RU2405837C1 - Early dna-diagnostic technique of prostate cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: blood serum is analysed for promoter region methylation of GSTP, p16 and N33 genes by methyl-sensitive PCR method. If observing abnormal methylation at least in two of said genes, the presence of a disease is concluded.

EFFECT: higher accuracy of early DNA-diagnosis of prostate cancer ensured by the use blood serum DNA to be analysed, technique exhibits higher specificity due to the correct gene specification in a test system and allows applying profit-proved detection methods.

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Description

The invention relates to medicine, in particular to oncology, and may find application in the diagnosis of prostate cancer (PCa).

One of the main genetic events necessary for the development of any type of tumor is the inactivation of tumor suppressor genes. Inactivation of these genes is an initiating event and is detected long before the appearance of atypical proteins used as biochemical markers of the tumor.

The most common mechanism of such inactivation is the abnormal methylation of CpG islands of the promoter and regulatory regions of these genes. Hypermethylation of genes involved in carcinogenesis is one of the earliest events of tumor formation and is often found in many precancerous conditions. However, such a mechanism of inactivation of tumor suppressor genes does not appear in acute and chronic inflammatory diseases. Hypermethylation of various suppressor genes can serve as a molecular marker for the early diagnosis, monitoring, and clinical prognosis of tumors.

There are several methods for analyzing abnormal methylation for diagnostic purposes, two of which are the most common.

1. Methyl-sensitive polymerase chain reaction (MCH-PCR) is based on the use of restriction endonucleases that are sensitive and insensitive to methylation of cytosine residues. For the analysis of methylation of specific specific sequences, methyl-sensitive restrictase can be used, the most effective are the often splitting HpaII and HhaI., Because regions of CpG islands have many recognition sites, which virtually eliminates the possibility of incomplete hydrolysis of DNA. If the DNA fragment does not contain modified bases, then the hydrolysis takes place completely, PCR does not occur and, accordingly, its product is not determined. At the same time, if methylcytosine is present in the restriction enzyme recognition site instead of cytosine, hydrolysis does not occur, and a fragment of a certain length is detected in the gel. The great advantage of the method is its high sensitivity, which allows one to analyze methylated alleles in the presence of a large excess of wild-type alleles (analytical sensitivity - 1 methylated sequence per 2000 unmethylated).

2. Methyl-specific polymerase chain reaction (MS-PCR) is a method for assessing the methylation status of individual CpG islands. The procedure consists in pretreating the tested DNA samples with sodium bisulfite, which under certain conditions leads to deamination of cytosine with the formation of uracil, while methylated cytosine residues remain unchanged. In subsequent PCR, uracil is replaced by thymine. Thus, it is possible to design primers that selectively amplify sequences containing or not containing methylated cytosine residues in CpG islands. The sensitivity of the method is: 1 methylated allele per 1000 unmethylated. The disadvantages include the not always efficiently performed DNA modification, problems of analysis with a small amount of DNA and a large investment of time in performing diagnostics.

To date, certified test systems and DNA diagnostic methods do not exist.

Several options for the determination of DNA markers in patients with precancerous and oncological diseases of the prostate gland are described.

The analysis of abnormal methylation of three genes (GSTP1, RASSF1A, RARβ2) involved in carcinogenesis of the prostate gland is described, a significant frequency of this phenomenon is shown. It is suggested that these genes can be used for early diagnosis and prognosis of prostate cancer (Perry AS, Foley R, Woodson K, Lawler M. The emerging roles of DNA methylation in the clinical management of prostate cancer. Endocr Relat Cancer. 2006 Jun; 13 (2): 357-77).

However, the authors do not analyze the methylation of other genes; they use the methylspecific polymerase reaction (MS-PCR) in their analysis. In addition, the analysis was carried out on tumor and biopsy material (which can lead to an underestimation of methylation frequencies if tumor cell microdissection is not used).

In another work, the authors attempted to collect various markers into the system (Papadopoulou E, Davilas E, Sotiriou V, Koliopanos A, Aggelakis F, Dardoufas K, Agnanti NJ, Karydas I, Nasioulas G. Cell-free DNA and RNA in plasma as a new molecular marker for prostate cancer. Oncol Res. 2004; 14 (9): 439-45). One of them is the total amount of free DNA in the blood plasma of patients with prostate cancer. Another marker is the methylation of the GSTP1 gene. The presence of free RNA of the CEA and PSMA genes, uncharacteristic for control individuals, is a molecular marker for patients with various stages of prostate cancer. The combination of an increased amount of free DNA and methylation of GSTP1 was determined in 83%, which is a fairly significant indicator.

