RU2340896C1 - Method of diagnostics of oncologic diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, namely to ways of diagnostics of oncologic diseases. The method provides universality of diagnostics of oncologic diseases of various organs of the person. Perform allocation from the sample taken from the patient, the general RNA, obtaining of cDNA and diagnosing of oncologic disease on presence of a fragment of sequence of mRNA gene HHLA1, shown on fig.1. Perform the analysis of mRNA, in particular, by it amplification with the help polymerase chain reaction at presence of primers, specific to a sequence fragment of mRNA gene HHLA1, shown on fig.1. For amplification use a straight primer with sequence 5'-TTATGCCCAAAGTGTCAAGC-3' and a return primer with sequence 5'-TGTTCAGGGTCTCCTCTGTTTC-3'.

EFFECT: maintenance of universality of diagnostics of oncologic diseases of various organs of the person.

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Description

The invention relates to medicine, namely to methods for diagnosing cancer.

Currently, the number of patients with malignant neoplasms of various organs and tissues is growing in the world. This situation is associated with a number of reasons: an increase in the general radiation background of the Earth, a deterioration of the ecological situation and the endoecological environment, as well as many others, which cause a decrease in the body's immunity and increase the ability of transformed cells to progressive growth.

The success of treatment of cancer patients largely depends on the stage at which the disease is detected. However, it is known that it is extremely difficult to identify patients in the early stages of the development of cancer, because, as a rule, the oncology patients see a doctor due to the presence of complaints (pain, bleeding, deformation of the tissue or organ, etc.) that indicate about the already developed clinical picture of the disease. In this regard, one of the main problems of clinical oncology is the diagnosis of malignant tumors in the initial stage of their development, when existing methods of treatment are most effective.

Modern oncological science has a number of methods for screening diagnosis of malignant neoplasms, which make it possible to judge the presence or absence of a tumor at the time of examination. In particular, prophylactic mammography has been widely used for the early diagnosis of breast cancer in the female population (A.G. Holleb. Review of Breast Cancer Screening Guidelines. Cancer Supplement, 1992; 69, pp. 1911-1912). Also known is a method of cytological screening for malignant tumors of the cervix uteri (Guzick DS Efficacy of screening for cervical cancer. A review. Am. J. Public Heath, 1978; 68, pp. 125-134) and a method for diagnosing lung cancer by determining the concentration of nonadecanoic acid in excretion products (RU 2088926, 1997).

However, the use of most of the known methods of screening diagnostics is aimed at identifying a specific type of tumor of a specific organ that cannot be used in the general diagnosis of oncology of body tissues.

A known method for the diagnosis of malignant neoplasms (RU 98112260, 2000), including determining the time of longitudinal nuclear magnetic resonance relaxation in the blood serum, quantifying homeostasis disorders according to biophysical, hematological and biochemical analyzes, which are used to judge the presence of malignant neoplasms.

The disadvantage of this method is its technical complexity, the inability to use in the clinical laboratories of hospitals, the difficulty of identifying an early stage of malignant growth.

A known method for the diagnosis of malignant neoplasms by determining cancer-embryonic antigen (CEA) by radioimmunoassay (SU 76333, 1960). However, this method also has low specificity, in particular, an increase in the titer of this antigen can occur in smokers, with inflammatory diseases and benign neoplasms.

A known method for detecting structural heterogeneities in biological tissues using liquid crystals (RF patent No. 2166194, 2001), which proposes to cut a tissue section with a solution of a liquid nematic crystal and, if it is not decorated with tissue sections, diagnose the presence of a malignant tumor.

The disadvantage of this method is its high invasiveness, the ability to detect disease only at the clinical stage of development.

Currently, it is assumed that the most promising for the general diagnosis of cancer are methods based on the determination of tumor markers in biological fluids of the body, in particular in the blood. In particular, there is a known method for the diagnosis of human malignant tumors, based on the detection of proteins specific for malignant growth in the blood of the patient (V. Korotorkuchko. Precipitation of cancer in the diagnosis of tumor disease. Kiev, Naukova Dumka. 1967). To identify them, the blood serum is kept in a solution of hydrochloric acid, after which the protein is precipitated with nitric acid and distilled water is added to it. The resulting precipitate of serum proteins of cancer patients, unlike healthy people, did not dissolve. The result of the reaction, called the sedimentary response to cancer (ORP), is fixed by visual indicator. This method is quite universal, however, it does not have sufficient specificity, because ORP is often positive for inflammatory and other non-tumor diseases.

