EA010571B1 - Method for diagnosis of non-small cell carcinoma of lung and a kit therefor - Google Patents

Abstract

The invention relates to medicine, in particular to oncology and molecular biology and discloses the use of transcriptional level of a TIMP3 gene in the form of a diagnosis marker for a non-small cell carcinoma of lung. The decreased transcriptional level in a human tissue which is invaded by cancer for a long time in comparison with the level thereof in a sound tissue is used in the form of a diagnosis character of a non-small cell carcinoma of lung. Said invention makes it possible to diagnosticate with a very high degree of confidence a non-small cell carcinoma of lung, including a squamous cell carcinoma, at a very early stage of the progression of a neoplastic aberration.

Description

The present invention relates to medicine, in particular oncology, and molecular biology and can be used for early diagnosis of non-small cell lung cancer, including squamous cell cancer.

State of the art

Lung cancer remains one of the leading causes of cancer death in the world. Mortality from lung cancer exceeds mortality from cancer of the breast, colon and prostate combined (1esh1 A., 8tede1 K., \ Uagy E., Miggau T., Chi 1., 8t1da1 S., Tyip M.1. 2006 Sapseg zSZzysysz, 2006. SA Sapseg 1. S1sh. 56, 6-30). In Russia, the mortality rate from lung cancer in men is 68.2 cases per 100 thousand of the population, in women - 6.8 cases (Zaridze DG 2004. Epidemiology and etiology of malignant neoplasms, pp. 29-85 - in the book Carcinogenesis / Edited by D.G. Zaridze, Moscow: Medicine). There are two main types of lung cancer (RL): small cell (MRL) and non-small cell (NSCLC), comprising 15-20 and 75-80%, respectively. NSCLC includes squamous cell lung cancer (PRL), lung adenocarcinoma (AKL), and large cell cancer. Among the various forms of lung cancer, in terms of the frequency of occurrence of PRL, it ranks first, and AKL is second.

The diagnosis in half of the cases of NSCLC is made at a far advanced stage of the tumor process, which makes radical cure unrealistic. Modern treatment methods increase the average five-year life expectancy of patients with NSCLC by no more than 15%. This indicator can be significantly improved only through the development of early diagnostic methods.

The main methods for diagnosing lung cancer are instrumental - x-ray and endoscopic. Sputum analysis for atypical cells, lung tissue biopsy, computed tomography, and fluorescence fibroscopy are also used. Immunological diagnostic methods have so far limited clinical use. The definition of tumor markers is of practical importance: CEA - cancer-embryonic antigen - tumor marker of colon cancer, but can be used in the evaluation of lung cancer; Ν8Ε - neuron-specific enolase - in some cases used in assessing the status of patients with lung cancer; tissue polypeptide antigen (TPA) is a fragment of cytokeratin, a more specific marker of lung cancer. These markers are used to control treatment, to identify relapses, but not to diagnose early stage RL. For the diagnosis of NSCLC, 8CC markers are widely used - squamous cell carcinoma antigen and a fragment of cytokeratin-19 (MURDLE 21.1) (Razlog A., Mepepyekh K., Sheshayez ML., Razlog V., Lloporz K., Ahpag 1. 1997. Ehadpozys aa1ie o £ £ 8CC, CEA apy SURGUE 21.1 w 1ipd sapseg: a Vauez1ap apauz1z.Eig Kezri 1. 10, 603-609; Wissyep S., TogsYo R., Retppo Ό. 2003. Syssa1 Es. | Schuepse o Т T \ s Suyukegaig Magk No. 11 Se11 yppd Sapseg. And 81iu o Т Tzzie Po1urerye Apdep apy Su1okega Р 19 Rgadshez Sziez1. 124, 622-632), but they are also unsuitable for the early diagnosis of RL.

Diagnostic signs of malignant transformation of cells can also be changes in the genome in tumor cells — point mutations in the coding and regulatory regions of genes, microsatellite instability, allelic losses, changes in the level of transcription or translation of genes, methylation of promoter regions of the gene, etc. In contrast to protein, molecular genetic markers are significantly more sensitive. Lung cancer is accompanied by a change in the functional activity of many genes. Among the promising potential marker genes involved in the carcinogenesis of various forms of NSCLC, the T1MP3 gene is considered (Keiipep E., ApS1a 8., 8erpep 1.K., Kada1ashep A., Eidgep N., Lzhyos I., 8a1ouaaga K., Mzzep A .M., 8a1o 1., Mayzop K., No11tep 1., Kpiiy1a 8., \ U | ktap N. 2004. E | Pseptsa11u exprzepsez dep pop popsta11 se11 1ipd sapseg: exprzzzup rgoGShpd o £ sapseg-gekIey depes t ztspatopz se11 1 sapseg sapseg. Sepe1. SuYudepek 149, 98-106; Eattapp K., 81gipshkoua M., 8syadyagzigeptd I., Kaz1eyeg M., Rargykh M., Nayepyogz! I.E., Nogtapp H. 8., 8 .E., Vigyasy 8., Napzep S. 2005. CpS 1z1apy te 1yuyayp apy exprzzyup oG 1itopg-azzos1a1 depoz sh 1ipd sagstota. Eig. 1. Sapseg. 41, 1223-1236).

