver cancer using a single gene, a sufficiently high diagnostic yield was demonstrated as to detect early liver cancer by combining two or three genes. Specifically, it was possible to detect liver cancer in early stages with

30 a sensitivity of 78% and a specificity of 95% with the combination of the BASP1 and SPINT2 genes and with a sensitivity of 89% and a specificity of 90% with the combination of the BASP1, SPINT2 and SRD5A2 genes. Therefore, it is suggested that the determination of the amount of methylated DNA of the methylated liver cancer specific genes discovered in the present invention is useful for the detection of early stage liver cancer.

35 Example 3: Evaluation of the diagnostic performance of specific methylated liver cancer genes with a high number of cases

The total DNA of 1 ml of serum extracted from one hundred and nineteen cases of patients with liver cancer was extracted

40 positive for HCV (including twenty-five cases of early-stage liver cancer whose stage classification corresponds to stage I (stage based on The General Rules for the Clinical DNA Pathologic Study of Primary Liver Cancer (“General rules for the clinical and pathological study of primary liver cancer, see non-patent literature 15) and tumor size less than 3 cm, and twenty-three cases of liver cancer in a well-differentiated early stage) and eighty cases of patients at high risk for liver cancer (chronic hepatitis and liver cirrhosis)

45 positive for HCV, using the DNA Extractor SP Kit for Serum and Plasma (Wako Pure Chemical Industries, Ltd.) and 50 µl of DNA solution was prepared. Said solution was treated with BIS using a reagent of its own preparation and 50 µl of BIS treated DNA was prepared.

Next, the methylation-specific PCR (MSP) of the seven specific methylated genes of liver cancer (BASP1, SPINT2, APC, CCND2, CFTR, RASSF1 and SRD5A2) genes were performed with 5 µl of said BIS-treated DNA solution . In said MSP, by adding the TaqMan probe specific to each gene concomitantly, quantification of the methylated DNA was performed by real-time PCR. On the other hand, according to the method described in non-patent literature 1, the amount of total serum DNA was determined at the same time with 1 µl of said BIS-treated DNA solution. Quantification of methylated DNA was actually performed according to the following protocol.

image13

BASP1, SPINT2, CCND2, CFTR and RASSF1 genes

Real-time PCR was performed using the LightCycler • TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler • II (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. For each gene, 5 µl of BIS-treated DNA solution was added to a PCR reaction mixture in which the primer groups and TaqMan probes had been mixed at the concentrations shown in Table 4 in a 1x Master mix, performing the PCR in a total volume of 20 µl. For the BASP1, SPINT2, CCND2 and RASSF1 genes, PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 63 ° C for 45 seconds and 72 ° C for 5 seconds in a row of a heating at 40 ° C for 30 seconds. For the CFTR gene, PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 64 ° C for 60 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the extension reaction at 72 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 1,000, 200, 40 and 4 pg / µl for the BASP1, SPINT2, CCND2 and RASSF1 genes and at 200, 50 and 20 pg / µl for the CFTR gene). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

APC and SRD5A2 genes

Real-time PCR was performed using a reagent of its own as an amplification reagent and LightCycler • 2.0 (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. For each gene, 5 µL of BIS-treated DNA solution was added after adding the primer group, the TaqMan probe, potassium acetate (pH 7.5) and Aptamer48 to the concentrations or amounts shown in Table 5 in the mixture PCR reaction composed of Tricine (pH 8.3) 50 mM, 3 mM magnesium acetate, 375 µM dNTPs, 2.5% glycerol and 0.15 units of ZO5 (thermostable DNA polymerase), volume PCR being performed total of 20 µl. For the APC gene, amplification was performed by initial denaturation at 95 ° C for 2 minutes followed by 50 cycles of 95 ° C for 10 seconds, 64 ° C for 60 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds .

For the SRD5A2 gene, the amplification was performed by initial denaturation at 95 ° C for 2 minutes followed by 50 cycles of 95 ° C for 15 seconds, 66 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS treated DNA using the standard curve performed with simultaneously measured standards (serial dilution of artificially methylated DNA at 200, 50, 20 and 4 pg / µl for the APC gene, and at 200, 40, 10 and 4 pg / µl for the SRD5A2 gene). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

The Fisher coefficient for each gene was calculated using the method reported by lizuka and others (see non-patent literature 14) using the amount of methylated DNA from these seven genes in the previous one hundred and nineteen cases of patients with HCV positive liver cancer and Eighty cases of patients at high risk for liver cancer. The results are shown in Table 7. The higher the Fisher coefficient, the more promising is the gene of the analyzed result. Therefore, it was demonstrated that the BASP1 gene and the SPINT2 gene were promising genes, and particularly that the BASP1 gene was a promising gene.

ROC analysis was performed using the amount of methylated DNA of said BASP1 and SPINT2 genes. Therefore, it was possible to detect liver cancer with a sensitivity of 50% and a specificity of 95% or a sensitivity of 62% and a specificity of 80% with the BASP1 gene only, and with a sensitivity of 38% and a specificity of 96% with the SPINT2 gene only. Therefore it is suggested that the determination of the amount of methylated DNA of the BASP1 and / or SPINT2 gene of the methylated liver cancer specific genes discovered by the present invention is useful for the detection of liver cancer.

image14

Table 7: Classification of the ability to discriminate between patients with liver cancer in early stages and patients with HCV high risk of liver cancer by Fisher's coefficient

Classification

Gen Fisher coefficient

one

BASP1 0.0704

2

CFTR 0.0412

3

SPINT2 0.0365

4

RASSF1 0.0344

5

CCND2 0.0155

6

SRD5A2 0.0145

7

APC 0.0110

5 Next, the diagnostic performance of the combination of these genes was evaluated by examining their ability to detect liver cancer by combining three of the seven genes using the FLC analysis (Fisher linear classifier) in one hundred and nineteen cases of patients with liver cancer, twenty-three cases of early stage liver cancer whose stage classification is stage I and a tumor size less than 3 cm, or twenty-one cases of early stage liver cancer well differentiated. Specifically, a data reduction algorithm was established using

10 the amount of methylated DNA of each gene by the method reported by Iizuka and others (see non-patent literature 14), the sensitivity, specificity and recognition rate for liver cancer detection were calculated using the same algorithm, and performed an RPC analysis based on the results of said FLC analysis. The results are shown in figures 2 to 4.

15 As a result of these FLC and ROC analyzes, it was possible to detect liver cancer with a sensitivity of 70%, a specificity of 80% and a recognition rate of 74% using the formula: 0.9586 x BASP1 value + 0.2843 x SPINT2 value - 0.0078 x CFTR value + 0.3180 (if the calculated value is <0, indicates liver cancer) combining these three genes, BASP1, SPINT2 and CFTR, in one hundred and nineteen cases of patients with liver cancer.

20 It was possible to detect liver cancer in early stages with a sensitivity of 52%, a specificity of 88% and a recognition rate of 79% using the formula: - 0.003091 x BASP1 value - 0.006448 x SPINT2 value - 0 , 000655 x CCND2 value + 0.350734 (if the calculated value is <0, indicates liver cancer) combining these three genes, BASP1, SPINT2 and CCND2, in twenty-five cases of patients with early liver cancer whose

25 stage classification is stage I with a tumor of less than 3 cm. It was possible to detect liver cancer in early stages with a sensitivity of 67%, a specificity of 80% and a recognition rate of 77% using the formula: - 0.009564 x BASP1 value - 0.004042 x SPINT2 value - 0, 001373 x RASSF1 + 0.595593 value (if the calculated value is <0, indicates liver cancer) by combining these three genes, BASP1, SPINT2 and RASSF1, in twenty-one patients with well-differentiated early liver cancer (tables 8 to 10).

30 As shown in Figures 2 to 4, said ability to detect liver cancer exceeds that of AFP or PIVKAII (for example, HCC (n = 115) vs. HCV carrier (n = 57); AFP (cutting heat 200); sensitivity 30%, specificity 100%, recognition rate 53%; PIVKAII (cut-off value 40%); sensitivity 60%, specificity 85%, recognition rate 68%), conventional tumor markers determined simultaneously. By

Therefore, it is suggested that the determination of the amount of methylated DNA of methylated liver cancer specific genes discovered in the present invention is useful for the detection of liver cancer, especially early stage liver cancer.

