RU2627440C2 - Method for c-kit gene l576p mutation diagnostic in melanoma tissue - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is intended to diagnose the presence of c-KIT gene L576P mutation in melanoma tissues. Estimation of the presence of mutation is carried out according to the ratios of the values of Ct threshold cycles in three real-time PCR reactions with allele-specific primers. Based on the results of three duplicate reactions, the mutant DNA proportion in the sample is calculated.

EFFECT: invention provides optimisation of real-time allele-specific PCR for diagnosis of c-KIT L576P tyrosine kinase receptor mutation in melanoma tissues.

6 dwg, 1 tbl, 4 ex

Description

The invention relates to molecular biology and medicine, in particular to dermatology, molecular oncology, and can be used to diagnose a mutation of the KIT gene, leading to the replacement of leucine with proline at position 576 in the c-KIT protein (hereinafter referred to as mutation L576 of the c-KIT gene), in laboratory centers.

Melanoma, like any other malignant neoplasm, is characterized by an individual genetic profile in each patient. For this reason, for the successful treatment of melanoma, it is very important not only to identify it in a timely manner, but also to evaluate all the features of a particular disease, allowing you to choose the appropriate therapy for this patient. This is the basis for the principle of personified medicine, which is receiving more and more support from oncologists today.

Among the parameters for assessing the individual characteristics of melanoma, it is important to diagnose the possible presence of a number of genetic mutations in tumor cells. The presence of these mutations determines the choice of a drug for the treatment of melanoma.

One of the diagnostically important mutations is the missense mutation, which leads to the replacement of leucine with proline at position 576 (L576P) in the c-KIT tyrosine kinase receptor polypeptide chain — the L576P mutation of the c-KIT gene. This mutation is located in exon 11 of the KIT gene and is found mainly in acral melanoma (4.4–8%), mucous membrane melanoma (5.3–7%), and skin areas subjected to prolonged ultraviolet irradiation (5.5%) [Beadling S., Jacobson-Dunlop FS Hodi C. Le et al. KIT gene mutations and copy number in melanoma subtypes // Clin. Cancer Res. - 2008. - Vol. 14, No. 21. - 6821-6828; Curtin J.A., Busam K., Pinkel D., Bastian B.C. Somatic activation of KIT in distinct subtypes of melanoma // J. Clin. Oncol. - 2006. - Vol. 24, No. 26. - P. 4340-4346; Torres-Cabala CA, Wang WL, Trent J., Yang D., Chen S., Galbincea J., Kim KB, Woodman S., Davies M., Plaza JA, Nash JW, Prieto VG, Lazar AJ, Ivan D. Correlation between KIT expression and KIT mutation in melanoma: a study of 173 cases with emphasis on the acral-lentiginous / mucosal type // Mod. Pathol. - 2009. - Vol. 22, No. 11. - p. 1446-1456].

To date, it has been shown that the presence of this mutation leads to continuous activation of the KIT tyrosine kinase receptor, which does not require the SCF ligand, which is manifested in accelerated growth and avoidance of apoptosis of melanocytes in the initial stages of melanoma (in primary melanoma) [Smalley K.S.M., Sondak V.K. and Weber J.S. C-KIT signaling as the driving oncogenic event in sub-groups of melanomas // Histol Hepathol. - 2009. - Vol. 24. - P. 643-650]. This increases the sensitivity of the tumor to therapy with drugs - tyrosine kinase inhibitors dasatinib, imatinib, nilotinib [Antonescu C.R., Busam K.J., Francone T.D., Wong G.C. et al. L576P KIT mutation in anal melanomas correlates with KIT protein expression and sensitive to specific kinase inhibition // Int. J. Cancer. - 2007. - Vol. 121. - P. 257-264]. Accordingly, for patients with this type of mutation, the chances of remission are significantly increased.

Thus, the detection of the L576P mutation in the c-KIT gene in clinical laboratories is important and relevant in the treatment of patients with melanoma.

