KR102041001B1 - Method for quantitative analysis of FLT3 gene mutation and kit - Google Patents

Method for quantitative analysis of FLT3 gene mutation and kit Download PDF

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KR102041001B1
KR102041001B1 KR1020150158956A KR20150158956A KR102041001B1 KR 102041001 B1 KR102041001 B1 KR 102041001B1 KR 1020150158956 A KR1020150158956 A KR 1020150158956A KR 20150158956 A KR20150158956 A KR 20150158956A KR 102041001 B1 KR102041001 B1 KR 102041001B1
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김명신
김용구
이건동
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가톨릭대학교 산학협력단
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Abstract

The present invention relates to a method for quantitative analysis of FLT3 gene mutation and a kit therefor. The FLT3-ITD quantitative analysis method using section analysis according to the present invention is inexpensive compared to the real-time quantitative PCR method, and has the effect of measuring the quantitative value relatively easily and efficiently in various variations such as FLT3-ITD. In addition, the sensitivity, specificity, and sensitivity are more sensitive than conventional PCR results, and can be useful for analyzing the quantitative value of FLT3-ITD in patients with acute myeloid leukemia. It is expected to be high.

Description

Method for quantitative analysis of FLT3 gene mutation and kit}

The present invention relates to quantitative analysis and analysis kit for FLT3 gene mutation.

In hematologic malignancies, high levels of FLT3 are expressed or FLT3 mutations cause uncontrolled induction of FLT3 receptors and downstream molecular pathways. Mutations in FLT3 are found in about 30% of patients with acute myelogenous leukemia and a small number of acute lymphomatic leukemia or myelodysplastic syndromes.

The FLT3 (fms-related tyrosine kinase 3) gene is located in the long arm (13q12) of chromosome 13 and produces a tyrosine kinase protein that regulates the differentiation and proliferation of hematopoietic stem cells. Patients with mutations in FLT3 tend to have a poor prognosis with reduced duration of remission and disease cure survival. Activated mutations of FLT3 are largely divided into internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations. Three quarters of the FLT3 mutations are ITD mutations, and the other 1/4 are TKD mutations. The FLT3-ITD mutation is a duplication of 4-40 amino acids in the juxtamembrane region of the receptor (25-30% of patients) and the FLT3-TKD mutation is a point mutation of the kinase domain (patient 5- 7%). These mutations are often associated with small tandem duplications of amino acids in the juxtamembrane domain of the receptor, resulting in the activity of tyrosine kinases.

Genetic mutation testing in acute myeloide leukemia (AML) is used as an indicator for predicting prognosis such as detection, recurrence, and survival, as well as diagnosis. Among these, FLT3 (fims-like tyrosine kinase) mutation is the most common mutation in AML, and the presence of FLT3 internal tandem duplication (FLT3-ITD) at diagnosis is poor in prognosis and affects recurrence and survival. Has been reported to be mad. In particular, it has been reported that the quantification difference of mutations is more closely related to the treatment outcome and the prognosis of patients than the presence or absence of FLT3-ITD. However, the quantitative analysis of FLT3-ITD is difficult to perform by real-time quantitative PCR because of the variable position and size of gene mutations.

United States Patent No. 7,858,333

In this regard, the present inventors have newly developed a quantitative analysis method of FLT3-ITD using a fragment analysis method, and verified and evaluated the FLT3-ITD by various methods, thus completing the present invention.

Accordingly, an object of the present invention is to provide a method for quantitative analysis of FLT3 gene mutation.

In addition, another object of the present invention to provide a kit for detecting the FLT3 gene mutation for FLT3 gene mutation quantitative analysis method.

Another object of the present invention is to provide a method for providing information for predicting the prognosis of acute myeloid leukemia.

In order to achieve the above object, the present invention comprises the steps of PCR amplifying a DNA sample to be analyzed using a FLT3 primer; Electrophoresis of the PCR amplification product; And it provides a method for quantitative analysis of FLT3 gene mutations comprising; quantitative analysis.

According to one embodiment of the present invention, the primer may be a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

According to one embodiment of the present invention, the primer may be labeled with a fluorescent material 5 '.

According to one embodiment of the invention, the PCR amplification, the reaction for 5 minutes at 95 ℃; 30 seconds at 95 ℃, 1 minute at 56 ℃ and 2 minutes at 72 ℃ repeated for 30 to 40 times the reaction: it may include a step of reacting for 30 minutes at 60 ℃.

According to an embodiment of the present invention, the quantitative analysis may be performed using GeneMapper software.

According to one embodiment of the present invention, the quantitative analysis may be calculated by Equation 1 below.

[Equation 1]

{Variable Peak Height or Area / (Wild Peak Height or Area + Variant Peak Height or Area) x 100}

According to one embodiment of the invention, the quantitative analysis minimum detection DNA copy number may be 10.