Tissue inhibitor of metalloproteinase 3 - T1MP3 - is involved in intercellular interactions and the induction of apoptosis, can inhibit the growth of many tumors, including NSCLC, angiogenesis, germination and metastasis (Egupya A., Opach R.N., No. shapp M., yai ya baap .N. Rar S., Eguiyah 8., Mesheske I., Kekote 1., Neitai ^., Nshkhtda T. ^., No-headquarters M., Koshd ^., Rar T. 2005. Cepé 1gapzGeg oG yzie 1pyyog o £ te1a11orgo1tasez-3 geujerz 1e bhjyogu eGGes1z o Т TM-a1rya op Raz-shyisey aor! z1z t gyeita1o1y aygShz zupou1a1 biblaz! z. 1. Gene inactivation was observed in cancers of the cervix, colon and rectum, prostate gland, and also with NSCLC, including PRL (Vgaesk1 ^ .M., Prepotasya 1., \ Uesh A., Kiskey 8., Roghpeg M., E | e1ta1eg ^., Kitte1e R., Sgopeg K.8., Vohegdeg R., Ksypeg T., Neuepiegdeg ^., Naip E.S., 1ppd A. 2005. A11ega (1opp t 1ie yzie 1py1yogo o £ te1a11orgo1etase-3 (T1MP1 -3) age Goipy Ggeschepuyu yitap soges! A1 Stoigz Ylzr1au1pd nyyeg Sh1sgoza1e11ye z! Ayyu (M88) og 1nz1a1N (y (M81). Sapseg Lei. 223, 137-42).

The T1MP3 gene is considered a potential tumor suppressor gene. The loss of activity of such genes occurs as a result of point mutations, allelic deletions, homozygous deletions or hypermethylation of CpC islets of genes, especially in the promoter regions (Weipzku 8.A., K1shde E.M., Eekkeg Ι.Ό., 8shy M. ^. , Vosk1ade T.1., Szhyapy Ρ.Ό., Sgo \\ 'e11 K.E., Kagr Ό.Ό., 8y1eu S.A., Ryusy M.A.

- 1 010571

2005. Sepa rgoto! E te! Yu1ayop ίη p1azta apb zhSt shsgeazez \ νί11ι 1ipd sapseg pzk. C1t. Sapseg Kez. 11, 6505-6511). For the T1MP3 gene, using mainly immunohistochemical staining and immunoblotting methods, a change in the amount of the corresponding protein was detected in NSCLC (Keiipep e! A1., 2004, zirga). It should be noted that the transcriptional activity of this gene in normal tumors remains poorly understood, although from a diagnostic point of view, the high stability of the resulting transcript plays a significant role in choosing a candidate for the role of a diagnostic marker.

Loss of transcriptional activity in NSCLC is indicated for several genes. In the work (TegaisP K., 8 Ytaba 1., Ieka ^ a Ν., Uao1 T., Magiuata Μ., RizKi1 8. 2006. Sapseg-azzoaaeb 1ozz oG TAK8N depez exezzyup sh Yitap rptagu 1ipd sazeg. 1. Sapsez Kez. Opso1. 132, 28-34) revealed a decrease in transcription of the TAK8H gene, one of the genes associated with cell aging, in tumor cell lines and NSCLC samples compared to the norm. Based on the results, the authors suggested that this gene could be used as a biomarker for the diagnosis of NSCLC. However, in this work, only 8 samples of PRL, the most common form of radar, were analyzed. This analogue is closest in technical essence to the claimed invention and adopted as a prototype.

From the foregoing, it is clear that in this area there is an urgent need to search for a new diagnostic marker for NSCLC, which can reliably and from the very early stages diagnose the disease, and to develop a simple, sensitive, reliable method applicable to it in clinical or outpatient medical institutions for the diagnosis of non-small cell lung cancer.

Disclosure of invention

This invention was made possible by the authors of a comparative analysis of the level of transcription of the T1MP3 gene in tumor tissues of the lungs of various types and at different stages of their malignant transformation and the unexpected discovery of the fact that already the early stages of development of malignant transformation are accompanied by a significant decrease in the level of transcription of the T1MP3 gene.

The present invention in its first aspect relates to a new marker for the diagnosis of non-small cell lung cancer, which is the level of transcription of the T1MP3 gene. The reduced level in the tissue of human tissue suspected of being cancer compared with its level in healthy tissue is a diagnostic sign of non-small cell lung cancer.

In one embodiment, lung cancer, the marker of which is the transcription level of the T1MP3 gene, is squamous cell lung cancer.

In a further aspect, the present invention relates to the use of a T1MP3 gene transcription level as a marker for the diagnosis of non-small cell lung cancer.

In one embodiment, lung cancer, for which the transcription level of the T1MP3 gene serves as a diagnostic marker, is squamous cell lung cancer.

In another aspect, the present invention relates to a method for the diagnosis of non-small cell lung cancer. This method includes the following steps:

a) obtaining an initial pair of tissue samples from a patient, where one of the samples was obtained from tissue presumably affected by cancer, and the second was obtained from adjacent histologically normal (conditionally normal) tissue;

b) isolation and purification of RNA preparations from the initial pair of samples;

c) synthesis of single-stranded or double-stranded cDNA on an RNA template using oligonucleotide primers;

d) normalization of the concentration of T1MP3 cDNA according to the control gene, the transcription level of which is constant in normal conditions and in lung cancer;

e) conducting a quantitative or semi-quantitative amplification reaction of a T1MP3 gene fragment using the cDNA obtained in step c) as a template and a pair of gene-specific oligonucleotide primers;

f) comparing the amount of the amplified T1MP3 DNA fragment for the sample obtained from the tissue presumably affected by the cancer with the amount of the amplified DNA fragment for the sample obtained from normal tissue, where the indicated amounts of the amplified DNA fragment reflect the transcription level of the T1MP3 gene, and the decrease in the transcription of the T1MP3 gene is diagnostic a sign of lung cancer.

In one of the embodiments of the claimed method is intended for the diagnosis of squamous cell lung cancer.

In one embodiment of the method of the invention, in step c), the oligonucleotide primers used to synthesize single-stranded or double-stranded cDNA are selected from oligo (DG) -containing primers, random hexamers or a combination thereof, as well as gene-specific primers.

In a further embodiment of the method of the present invention, oligonucleotide primers selected in such a way that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA are used to amplify the cDNA in step d).