-интерлейкина 2 (RU 2144675, 2000), полинуклеотиды (RU 2174409, 2001) и некоторые другие вещества. В частности, перспективными являются методы диагностики онкологических заболеваний по присутствию в биологических жидкостях организма внеклеточных нуклеиновых кислот, специфических для клеток злокачественных новообразований (Y.M.Dennis Lo and Rossa W.K. Chiu. The biology and diagnostic application of plasma RNA // Ann. N.Y. Acad. Sci., 2004, 1022: 135-139), или базирующиеся на обнаружении раковых клеток, экспрессирующих специфический ген (A.M.Gilbey, D.Burnett, R.E.Coleman, I.Holen. The detection of circulating breast cancer cells in blood // J. Clin. Pathol. 2004; 57: 903-911). В частности, в качестве одного из вариантов предлагается (RU 2251696, 2005) определять концентрацию нуклеиновых кислот (НК), связанных с клеточной поверхностью форменных элементов крови, и на основе данного показателя диагностировать наличие или отсутствие онкологического заболевания.The receptor was also used as a tumor marker for the diagnosis of cancer.

-interleukin 2 (RU 2144675, 2000), polynucleotides (RU 2174409, 2001) and some other substances. In particular, methods for diagnosing cancer by the presence of extracellular nucleic acids specific for malignant neoplasm cells (YM Dennis Lo and Rossa WK Chiu. The biology and diagnostic application of plasma RNA // Ann. NY Acad. Sci., 2004, 1022: 135-139), or based on the detection of cancer cells expressing a specific gene (AMGilbey, D. Burnett, REColeman, I. Holen. The detection of circulating breast cancer cells in blood // J. Clin. Pathol. 2004 ; 57: 903-911). In particular, as one of the options it is proposed (RU 2251696, 2005) to determine the concentration of nucleic acids (NK) associated with the cell surface of blood cells, and on the basis of this indicator to diagnose the presence or absence of cancer.

The closest in technical essence to the claimed method is a method for the diagnosis of gastric cancer (RU 2204835, 2005), which consists in examining a patient’s tissue sample containing cells of a problem zone in order to determine cyclooxygenase-2 or protein COX-2 in the mRNA sample. For this, total RNA was isolated using RNAzol 1 reagent (Tel-Test). The RNA sample was denatured and transferred to nylon membranes, which were then irradiated with UV. Fragments of purified cDNA cyclooxygenase-1 (COX-1), COX-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were labeled, and then purified on nick columns. Total RNA was converted to cDNA on a Superscript II instrument using oligo-dT and random hexamers (Life Technology). cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using specific COX-2 primers in the presence of DNA polymerase. Amplified cDNA was stained with ethidium bromide and quantified by electrophoresis on a 2% agarose gel using a high-resolution CCD camera. The results were compared with data obtained in a similar study of healthy tissues and obviously cancer cells, and on the basis of such a comparison, the presence or absence of cancer was diagnosed.

The disadvantage of this method is the limited scope due to the lack of reliable data on the presence of COX in other tissues of the body.

The technical problem solved by the authors was the creation of a more universal method for the diagnosis of cancer, suitable for detecting cancer of various organs of the human body.

The technical result was achieved as a result of the discovery by the authors during research in tumor tissues of fragments of the HHLA1 gene that are absent in healthy tissues of the human body. This made it possible to create a method for the diagnosis of cancer, including the analysis of tissues of the human body and the diagnosis of cancer when the HHLA1 gene is detected in mRNA tissues. The mRNA sequence of the HHLA1 gene is shown in figure 1.

HHLA1 gene mRNA can be detected using traditional mRNA analysis methods, such as dot hybridization, reverse transcriptase polymerase chain reaction (RT-PCR), etc. The best results were achieved during detection by RT-PCR. As part of its implementation, the patient’s cDNA obtained by known methods was amplified in the presence of primers specific for the HHLA1 gene mRNA fragment. In this case, an oligonucleotide with the sequence: 5'-TTATGCCCAAAGTGTCAAGC-3 'was used as a direct primer, and 5'-TGTTCAGGGTCTCCTCTGTTTC-3' as the opposite.

The results showed that the inventive method has a greater breadth of application and allows to obtain sufficiently reliable and selective results, which is illustrated by the following examples.