According to one embodiment of the invention, the concentration detectable by the quantitative analysis may be from 3 to 100%.

According to one embodiment of the invention, the DNA sample may be isolated from the bone marrow.

In addition, the present invention provides a buffer solution; DNA polymerase; DNTPs; And it provides a kit for detecting FLT3 gene mutations comprising a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention, PCR amplify the DNA sample to be analyzed using a FLT3 specific primer; Capillary electrophoresis of the PCR amplification product; And it provides a method for providing information for predicting the prognosis of acute myeloid leukemia, comprising the step of quantitatively analyzing the FLT3 gene mutation.

In one embodiment of the present invention, the primer may be a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In one embodiment of the present invention, when the quantitative analysis results that the quantitative value of the FLT3 gene mutation contained in the DNA sample to be analyzed is 50% or more or the mutation length is 70bp or more, it is determined that there is a high possibility of recurrence of acute myeloid leukemia as a risk group. However, if the quantitative value of the FLT3 gene mutation is less than 50% and the mutation length is less than 70 bp, the risk of recurrence is low.

According to the present invention, FLT3-ITD quantitative analysis using section analysis is inexpensive compared to real-time quantitative PCR, and has the effect of measuring quantitative values relatively easily and effectively in various variations such as FLT3-ITD. In addition, the sensitivity, specificity, and sensitivity are more sensitive than conventional PCR results, and can be useful for analyzing the quantitative value of FLT3-ITD in patients with acute myeloid leukemia. It is expected to be high.

Figure 1 shows the linearity evaluation results for the FLT3 wild-type and mutant mixed sample according to an embodiment of the present invention.
Figure 2 shows the correlation between the height value of the peak and the area value of the peak when calculating the quantitative percentage according to an embodiment of the present invention.
Figure 3 shows the rate of FLT3-ITD mutation of the patient requested for FLT3-ITD test during the initial diagnosis of AML according to an embodiment of the present invention.
4 is a result of analyzing the effect of FLT3-ITD quantification according to an embodiment of the present invention on overall survival, disease free survival.
5 is a result of analyzing the effect of FLT3-ITD quantification on the survival rate after hematopoietic stem cell transplantation according to an embodiment of the present invention.
FIG. 6 shows the results of analyzing the effects of FLT3-ITD quantification and mutation length on overall survival and event free survival according to an embodiment of the present invention.
Figure 7 analyzes the effect of FLT3-ITD quantification and mutation length on overall survival and event free survival in patients with acute myeloid leukemia showing normal chromosomes according to an embodiment of the present invention One result.
8 is a result of analyzing the effect of FLT3-ITD quantification and mutation length on overall survival and event free survival in the consolidation therapy treatment group according to an embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, the detailed description can be omitted for techniques well known to those skilled in the art. In addition, in describing the present invention, when it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. Also, the terminology used herein is a term used to properly express a preferred embodiment of the present invention, which may vary depending on a user, an operator's intention, or customs in the field to which the present invention belongs.

Therefore, the definitions of the terms should be made based on the contents throughout the specification. Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

The present invention relates to a method for quantitative analysis of FLT3 gene mutation and a kit for the same, and more specifically, a kit for detecting FLT3 gene mutation for FLT3-ITD quantitative analysis method and FLT3 gene quantitative analysis method using fragment analysis method It is about.

The present invention has developed a quantitative assay for FLT3-ITD gene mutation in acute myeloide leukemia (AML), (1) PCR using FLT3 primers, and (2) ABI 3130 Genetic Analyzer. Capillary electrophoresis, (3) fragment analysis, and (4) quantitative analysis using GeneMapper v4.1 software to more accurately diagnose the prognosis, survival, and recurrence rate of AML. It was found possible.

In the present invention, "fms-related tyrosine kinase 3" (FLT3) refers to FMS-like receptor tyrosine kinase. Several mutations of FLT3 have been identified and have been shown to cause structural activation of the receptor. In acute myeloid leukemia, this mutation is the most common genetic modification associated with the disease and accounts for approximately 25% of patients with AML. FLT3 has been shown to phosphorylate STAT5 and regulate activity.

Preferably, the FLT3 gene to be analyzed in the present invention is a gene of GenBank No. NG_007066.1. The gene includes various mutations present in FLT3.

In addition, in the present invention, the "FLT3-ITD" refers to the internal tandem duplication of FLT3 FLT3-ITD is a somatic mutation of acute myeloid leukemia with a change in the location, length and number of the overlap of the FLT3 gene FLT3-ITD may have its mutation in the gene of GenBank No. NG_007066.1.

Accordingly, the present invention provides a method for quantitative analysis of FLT3 gene mutation, and the method for quantitative analysis of FLT3 gene mutation comprises: (a) PCR amplifying a DNA sample to be analyzed using FLT3 primers; (b) electrophoresis of the PCR amplification products; And (c) quantitative analysis.