In addition, AFP and PIVKAII determined simultaneously were combined with the previous seven genes giving

40 place to nine factors, and the diagnostic performance of said nine factors was evaluated by examining the ability to detect liver cancer from combinations of two or three of said nine factors. As a result, it was possible to detect liver cancer with a sensitivity of 75%, a specificity of 82% and a recognition rate of 78% using the formula: - 0.000478 x BASP1 value - 0.000011 x AFP + 0 value, 001089 (if the calculated value is <0, indicates liver cancer) combining these two factors, BASP1 and AFP gene. It was also

It is possible to detect liver cancer with a sensitivity of 79%, a specificity of 82% and a recognition rate of 80% using the formula: - 0.000476 x BASP1 value - 0.000017 x SPINT2 value - 0.000010 x value AFP + 0.001789 (if the calculated value is <0, indicates liver cancer) combining, the BASP1 gene, the SPINT2 gene and AFP.

50 It was possible to detect liver cancer in early stages with a sensitivity of 60%, a specificity of 82% and a recognition rate of 74% using the formula: - 0.002106 x BASP1 value - 0.005210 x SPINT2 value + 0 , 000069 x PIVKAII value + 0.001390 (if the calculated value is <0, indicates liver cancer) by combining these three genes, BASP1, SPINT2 and PIVKAII, in twenty-five cases of patients with early-stage liver cancer whose stage classification is stage I with a tumor of the tumor less than 3 cm. It was possible to detect liver cancer

55 in early stages with a sensitivity of 67%, a specificity of 88% and a recognition rate of 81% using the formula: - 0.002538 x APC value - 0.003626 x SPINT2 value - 0.013544 x AFP + value 0.852581 (if the calculated value is <0, indicates liver cancer) by combining these 3 markers, APC gene, SPINT2 gene and AFP, in twenty-one patients with well-differentiated early stage liver cancer (tables 8 to 10). Therefore, it is suggested that the determination of the amount of methylated DNA in specific methylated liver cancer genes discovered in the present invention, and the combination of the determination of said values with conventional AFP or PIVKAII markers may further increase the ability to detect liver cancer,

image15

5 especially in early liver cancer.

In addition, the amount of serum DNA determined simultaneously with AFP, PIVKAII and the previous seven genes was combined, giving rise to ten factors, the diagnostic performance of said combination of factors was evaluated by examining the ability to detect liver cancer combinations of three of these ten 10 factors. As a result, it was possible to detect liver cancer with a sensitivity of 87%, a specificity of 80% and a recognition rate of 85% using the formula: - 0.000219 x BASP1 value - 0.000016 x SPINT2 value - 0, 015627 x amount of serum DNA + 0.618672 (if the calculated value is <0, indicates liver cancer) combining the BASP1 gene, the SPINT2 gene and the amount of serum DNA, with the formula: - 0.000220 x value BASP1

- 0.000564 x SRD5A2 value - 0.015633 x amount of serum DNA + 0.613763 (if the calculated value is <0, it indicates

15 liver cancer) combining, the BASP1 gene, the SRD5A2 gene and the amount of serum DNA, and with the formula: 0.000224 x BASP1 value - 0.000010 x AFP value - 0.015610 x amount of serum DNA + 0.617716 (if the calculated value is <0, indicates liver cancer), combining the BASP1 gene, the AFP and the amount of serum DNA.

It was possible to detect liver cancer in early stages with a sensitivity of 84%, a specificity of 88%

20 and a recognition rate of 86% using the formula: - 0.021150 x SRD5A2 value - 0.007102 x SPINT2 value - 0.052594 x amount of serum DNA + 2.091284 (if the calculated value is <0, indicates liver cancer) by combining these three markers, SRD5A2 gene, SPINT2 gene and amount of serum DNA, in twenty-five cases of patients with early-stage liver cancer whose stage classification is stage I with a tumor of tumor less than 3 cm. It was possible to detect liver cancer in early stages with a sensitivity of 86%, a specificity of

25 90% and a recognition rate of 89% using the formula: - 0.001553 x SRD5A2 value - 0.002923 x SPINT2 value - 0.029064 x amount of serum DNA + 1.305493 (if the calculated value is < 0, indicates liver cancer), the formula: - 0.003378 x SPINT2 value - 0.009112 x AFP value - 0.027178 x amount of serum DNA + 1.393957 (if the calculated value is <0, indicates liver cancer ) and the formula: - 0.002833 x SPINT2 value + 0.000061 x PIVKAII value - 0.029513 x amount of serum DNA + 1.384308 (if the calculated value is <0, indicates liver cancer) combining

30 the three markers, SRD5A2 gene, SPINT2 gene and quantity of serum DNA, the three SPINT2 gene markers, AFP and quantity of serum DNA and the three SPINT2 gene markers, PIVKAII and quantity of serum DNA in twenty-one patients with liver cancer in early stages well differentiated (tables 8 to 10).

Therefore, it is suggested that the determination of the amount of methylated DNA in specific methylated genes

35 of liver cancer discovered in the present invention, and the combination of the determination of said values with the amount of serum DNA, of the determination of said values with the amount of serum DNA and the values of AFP or the combination of the Determining these values with the amount of serum DNA and PIVKAII can further increase the ability to detect liver cancer, especially in early-stage liver cancer.

40 Table 8; Ability to detect liver cancer by combining the specific methylated genes of liver cancer, with tumor markers used conventionally and with the amount of DNA in the serum

HCC119 cases

7 methylated DNA markers (selection from the 7 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

CFTR BASP1 SPINT2

70 80 74 - 0.958600 x BASP1 -0.284300 x SPINT2 + 0.007800 x CFTR + 0.318000 If <0, indicates HCC

2 conventional markers + 7 methylated DNA markers (selection from the 9 markers)

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Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

AFP BASP1 SPINT2

79 82 80 - 0.000476 x BASP1 -0.000017 x SPINT2 0.000010 x AFP + 0.001789 If <0, indicates HCC

AFP BASP1

75 82 78 - 0.000478 x BASP1 -0.000011 x AFP + 0.001089 If <0, indicates HCC

Amount of DNA + 2 conventional markers + 7 methylated DNA markers (selection from the 10 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

BASP1 SPINT2 DNA

87 80 85 -0,000291 x BASP1 -0.000016 x SPINT2 0.015627 x DNA + 0.618672 If <0, indicates HCC

BASP1 DNA SRD5A2

87 80 85 - 0.000220 x BASP1 -0,000564 x SRD5A2 - 0.015633 x DNA + 0.613763 If <0, indicates HCC

AFP BASP1 DNA

81 80  85 -0,000224 x BASP1 -0.000010 x AFP 0.015610 x DNA + 0.617716 If <0, indicates HCC

Table 9; Ability to detect liver cancer by combining the specific methylated genes of liver cancer, with tumor markers used conventionally and with the amount of DNA in the serum

Early HCC (TNM stage I classification and tumor size less than 3 cm) 25 cases

7 methylated DNA markers (selection from the 7 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

CCND2 BASP1 SPINT2

70 80 74 - 0.003091 x BASP1 -0.006448 x SP1NTT2 0.000655 x CCND2 + 0.350734 If <0, indicates HCC

2 conventional markers + 7 methylated DNA markers (selection from the 9 markers)

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Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

PIVKA BASP1 SPINT2

60 82 74 + 0.000069 x PIVKA 0.002106 x BASP1 -0.005210 x SPINT2 + 0.001390 If <0, indicates HCC

Amount of DNA + 2 conventional markers + 7 methylated DNA markers (selection from the 10 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

SRD5A2 SPINT2 DNA

87 80 85 - 0.052594 x DNA + 0.021150 x SRD5A2 - 0.007102 x SPINT2 + 2.09128 If <0, indicates HCC