Example 1. Detection of mRNA of the HHLA1 gene by RT-PCR. Diagnosis was as follows. Isolation of total RNA from tumor tissues was performed according to the Chirgwin method (Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease // Biochemistry. 1979 Nov. 27; 18 (24): 5294-9 ) For this, 1 g of tissue frozen in liquid nitrogen was ground in a ceramic mortar with liquid nitrogen, 10 ml of a solution containing 5.5 M GuSCN, 50 mM sodium acetate, 50 mM ethylenediaminetetraacetate (EDTA), 0.5% lauryl sarcosylate, 0 were added to it. , 1M beta-mercaptoethanol (calculated as 10 ml per 1 g of tissue). The mixture was left at room temperature until completely thawed, after which it was transferred to 15 ml plastic tubes and centrifuged in an MLW K23 centrifuge for 13 minutes at a speed of 12,000 rpm. The supernatant was then transferred to clean tubes and mixed with 2.6 ml of a solution containing 5.7 M CsCl, 25 mM sodium acetate, 50 mM EDTA.

The resulting mixture was layered on 4 ml of the same solution in a test tube for the SW41 Ti rotor, after which it was centrifuged for 16 hours at a temperature of 20 ° C and 28,000 rpm. The supernatant was drained, the samples were washed with 70% ethanol and dried at -20 ° C, after which the precipitate was dissolved in water treated with dimethyl pyrocarbonate (WPC). The concentration and purity of the isolated RNA was determined using an Ultrospec® 3100 pro spectrophotometer. RNA purity was considered suitable for operation at a ratio of A ₂₆₀ / A ₂₈₀ ≥1.7 (aqueous solution). Next, RNA quality analysis was performed by gel electrophoresis, according to the ratio of 28s and 18s rRNA in 1% agarose gel. Before electrophoresis, sodium dodecyl sulfate (SDS) was added to RNA preparations containing 1-2 μg of total RNA (in a volume of 5-10 μl) to a concentration of 0.3%. After that, RNA samples were denatured at 65 ° С for 10 minutes, quickly cooled on ice, 0.1 volume of the sample volume of 10-fold buffer solution containing 50% glycerol and 0.4% bromophenol blue was added, and applied to the gel.

Electrophoresis was carried out in horizontal devices in TAE buffer (40 mM Tris-acetate; 2 mM EDTA, pH 8.0; 0.5 μg / ml ethidium bromide) at a voltage of 5-14 V / cm of gel length. The result of RNA separation was recorded in transmitted ultraviolet light of the transilluminator (Macrovue, LKB, Sweden).

CDNA synthesis was performed using the Revert Aid® First Strand cDNA Synthesis Kit (Fermentas). For this, 5 μg of total RNA and 0.2 μg of hexanucleotide primers were mixed on ice and the mixture volume was adjusted to 12 μl with water. To denature RNA, the mixture was incubated for 5 minutes at 70 ° C and transferred to ice. Then added 4 μl of 5-fold buffer, 1 μl of ribonuclease inhibitor, 2 μl of a mixture of 10 mm dNTP. After this, the mixture was incubated at 25 ° C for 5 minutes and 1 μl of Revert Aid® M-MuLV reverse transcriptase was added. The volume of the resulting mixture was 20 μl. The reaction mixture was incubated for 10 minutes at 25 ° C, and then 60 minutes at 42 ° C. Then the reaction was stopped by heating to 70 ° C for 10 minutes and put on ice. The resulting cDNA was stored at −20 ° C. until use.

To identify independent HHLA1 transcripts in human tissues, a series of PCR experiments was performed on cDNA panels from various normal and tumor tissues.

The polymerase chain reaction (PCR) was carried out in a solution containing 2.5 μl of cDNA, 1-time PCR buffer, MgCl ₂ (4 mM), dNTP (200 μM each), 0.4 μM of each primer and 1 Taq DNA unit polymerase in a total volume of 25 μl under the following conditions: 1 min at 95 ° C, 35 cycles, including 30 s at 95 ° C, 30 s at 52 ° C and 1 min at 72 ° C. At the end, the fragments were completed for 5 min at 72 ° C. PCR products were separated by electrophoresis in 2% agarose and stained with ethidium bromide. The presence of the desired mRNA in the sample was determined by the presence of an amplification product with a size of 227 bp

To check the quality of cDNA panels, PCR was performed using primers for the G3PDH gene (Direct 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3 ', reverse 5'-CATGTGGGCCATGAGGTCCACCAC-3', Tm 68 ° C, 30 cycles, fragment amplifiable 9). 9 fragment.

Figure 2 shows the results of PCR with primers specific for the HHLA1 gene on a cDNA panel of normal tissues (Human MTS Panel I, Clontech). Figure 3 shows the results of PCR with primers specific for the HHLA1 gene on a cDNA panel of normal tissues (Human MTS Panel II, Clontech). Figure 4 shows the results of PCR with primers specific for the HHLA1 gene on the cDNA panel of embryonic tissues (Human Fetal MTS Panel, Clontech). Figure 5 shows the results of PCR with primers specific for the HHLA 1 gene on the cDNA panel of tissues of the immune system (Human Immune System MTC Panel, Clontech). Figure 6 shows the results of PCR with primers specific for the HHLA1 gene on the cDNA panel of tumor tissues (BioChain). Ha 7 shows the results of PCR with primers specific for the HHLA1 gene on a cDNA panel from clinical samples of tumor tissues (Biomedical Center).

