RU2454464C2 - Molecular genetic method of tumour sensitivity test in patients with lung cancer to gefitinib therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves identifying in the 19^th exon of the EGFR gene by polymerase chain reaction (PCR) with using upstream 5'-CTGTCATAGGGACTCTGGAT-3' and downstream 5'-CAGCAAAGCAGAAACTCACAT-3' primers that is followed by measuring a length of an amplified fragment by means of polyacrylamide gel electrophoresis. The L858R substitution in the 21^st exon of the gene is detected by allel-specific PCR with using 5'-CACCCAGCAGTTTGGCCA-3', 5'-CACCCAGCAGTTTGGCCC-3' and 5'-GCATGAACTACTTGGAGGAC-3' primers. If observing in patients any of two types of mutations in the EGFR gene, the gefitinib therapy is prescribed.

EFFECT: invention provides high information value and reliability of tumour sensitivity test in the patients suffering lung cancer to gefitinib therapy with underlying simplicity and availability of the method.

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Description

The invention relates to medicine, in particular to molecular oncology, molecular diagnostics.

The proposed test is aimed at selecting patients with a high sensitivity of the tumor to Iressa therapy by detecting the most frequent EGFR mutations. A reasonable selection of patients for treatment will reduce the ineffective use of an extremely expensive drug (the cost of 1 month of treatment with gefitinib is approximately 100,000 rubles).

To date, a number of methods for detecting intragenic mutations of EGFR have been described. Most often, direct sequencing of 18-21 exons of a gene is used to search for EGFR genetic disorders, which allows to reveal the whole spectrum of known mutations [Tokumo et al. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers, Clin. Cancer Res. 2005 Feb 1; 11 (3): 1167-73]. This method has several significant drawbacks that prevent its widespread use in clinical practice. Firstly, this is a relatively low sensitivity due to the frequent contamination of tumor samples with normal tissue that does not contain mutations. To detect a mutation by sequencing, the sample must contain at least 30% mutant DNA [Bosari et al. Detection of p53 mutations by single-strand conformation polymorphisms (SSCP) gel electrophoresis. A comparative study of radioactive and nonradioactive silver-stained SSCP analysis. Diagn Mol Pathol. 1995 Dec; 4 (4): 249-55]. Secondly, this technique is quite expensive and time-consuming.

Another approach to detecting EGFR genetic defects is to use a variety of modifications of the polymerase chain reaction (PCR): PCR using modified base oligonucleotides (PNA-LNA PCR clamp) [Nagai et al. Genetic heterogeneity of the epidermal growth factor receptor in non-small cell lung cancer cell lines revealed by a rapid and sensitive detection system, the peptide nucleic acid-locked nucleic acid PCR clamp. Cancer Res. 2005 Aug 15; 65 (16): 7276-82], TaqMan probes [Suzuki et al. Expression and mutation statuses of epidermal growth factor receptor in thymic epithelial tumors. Jpn J Clin Oncol. 2006 Jun; 36 (6): 351-6], Scorpion primers [Kimura et al. Detection of epidermal growth factor receptor mutations in serum as a predictor of the response to gefitinib in patients with non-small-cell lung cancer. Clin Cancer Res. 2006 Jul 1; 12 (13): 3915-21], PCR with enrichment of a mutant variant of DNA (mutant-enriched PCR) [Asano et al. Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay. Clin Cancer Res. 2006 Jan 1; 12 (1): 43-8] et al. This group of methods is aimed at finding individual, most common EGFR lesions. These methods have higher sensitivity, but require the use of combinations of expensive fluorescently-labeled oligonucleotides or additional analysis procedures (restriction analysis with mutant-enriched PCR).

Another option for detecting EGFR mutations is high resolution melting [Heideman et al. A panel of high resolution melting (HRM) technology-based assays with direct sequencing possibility for effective mutation screening of EGFR and K-ras genes. Cell Oncol. 2009; 31 (5): 329-33] - is characterized by high sensitivity, but cannot be recommended for routine use due to the high cost and small distribution in our country of equipment for analysis.

Also, among methods based on PCR, it is necessary to note the method described by Pan et al., 2005 [Pan et al. Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas. J Mol Diagn. 2005 Aug; 7 (3): 396-403]. The Pan method allows detecting the entire spectrum of deletions in exon 19, but it is proposed to additionally use the restriction analysis step to detect L858R substitution.

As a prototype, among the known analogues of the invention, the closest method for detecting the most frequent deletions in exon 19 of EGFR (delE746-A750) and missense mutation L858R in exon 21 described in OHNISHI H. et al. A simple and sensitive method for detecting major mutations within the tyrosine kinase domain of the epidermal growth factor receptor gene in non-small-cell lung carcinoma. Diagn Mol Pathol. 2006 Jun; 15 (2): 101-8. The claimed method differs from the known one in that PCR is used to determine deletions in exon 19, followed by determination of the length of the amplified fragment by polyacrylamide gel electrophoresis, and allele-specific PCR is used to detect replacement of L858R in exon 21 of the gene. If a patient identifies either of the two types of mutations in the EGFR gene, hephitinib therapy is prescribed.

The technical result of the claimed invention is to increase the information content and reliability of the detection of mutations in the EGFR gene in patients with lung cancer, which allows us to determine the sensitivity of the tumor to gefitinib therapy.