Table 10; Ability to detect liver cancer by combining the specific methylated genes of liver cancer, with tumor markers used conventionally and with the amount of DNA in the serum

Early HCC (well differentiated) 21 cases

7 methylated DNA markers (selection from the 7 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

RASSFI BASP1 SPINT2

67 80 77 - 0.009564 x BASP1 -0.004042 x SPINT2 0.001313 x RASSFI + 0.595593 If <0, indicates HCC

2 conventional markers + 7 methylated DNA markers (selection from the 9 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

AFP APC SPINT2

67 88 81 - 0.013644 x AFP 0.002538 x APC -0.003626 x SPINT2 + 0.852581 If <0, indicates HCC

Amount of DNA + 2 conventional markers + 7 methylated DNA markers (selection from the 10 markers)

Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

SRD5A2 SPINT2 DNA

86 90 89 - 0.029064 x DNA -0.001553 x SRD5A2 - 0.002923 x SPINT2 + 1.305493 If <0, indicates HCC

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Combination of factors

Sensitivity (%) Specificity (%) Recognition Rate (%) Sorter

AFP SPINT2 DNA

86 90 89 - 0.009112 x AFP 0.027118 x DNA -0.003378 x SPINT2 + 1.393957 If <0, indicates HCC

PIVKA SPINT2 DNA

86 90 89 +0.000061 x PIVK4 -0.029513 x DNA -0.002833 x SPINT2 + 1.3243087 If <0, indicates HCC

Example 4: Quantification of methylated DNA by direct quantitative sequencing

5 In the seven specific methylated liver cancer genes, the BASP1, SRD5A2, SPINT2, APC CCND2 CFTR and RASSF1 genes, methylation analysis was performed by direct quantitative sequencing of regions that include CpG islands in a pair of DNAs from Tumor and non-tumor tissue extracted from livers from twenty cases of patients with liver cancer.

In particular, 1 µg of genomic DNA extracted from the tissues was treated with BIS and the regions including CpG islands of the previous seven genes were amplified with the primers described in Table 11 using as template said BIS treated DNA. The PCR reaction solution was composed of 26.7 ng of BIS treated DNA, two units of thermostable DNA polymerase (TOYOBO CO., LTD) previously treated with an equal volume

15 of the TaqStart ™ antibody (Clontech Laboratories, Inc.) for 5 minutes at room temperature, Tirs-HCl (pH 8.8) 67 mM, 16.6 mM ammonium sulfate, 0.01% Tween 20, 200 µM dNTPs, 1 µM of each primer, and 1.5 or 3 mM magnesium chloride in a final volume of 100 µl.

DNA amplification was performed using the GeneAmp PCR system 9600 system (Applied Biosystems) as

20 PCR amplification instrument by initial denaturation at 95 ° C for 2 minutes followed by 5 cycles of denaturation at 95 ° C for 25 seconds, hybridization at 70 ° C for 45 seconds, extension at 72 ° C for 45 seconds, and followed by 40 denaturation cycles at 95 ° C for 25 seconds, hybridization at 65 ° C for 50 seconds, extension at 72 ° C for 45 seconds. The PCR products were concentrated and subjected to agarose gel electrophoresis. The target bands of the amplified products were cut from the gel and

25 were isolated using the QIAquick Gel Extraction Kit (QIAGEN GmbH). Next, the state of methylation of the regions with CpGs was examined by direct quantitative sequencing using the PSQ96MA pyrosequencer and Pyro gold Reagents (Biotage AG) reagents using these isolated products as a template.

Table 11: Composition of the PCR reaction to amplify regions that include CpG islands of liver cancer-specific methylated genes

Gen

Primer group Concentration of magnesium chloride (mM)

BASP1

8 1.5

SPINT2

9 1.5

APC

10 1.5

CCND2

eleven 1.5

CFTR

12 1.5

RASSF1

13 1.5

SRD5A2

14 3

The results of the analysis by quantitative direct sequencing are shown in tables 12 to 14. The amount of methylation is shown by adding "*** (3 asterisks)", "** (2 asterisks)", "* (1 asterisk)" , and no 35 asterisk in cases more than 75%, less than 75% and more than 50%, less than 50% and more than 25%, and less than 25%, respectively. As shown in Tables 12 to 14, it was possible to clearly distinguish between tumor and non-tumor tissues of patients with liver cancer and tissues of HCV carriers (patients

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high risk of liver cancer) by performing quantitative methylation analysis of regions that include CpG islands of the seven specific methylated liver cancer genes.

That is, it was demonstrated that the analysis of the methylation of the regions that include CpG islands in said seven methylated liver cancer-specific genes in tissue DNAs by said direct sequencing is useful for the identification and discrimination of liver cancer.

In addition, by comparing the amount of methylated DNA of the CFTR gene shown in Tables 12 to 14 between the well-differentiated HCC and the poorly differentiated HCC, the differentiation between stages (malignancy) is positively correlated with the amount of methylated DNA (p <0 , 05). Therefore, it is suggested that the quantitative methylation analysis would be usable as an indicator of liver cancer malignancy.

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Table 12; Results of methylation analysis by quantitative direct sequencing Table 13; Results of methylation analysis by quantitative direct sequencing

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Patients with liver cancer / tumor tissues

ID

Type of Differentiation HCV / HBV infection Tumor size (cm) TNM classification stage Amount of methylated DNA (%)

BASP1

SPINT2 APC CCND2 CFTR RASSF1 SRD5A2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Good Good Good Moderate Moderate Moderate Moderate Good Moderate Good Good Good Moderate Moderate Moderate Good Good Moderate Good Bad VHC VHC VHC -VHC -VHC VHC VHC / VHB VHC VHC VHC VHC VHC -VHC / VHB VHB VHC VHC VHC 2.4 3.5 3.4 16 3 2.5 4.2 1.8 3.4 1.4 3.7 5.4 1.2 8.1 3.5 2.4 2.3 4.5 3.8 1.2 II III II IV II II II I * N I II III I II III II III III III III 27.6 * 34.7 * 31.1 * 42.5 * 36.4 * 34.8 * 31.4 * 33.7 * 42.4 * 34.6 * 25.7 * 55.6 ** 30 , 3 * 26.7 * 18.5 21.1 22.4 34.3 * 41.3 * 34.3 * 37.2 * 40.4 * 31.7 * 48.0 * 9.7 16.3 28.2 * 22.1 39.9 * 20.6 9.0 56.0 ** 39.4 * 48, 7 * 16.5 24.4 23.8 48.7 * 44.0 * 42.2 * 51.1 ** 63.3 ** 53.8 ** 36.1 * 62.1 ** 42.1 * 50.0 ** 61.7 ** 68.1 ** 14.3 32.7 * 69.4 ** 51.4 ** 60.9 ** 21.1 33.2 * 31.6 * 72.2 *** 57.6 ** 53.8 ** 43.8 * 57.1 ** 48.8 * 46.9 * 40.3 * 35.4 * 40.5 * 49.9 67.8 ** 41.3 * 18.3 41.5 * 50, 6 ** 45.1 * 30.3 * 39.3 * 31.4 * 78.5 *** 60.8 ** 46.3 * 20.3 36.6 * 42.8 * 62.1 ** 30.9 * 36.6 * 24.8 44.9 * 29.3 * 31.3 * 22.7 68.8 ** 33.4 * 60.2 ** 16.8 32.1 * 26.8 * 45.4 * 43.9 * 56.2 ** 41.7 * 44.5 * 48.2 * 60.9 ** 49.1 * 29.8 * 39.6 * 55.0 ** 64.7 ** 55.9 ** 21.4 60.3 ** 46.9 * 59.3 ** 28.8 * 37.2 * 53.7 ** 66.6 ** 54.7 ** 54.9 ** 23.6 24.8 27.2 * 48.3 * 20.1 21.4 35.8 * 49.5 * 48.1 * 20.6 32.9 * 28.7 * 23.7 32.8 * 28.8 * 36.4 * 27.4 * 19.4 47.3 * 47.6 *

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Patients with liver cancer / non-tumor tissues