The disadvantages of this method include the following facts:

1) the presence of free DNA is not a specific marker, because takes place at all stages of carcinogenesis and at various locations of the tumor;

2) used methyl-specific PCR to determine the methylation of GSTP;

3) free RNA of the CEA and PSMA genes can be a molecular marker of any stages of prostate cancer.

Another study carried out significant work on the quantification of methylation of a number of genes (GSTP1, MGMT, p14, p16, RASSF1A, APC, TIMP3, S100A2, CRBP1) in paired samples of carcinomas, intraepithelial neoplasias, and benign prostatic hyperplasias (Jeronimo C, R, Hoque MO, Mambo E, Ribeiro FR, Varzim G, Oliveira J, Teixeira MR, Lopes C, Sidransky D. A quantitative promoter methylation profile of prostate cancer. Clin Cancer Res. 2004 Dec 15; 10 (24): 8472- 8). It was found that methylation of the GSTP and APC genes in carcinomas was significantly different from hyperplasia and showed a high sensitivity of about 98.3%.

In the described diagnostic method for analysis, biopsy material is used, which reduces the accuracy of the diagnosis. In addition, the authors propose to determine methylation using the method of methyl-specific PCR, which complicates and increases the cost of diagnosis.

The objective of the invention is to improve the accuracy of early DNA diagnosis of prostate cancer due to the use of DNA from blood serum for analysis.

The problem is solved by the method of early DNA diagnosis of prostate cancer, which consists in the study of blood serum for methylation of the promoter regions of the GSTP, p16 and N33 genes by methyl-sensitive PCR and if abnormal methylation of at least two of these genes is detected, they conclude the presence of the disease.

The method is based on the pathogenetic factor of carcinogenesis - methylation of regulatory regions of genes involved in the malignant process, which leads to their complete inactivation without disrupting the DNA structure. In addition, an approach was used to determine these markers in the blood serum of patients, which is based on the well-established fact of an increase in the amount of free DNA in the blood serum of cancer patients containing DNA markers of the corresponding tumor.

The method is as follows.

A peripheral venous blood sample of 10 ml is taken from the patient. The whole blood sample is not frozen and stored for no more than 2 days at a temperature of + 4 ° C.

Isolation of genomic DNA from blood serum

To obtain DNA from blood serum, the following DNA isolation method is used:

1. Whole blood is centrifuged for 5 min at 3000 rpm, about 5 ml of serum in the upper phase is taken.

2. Serial DNA extraction is carried out sequentially in equal volumes of phenol, a mixture of phenol-chloroform and chloroform.

3. DNA is precipitated with 2.5 volumes of cold 96% ethanol, the sample is kept for 30 minutes at a temperature of -70 ° C.

4. The sample is centrifuged at 0 ° C for 15 min at 12000 rpm, the DNA residue is dried in air and dissolved in 50 μl of deionized sterile water or 1x TE buffer.

5. After complete dissolution of the DNA, its concentration is measured on a spectrofluorimeter or spectrophotometer and the absorption spectrum is taken in the range from 220 to 320 nm, in order to determine the purity of the DNA. The absorption maximum is observed at 260 nm.

Hydrolysis of DNA obtained from the sample with methyl sensitive restriction enzyme HpaII (CCGG).

To 2 μl of dissolved genomic DNA (100-200 ng / μl) add 5 units of act. HpaII restriction enzymes (CCGG) and 2 μl of 10X hydrolysis buffer for HpaII. To the final volume of the incubation mixture, 20 μl was adjusted with deionized or sterile water and incubated overnight at 37 ° C. After hydrolysis, the tube is stored in a freezer at -18-20 ° C.

Determination of abnormal methylation of CpG islands of promoter regions of p16, GSTP, and N33 genes

To determine the methylation of CpG islands of the promoter regions of the studied genes, the method of methyl-sensitive PCR (MCH-PCR) is used. The method is based on the ability of methyl-sensitive restrictase to hydrolyze DNA that does not contain modified bases and leave unhydrolyzed sections containing methylcytosine. As a matrix for the polymerase chain reaction, DNA obtained from the patient’s blood plasma, previously hydrolyzed with methyl sensitive restriction enzyme HpaII (CCGG), is used.