In a separate preferred embodiment of the present invention, the sequence of primers

- 2 010571 represented by 8ES ΙΌ N0: 1 and 2.

In yet another embodiment of the method of the present invention, in step e), a quantitative or semi-quantitative amplification reaction of gene fragment # 3 is a real-time polymerase chain reaction (PCR) or a standard reverse transcription polymerase chain reaction (RT-PCR).

In one embodiment of the inventive method, in step d), the gene ΟΑΡΌΗ encoding glyceraldehyde-3-phosphate dehydrogenase protein is used as a control gene.

Another aspect of the present invention is a set of primers for the implementation of the polymerase chain reaction to determine the level of transcription of the gene ΤΙΜΡ3 having the sequence 8EC ΙΌ N0: 1 and 2.

List of figures

The invention will now be described in more detail with reference to separate illustrative examples and figures, where FIG. 1 shows the results of the selection of conditions for determining the level of transcription of gene ΤΙΜΡ3 in lung tissue samples. Electrophoretic separation on an agarose gel of PCR products (size 279 bp) obtained after 27, 30 and 34 cycles was carried out on a 1.8% agarose gel. M is a marker of molecular masses of DNA (DNA of plasmid pBK222 / A1i1);

FIG. Figure 2 shows the results of an RT-PCR analysis of the level of transcription of the ΤΙΜΡ3 gene in PRL with central localization of the tumor (after 30 cycles). Sample numbers are indicated above the tracks. T - tumor, N - conditional norm (tissue adjacent to the tumor). Ν1-Ν3 is the norm. Gene ΟΑΡΌΗ (28 PCR cycles) was used as an internal control. Electrophoretic separation of PCR products was performed on a 1.8% agarose gel.

The implementation of the invention

This invention provides a new genetic marker for the diagnosis of non-small cell lung cancer and based on the determination of the level of transcription of this marker, a simple, reliable method for the diagnosis of NSCLC at various stages of the development of malignant transformation, including the initial ones. A reliably detectable difference in transcription of the ΤΙΜΡ3 gene in normal and tumor tissues can be used to detect lung cancer.

Lung tissue samples for analysis

As samples for analysis, biopsy samples, punctate, including material obtained by bronchoscopy with direct biopsy (central lung cancer) and transthoracic (percutaneous) tumor puncture (peripheral cancer), can be used.

Isolation of RNA from lung tissue samples

Methods of isolating total RNA from mammalian tissue samples are well known to specialists and, as a rule, include the following stages: grinding of tumor and normal tissue samples in liquid nitrogen, cell lysis, RNA isolation and purification, RNA quality control by electrophoresis in 1% agarose gel in the presence of dye of ethidium bromide or denaturing polyacrylamide gel, as well as spectrophotometric determination of the amount of RNA. Homogenization of tissue pieces can be carried out manually, grinding with a pestle in a ceramic mortar, or with the help of mechanical homogenizers, for example, M1kheg or M1sgo-O18teshba1og and the company Sag1opi5 (Germany). Various protocols can be used to isolate RNA that are well known to those skilled in the art. In classical RNA isolation methods, strong chaotropic agents are used, such as guanidinochloride and guanidinoisothiocyanate, which dissolve proteins and sequential extractions with phenol and chloroform to denature and remove proteins (8th Century 1, Grigy EG, Mashayk T., Molybdenum Sulfur. 2pb Ebayup eb. 1989, Co1b 8rppu Nagobig: C8Hb Ь1Ό55). The method with the use of the reagent Txo1 (CBSCO / LrHe Teslofo5e5) is also widely used. In order to prevent RNA degradation by RNases, inhibitors can be used, such as a ίδίη inhibitor of placental or recombinant origin or, for example, a vanadyl-riboside complex, a non-specific broad-spectrum RNAse inhibitor.

Rapid and high-quality RNA isolation can be carried out using a number of commercially available kits (Klonogen, St. Petersburg; Vaa8yu ky (O | ayep); 8U To1a1 ΚΝΑ [8o1aNop 8y1et, Bhotua (USA), etc.).

In order to exclude work with aggressive agents, to speed up and simplify the isolation of RNA, various devices are used, for example, OshskSepe-810 (YGe 8aepse, Japan). For the extraction of RNA, this device uses an 80 μm porous membrane, which is 12.5 times thinner than the glass filter commonly used in such devices (1000 μm). This allows you to reduce the degradation of RNA and increase its output.

Reverse transcription reaction: cDNA synthesis on an RNA matrix isolated from lung tissue samples and its conversion into double-stranded form

The reverse transcription process, as a result of which a single-stranded DNA chain is synthesized on an RNA matrix, if necessary, with the completion of the second chain, allows us to switch from unstable RNA molecules to more stable DNA molecules and amplify using polymerase chain re

- 3 010571 shares to the quantities required for detection. RT-PCR amplification allows the use of very small amounts of the original RNA (at the level of 1 ng), and, therefore, the amount of the lung tissue under study, from which RNA is isolated.

The reverse transcription reaction can be carried out using a number of commercially available reverse transcriptase preparations, such as the reverse transcriptase of Moloni mouse leukemia virus (M-MI), avian myeloblastosis virus (AMU), Po \\ cgZepr1 (point mutation M-MYU-JT), S. TNegsh Ro1utegake et al., With the help of which it is possible to obtain amplification products up to several thousand and even several tens of thousands of nucleotide pairs (kb). Thermostable DNA polymerase Tyttic 1yettor L1i8 (T1H) can be used. possessing reverse transcriptase activity in the presence of Mn ^{2+ ions} .