To date, one of the common methods for detecting mutations in DNA sites is Sanger sequencing [Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors // Proc. Natl. Acad. Sci. U.S.A. - 1977. - Vol. 74, No. 12. - P. 5463-5467]. This method involves the formulation of the polymerase reaction in four tubes in the presence of terminating dideoxynucleotide triphosphates (ddNTPs: ddATP, ddTTP, ddCTP, ddGTP — one species in each mixture), which, when inserted into the chain, leads to its termination. As a result, DNA fragments of various lengths ending in identical nucleotides are formed in each mixture. The separation of molecules in the mixture by gel electrophoresis allows you to identify the lengths of the fragments, and when comparing the lengths in four mixtures, a nucleotide sequence in the original DNA molecule is built. This method can be successfully used to detect several mutations in a DNA region, but it can be unprofitable to detect a single mutation, since it requires highly qualified personnel, rather high labor costs associated with both research and data analysis, acquisition of expensive equipment and reagents .

Another frequently used method for the detection of single nucleotide substitutions in DNA is the analysis of high resolution melting analysis (HRM analysis [US 2008/0003603 Al, 01/03/2008]. It is based on the ability of double-stranded nucleic acid molecules to slowly “melt” with the formation of single-stranded with a gradual increase in temperature. In this case, the melting rate depends on the nucleotide composition of the molecule. Thus, molecules with and without mutations will have different melting profiles [Montgomery J., Wittwer C.T., Palais R., Zhou L. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis // Nature Protocols . - 2007. - Vol. 2, No. 1. - P. 59-66]. This method is suitable for detecting single nucleotide substitutions, however, it requires special add-ons or equipment functions, which makes its implementation less accessible.

Closest to the proposed is a method of genotyping of single nucleotide polymorphisms based on real-time polymerase chain reaction (patent US 007018816, 03/28/2006). The authors proposed a polymerase chain reaction in which the separation between the wild type and the mutant allele is carried out using specific primers containing a consciously introduced non-complementary nucleotide at the 3'-end. Moreover, the incompleteness of the target nucleotide to the primer in combination with a known unpaired base significantly reduces the efficiency of the reaction up to its complete absence. For the detection of reaction products in the described method, it is proposed to use fluorescent dyes, the method of melting curves or gel electrophoresis. However, the method does not take into account that the method of allele-specific polymerase chain reaction (PCR) is far from always applicable for tissues fixed with formalin and enclosed in paraffin blocks - formalin fixed paraffin embaged (FFPE). The reason for this is the fact that when tissue is fixed with formalin, nucleic acids in cells are partially damaged, distorting the result of PCR, for example, leading to a decrease in the efficiency of the reaction not associated with a mutation. Thus, the results of allele-specific PCR can be distorted and difficult to interpret. In addition, when working with tissues of malignant neoplasms, the problem of heterogeneity arises when some of the cells contain a mutation, while the other part has a wild variant of polymorphism. It can also lead to dubious results.

The objective of the proposed method is the optimization of the allele-specific polymerase chain reaction in real time to diagnose the mutation L576 of the c-KIT gene in FFPE melanoma tissues for further selection of individual treatment.

) - прямой праймер первого комплекта; ASF2

- прямой праймер второго комплекта; QF

- прямой праймер третьего комплекта; ASR

- обратный праймер; ASP

- зонд; по результатам трех дублей реакции рассчитывают среднее значение Ct_QF, Ct_ASF1, Ct_ASF2, где Ct_QF, - значение пороговых циклов Ct в реакции QF, Ct_ASF1 - значение пороговых циклов Ct в реакции ASF1, Ct_ASF2 - значение пороговых циклов Ct в реакции ASF2; при (Ct_ASF1-Ct_QF)≤1,16 и (Ct_ASF2-Ct_QF)≥3,2 доля мутантной ДНК в образце приближена к 0, при (Ct_ASF1-Ct_QF)≈(Ct_ASF2-Ct_QF) доля мутантной ДНК составляет 50%, при (Ct_ASF1-Ct_QF)≥3,2, a (Ct_ASF2-Ct_QF)≤1,16 доля мутантной ДНК приближена к 100%.The problem is solved due to the fact that the assessment of the presence of mutations is carried out according to the ratios of the threshold cycles Ct in three reactions, mixtures for setting three reactions are prepared in separate tubes and primers and probes are added in the following combinations: the first mixture is ASF1, ASR, ASP; the second mixture is ASF2, ASR, ASP; the third mixture is QF, ASR, ASP, where ASF1 (sequence