ID

Type of Differentiation HCV / HBV infection Tumor size (cm) TNM classification stage Amount of methylated DNA (%)

BASP1

SPINT2 APC CCND2 CFTR RASSF1 SRD5A2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Good Good Good Moderate Moderate Moderate Moderate Good Moderate Good Good Good Moderate Moderate Moderate Good Good Moderate Good Bad VHC VHC VHC -VHC -VHC VHC VHC / VHB VHC VHC VHC VHC VHC -VHC / VHB VHB VHC VHC VHC 2.4 3.5 3.4 16 3 2.5 4.2 1.8 3.4 1.4 3.7 5.4 1.2 8.1 3.5 2.4 2.3 4.5 3.8 1.2 II m II IV II II II I IV I II III I II III II III m III m 23.2 28.1 * 25.6 * 29.2 * 20.4 29.5 * 20.8 23.7 24.7 29.2 * 27.6 * 19.6 21.0 20.8 22, 8 25.5 * 17.8 19.7 21.4 * 19.0 8.0 7.5 6.3 12.6 6.9 6.7 9.2 9.1 11.2 10.6 6.9 10.0 9.4 10.8 11.1 11.8 10, 7 5.8 11.1 9.4 24.3 16.1 17.3 31.1 * 18.6 15.4 17.7 16.6 21.3 23.8 13.3 14.6 18.6 19.8 15.2 14.8 18 , 8 13.1 11.9 14.8 32.0 * 17.9 30.5 * 25.7 * 22.3 22.1 25.7 * 21.1 36.7 * 19.8 24.8 19.0 24.8 14.9 28.5 * 26.1 * 20.7 31.9 * 27.8 42.7 * 18.0 20.1 22.7 17.9 15.1 9.2 14.1 16.2 23.0 14.9 17.8 21.4 21.3 18.7 14.4 22.9 17, 5 16.8 20.0 15.0 19.5 19.3 12.5 39.7 * 28.2 * 23.8 18.8 27.8 * 22.9 33.3 * 18.5 19.9 19.9 40.6 * 37.1 * 21.6 19.9 17.2 16.0 24.7 20.8 18.9 18.0 22.2 17.4 15.9 23.0 19.2 26.5 * 21.9 21.1 21.8 17.5 23.2 21.2 27.5 * 24.8 18.6 24.7 25.3

Table 14; Results of methylation analysis by quantitative direct sequencing

HCV carrier (patients at high risk for liver cancer) / liver tissues

ID

HCV / HBV infection Amount of methylated DNA (%)

BASP1

SPINT2 APC CCND2 CFTR RKSSF1 SRD5A2

21 22

HCV HCV 16.9 9.4 13.0 30.7 * 20.1 23.5 19.2

17.4

9.4 16.4 37.2 * 18.3 28.3 * 23.7

Example 5. Quantification of methylated DNA by PCR-HM (heavy-methyl)

5 Of the seven methylated genes specific to liver cancer, the methylation analysis of the region with CpG islands of the SPINT2 gene was performed in genomic DNA of tumor tissue obtained from ten cases of patients with liver cancer and in genomic DNA of non-tissue tumor obtained from nine cases of patients with liver cancer, using PCR-HM. Specifically, BIS DE 1 µg of DNA extracted from the tissues was performed and

10 prepared 75 µl of BIS treated DNA. Next, the PCR-HM was performed using a group of primers (sequence identifier No. 29 and 30) and two blocking probes (sequence identifier No. 38 and 39) using as template 1 µl (13.3 ng) of said solution of DNA treated with BIS.

During said PCR-HM, quantification of the methylated DNA was performed by real-time PCR using the

15 TaqMan probe (sequence identifier # 32) specific to the SPINT2 gene. Real-time PCR was performed using the LightCycler • TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler • 2.0 (Roche Diagnostics GmbH) as an amplification instrument. 1 µl of BIS-treated DNA solution was added to a PCR reaction mixture containing 0.5 µM of the primer group, 20 µM of blocking probes and 0.1 µM of TaqMan probe in 1X Master mix, performing the PCR in a total volume of 20 µl. The

20 PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds and 58 ° C for 60 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the 58 ° C extension reaction of each cycle. The amplification of the target gene

25 was monitored with the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the concentration of methylated DNA in 75 µl of BIS-treated DNA solution was calculated using the standard curve performed with simultaneously measured standards (serial dilution of artificially methylated DNA at 1,000, 200, and 40 pg / µl As shown in Table 15, it was possible to discriminate between tumor and non-tumor tissues with a sensitivity of 80%, a specificity of 88.9% using a cut-off value of 100 pg / µL.

Therefore, it is suggested that the analysis of the methylation of the region containing CpG islands of the specific methylated gene of liver cancer SPINT2 in DNA from liver tissue by PCR-HM is useful for detecting and distinguishing liver cancer.

35

Table 15: Results of methylation analysis using PCR-HM

ID

Cp Conc (pg / µL) Met (%)

HCC 1

31.91 5.98E + 02 60.6

HCC 5

32.55 3.95E + 02 40.1

HCC 6

33.51 2.13E + 02 21.6

HCC 7

32.09 5.34E + 02 54.2

HCC 11

33.35 2.36E + 02 23.9

HCC 12

HCC 27

34.15 1.41 E + 02 14.3

HCC 43

34.55 1.09E + 02 11.1

HCC 64

HCC 119

34.27 1.30E + 02 13.2

non-HCC

1 39.54 4.32E + 00 0.4

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non-HCC 5

non-HCC 6

non-HCC 7

33.19 2.61E + 02 26.5

non-HCC 12

non-HCC 27

non-HCC 43

non-HCC 64

non-HCC 119

std 5ng

31.14 9.86E + 02 1000

std 1ng

33.57 2.05E + 02 200

std 200pg

37.18 1.98E + 01 40

CP: Met cut-off point: Methylation rate

Example 6. Detection of liver cancer risk by determining methylated genes

liver cancer specific

5 The total DNA of 1 ml of serum extracted from thirty-one cases of HCV positive liver cancer patients (patients monitored more than two years after serum extraction and curative resection of liver cancer) was extracted using the Kit DNA Extractor SP Kit for Serum and Plasma (Wako Pure Chemical Industries, Ltd.) and 50 µl of DNA solution were prepared. Said solution was treated with BIS using a preparation reagent

10 own preparing 50 µl of DNA solution treated with BIS.

Next, the specific methylation PCR (MSP) of each of the seven methylated liver cancer specific genes (BASP1, SPINT2, APC, CCND2, CFTR, RASSF1 and SRD5A2) genes was performed with 5 µl of said DNA solution treated with BIS. In said MSP, quantification of methylated DNA was performed by PCR

15 in real time by concomitantly adding the specific TaqMan probes for each gene. On the other hand, according to the method described in the patent literature 1, the amount of total DNA per 1 ml of serum was determined concomitantly with 1 µl of said BIS-treated DNA solution. Specifically, the quantification of methylated DNA was performed following the following procedure:

20 BASP1 and SPINT2 genes

Real-time PCR was performed using the LightCycler • TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler • II (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. For each gene, 5 µl of BIS treated DNA solution was added to a PCR reaction mixture

25 containing the primer group and the TaqMan probe at the concentrations shown in Table 4 in a 1x Master mix, the PCR being performed in a total volume of 20 µl. PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 63 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

30 The fluorescent signal was detected after the extension reaction at 72 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 1,000, 200, 40, and 4 pg / µL In addition, said amount of methylated DNA in solution was multiplied by 50 and converted to amount of DNA

35 methylated per ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

SRD5A2 gene

Real-time PCR was performed using the LightCycler • TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler • II (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. 5 µl of BIS-treated DNA solution was added after mixing the primer group, the probe

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TaqMan, potassium acetate (pH 7.5) and Aptamer48 at the concentrations and amounts shown in Table 5, in a PCR reaction mixture composed of Tricine (pH 8.3) 50 mM, 3 mM magnesium acetate, 375 µM dNTPs , 2.5% glycerol and 0.15 units of ZO5 (thermostable DNA polymerase), the PCR being performed in a total volume of 20 µl. PCR amplification was performed by initial denaturation at 95 ° C for 2 minutes followed by 50 cycles of 95 ° C for 15 seconds, 66 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the extension reaction at 66 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 200, 40, 10 and 4 pg / µL In addition, said amount of methylated DNA in solution was multiplied by 50 and converted to amount of methylated DNA per 1 ml of serum. Serum was used for statistical analysis and clinical evaluations.