During PCR, the presence of internal recognition sites for HpaII in the amplified region is taken into account (for the P16 gene - 4 sites, N33 - 10 sites, GSTP - 4 recognition sites for the HpaII enzyme). In the event that cytosines are modified to methylcytosine (abnormal methylation), DNA hydrolysis does not occur, and the PCR product of a specific molecular weight corresponding to each gene can be detected in the gel. In the absence of abnormal methylation, DNA is completely hydrolyzed, and the PCR product is not detected. Specific primers per gene fragment are used as a control for the PCR reaction. XLISS, which does not contain recognition sites for the HpaII enzyme.

Multilocus PCR is performed for two pairs of primers (test gene and control). The following scheme is used: 0.05 μM of each oligoprimer, 200 μM of each deoxynucleotide triphosphate, 1-2 units of Taq polymerase, 50 μl of a single PCR buffer (50 mM KCl, 10 mM) are added to 2 μl of hydrolyzed DNA (50-200 ng) Tris-HCl pH 8.4, 5 mM MgCl ₂ , 10% DMSO). 2 drops of liquid paraffin are added to the resulting mixture, the mixture is heated at 95 ° C for 10 minutes and 33 cycles are carried out with the following parameters: denaturation 95 ° C for 30 seconds, annealing and elongation 58-62 ° C for 2 minutes 30 seconds. The nucleotide sequence of the primers and annealing modes are presented in the table. After the end of the PCR reaction, the product is stored at 4 ° C in the refrigerator.

The sequence of primers for amplification of regions of the promoter regions of genes Gene Primer sequence Annealing temperature, ° С Fragment Length P16 F: gaa cgc act saa asa cgc 60 352 bp R: agc cag ccc with ctc ttt ctt s Gstp1 F: acc tgg gaa aga ggg aaa ggc tt 60 609 bp R: gaa gac tgc ggc ggc gaa act N33 F: tgg gtt cag agc agg atg gc 60 336 bp R: gca gga gaa gga agg gaa agc XL3.2 control F: ttg gct agc agc aac agt gc 60 180 bp R: agt ttg atg gct tct gtg at

Separation of PCR products in an 8% polyacrylamide gel by vertical electrophoresis

The separation of PCR products by molecular weight in the methylation analysis is carried out using vertical electrophoresis in 8% PAG with the following composition: 8.4 ml of a 30% solution of acrylamide, 0.6 ml of 10% ammonium persulfate, 26 μl of TEMED, up to 30 ml of diluted buffer TBE. Ammonium persulfate and TEMED are added after all other components and the resulting mixture is thoroughly mixed. Electrophoresis is carried out at a voltage of 400 V using a camera for vertical electrophoresis (Helikon, Moscow) for 2.0-3.0 hours.

As a control of the molecular weight of DNA, DNA of standard molecular weight pUC19 / Msp1 is used. Visual control of the mileage of DNA samples is carried out on xylencyanol and bromphenol blue. Gel staining with silver nitrate to visualize PCR products is carried out according to the following procedure.

Ultrafine silver nitrate staining.

Gel staining is carried out according to an advanced Liberman technique. After electrophoresis, the gel is incubated in a solution of 0.011 M AgNO ₃ for 10-15 minutes, then washed three times with distilled water. The manifestation is carried out in a modified developer solution (increased concentration of HCNO) of the following composition: 0.75 M NaOH; 0.5 M HCNO; 2.3 mM Na (BH ₄ ) for about 10-15 minutes, depending on the intensity of the gel.

Optimal criteria for the interpretation of research results

If abnormal methylation of promoters of the N33, p16, and GSTP1 genes is present in tumor DNA, then the cytosines in the composition of CpG dinucleotides are replaced by 5-methylcytosines, including in HpaII recognition sites. In this case, the restriction enzyme HpaII is not able to hydrolyze the genomic DNA at recognition sites, the template for PM-PCR remains intact. As a result, fragments corresponding to the promoter regions of N33, p16, and GSTP1 are visible in stained PAG.

In those samples where there is no gene methylation, genomic DNA is hydrolyzed at HpaII recognition sites. At the first denaturation, the PCR matrix is destroyed and the PCR product corresponding to the gene promoter is absent in the PAGE. Internal control MCH-PCR that does not contain HpaII recognition sites should be present in all samples analyzed. The negative control should not contain PCR products; the positive control should contain PCR products of internal control and promoters of the studied genes.