) - direct primer of the first kit; ASF2

- direct primer of the second set; QF

- direct primer of the third set; ASR

- reverse primer; ASP

- probe; using the results of three reaction doubles, the average value of Ct _QF , Ct _ASF1 , Ct _{ASF2 is calculated} , where Ct _QF is the value of the threshold threshold cycles Ct in the reaction QF, Ct _ASF1 is the value of the threshold threshold cycles Ct in the reaction ASF1, Ct _ASF2 is the value of the threshold threshold cycles Ct in the reaction ASF2; at (Ct _ASF1- Ct _QF ) ≤1.16 and (Ct _ASF2- Ct _QF ) ≥3.2, the fraction of mutant DNA in the sample is close to 0; at (Ct _ASF1- Ct _QF ) ≈ (Ct _ASF2- Ct _QF ) mutant DNA is 50%, with (Ct _ASF1- Ct _QF ) ≥3.2, and (Ct _ASF2- Ct _QF ) ≤1.16, the proportion of mutant DNA is close to 100%.

The proposed method is as follows.

A formalin-fixed melanoma biopsy specimen embedded in paraffin is macrodissected to remove surrounding healthy tissue. Next, dewaxing of the tissue with xylene and ethanol is carried out according to a standard method. The predominant presence of tumor cells in the sample is checked by making a slice stained with hematoxylin and eosin, followed by evaluation using light microscopy. Using a microtome, a series of sections are obtained from a dissected area of paraffin embedded tissue. Next, dewaxing of the tissue with xylene and ethanol is carried out according to a standard method.

DNA isolation is carried out using the available DNA isolation kit, for example, DNA-Sorb B (AmpliSens, Federal State Institution of Scientific Research at the Federal Agency for Consumer Rights Protection and Human Welfare, Russia) using the attached protocol. For analysis, it is necessary to obtain at least 25 μl of purified DNA at a concentration of at least 3 ng / μl.

A portion of purified DNA (6 μl) is used to set up a preliminary real-time PCR reaction containing a special set of primers and a probe in which the direct primer is complementary to the same DNA region as the allele-specific primers, but does not affect the target mutation. Primer nucleotide sequence:

Direct Primer (QF):

Reverse Primer (ASR):

Fluorescence Probe (ASP):

For the reaction, a reaction mixture containing Tris-HCl (pH 8.8), KCl, Tween 20, MgCl ₂ at a concentration of 5.5 mM, deoxynucleoside triphosphates, glycerol, Taq polymerase (SynTaq polymerase (Syntol, Russia) was used) was used. The concentration of each primer and probe is 350 nM. The real-time PCR reaction is carried out on a StepOne ™ device (Applied Biosystems, Singapore). Reaction conditions: preliminary denaturation of 95 ° C for 5 minutes; 40 cycles of the reaction in 95 ° C mode for 15 sec, 55 ° C - 1 min (fluorescence detection) .At the end of the reaction, the graphs of the level of the fluorescence signal in the samples are analyzed The instrument sets the threshold level of fluorescence above which the signal is considered positive. The cycle number in which the fluorescence intensity graph for a given sample crossed the threshold level and became positive is called the threshold cycle — Ct. Estimate the Ct values for each sample. If the Ct value does not exceed 35 cycles, the purified sample DNA is considered suitable for further analysis. This allows us to evaluate the effectiveness of the real-time PCR reaction for each sample, the absence of obstacles to amplification in the form of a low DNA concentration, additional mutations or DNA breaks in the studied gene region.

If the sample is suitable for mutation analysis, allele-specific PCR with three sets of primers is performed.