For reverse transcription, various primers can be used, for example:

1) oligo (bT) - containing primers that bind to the endogenous polyA tail at the 3 'end of the mRNA (the number n is usually 12-18, but can reach a larger value). These primers are most often used to obtain full-size cDNA. Nucleotides A, C, or C are often added to the oligo (bT) sequence at the 3'-end to anchor the primer to the border of the transcript and the poly-A tract;

2) random hexanucleotide primers (statistical primers) that hybridize with RNA in numerous sites. When reverse transcription with these primers receive short cDNA. Random hexamers are used to overcome the difficulties associated with the strong secondary structure of RNA, they are more effective in reverse transcription of 5'-regions of mRNA;

3) hexamers or other short oligonucleotides (10-12 nucleotides) of random composition can also be used in combination with oligo (bT) -containing primers;

4) specific oligonucleotide primers that are used to transcribe the mRNA region of interest for the study. These primers are successfully used for diagnostic purposes.

Gene transcription analysis can be performed using single-stranded or double-stranded and amplified cDNA. Specific primers are most often used for the synthesis of the second chain and its amplification. Commercially available kits for cDNA synthesis, based on the use of various reverse transcriptases and various primers for seed. To obtain cDNA, the ZMAT method was also developed (8 \\ 'Cs1ipd testyakt! Not 5' epb oG KIA Getr1aGek oG geuegke GgpksprGake), which is based on the property of reverse transcriptases to add several nucleotide residues to the 3'-end of the synthesized first cDNA chain, mostly BS. This oligo (bS) sequence serves as the annealing site for the oligonucleotide adapter having a complementary oligo (bS) sequence at the 3'-end. Reverse transcriptase perceives the primer as an extension of the RNA template and continues the synthesis of the first strand (Zc1ishb1 ^ .M., Mie11eg M. \ U. 1999. SarZe1esG: ayu kepkuyte teGyob Gog 5 'SAR-bebbep! This is the first cDNA strand flanked on one side by a 3'-primer sequence with oligo (bT) at the 3'-end, and on the other hand, by a sequence of cDNA-teBaGeB apA1u818 oG tKIAK. complementary to the adapter These primers have the same external sequences, differing only at the 3'-end. The first strand is amplified by PCR with a primer corresponding to the outer part of the 3'-primer and adapter.The nucleotide sequence of the common part of these primers is selected depending on further purposes, for example, obtaining clonotech, using subtractive hybridization, etc. As a result, double-stranded DNA enriched with full-sized sequences.With the use of an adapter with a blocked 3'-end, a significant reduction in background amplification is achieved. When using the modified HMAVT method, amplification of the starting material by more than 10 ⁵ times takes place in a short time, therefore, it is possible to work with very small amounts of RNA (less than 1 ng), and, therefore, with a small amount of the studied tissue (Ζ1ιιι Υ.Υ. , Masyebet, E.M., Syepsyk, A., L1, K., ZeyeP, St. Petersburg, 2001. Kueuetke GgapksprGake 1tr1a1e 8 \ uSs1ipd: and ZMAK.T arrgoask Gogi11-1epd111 sEyA Eztu2b2b2. Kits for obtaining cDNA by this method are produced by various companies, for example, Evrogen, Russia (a set of MJT), S1oy1esy, USA, etc.

Analysis of the level of transcription of the T1MRZ gene using PCR

Using the first or second cDNA strand as a template commercially available from a wide range of manufacturers, thermostable DNA polymerase (for example, Tad, RGi, TG1, TGy, Tta, etc.) and specific primers, sites for which are located in the transcript of interest, conduct standard, semi-quantitative PCR, in which the amplified transcript fragment is detected by simple gel electrophoresis, or real-time quantitative PCR. The selection of specific primers is carried out by a method well known to specialists in this field. To select primers and annealing temperatures, it is advisable to use commercially available programs or programs that are freely available on the Internet. Among such programs, we can mention ONDO (version 6.42), РптетЗе1есГ from the Eaketdepe package (\ ν \ ν \ ν.bηa8ιa ^. Hundred). Рптет Ртет1ет 5, рптткЗ (Лбр: // Гбобо; et1.тё.бей / сд1-Л1п / рг1teГ / рг1teГ_тетете.сд1С), ЕакЕхеПрпteg (\ Уи X., Мпге Епепепепепепепепепепепепепепепе Arr1. Vyut Gogtaysk. 5, 119-120), ExRpteg

- 4 010571 (ZapbNi Κ.8., Asyagua Κ.Κ. 2005. Exxp: ΐο άοδίβη rptegte Ggot опhop-опhop) υηοΙίοη5. Vyut Gogtabsk. 21, 2091-2092), Reg1Rpteg. RACHRSK (1Br: // \ y \ y \ u. Yosep1eg.11e15tk | .P / Y / Rogodgat5 / Ga51rsg.1it). RPTEGCSPEC (1Wr: // 5Syoo15. | Shk1a.sot / RPTegs. | IE51 /). Given the complexity of the analyzed genome, the length of the primers can be selected in the range from 18 to 25 bp

In order to simplify the procedure for the implementation of the method for the purposes of clinical analysis and to ensure maximum preservation of the RNA contained in the sample, it is necessary to minimize manipulations with the tissue sample, which could potentially lead to the destruction of RNA. In this regard, in one of the preferred variants of obtaining the RNA preparation in this invention do not use RNAse-free DNase. Moreover, primers for PCR are selected so that they specifically hybridize with cDNA even in the presence of an impurity of genomic DNA in the preparation. This can be achieved, for example, by selecting primers for different exons of the T1MP3 gene. When using gene-specific primers complementary to regions of different exons, the length of the PCR product amplified with impurity genomic DNA will be significantly longer than the expected PCR product amplified with cDNA. If at least one of the selected primers overlaps the boundary between exons, impurities of genomic DNA will not affect the results of the amplification reaction.

In a preferred embodiment, primers are used.