In case of abnormal methylation of the promoter regions of the N33, p16 and GSTP1 genes, both all at once and in paired combinations in DNA samples from the blood serum of patients, a high probability of a malignant process in the prostate gland is found, and in the case of cancer already diagnosed, the diagnosis .

The effectiveness of the proposed diagnostic method

In patients with prostate cancer, we found a high percentage of methylation of malignant epithelium. High methylation frequencies were found for adenocarcinoma: p16 - 50%, GSTP1 - 93%, N33 - 27%. In the glands of prostatic intraepithelial neoplasia (IDU) of a high degree in patients with prostate cancer, the methylation frequencies of the studied genes are close to those in the glands of adenocarcinoma: p16 - 53%; GSTP1 - 73%; N33 - 27%. Molecular genetic changes in IDUs are consistent with those in early invasive cancer, although less pronounced.

Methylation of the p16, N33, and GSTP1 genes was analyzed in 102 biopsy samples of pancreatic tissue. Pancreatic tissue samples were conditionally divided according to the presence of a certain histological picture in the patient. So patients with at least one of the 12 biopsy fragments of an adenocarcinoma belonged to the group of prostate cancer. Patients with one or more IDU sites in any of the 12 biopsy fragments were in the IDU group, a similar principle was used for patients with benign prostatic hyperplasia (BPH).

Hypermethylation of the promoter region of the p16 gene was found in 18% of cases of hyperplasia, 48% of IDUs and 42% of adenocarcinomas. A similar frequency of methylation of the GSTP1 gene was found for adenocarcinoma (41%). However, if the frequency of methylation of the p16 gene for IDUs is as high as for an adenocarcinoma, a significant difference was found between IDUs and adenocarcinoma for GSTP1 (p = 0.007). In precancerous states, methylation of the N33 gene was not detected, while the frequency of methylation of this gene in an adenocarcinoma was 22% (p = 0.05).

To study the possibility of non-invasive diagnosis, methylation of the P16, N33, and GSTP genes was analyzed in 112 plasma samples of patients with prostate cancer and patients with BPH in order to assess the diagnostic potential of the selected epigenetic markers. Blood samples were obtained from patients with prostate cancer before performing a prostatectomy.

No methylation of the GSTP1 gene was detected in patients with BPH, while for patients with adenocarcinoma, the frequency of methylation of the GSTP1 gene in plasma was (28%) (p = 0.05). In the plasma of patients with precancerous conditions, methylation of the P16 gene was not detected, while the frequency of methylation of this gene in the plasma of patients with adenocarcinoma was 19%. The methylation frequency for the N33 gene was 30%.

The effectiveness of any biological marker is determined by the parameters of sensitivity and specificity. These criteria are very important in assessing the diagnostic significance of markers for population screening of a given disease or for clinical observation of an increased risk group. To assess the potential significance of the selected methylation markers (p16, GSTP, and N33 genes), the sensitivity and specificity of these markers were calculated. It was shown that GSTP has a sensitivity of 36%, P16 - 25%, and N33 - 27%, at the same time, all markers have 100% specificity. Thus, the molecular marker system, consisting of three genes, has a sensitivity of 75% and a specificity of 100%.

Since the system of DNA markers is technologically open, there is a real possibility of increasing its sensitivity by adding another 2-3 markers to it.

The following example illustrates the method of the invention.

In a study using traditional instrumental and biochemical diagnostic methods, a 52-year-old patient was diagnosed with benign prostatic hyperplasia. Using a system of molecular DNA markers in blood serum, methylation of the GSTP gene was determined in a patient. In a detailed study using a 12-needle biopsy, a high-grade IDU / carcinoma in situ was detected in one of the tissue columns. In this sample, in addition to the GSTP gene, methylation of the N33 gene was detected, which allowed timely treatment of the patient.

The method according to the invention improves the accuracy of early DNA diagnosis of prostate cancer through the use of DNA from blood serum for analysis. The proposed method has a higher specificity due to the correct selection of genes in the test system, and also allows the use of economical detection methods.

Claims (1)

A method for early DNA diagnosis of prostate cancer, which consists in examining blood serum for methylation of the promoter regions of the GSTP, p16 and N33 genes by methyl-sensitive PCR and, when abnormal methylation is detected, at least two of these genes conclude that the disease is present.