In the thirty-one cases of patients with HCV-positive liver cancer (patients being followed up for more than two years after the extraction of the sample and the curative resection of liver cancer), the clinical evaluation of the risk detection of liver cancer using the data reduction algorithm established during the evaluation of the diagnostic performance of the methylated genes specific to liver cancer with a large number of cases (example 3). Specifically, after the logarithmic transformation of the amount of methylated DNA of each gene and the amount of serum DNA determined simultaneously followed by the calculation of the score value by means of said data reduction algorithm, the difference of said punctuation values was analyzed. among eight cases that developed a new liver cancer (“de novo” HCC) in the first two years after surgery (population with multicentric hepatocarcinogenesis) and twenty-three cases that did not develop metastases in any organ in the first two years (free population of recurrence) by means of the t-test of the means.

As a result, it has been shown that the population with multicentric hepatocarcinogenesis has significantly lower scoring values (p <0.05) than the recurrence-free population using the formula: 0.042965 x BASP1 value - 0.1887008 x SPINT2 - 2 value , 600843 x amount of serum DNA + 9.331859 combining the 3 markers gene BASP1, gene SPINT2 and amount of DNA in serum (table 16). Likewise, it has been shown that the population with multicentric hepatocarcinogenesis has significantly lower score values (p <0.01) than the recurrence-free population using the formula: - 0.112025 x SRD5A2 value -0.199992 x SPINT2 value - 2 , 696542 x amount of serum DNA + 9,867470 combining the 3 markers SRD5A2 gene, SPINT2 gene and amount of serum DNA (table 16). There are no significant differences between the follow-up period of both populations (p = 0.741).

In addition, when the risk of liver cancer of the same thirty-one cases of liver cancer was investigated by dividing the cases between those with score values of 0 and higher (twelve cases) and those with score values below 0 (nineteen cases) according to the formula: <0.042965 x BASP1 value - 0.1887008 x SPINT2 value - 2.600843 x amount of serum DNA + 9.331859 combining the 3 markers BASP1 gene, SPINT2 gene and amount of serum DNA, none of the twelve cases with score values of 0 or higher developed a new liver cancer, while eight (42.1%) of the nineteen cases with score values below 0 developed a new liver cancer (p <0.05 by Fisher's exact test). In addition, the same result was obtained with the formula: - 0.112025 x SRD5A2 value - 0.1999992 x SPINT2 value - 2.696542 x amount of serum DNA + 9.867470 combining the 3 markers SRD5A2 gene, SPINT2 gene and quantity of serum DNA.

Therefore, it is suggested that the determination of the amount of methylated DNA of the methylated liver cancer specific genes discovered in the present invention and the combination of the determination of said values with the amount of serum DNA allows us to detect the risk of developing a new liver cancer, a new HCC.

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Table 16: Clinical evaluation of liver cancer risk detection by combining specific methylated liver cancer genes and the amount of serum DNA

Combination

 Multicentric hepatocarcinogenesis (HCC "de de novo" N Half* Standard deviation

BASP + SPINT + DNA (logarithmic transformation)

no Yes 23 8 0.038832 -1.635407 2,0777707 1,2643829

* p <0.05

Combination

 Multicentric hepatocarcinogenesis (HCC "de de novo" N Half** Standard deviation

SRDSA + SPINT + DNA (logarithmic transformation)

no Yes 23 8 0.138446 -1.641973 1,9552844 1.2470458

** p <0.01

Example 7. Detection of the risk of recurrence of liver cancer by determining the methylated genes 5 specific liver cancer

The total DNA of 1 ml of serum extracted from eighty and one cases of patients with HCV positive liver cancer (patients monitored more than one year after serum extraction and curative resection of liver cancer) was extracted using the kit DNA Extractor SP Kit for Serum and Plasma (Wako Pure Chemical Industries, Ltd.)

10 and 50 µl of DNA solution were prepared. Said solution was treated with BIS using a reagent of its own preparation and a solution of 50 µl of BIS treated DNA was prepared.

Next, the specific methylation PCR (MSP) of each of the three methylated liver cancer specific genes (BASP1, SPINT2 and SRD5A2 genes) was performed with 5 µl of said DNA solution treated with

15 BIS. In said MSP, the quantification of the methylated DNA was performed by real-time PCR by concomitantly adding the TaqMan probes specific to each gene. On the other hand, according to the method described in patent literature 1, the amount of total DNA per 1 ml of serum was determined concomitantly with 1 µl of said BIS-treated DNA solution. Specifically, the quantification of methylated DNA was performed following the following procedure.

twenty

BASP1 and SPINT2 genes

Real-time PCR was performed using the LightCycler TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler II (Roche Diagnostics GmbH) as an acid amplification instrument

25 nuclei For each gene, 5 µl of BIS-treated DNA solution was added to a PCR reaction mixture containing the primer group and the TaqMan probe at the concentrations shown in Table 4 in a 1x Master mix, the PCR being performed in a total volume of 20 l. PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 63 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

30 The fluorescent signal was detected after the extension reaction at 72 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 1,000, 200, 40 and 4 pg / µl).

In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

SRD5A2 gene

40 Real-time PCR was performed using a proprietary reagent as an amplification reagent and LightCycler 2.0 (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. 5 µl of BIS-treated DNA solution was added after mixing the primer group, the TaqMan probe, potassium acetate (pH 7.5) and Aptamer48 at the concentrations and amounts shown in Table 5, in a PCR reaction mixture

45 composed of Tricine (pH 8.3) 50 mM, 3 mM magnesium acetate, 375 µM dNTPs, 2.5% glycerol and 0.15 units of ZO5 (thermostable DNA polymerase), performing the PCR in a total volume of 20  l. PCR amplification was performed by initial denaturation at 95 ° C for 2 minutes followed by 50 cycles of 95 ° C for 15 seconds, 66 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

image26

The fluorescent signal was detected after the extension reaction at 66 ° C of each cycle. Amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with

5 standards measured simultaneously (serial dilution of artificially methylated DNA at 200, 40, 10 and 4 pg / µl). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

10 In the eighty-one cases of patients with HCV-positive liver cancer (patients being followed up for more than 1 year after sample extraction and curative resection of liver cancer), the clinical evaluation of risk detection was implemented of liver cancer using the data reduction algorithm established during the evaluation of the diagnostic performance of the methylated genes specific to liver cancer with a large number of cases (example 3). Specifically, after the logarithmic transformation of the quantity

15 of methylated DNA of each gene and the amount of serum DNA determined simultaneously followed by the calculation of the scoring value by means of said data reduction algorithm, the difference of said scoring values between sixty-eight cases that did not develop recurrence was analyzed. in the year following the surgical intervention of liver cancer (population free of recurrence of liver cancer) and thirteen cases that developed recurrence in the year following the surgical intervention of liver cancer (population with early recurrence of

20 liver cancer) by means of the t-test of the means.

As a result, it has been shown that the population with early recurrence has significantly lower scoring values (p <0.05) than the recurrence-free population using the formula: - 0.042965 x BASP1 value - 0.1887008 x SPINT2 value - 2,600843 x amount of serum DNA + 9.331859 combining the 3

25 markers BASP1 gene, SPINT2 gene and quantity of serum DNA (table 17).