Т1МР3_Р (5ЕЕ) ΙΌ N0: 1) 5'-ASSATSAASSASATSAASATSTASS-3 'and

Т1МР3_В (5ЕЕ) GO N0: 2) 5'-ASTTSATSTSTSSASTTASAASSA-3 '.

One skilled in the art will understand that other pairs of primers can be selected that differ, for example, in length, in their localization relative to the transcript sequence of the T1MP3 gene, which will provide specific and effective amplification of the transcript fragment of the T1MP3 gene even in the presence of an impurity of genomic DNA .

Quantification of the transcription level is achieved using parallel PCR with the test transcript and the control / standard transcript. As an endogenous internal control, relative to which the amplification products of the studied T1MP3 gene were normalized, the household gene SAREN encoding glyceraldehyde-3phosphate dehydrogenase was selected. The expression of housekeeping genes (yoekekerdd depex) is the same in all cells. For the SAREN gene in the case of NSCLC, the smallest spread of transcription levels in normal and tumor tissues is shown. (TOLU. Lh, 8.T. SNep, N.R. Lsh. Syoyu OG epbedeoekik sop! Goog Goge depe exherkschop ίη popkshaP 1ipd sapseg. 2005. Eig. Kekry. 1. 26, 1002-1008.)

To analyze the level of gene transcription, not only standard PCR but also real-time PCR (PCR-RV) can be used. Unlike the standard method, where only the final reaction products are recorded, real-time PCR uses fluorescently labeled oligonucleotide probes to detect DNA during its amplification; therefore, it allows one to observe the accumulation of amplified fragments in the exponential phase of the reaction, which increases the sensitivity of the method. The probe, complementary to the middle part of the amplified fragment, contains a fluorophore and a quencher at the ends. When the fluorophore and quencher are associated with an oligonucleotide probe, only slight fluorescence emission is observed. During the amplification process, due to the 5'-exonuclease activity of the Tac polymerase, the fluorescent label passes into solution, being released from the vicinity of the quencher, and generates a fluorescent signal that amplifies in real time in proportion to the accumulation of the amplification.

The selection of probes for PCR-RV can be carried out in accordance with the standard recommendations of the manufacturer of devices for PCR-RV. If there is no need for multiplex analysis of several genes simultaneously, an economical alternative may be a system using the double-stranded DNA specific dye ZVVK Skheep, whose fluorescence intensity increases in real time in proportion to the increase in the number of amplicons. In this embodiment, primers without a probe can be used.

To carry out real-time PCR, various companies have developed amplifiers, for example, AB1 Рпп 7000 Zetsnepse Ее1ес11оп ЗУЧЕТ of the company ArrNeb VuuuChep (USA), SNgoto4, MshYurysop or 1Cus1eg 1Ц5 M1 KekeagnsN (Vyu-K.ab), as well as 32 domestic DNA devices ДТ -Technology), ANK32 (Institute of Analytical Instrumentation of the Russian Academy of Sciences, Y1p: //tetet.8up1o1.ts/gobis1apk.t), etc. The use of PCR-RV also reduces the risk of contamination and automates the diagnostic process. The process lasts 2-3 hours (50 or 60 PCR cycles, respectively) and includes simultaneous PCR, detection of a fluorescent signal, data processing and presentation in graphical form (full kinetic curve) using special software. The results of the analysis become available immediately after the completion of the process and do not require additional purification and analysis of PCR products. As the main method for measuring the level of gene transcription, the comparative method (relative measurement method, CC method) is usually chosen, based on the relative measurement of the number of studied polynucleotide sequences, which allows a double comparison of the results for control and target genes, as well as for normal and tumor samples cDNA

Further, the present invention will be illustrated in detail with reference to specific when

- 5 010571 measures representing the most preferred embodiments of the present invention. It should be understood that the invention is not limited to these described embodiments. On the contrary, it is intended that it includes any alternatives, modifications, or equivalents that are acceptable given the nature and scope of the invention.

Example 1. Samples of lung tissue.

We analyzed 29 pairs of pulmonary tissue samples (tumor is a conditional norm) of patients with NSCLC: 25 pairs of PRL samples (19 samples with central tumor localization and 6 with peripheral) and 4 pairs of AKL samples. The histologically normal lung tissue taken from the tissue adjacent to the tumor closer to the edge of the resection was taken as the conditional norm. In addition, we used lung tissue obtained postmortally from 10 people who did not have a history of cancer (normal): 5 samples taken in the central regions of the lungs and 5 in peripheral regions. The average age of patients, including 28 men and 1 woman, is 61 years old (range 34-76 years). The diagnosis in each case was established on the basis of the results of clinical, morphological, endoscopic and radiological examinations. All tumor samples were characterized according to the international system of clinical and morphological classification of tumors ΤΝΜ, where Т Дцшот) - Т0-Т4 - categories reflecting the increase in the size and / or local distribution of the primary tumor, N (bib1i8) - Ν0-Ν3 - categories reflecting different degrees metastatic lesions of the regional lymph nodes; M (bs1ak1ak1k) - M0-M1 - characterizes distant metastases. All possible combinations ΤΝΜ are combined into larger groups - stages that reflect the course of the tumor process. Stage I disease was found in 6 patients, II - in 18, III - in 5. Signs of distant metastases were not observed in any of the patients. None of the patients were exposed to radiation therapy and chemotherapy before surgery.

Example 2. The selection of RNA from tissue samples.