When stating PCR on a set of normal human tissues, Human MTS1 Panel HHLA1-specific fragment was not formed in any sample (figure 2). Amplification of the HHLA1-specific fragment was not observed in any sample from the Human MTS II Panel kit (Fig. 3) and embryonic tissue panel (Fig. 4).

In contrast, analysis of cDNA samples from tumors showed the presence of HHLA1 mRNA in samples of malignant tumors of various origins. On the cDNA panel of tumor tissues manufactured by Biochain (USA), signals are observed on tracks corresponding to tumors of the esophagus, stomach, large intestine, bladder, uterus, brain, breast, lung, and kidney (Fig. 6). HHLA1 gene expression was also detected in all clinical tumor samples (Fig. 7).

Example 2. Detection of mRNA of the HHLA1 gene by dot hybridization. A double-stranded cDNA fragment of the HHLA1 gene obtained by RT-PCR was used as a sample. Labeling of the probe for hybridization with radioactive phosphorus [α- ³² P] was performed using the HexaLabel ™ DNA Labeling Kit (Fermentas, Lithuania) according to the manufacturer's protocol. The resulting probe was stored at -70 ° C until use, but not more than 3 days.

Isolation and quality assessment of RNA was performed using the technology described in example 1. To transfer RNA to the membrane, 5 μg of each RNA sample diluted in 10 μl of water was taken and mixed with 30 μl of denaturing solution (660 μl of formamide, 210 μl of formaldehyde, 130 μl of 10-fold electrophoresis buffer (MOPS pH = 7.0), then the mixture was incubated at 65 ° C for 5 minutes and cooled on ice. Next, an equal volume of a 20-fold SSC solution (3 M NaCl, 0.3 M sodium citrate) was added to the mixture, and RNA was spotted onto the nylon membrane moistened in a 10-fold SSC solution using a vacuum transfer device. After that, all points were washed twice with a 10-fold SSC solution and RNA was fixed on the membrane, irradiating it with UV rays on a transilluminator for 3 minutes. The membrane was placed for 2 hours in 15 ml prehybridization solution (0.5 M sodium phosphate pH = 7.2, 7% SDS, 1 mm EDTA pH = 7.0) at a temperature of 68 ° C. Immediately before use, the hybridization probe was denatured by boiling for 10 minutes and subsequent rapid cooling on ice. The denatured probe was added to the prehybridization solution and incubated for 14 hours at 68 ° C. Then, the following washes were performed: once in 150 ml of the first washing solution (1x SSC and 1% SDS) for 10 minutes at room temperature and 3 times in 150 ml of the second washing solution (0.5x SSC, 0.1 % SDS) for 10 minutes at 68 ° C. Next, the membrane was dried and exposed with an x-ray film for 48 hours at -70 ° C, after which the film was developed according to the standard procedure. According to the results of hybridization, the signal strength was visually analyzed at points corresponding to healthy tissues and at points corresponding to tumor tissues.

The clinical samples of the tumors described in Example 1 were taken for analysis. During their study, hybridization signals were detected in samples 7 (testicular cancer), 14 (lung cancer), 17 (bronchial cancer), 21 (breast cancer), 31, 92 ( lymphomas), 88, 108 (stomach cancer).

Comparison of the results of the detection of HHLA1 mRNA by both methods showed their similarity, however, this method is less sensitive and longer.

The above experimental data indicate that the inventive method can be used with a high degree of reliability to detect cancer of various organs.

Claims (3)

1. A method for the diagnosis of cancer, which includes examining a sample taken from a patient to identify an oncological marker, and diagnosing an oncological disease, characterized in that the tissue is examined for the presence of HHLA1 mRNA in it, the sequence of which is shown in FIG. 1, and they diagnose a high probability of cancer when it is detected. 2. The method for the diagnosis of cancer according to claim 1, characterized in that when examining a sample taken from a patient, total RNA is isolated, cDNA is obtained and amplified by polymerase chain reaction with primers specific for the fragment of the nucleotide sequence of the HHLA1 gene indicated on figure 1, followed by analysis of the amplified product. 3. The method according to claim 2, characterized in that the amplification is carried out using a forward primer with the sequence 5'-TTATGCCCAAAGTGTCAAGC-3 'and a reverse primer with the sequence 5'-TGTTCAGGGTCTCCTCTGTTTC-3'.

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