In addition, when the risk of liver cancer of the same eighty-one cases of liver cancer was investigated by dividing the cases between those with score values of 0 and higher (seventeen cases) and those with score values less than 0 (sixty-four cases) according to the formula: -0.042965 x BASP1 value - 0.1887008 x 30 SPINT2 value - 2,600843 x amount of serum DNA + 9.331859 combining the 3 markers BASP1 gene, SPINT2 gene and amount of serum DNA , none of the seventeen cases with score values of 0 or higher developed a new liver cancer, while thirteen (20.3%) of the sixty-four cases with score values below 0 developed a new liver cancer (p < 0.06 by Fisher's exact test). In addition, the same result was obtained with the formula: - 0.112025 x value SRD5A2 - 0.1999992 x value SPINT2 -

35 2.696542 x quantity of serum DNA + 9.867470 combining the 3 markers SRD5A2 gene, SPINT2 gene and quantity of serum DNA.

Accordingly, it is suggested that the determination of the amount of methylated DNA of the methylated liver cancer specific genes discovered in the present invention and the combination of the determination of

40 these values with the amount of DNA in serum allows us to detect the risk of early recurrence of liver cancer.

Four. Five

Table 17: Clinical evaluation of the detection of the risk of recurrence of liver cancer by combining the specific methylated genes of liver cancer and the amount of serum DNA

Combination

 Early recurrence of liver cancer N Half* Standard deviation Standard error of the mean

BASP + SPINT + DNA (logarithmic transformation)

no Yes 68 13 -1,622687 -3,806198 2,4166428 1,8854819 0.3003370 0.5229403

* p <0.005

Example 8. Monitoring the survival rate over time of liver cancer by

determination of the specific methylated genes of liver cancer

50 Total DNA of 1 mL of serum extracted from eighty-seven cases of HCV positive liver cancer patients (patients with curative resection of liver cancer) was extracted using the DNA Extractor SP Kit for Serum and Plasma kit (Wako Pure Chemical Industries , Ltd.) and 50 µl of DNA solution were prepared. Said solution was treated with BIS using a reagent of its own preparation and a solution of 50 µl of BIS treated DNA was prepared.

55 Next, the specific methylation PCR (MSP) of each of the three methylated liver cancer specific genes (BASP1, SPINT2 and SRD5A2 genes) was performed with 5 µl of said BIS-treated DNA solution. In said MSP, the quantification of the methylated DNA was performed by real-time PCR by concomitantly adding the TaqMan probes specific to each gene. On the other hand, according to the method described in patent literature 1, the amount of total DNA in 1 ml of serum was determined concomitantly with 1 µl of said BIS-treated DNA solution. Specifically, the quantification of methylated DNA was performed following the following procedure:

image27

BASP1 and SPINT2 genes

Real-time PCR was performed using the LightCycler TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler II (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. For each gene, 5 µl of BIS-treated DNA solution was added to a PCR reaction mixture containing the primer group and the TaqMan probe at the concentrations shown in Table 4 in a 1x Master mix, the PCR being performed in a total volume of 20 l. PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 63 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the extension reaction at 72 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 1,000, 200, 40 and 4 pg / µl). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. Said amount of serum methylated DNA was used for statistical analysis and clinical evaluation.

SRD5A2 gene

Real-time PCR was performed using a proprietary reagent as an amplification reagent and LightCycler II (Roche Diagnostics GmbH) for a nucleic acid amplification instrument. 5 µl of BIS-treated DNA solution was added after mixing the primer group, the TaqMan probe, potassium acetate (pH 7.5) and Aptamer48 at the concentrations and amounts shown in Table 5, in a PCR reaction mixture composed of Tricine (pH 8.3) 50 mM, 3 mM magnesium acetate, 375 µM dNTPs, 2.5% glycerol and 0.15 units of ZO5 (thermostable DNA polymerase), the PCR being performed in a total volume of 20 µl . PCR amplification was performed by initial denaturation at 95 ° C for 2 minutes followed by 50 cycles of 95 ° C for 15 seconds, 66 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the extension reaction at 66 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 200, 40, 10 and 4 pg / µl). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

In the eighty-seven previous cases of patients with liver cancer positive for HCV (patients with curative resection of liver cancer), the clinical evaluation on monitoring the survival rate over time was implemented using the data reduction algorithm established during the evaluation of the diagnostic performance of the methylated genes specific to liver cancer with a large number of cases (example 3). Specifically, after the logarithmic transformation of the amount of methylated DNA of each gene and the amount of serum DNA determined simultaneously followed by the calculation of the score value by means of said data reduction algorithm, the survival rate over time was compared. after liver cancer surgery between cases with a score value of 0 or higher (twenty-two cases) and those with a score value of less than 0 (sixty-five cases).

As a result, as shown in Figure 5, using the formula: - 0.042965 x BASP1 value - 0.1887008 x SPINT2 value - 2.600843 x amount of serum DNA + 9.331859 combining the 3 BASP1 gene markers , SPINT2 gene and quantity of serum DNA, twenty-two cases with score values of 0 or higher had a survival rate greater than sixty-five cases with a score value of less than 0 (overall survival rate: p = 0, 0289; disease-free survival rate; p = 0.0372).

The overall survival rate is a method that expresses the survival rate during the follow-up period of patients regardless of the cause of death including recurrence of liver cancer (including patients who died of other diseases, by age, rupture of varicose veins esophageal secondary to liver cirrhosis but without liver cancer, other cancers, traffic accidents or suicide). The disease-free survival rate (recurrence-free survival rate) is a method that expresses whether or not metastases or recurrence of liver cancer have been observed during the follow-up period of surviving patients.

image28

Overall survival rate: Percentage of surviving patients in a given day (time) after surgical intervention. Disease-free survival rate: Percentage of patients without metastases or cancer recurrence Hepatic in a given day (moment) after surgery.

Therefore, it is suggested that the determination of the amount of methylated DNA of the methylated liver cancer specific genes discovered in the present invention and the combination of the determination of said values with the amount of serum DNA is useful for monitoring the rate of time survival after liver cancer surgery.

Example 9. Detection of liver cancer progression by determining specific methylated liver cancer genes

Total DNA of 1 mL of serum extracted from one hundred and forty-nine cases of HCV positive liver cancer patients was extracted using the DNA Extractor SP Kit for Serum and Plasma kit (Wako Pure Chemical Industries, Ltd.) and 50 µl were prepared of DNA solution. Said solution was treated with BIS using a reagent of its own preparation and a solution of 50 µL of BIS treated DNA was prepared.

Next, the specific methylation PCR (MSP) of each of the 3 methylated liver cancer specific genes (BASP1, SPINT2 and SRD5A2 genes) was performed with 5 µl of said BIS-treated DNA solution. In said MSP, the quantification of the methylated DNA was performed by real-time PCR by concomitantly adding the TaqMan probes specific to each gene. On the other hand, according to the method described in the patent literature 1, the amount of total DNA per 1 ml of serum was determined concomitantly with 1 µl of said BIS-treated DNA solution. Specifically, the quantification of methylated DNA was performed following the following procedure.

BASP1 and SPINT2 genes

Real-time PCR was performed using the LightCycler TaqMan Master Kit (Roche Diagnostics GmbH) as an amplification reagent and the LightCycler II (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. For each gene, 5 µl of BIS-treated DNA solution was added to a PCR reaction mixture containing the primer group and the TaqMan probe at the concentrations shown in Table 4 in a 1x Master mix, the PCR being performed in a total volume of 20 l. PCR amplification was performed by initial denaturation at 95 ° C for 10 minutes followed by 50 cycles of 95 ° C for 10 seconds, 63 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the extension reaction at 72 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 1,000, 200, 40 and 4 pg / µl). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

SRD5A2 gene

Real-time PCR was performed using a proprietary preparation reagent as an amplification reagent and LightCycler 2.0 (Roche Diagnostics GmbH) as a nucleic acid amplification instrument. 5 µl of BIS-treated DNA solution was added after mixing the primer group, the TaqMan probe, potassium acetate (pH 7.5) and Aptamer48 at the concentrations and amounts shown in Table 5, in a PCR reaction mixture composed of Tricine (pH 8.3) 50 mM, 3 mM magnesium acetate, 375 µM dNTPs, 2.5% glycerol and 0.15 units of ZO5 (thermostable DNA polymerase), the PCR being performed in a total volume of 20 µl . PCR amplification was performed by initial denaturation at 95 ° C for 2 minutes followed by 50 cycles of 95 ° C for 15 seconds, 66 ° C for 45 seconds and 72 ° C for 5 seconds followed by heating at 40 ° C for 30 seconds.