Total RNA was isolated from frozen samples of tumor and normal tissues, crushed in liquid nitrogen. Tissue samples were homogenized on a Muto-Oysssshtact and instrument (Zaiotshk, Germany). RNA was purified by the standard method using guanidinisothiocyanate and phenol, followed by precipitation with ethanol (Zashbok s1 a1., 1989, kirg). To remove impurities glycoproteins, which are rich in lung tissue was used additional precipitation of RNA with brine (S11ots / UPCC | R., K. 1995. Maskeu Mobbtsabop Og 1ns TS gsadsgI rtossbits Gogh 1ko1abop Og ΡΝΆ Ggosh ro1ukass11apbs- rgo1sod1usap APB-ps11 koigssk Vyu1ss11pk.. | claim 19, 942-945). After salt precipitation, all RNA preparations were purified using the Yapsaku M1sh kit (Tskhadep, USA) according to the protocol attached by the manufacturer. Such a three-stage RNA purification procedure allowed us to effectively get rid of not only insoluble glycoprotein precipitates, but also low molecular weight RNA. The quality of the RNA preparation was checked by electrophoresis on a 1% agarose gel in the presence of ethidium bromide. The amount of RNA was determined spectrophotometrically (Zashbok s1 a1., 1989, Kirg).

Example 3. The reaction of reverse transcription.

Synthesis of the first strand of cDNA.

Single-stranded cDNA was synthesized on an RNA matrix isolated as described in Example 2. To obtain cDNA, we used the modified ZMAT method (Ζ1ιι.ι Υ.Υ., MasYsbst Ε.Μ., SNepsyk A., Ya Ya., 81sbcg (Ρ.Ό. 2001. Yasustks 1gapkspr1aks 1sshr1a1s k \\ ys1ipd: a Gog Gi11-1spd (H c ^ NA 11Btatu sopk1gis0op. Vyu1ss11pk. | Claim 30, 892-897), 2 ng-1 μg of total RNA, primers

ZMAYAT (5'-AA6SA6T66TATSAAS6SA6A6TAS6Sg6g6g0-3 ') and

SE8 (5'-A6CA6T66TATSAA6CA6AOTAC (T) zOK ^ -3 '), and reverse transcriptase Po \\' si8spr1 (C1o1ssy, United States).

Reaction conditions

The composition of the reaction mixture, 10 μl:

RNA, 1 μg / μl 1.0 μl

Primer ZMAT, 6 μm 2.0 μl

Primer SE8, 10 μm 1.0 μl

Sterile deionized water 1.0 μl

The sample was heated at 72 ° C for 3 min and placed on ice. 5 μl of the mixture was added:

Buffer (Cl1cbcb) for constructing the 1st chain (5x) 2.0 μl

6ΝΓΡ, 10 mM 1.0 μl

OTT, 20 mm 1.0 μl

Reverse transcriptase Po \\ 'cr8spr1 (C1op1esb) 1.0 μl

Incubated at 42 ° C, 60 min. The reaction was stopped by heating at 65 ° C for 5 min, 2 μl of 60 mM EDTA was added, and the sample volume was adjusted to 20 μl.

Example 4. The synthesis of the second strand of cDNA.

For the synthesis of the second cDNA chain and amplification, 1/10 part of the volume of the reaction mixture obtained in Example 3 was taken. The synthesis was carried out using AbuAscchad2 ΩΝΛ Po1uschsgax with primer 5'AA0CA6T66TATcAaC6CA6A0T-3 'according to the protocol AbgAcchad 2 RSY ky (Shoksb1

- 6 010571

Segtapu).

Conditions for PCR.

The composition of the reaction mixture, 50 μl:

AbsAchade 2 PCR buffer (C1op1ess) (10x) 6), 2.5 mM Primer, 10 μM cDNA (single stranded) AbsaShade 2 DNA polymerase (C1O1) Sterile deionized water 5.0 μl 5.0 μl 1.0 μl 1.0 μl 1.0 μl 37.0 μl

Amplification conditions:

95 ° C, 1.5 min 95 ° C, 20 s; 65 ° C, 20 s; 72 ° C, 3 min 1 cycle 14-17-20-23 cycle

For each sample, the number of cycles was selected, allowing to obtain the same amount of amplified material.

Example 5. Selection of conditions for determining the level of transcription of gene ΤΙΜΡ3 in lung tissue samples. Protocol for determining the level of transcription.

When selecting conditions for determining transcription levels for the amplification of double-stranded cDNA, gene-specific primers were used.

ΤΙΜΡ3_Ε (HEC) ΙΌ N0: 1) and

ΤΙΜΡ3_Κ (HEC) ΙΌ N0: 2), which were matched to different exons of the ΤΙΜΡ3 gene, moreover, one of the primers overlaps the boundary between exons. Under these conditions, genomic DNA impurities do not affect the amplification results.

The size of the PCR fragment was 279 bp The selection of PCR conditions was carried out on several cDNA samples. All primers are selected using the program Ρπιικγ Ekekspeg. developed at the IMB RAS. Amplification was carried out in 25 μl of a mixture containing 67 mM Tris-HC1, pH 8.8, 16.6 mM (ΝΗ ₄ ) ₂ δΟ ₄ , 0.1% ^^ 11-20, 2.5 mM MdC1 ₂ , 0, 2 mM each of 6ΝΤΡ, 0.1 μg cDNA, 0.2 μM each of the primers, 2 units DNA polymerase activity Βίο-Τας (Ωίαΐαΐ L1b., Moscow).

Conditions for PCR. The composition of the reaction mixture, 25 μl: PCR buffer (10x) 2.5 μl MDS1 ₂ , 25 mm 2.5 μl 6ΝΤΡ, 10 mm 0.5 μl Primer ΤΙΜΡ3_Ε, 10 μM 0.5 μl Primer ΤΙΜΡ3_Β, 10 μM 0.5 μl cDNA, 0.1 μg / μl 1.0 μl ΒίοΤ; · κ. | DNA polymerase, 5 units / μl 0.3 μl Sterile deionized water 17.2 μl Amplification conditions: 94 ° C, 2 min 1 cycle 94 ° C, 30 s; 56 ° C, 30 s; 72 ° C, 45 s 27, 30 and 34 cycles 72 ° C, 5 min 1 cycle

The cDNA samples were normalized by the SABON control gene encoding glyceraldehyde 3-phosphate dehydrogenase protein. Used primers selected for different exons of the SAGON gene, and one of the primers overlaps the border between exons:

SAOSN E: 5'-SSACΤSAASSSSΤΤΤSSΤS-3 'and

SAOSN I: 5'-ΤССССΤССААΤСАΤАΤΤССААСАΤ-3 '.