The first direct allele-specific primer designated ASF1 is complementary to the wild version of the c-KIT gene lacking the L576P mutation, with the exception of the 3'-terminal nucleotide:

ASF1:

where the studied nucleotide is marked in bold with underlining, in this case, unchanged by the thymine mutation.

The second allele-specific primer designated ASF2 is complementary to the c-KIT gene variant containing the L576P mutation:

ASF2:

where the studied nucleotide is marked in bold with underlining, in this case, modified by a mutation of cytosine.

The third reaction is carried out with QF primer (the sequence is presented above).

The ASR reverse primer and ASP fluorescence probe remain the same for all three reactions.

Thus, for the formulation of the three real-time PCR reactions, mixtures with the following combinations of primers and probe are prepared in separate test tubes:

the first mixture is ASF1, ASR, ASP; the second mixture is ASF2, ASR, ASP; the third mixture is QF, ASR, ASP. Other components of the mixtures are Tris-HCl (pH 8.8), KCl, Tween 20, MgCl ₂ at a concentration of 5.5 mM, deoxynucleoside triphosphates, glycerol, Taq polymerase (SynTaq polymerase (Syntol, Russia) was used.

Reaction temperature: 95 ° C - 5 minutes; 10 alternating cycles of 95 ° C for 15 sec and 55 ° C for 1 min, then 40 alternating cycles of 95 ° C for 15 sec and 55 ° C for 1 min (detection).

At the end of the reaction, the difference values of the threshold cycles Ct (ΔCt) are calculated in the following reactions:

ΔCt ₁ = (Ct _ASFl -Ct _QF ),

ΔCt ₂ = (Ct _ASF2 -Ct _QF ),

where Ct _ASF1 is the value of the threshold cycle Ct in the reaction with primer ASF1, Ct _ASF2 is the value of the threshold cycle Ct in the reaction with primer ASF2, Ct _QF is the value of the threshold cycle Ct in the control reaction with primer QF.

27 samples of melanoma enclosed in paraffin blocks were analyzed, and reactions with artificially synthesized DNA fragments of exon 11 of the KIT gene containing and not containing the studied mutation were carried out. Table 1 presents the calculation results of ΔCt ₁ and ΔCt ₂ for the samples studied.

Table 1 shows that in the absence of a mutation in the tissues, their Ct values almost coincide, being limited only by the difference in one artificially unpaired nucleotide ΔCt ₁ ≤1.16. This means that the reaction with the ASF1 primer proceeds on average with the same efficiency as in the control reaction: The reaction efficiency with the ASF2 primer is noticeably reduced: ΔCt ₂ ≥3.2.

If there is a mutation in 50% of tissue cells, the amplification efficiency in the reaction with ASF2 primer increases: ΔCt ₂ ≈ΔCt ₁ .

If there is a 100% mutation, according to theoretical calculations, the opposite picture should be noted with the absence of mutation: ΔCt ₁ ≥3.2, ΔCt ₂ ≤1.16, and in the studied control sample the data fall into the indicated ranges.

The proposed method allows the detection of the L576P mutation of the c-KIT gene in melanoma tissue, fixed with formalin and enclosed in a paraffin block, within one working day.

Example 1

A sample of tumor tissue of a patient with a diagnosis of acral-lentiginous melanoma with localization on the wrist. This type of melanoma is a type of melanoma capable of containing the L576P mutation of the c-KIT gene.

Figure 1A shows graphs of changes in the level of fluorescence in samples during the formulation of the reactions of the proposed method.

According to the results of the analysis, the threshold cycle values were: Ct _QF = 22.6 (black fluorescence plots), Ct _ASF1 = 23.3 (dark gray fluorescence plots), Ct _ASF2 > 40 (light gray fluorescence plots).

ΔCt ₁ = (23.3-22.6) = 0.7 <1.16

ΔCt ₂ > 3.2

According to the proposed method in this sample, the proportion of mutant DNA is close to 0%.

The data obtained were confirmed by sequencing (Figure 1B).

Thus, further molecular genetic research is recommended for this patient.