The fluorescent signal was detected after the extension reaction at 66 ° C of each cycle. The amplification of the target gene was monitored by the F1 / F3 analysis mode of the LightCycler program (Roche Diagnostics GmbH), and the methylated DNA was quantified in 50 µl of BIS-treated DNA solution using the standard curve performed with simultaneously measured standards ( serial dilution of artificially methylated DNA at 200, 40, 10 and 4 pg / µl). In addition, said amount of methylated DNA in solution was multiplied by 50 and converted into amount of methylated DNA per 1 ml of serum. This amount of serum methylated DNA was used for statistical analysis and clinical evaluations.

In the one hundred and forty-nine previous cases of HCV positive liver cancer patients, the clinical evaluation of liver cancer progression was implemented using three specific methylated liver cancer genes, the amount of serum DNA and the data reduction algorithm established during the evaluation of the diagnostic performance of the methylated genes specific to liver cancer with a large number of cases (example 3). Specifically, it was assessed using the Pearson coefficient correlation test, the correlation between tumor size and the amount of methylated DNA, the amount of serum DNA or the score values

image29

5 calculated by said data reduction algorithm after the logarithmic transformation of the amount of methylated DNA of each gene and the amount of DNA in serum.

As a result, the amount of methylated DNA of the BASP1 gene tended to positively correlate with the size of the tumor (r = 0.216, p <0.01) (table 18). In addition, the scoring values calculated using the formula:

10 0.042965 x BASP1 value - 0.1887008 x SPINT2 value - 2,600843 x amount of serum DNA + 9.331859 combining the 3 markers gene BASP1, gene SPINT2 and amount of DNA in serum or by the formula: - 0 112025 x SRD5A2 value - 0.1999992 x SPINT2 value - 2.696542 x amount of serum DNA + 9.867470 combining the 3 markers SRD5A2 gene, SPINT2 gene and amount of serum DNA tended to correlate negatively with tumor size (r = - 0.235, p <0.005, r = - 0.244, p <0.005) (table 18).

Therefore, it is suggested that the determination of the amount of methylated DNA of the methylated liver cancer specific genes discovered in the present invention or the combination of the determination of said values with the amount of serum DNA are useful for the detection of tumor size and liver cancer progression.

20 Table 18: Clinical evaluation in the detection of liver cancer progression with a specific amount of methylated liver cancer gene with methylated DNA, or in combination with specific methylated liver cancer genes and an amount of serum DNA

N = 149

Tumor size Amount of serum DNA BASP1 SPINT2 SRD5A2 BASP + SPINT + DNA (logarithmic transformation) SRD5A + SPINT + DNA (logarithmic transformation)

Pearson correlation coefficient

1,000  0.121 0.216 -0.028 -0.036  -0,235 -0.244

Probability of tumor size (2 tails)

- 0 0.008 0.136 0.660  0.004 0.003

25 Industrial Applicability

The present invention is used to analyze and diagnose liver cancer, especially for carrying out liver cancer screening tests, the detection of precancerous lesions, the detection of the risk of

30 recurrence after liver cancer treatment, detection of liver cancer malignancy and monitoring of liver cancer progression over time, and can be used in clinical diagnostic industries, pharmaceutical reagents and medical instruments.

BRIEF DESCRIPTION OF THE DRAWINGS

35 Figure 1. Results of the evaluation of the diagnostic performance of the methylated genes specific to liver cancer by ROC analysis.

Figure 2. Results of the evaluation of the diagnostic performance of the methylated genes specific to liver cancer by FLC and ROC analysis: Liver cancer detection capacity (BASP1 + SPINT2 + CFTR, sensitivity: 70%, specificity: 80%, rate of recognition: 74%).

Figure 3. Results of the evaluation of the diagnostic performance of the specific methylated genes of liver cancer (early stage liver cancer: stage I and less than 3 cm) by FLC and ROC analysis: Ability to detect early stage liver cancer ( BASP1 + SPINT2 + CCND2, sensitivity: 52%, specificity: 86%, recognition rate: 78%).

Figure 4. Results of the evaluation of the diagnostic performance of the specific methylated genes of liver cancer (early stage liver cancer: well differentiated) by FLC and ROC analysis: 50 liver cancer detection capacity (BASP1 + SPINT2 + RASSF1, sensitivity: 67%, specificity: 80%, recognition rate: 77%) in early stage.

image30

Figure 5. Results of the clinical evaluation of the association between the determined values of the methylated genes specific to liver cancer and the amount of serum DNA and the survival rate (overall survival rate and disease-free survival rate) after liver cancer surgery; A: overall survival, p = 0.0289 using the “log-rank” test; B: disease-free survival, p 0.0372 using the log-rank test.

image31

SEQUENCE LIST

<110> F. HOFFMANN-LA ROCHE AG ROCHE DIAGNOSTICS GMBH

<120> METHOD FOR DETECTION OF HEPATIC CANCER, AND KIT AND REAGENT FOR THE SAME

<130> 24105 WO

<150> JP 2007-53312

<151> 02.03.2007

<150> JP 2008-021438

<151> 31.01.2008

<160> 39

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer BASP1_MSF

<400> 1 tgttcgtttt tttagggtat tc 22

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer BASP1_MSR

<400> 2 aattaaccga aacaacccg 19

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> SPINT2_MSF primer

<400> 3 tcggttattt tcgggagtc 19

<210> 4

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SPINT2_MSR

<400> 4 cgcctacgac actcaacga 19

<210> 5

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> APC_MSF primer

<400> 5 agtgcgggtc gggaagc 17

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221> <222>

image32

<223> APC_MSR primer

<400> 6 aaccacatat cgatcacgta cg 22

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer CCND2_MSF

<400> 7 tttgatttaa gtatgcgtta gagtacg 27

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer CCND2_MSR

<400> 8 actttctccc taaaaaccga ctacg 25

<210> 9

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> CFTR_MSF primer

<400> 9 aatcgggaaa gggaggtgc 19

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> CFTR_MSR primer

<400> 10 accgaatact acctaatccg 20

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer RASSF1_MSF

<400> 11 gcgttgaagt cggggttc 18

<210> 12

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer RASSF1_MSR

<400> 12 cccgtacttc gctaacttta aacg 24

<210> 13

<211> 21 <212> DNA

image33

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SRDSA2_MSF

<400> 13 aatcgcgtta gggttggacg c 21

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SRDSA2_MSR

<400> 14 aacgccaaac gccacccg 18

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer BASP1_F

<400> 15 tttgttaata ggtaagtaaa gtgaa 25

<210> 16

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer BASP1_R

<400> 16 ttaaaataaa ccccaactac aaac 24

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SPINT2_F

<400> 17 ggaagggtgg taggtgttta g 21

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SPINT2_R

<400> 18 tacctaaatc tactcctcac tc 22

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer APC_F <400> 19 ttgtttgttg gggattgggg t 21