The invention is directed to the selection of patients with lung cancer for gefitinib therapy. This goal is achieved by testing two types of disorders: all possible deletions in exon 19 and replacement of L858R in exon 21 of EGFR. These mutations account for 80-90% of all EGFR lesions found in lung cancer. Deletion in exon 19 is detected by PCR followed by separation of the amplification products in a polyacrylamide gel, and the variant is detected by the L858R method by allele-specific PCR. Due to the small size of the PCR fragments used, the proposed test can be effectively used to determine the status of EGFR in archival pathomorphological samples of tumor tissue.

The inventive step of the proposed diagnostic test is confirmed, firstly, by the spectrum of detected EGFR lesions, and, secondly, by using a combination of two inexpensive and simple PCR methods to detect mutations.

Description of the invention

Testing is recommended for all patients with lung adenocarcinoma, and can also be used in patients with other histological variants of lung tumors.

Deletion in exon 19 of EGFR is detected by PCR amplification of the entire exon sequence using direct 5'-CTGTCATAGGGACTCTGGAT-3 'and reverse 5'-CAGCAAAGCAGAAACTCACAT-3' primers, followed by electrophoretic separation of PCR products. The reaction mixture consists of 1 μl of a DNA solution (10-100 ng of DNA) and 9 μl of a mixture for PCR. The PCR mixture contains 1-fold PCR buffer, 2.5 mM MgCb, 200 μM dTTP, 200 μM dTTP, 200 μM dGTP, 200 μM dATP, 1 μM of each primer (final concentration 0.1 OU / L or 0.5 pmol / μl) and 0.5 units "hot-start" DNA polymerase. The PCR reaction begins with a 10-minute activation of DNA polymerase at 95 ° C; To accumulate the PCR product, 45 amplification cycles are carried out (denaturation: 15 sec at 95 ° C; annealing: 30 sec at 57 ° C; synthesis: 30 sec at 72 ° C). Each amplification set should include a deletion control sample as well as a negative control. The resulting product was separated by electrophoresis in 10% polyacrylamide gel (PAAG) in a vertical chamber. A wild-type allele corresponds to a 127 bp fragment, and in the case of division, an additional fragment of a smaller size is observed (the size of the additional fragment is determined by the type of deletion). Figure 1 shows examples of the detection of an EGFR deletion.

Testing the missense mutation EGFR L858R in exon 21 is carried out by allele-specific real-time PCR using the following primers: 5'-CACCCAGCAGTTTGGCCA-3 '(primer specific for the wild-type sequence), 5'-CACCCAGCAGTTTTGGCCC-3' (primer specific for the sequence with the mutation), and 5'-GCATGAACTACTTGGAGGAC-3 '(total primer) (the size of the PCR product is 120 bp). Real-time PCR ("real-time PCR") is performed on iCycler iQ Real-Time PCR Detection System equipment (Bio-Rad Laboratories, Hercules) and consists of 50 amplification cycles (denaturation: 15 sec at 95 ° C; annealing: 30 sec at 63 ° C; synthesis: 30 sec at 72 ° C). The PCR mixture (total volume 20 μl) contains 1 μl DNA solution (10-100 ng DNA), 1.0 unit DNA polymerase, 1-time PCR buffer (pH 8.3), 2.5 mM MgCl ₂ , 200 μM of each nucleotide triphosphate, 0.3 μM of each primer, SYBR green I at a concentration of 0.2x (stock solution 10,000x; Molecular Probes) . Each amplification set should include a control sample (heterozygous for the studied mutation), as well as a negative control.

To control the specificity of the fragments, analysis of melting curves is used. Figure 2 shows examples of the detection of the EGFR L858R mutation.

Detection of the L858R substitution is based on the dynamics of amplification of fragments with oligonucleotides specific for the normal and mutant alleles, which is reflected in a certain value of the Ct (Cycle threshold, point of intersection of the threshold line) amplification curves corresponding to the normal (wt) and mutant (mt) alleles. The genotype of the test sample is established by the formula: ΔCt = Ct (mt) -Ct (wt). If the sample is homozygous for the normal allele, ΔCt is 6 cycles or more, in the case of heterozygotes for the mutation ΔCt≤l. Intermediate ΔCt values indicate non-optimal PCR conditions.

The advantages of the test are simplicity in performance, accessibility, as well as high information content and reliability. Using two simple techniques (conventional PCR combined with electrophoresis and allele-specific PCR), up to 90% of the EGFR mutations encountered can be detected. The use of short PCR fragments allows the use of this test to search for mutations in archival pathomorphological samples of lung tumors.

Claims (1)

Molecular genetic method for determining the sensitivity of a tumor in patients with lung cancer for gefitinib therapy, including the study of tumor tissue, characterized in that the polymerase chain reaction (NDP) using direct 5'-CTGTCATAGGGACTCTGGAT-3 'is used to determine the division in the 19 exon of the EGFR and reverse 5'-CAGCAAAGCAGAAACTCACAT-3 'primers, followed by determination of the length of the amplified fragment by polyacrylamide gel electrophoresis, and allele-specific PCR using iso-PCR is used to detect L858R substitution in the exon of the gene by applying 5'-CACCCAGCAGTTTGGCCA-3 ', 5'-CACCCAGCAGTTTTGGCCC-3' and 5'-GCATGAACTACTTGGAGGAC-3 'primers, and if any of the two types of mutations in the EGFR gene are detected in patients, hephitinib therapy is prescribed.

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