Example 2

A sample of melanoma obtained from a patient with a diagnosis of surface-spreading melanoma. Graphs of changes in the fluorescent signal in real-time PCR reactions are presented in Figure 2A. In this case, Ct _QF = 22.8 (black graphics), Q _ASF1 = 22.9 (dark gray graphics), Ct _ASF2 = 26.1 (light gray graphics).

ΔCt ₁ = (22.9-22.8) = 0.1 <1.16

ΔCt ₂ = (26.1-22.8) = 3.3> 3.2

According to the proposed method in this sample, the proportion of mutant DNA is close to 0%. The result was confirmed by sequencing (Figure 2B).

This patient is recommended for further molecular genetic research.

Example 3

An artificially synthesized DNA sample containing a fragment of exon 11 of the KIT gene with a target nucleotide. In the prepared mixture, 50% of the sample DNA contained the L576P mutation, while the other 50% did not contain the mutation. When real-time PCR was performed using the proposed method, the fluorescence growth graphs were obtained, which are presented in Figure 3. Based on the results of three duplicates of the reaction, the average value of Ct _QF was 20.5, Ct _ASF1 = 21.4, Ct _ASF2 = 21.2.

ΔCt ₁ = (21.4-20.5) = 0.9

ΔCt ₂ = (21.2-20.5) = 0.7

This patient is recommended treatment with tyrosine kinase inhibitor drugs.

Example 4

Artificially synthesized exon 11 DNA sample of the KIT gene containing the L576P mutation. When carrying out the reaction by the proposed method, graphs of changes in the fluorescence level were obtained, presented in Figure 4.

According to the results of three takes of the reaction, the average value of Ct _QF was 20.4, Ct _ASF1 = 25.1, Ct _ASF2 = 20.7

ΔCt ₁ = (25.1-20.4) = 4.7

ΔCt ₂ = (20.7-20.4) = 0.3

This patient is recommended treatment with tyrosine kinase inhibitor drugs.

Thus, the results of the proposed method can be interpreted even in the presence of tissue heterogeneity by the L576P mutation of the c-KIT gene or in the case of a heterozygous mutation.

Claims (1)

A method for diagnosing the presence of a c5-KIT gene L576P mutation in melanoma tissues, including real-time polymerase chain reaction with allele-specific primers, characterized in that the presence of a mutation is evaluated by the ratio of the threshold cycles Ct in three reactions, mixtures for setting three reactions prepared in separate tubes and add primers and probes in the following combinations: the first mixture is ASF1, ASR, ASP; the second mixture is ASF2, ASR, ASP; the third mixture is QF, ASR, ASP, where ASF1 (sequence 5'-TTATGTTTACATAGACCCAACACAACTTT-3 ') is a direct primer of the first kit; ASF2 (5'-TTATGTTTACATAGACCCAACACAACCTT-3 ') - direct primer of the second set; QF (5'-CAATTATGTTTACATAGACCCAACACAAC-3 ') - direct primer of the third set; ASR (5'-GAACAAAACAAAGGAAGCCACTG-3 ') - reverse primer; ASP (5'-FAM-CCAGAAACAGGCTGAGTTTTGGTCAGT-BHQ1-3 ') - probe; using the results of three reaction doubles, the average value of Ct _QF , Ct _ASF1 , Ct _{ASF2 is calculated} , where Ct _QF is the value of the threshold threshold cycles Ct in the reaction QF, Ct _ASF1 is the value of the threshold threshold cycles Ct in the reaction ASF1, Ct _ASF2 is the value of the threshold threshold cycles Ct in the reaction ASF2 ; at (Ct _ASF1- Ct _QF ) ≤1.16 and (Ct _ASF2- Ct _QF ) ≥3.2, the fraction of mutant DNA in the sample is close to 0; at (Ct _ASF1- Ct _QF ) ≈ (Ct _ASF2- Ct _QF ) mutant DNA is 50%, with (Ct _ASF1- Ct _QF ) ≥3.2, and (Ct _ASF2- Ct _QF ) ≤1.16, the proportion of mutant DNA is close to 100%.

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