image34

<210> 20

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer APC_R

<400> 20 tacaaaaaat ccatctccaa tact 24

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer CCND2_F

<400> 21 tttttggagt gaaatatatt aaagg 25

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer CCND2_R

<400> 22 cccctacatc tactaacaaa cc 22

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer CFTR_F

<400> 23 gaggaggagg aaggtaggtt 20

<210> 24

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer CFTR_R

<400> 24 ccaccccttc cttttactct t 21

<210> 25

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer RASSF1_F

<400> 25 tagtttggat tttgggggag g 21

<210> 26

<211> 21

<212> DNA

<213> Artificial sequence <220>

image35

<221>

<222>

<223> primer RASSF1_R

<400> 26 ctacccctta actacccctt c 21

<210> 27

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SRD5A2_F

<400> 27 taagttatgg aaggatagtt taag 24

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SRD5A2_R

<400> 28 aacaactcct acaaaaacca aac 23

<210> 29

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> SPINT2_HMF primer

<400> 29 gttttttgtt tgttyggtta tt 22

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> primer SPINT2_HMR

<400> 30 acctaaatct actcctcact 20

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> BASP1_TMP probe

<400> 31 acgctactac ttacgaacgc tcgaa 25

<210> 32

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> SPINT2_TMP probe

<400> 32 ccaacgcgcg aaaatcgcca aaa 23

image36

<210> 33

<211> 25

<212> DNA

<220>

<221>

<222>

<223> APC_TMP probe

<400> 33 aaaacgccct aatccgcatc caacg 25

<210> 34

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> CCND2_TMP probe

<400> 34 aatcgccgcc aacacgatcg acccta 26

<210> 35

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> CFTR_TMP probe

<400> 35 tccacccact acgcaccccc g 21

<210> 36

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> RASSF1_TMP probe

<400> 36 acaaacgcga accgaacgaa acca 24

<210> 37

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> probe SRD5A2_TMP

<400> 37 ctcgacctta actcccgccc ct 22

<210> 38

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<221>

<222>

<223> SPINT2_HMBF probe

<400> 38 ttggttattt ttgggagttg tt 22

<210> 39

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<221>

<222> <223> SPINT2_HMBR probe

image37

<400> 39 tcctcactca caccctcacc a 21

image38

Claims (14)

1. Method for detecting the presence and / or quantity of liver cancer specific methylated cytosines in a region containing CpG sequences in the BASP1 gene and optionally in the SRD5A2 gene comprising the following steps (a) to (d):

(to)

isolate genomic DNA from a patient sample;

(b)

contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines;

(C)

amplifying a region containing methylated cytosines of the BASP1 gene and optionally of the SRD5A2 gene in the genomic DNA using a PCR method; Y

(d)

determining the presence and / or quantity of liver cancer specific methylated cytosines in a region containing CpG sequences of the BASP1 gene and optionally of the SRD5A2 gene in the sample of said patient,

so that the presence and / or quantity of methylated cytosines specific to liver cancer indicate that the patient is affected by liver cancer, has an increased risk of liver cancer, presents a risk of recurrence after treatment of liver cancer, presents liver cancer malignant or has liver cancer that is progressing over time. 2. Method to indicate that a patient is affected by a liver cancer, has an increased risk of liver cancer, presents a risk of recurrence after liver cancer treatment, has a malignant liver cancer or has a liver cancer that is progressing in the time by detecting the presence and / or amount of liver cancer specific methylated cytosines in regions containing CpG sequences in at least the BASP1 gene and the SPINT2 gene, optionally in an additional gene between the SRD5A2, CFTR, CCND2, APC genes and RASSF1 in the sample of a patient, said method comprising the following steps (a) to (d):

(to)

isolate genomic DNA from a patient sample;

(b)

contacting a reagent for chemical or enzymatic treatment with genomic DNA to discriminate between methylated and unmethylated cytosines;

(C)

amplifying a region containing methylated cytosines of the BASP1 gene and the SPINT2 gene, optionally of an additional gene between the SRD5A2, CFTR, CCND2, APC and RASSF1 genes in the genomic DNA using a PCR method; Y

(d)

determine the presence and / or quantity of liver cancer-specific methylated cytosines in a region containing CpG sequences of the BASP1 gene and the SPINT2 gene, and optionally of an additional gene between the SRD5A2, CFTR, CCND2, APC and RASSF1 genes in the sample of the patient.

3.

Method according to claims 1 or 2, wherein at least one nucleotide sequence of a pair of PCR primers is complementary to a nucleotide sequence in a region containing cytosine residues that can be methylated from genomic DNA, said primer hybridizing specifically to said genomic DNA nucleotide sequence when said cytosines are methylated.

Four.

Method according to claims 1 or 2, wherein at least one primer of a pair of PCR primers that specifically hybridize to a region that does not contain cytosine residues that can be methylated and oligonucleotides are used to block amplification by said claims PCR primers, in which said oligonucleotides partially overlap the nucleotide sequences of said PCR primers, are complementary to nucleotide sequences in other parts of the genomic DNA in a region containing residues that can be methylated and in which said oligonucleotides they can specifically hybridize to said genomic DNA nucleotide sequences when said cytosines are not methylated.

5.

Method according to claims 1 or 2, wherein a pair of primers can specifically hybridize to genomic DNA nucleotide sequences that do not contain cytosine residues that can be methylated at at least their 3 'ends, and in which the determination of the presence and / or quantity of methylated cytosines in the products according to step (d) is performed by sequencing analysis or mass spectrometry.

6.

Method according to claims 1 or 2, wherein the chemical treatment according to step (b) is carried out by providing the genomic DNA with a reagent for the conversion of unmethylated cytosines to uracils, so that essentially all non-methylated cytosines of said DNA is converted to uracils.

7.

Method according to any one of claims 1 to 6, wherein the concentrations of conventional tumor marker proteins such as AFP and / or PIVKA-II and / or the amount of free circulating DNA are determined simultaneously in the sample.

8.

Method according to any one of claims 1 to 7, wherein the liver cancer is an early stage liver cancer.

9.

Method according to any one of claims 1 to 8, wherein the sample is derived from serum, plasma, whole blood, tissues, blood cells, excretions or human internal / external secretions.

10.

Pair of primers or group of primers comprising the following two primers: 1) BASP1_MSF (Sequence Identifier #: 1) and BASP1_MSR (Sequence Identifier No.: 2), and optionally at least one pair of primers selected from the following group of pairs from

image 1 primers: 2) SPINT2_MSF (sequence identifier #: 3) and SPINT2_MSR (sequence identifier #: 4), 3) APC_MSF (sequence identifier No.: 5) and APC_MSR (sequence identifier No.: 6), 4) CCND2_MSF ( sequence identifier no .: 7) and CCND2_MSR (sequence identifier no .: 8), 5) CFTR_MSF (sequence identifier no .: 9) and CFTR_MSR (sequence identifier no .: 10), 6) RASSF1_MSF (sequence identifier no .: 11 and RASSF1_MSR (sequence identifier #: 12), and 7) SRDSA2_MSF (sequence identifier #: 13) and SRD5A2_MSR (sequence identifier No.: 14).

eleven.

Probe or combination of probes consisting of: 1) BASP1_TMP (sequence identifier #: 31), and optionally at least one probe

selected from the following group of probes: 2) SPINT2_TMP (sequence identifier #: 32), 3) APC_TMP (sequence identifier #: 33), 4) CCND2_TMP (sequence identifier #: 34), 5) CFTR_TMP (sequence identifier #: 35), 6) RASSF1_TMP (sequence identifier #: 36), and 7) SRDSA2_TMP (sequence identifier #: 37).

12.

Kit for the method of detection of liver cancer, according to any of claims 1 to 9, consisting of:

(to)

a pair of primers or a combination of primer pairs formed by at least the sequence identifier #: 1 and the sequence identifier #: 2 according to claim 10 and / or

(b)

a probe or combination of probes formed at least by the sequence identifier no .: 31 according to claim 11, and

(C)

reagents for DNA extraction.

13.

Reagent for the method of detection of liver cancer, according to any of claims 1 to 9, formed by:

(to)

a pair of primers or a combination of primer pairs formed by at least the sequence identifier #: 1 and the sequence identifier #: 2, according to claim 10 and / or

(b)

a probe or combination of probes formed at least by the sequence identifier no .: 31 according to claim 11, and

14.

Use of a pair of primers or a group of pairs of primers formed by sequence identifier #: 1 and sequence identifier #: 2 or a probe or combination of probes formed by sequence identifier No.: 31, according to the claims 10 or 11, to detect the presence and / or amount of methylated cytosine bases specific to liver cancer in a region containing CpG sequences of a HCC-related gene in a sample of a patient, or to detect liver cancer, a risk increased liver cancer, a risk of recurrence after liver cancer treatment, a malignant liver cancer or to monitor that a patient is developing a liver cancer that is progressing over time.