The size of the PCR fragment is 139 bp The selection of PCR conditions was carried out on several cDNA samples.

Amplification conditions:

preliminary heating at 94 ° C, 2 min; 94 ° C, 30 s; 56 ° C, 30 s and 72 ° C, 30 s 72 ° C, 5 min 28 and 30 cycles 1 cycle

PCR was performed on a McIegCus1eg amplifier, ErrepbogG (Germany) with a heating lid or Tertsik amplifier, DNA technology (Russia). Amplification products were analyzed on a 1.8% agarose gel with 0.5 μg / ml ethidium bromide (see FIG. 1). As a result, optimal RT-PCR conditions were selected (27-30-33 cycles), under which a linear relationship was obtained between the number of cycles and

- 7 010571 the number of PCR products. All amplification reactions were repeated three times.

The fluorescence intensity of the bands after electrophoretic separation of the PCR products was quantified using the SeieRgoD1eg photosensitometry program (1Bp: / Lu \ y \ g / 8 sapa1uis8.ot) and expressed as normal / tumor relative intensity values (Ν / Τ).

The amplified fragments of the ΤΙΜΡ3 gene were cloned into the pCEM®-T vector of Eacu (Rgoted) and sequenced. Their nucleotide sequences completely coincided with the sequences of the corresponding cDNA fragment. Sequencing was carried out using a set of reagents ΑΒΙ ΡΚΙ8 В® V1dEue ™ TegstaYug ν. 3.1 followed by analysis of the reaction products on an automatic DNA sequencer ΑΒΙ ΡΒΙ8Μ 3100-Atap1.

Example 6. Determination of the level of transcription of gene ΤΙΜΡ3 in samples of normal and tumor tissues.

Protocol for determining the level of transcription.

1. Prepare tayyeg-pykh by mixing all the PCR components except the matrix. Mayeg-Tih should be prepared from the calculation of 1 reaction with a volume of 25 μl for each sample + 1 additional reaction with a volume of 25 μl.

The composition of the reaction mixture, 25 μl:

PCR buffer (10x) 2.5 μl

MDS1 ₂ , 25 mm 2.5 μl

6ΝΤΡ, 10 mM 0.5 μl

Primer ΤΙΜΡ3_Ε, 10 μM 0.5 μl

Primer ΤΙΜΡ3_Β, 10 μM 0.5 μl cDNA 1.0 μl

ΒίοΤας DNA polymerase (Όί; · ι1; · ιΙ 11b., Moscow), 5 units / μl 0.3 μl

Sterile deionized water 17.2 μl

2. Add to the 0.6 ml tubes (labeled for 30 amplification cycles) add 24 μl of tak1eg-t1x.

3. Add 1 μl of the matrix to the tubes, mix by pipetting several times.

4. In a separate tube (control) mix 24 µl of the same and 1 µl of sterile deionized water.

5. Add 1 drop of mineral oil to each test tube (MP VyutebVaI, bc) and close the caps of the tubes (if PCR was performed on a Tertsik amplifier, DNA technology). In the event that PCR was performed on a лиι ^ ΚιΌλΌΓΓ Errepbog amplifier. no oil was added.

6. Place the tubes in the amplifier and carry out 27 cycles of the amplification reaction.

7. After completion of the amplification reaction, take 4 μl of amplification products (27 cycles), continue the amplification reaction for another 3 cycles, take 4 μl (30 cycles) and mix with 2 μl of paint 6x Ogapde Loobschd Eue. The reactions were carried out in the same manner at 30 and 33 cycles. Samples were applied on a 1.8% agarose gel containing 0.5 mg / L ethidium bromide. A molecular weight marker of DNA was also applied to the gel, which made it possible to estimate the size of amplification products (DNA of plasmid pBB222 / A1iE from Sibenzym, Russia was used). Gel electrophoresis was performed in TBE buffer containing 10 mg / L ethidium bromide. The separation length is 3-5 cm of the gel. After gel electrophoresis, visualization of the amplification products and their documentation should be carried out in ultraviolet at a wavelength of 302 nm.

The results of the analysis of the level of transcription of the gene ΤΙΜΡ3.

The average values of changes in the transcription levels of the studied genes and standard errors were calculated using the computer program 8–88 13.0 (8–88 Sun, USA). The reliability of the observed changes was evaluated based on the normal distribution of data. The data were considered reliable at P <0.05, where P is an indicator of the statistical significance (reliability) of the data.

We have shown that in 76% of NSCLC samples, the level of transcription is reduced by 3 or more times in tumors compared to the conventional norm (p <0.0001) (Fig. 2 and table). In 9 of 29 samples, gene transcription in tumors is practically absent. In 2 samples, gene transcription was not detected either in tumors or in adjacent tissues. The absence of gene transcription in tissue samples adjacent to dysplasia, but morphologically normal, shows the possibility of the spread of tumor cells into normal cells. In 6 samples, the level of gene transcription in tumors is approximately equal to the level in the conditional norm. Gene transcription was found in all 10 studied samples of normal lung tissue. No correlation was found between the change in the level of transcription of the ΤΙΜΡ3 gene, histological differences, and the stages of tumor progression; however, a lesser change in the level of gene transcription compared to normal was observed in peripheral localized PRL tumors than in the central PRL samples (table). It is possible that this is due to the greater dependence of the development of central BPD on smoking patients, harmful working conditions and other adverse factors.

The present invention also provides that the determination of the level of transcription of gene ΤΙΜΡ3

- 8 010571 will be useful in monitoring the effectiveness of anti-cancer therapy, and the analysis by the method of the present invention should be carried out before and after the course of treatment, and if necessary during the course of treatment. An increase in the level of transcription of the ΤΙΜΡ3 gene during or at the end of the course of treatment will indicate the restoration of the activity of the tumor suppressor gene ΤΙΜΡ3, which may indicate positive changes in the treatment. A decrease in the level of transcription of the ΤΙΜΡ3 gene will indicate a further suppression of gene activity, which may indicate a lack of treatment effectiveness.

Characterization of clinical samples and a change in the level of transcription of the ΤΙΜΡ3 gene in PRL samples with central and peripheral localization of the tumor and AKL compared to the conventional norm

tumor type Sample No. Description of tumor samples Change in the level of transcription in the tumor compared to the conditional norm ΤΝΜ Stage Keratinization ΤΙΜΡ3 central PRL 4 nine 80/05 Τ1Ν0Μ0 I - 98/05 ΙΑ + 21 26 497 300/05 Τ3Ν0Μ0 II -

6 pv 90/05 Τ2Ν1Μ0 pv - 7 Τ3Ν0Μ0 II + thirteen fifteen 77/05 5 pv 228/05 Τ3Ν1Μ0 III - 290/05 + 466 14 Τ3Ν2Μ0 peripheral PRL 475 Τ1Ν0Μ0 I - 2 Τ2Ν0Μ0 ΙΒ - 451 Τ3Ν0Μ0 II + 476 II - 452 II + 301/05 Τ3Ν1Μ0 III + AKL 311/05 Τ2Ν1Μ0 II N.D. 304/05 Τ3ΝΟΜΟ II N.D. 406 II n.d. 477 II N.d.

- 9 010571

Explanations for the table

О decrease (> 3 times) in the level of transcription of the ΤΙΜΡ3 gene.

the level of transcription did not change or decreased slightly (<3 times).

lack of transcription in the tumor and in the normal norm.

ΤΝΜ - clinical and morphological classification of tumors:

T0-T4 - categories reflecting the local distribution of the primary tumor;

Ν0-Ν3 - categories reflecting a different degree of metastatic lesion of the regional lymph nodes;

M0-M1 - characterize the presence of distant metastases;

A - lack of metastases;

B - lesion of single lymph nodes;

n D. - no data.

The above detailed description of the invention and its specific embodiments given in the examples with reference to the figures is intended solely to more fully clarify the essence of the claimed invention, but not to limit it. It will be apparent to one skilled in the art that various changes may be made, which, however, will be consistent with the spirit and scope of the present invention, which are defined by the appended claims.

List of sequences <110> INSTITUTE OF MOLECULAR GENETICS OF THE RUSSIAN ACADEMY

SCIENCE

ΙΝδΤΙΤυΤ ΜΟΙΈΚυΤΎΑΕΝΟΙ ΟΕΝΕΤΙΚΙ Κ088ΙΙ8ΚΟΙ AKAOEMP ΝΑΙΙΚ <120> METHOD FOR DIAGNOSTIC OF NON-SMALL CELL LUNG CANCER AND KIT FOR ITS IMPLEMENTATION Ag11G1S1a1 aeciepse <220>

<223> Ropuagy RPTTG T1MRZ_G <400> 1 AssaSsaats Aga1§aaga1 eDass 25 <210> 2 <211> 24 <212> OMA <213> Ag11G1s1a1 keiyeepse <220>

<223> Reuega rpteg T1MRZ_K <400> 2 ac ((§a! S (1

Claims (6)

CLAIM 1. A method for the diagnosis of non-small cell lung cancer, comprising the following stages: a) obtaining an initial pair of tissue samples from a patient, where one of the samples was obtained from tissue presumably affected by cancer, and the second was obtained from adjacent histologically normal (conditionally normal) tissue; b) isolation and purification of RNA preparations from the initial pair of samples; c) synthesis of single-stranded or double-stranded cDNA on an RNA template using oligonuclei - 10 010571 leotide primers; d) normalization of the concentration of T1MP3 cDNA according to the control gene, the transcription level of which is constant in normal conditions and in lung cancer; e) conducting a quantitative or semi-quantitative amplification reaction of the T1MP3 gene fragment using the cDNA obtained in step c) as a matrix and a pair of gene-specific oligonucleotide primers, the sequence of which is represented by 8EC GO N0: 1 and 2; f) comparing the amount of the amplified T1MP3 cDNA fragment for the sample obtained from the tissue presumably affected by the cancer with the amount of the amplified cDNA fragment for the sample obtained from normal tissue, where the indicated amounts of the amplified cDNA fragment reflect the transcription level of the T1MP3 gene, and a decrease in the transcription of the T1MP3 gene is diagnostic a sign of lung cancer. 2. The method of claim 1, wherein the lung cancer is squamous cell lung cancer. 3. The method according to claim 1, wherein in step c) the oligonucleotide primers used for the synthesis of single-stranded or double-stranded cDNA are selected from among long (bT) -containing primers, random hexamers or a combination thereof, as well as gene-specific primers. 4. The method according to claim 1, wherein in step e), the quantitative or semi-quantitative amplification reaction of the T1MP3 gene fragment is a real-time polymerase chain reaction (PCR) or a standard reverse transcription polymerase chain reaction (RT-PCR). 5. The method according to claim 1, wherein in step d), the SARIN gene encoding the glyceraldehyde-3-phosphate dehydrogenase protein is used as a control gene. 6. A set of primers for the implementation of the polymerase chain reaction to determine the level of transcription of the T1MP3 gene in the method according to claim 1, having the sequence of 8EC GO N0: 1 and 2.