In order to quantitatively analyze the FLT3 gene mutation in the present invention, first, the DNA sample to be analyzed is PCR amplified using the FLT3 primer.

In the present invention, the DNA sample can be separated from the biological sample. Such biological samples include solid tissue samples such as blood and other liquid samples of biological origin, biopsy samples, hair roots, tissue cultures or cells derived therefrom and their progeny. The samples also include reagent treatment, solubilized samples or cultured cells, cell supernatants and cell lysates. Preferably, the DNA sample may be isolated from the bone marrow. Isolation of DNA from the biological sample described above can be performed according to methods known in the art.

In the present invention, the primer is a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions and in suitable buffers (ie four different nucleoside triphosphates and polymerases). Means. Suitable lengths of primers can vary depending on various factors, such as temperature and the use of the primer. In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can hybridize with the template to perform the primer-specific action. Therefore, the primer in the present invention does not need to have a sequence that is perfectly complementary to the nucleotide sequence of the gene that is a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the gene sequence and acting as a primer. In addition, it is preferable that the primer according to the present invention can be used for gene amplification reactions.

The amplification reaction refers to an amplification reaction of a nucleic acid molecule, and the amplification reactions of such genes are well known in the art, for example, polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), and Liga. Aze chain reaction (LCR), electron mediated amplification (TMA), nucleic acid sequence substrate amplification (NASBA), and the like.

The FLT3 primer used for PCR amplification in the present invention may be a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

FLT3 forward primer: 5 6-FAM-TTTAGGTATGAAAGCCAGCTACA 3 (SEQ ID NO: 1)

FLT3 reverse primer: 5 AGCATTTTGACGGCAACCT 3 (SEQ ID NO: 2)

In addition, a DNA-binding fluorophore used in a real-time gene detection method is used as a fluorescent material for identifying gene amplification products, and there is no particular limitation on the type thereof. Preferably, a fluorescent substance may be labeled and used on the FLT3 forward primer 5 '.

PCR amplification in the present invention is a step of reacting once for 3 to 5 minutes at 90 ~ 100 ℃; 20 to 40 seconds at 90 to 100 ℃, 30 to 1 minutes at 55 to 65 ℃ and 1 to 3 minutes at 70 to 75 ℃ repeated for 30 to 40 times: and the reaction for 30 minutes at 60 ℃ It may include, in one embodiment of the present invention reacting for 5 minutes at 95 ℃; 30 seconds at 95 ℃, 1 minute at 56 ℃ and 2 minutes at 72 ℃ repeated for 30 to 40 times: and the reaction was carried out for 30 minutes at 60 ℃.

Next, the PCR amplification product is electrophoresed. Preferably capillary electrophoresis can be performed.

After electrophoresis, quantitative analysis can be performed, and fragment analysis can be further performed before quantitative analysis. At this time, the fragment analysis can be used as long as it is a method for fragment analysis of PCR products known in the art and in one embodiment of the present invention was analyzed using GeneMapper Software v4.1.

In addition, the quantitative analysis can also be performed using GeneMapper software, the quantitative analysis of the present invention can be calculated by the following equation (1).

[Equation 1]

{Variable Peak Height or Area / (Wild Peak Height or Area + Variant Peak Height or Area) x 100}

In the quantitative analysis method of FLT3 gene mutation according to the present invention, the minimum number of DNA detection is 10, and the detectable concentration is preferably 3 to 100%.

When the FLT3 gene mutation quantitative analysis method according to the present invention is used, it is inexpensive compared to the real-time quantitative PCR method used in the prior art, and it is possible to measure the quantitative value relatively easily and efficiently for the variation showing various aspects such as FLT3-ITD. . In addition, the sensitivity, specificity, and sensitivity are more sensitive than conventional PCR results, and can be useful for analyzing the quantitative value of FLT3-ITD in patients with acute myeloid leukemia. It is expected to be high.

On the other hand, the present invention provides a kit for detecting the FLT3 gene mutation for FLT3 gene mutation quantitative analysis method.

Kits according to the invention comprise primers having sequences complementary to the nucleotide sequence of the FLT3 gene. The primer included in the kit according to the present invention may be composed of at least one nucleotide selected from the group consisting of the base sequences of SEQ ID NO: 1 and SEQ ID NO: 2 or base sequences complementary to these base sequences, respectively.

If the kit for detecting the FLT3 gene mutation of the present invention is applied to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus). thermophilus (Tth), Thermus filiformis, Thermis flavus, thermococcus literalis or thermally stable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs, the kit for detecting FLT3 gene mutations of the present invention When is applied to an immunoassay, the kits of the invention may optionally comprise a secondary antibody and a substrate of the label. Furthermore, the kits according to the invention can be produced in a number of separate packaging or compartments comprising the reagent components described above.

According to one embodiment of the invention, the kit for detecting the FLT3 gene mutation is a buffer solution; DNA polymerase; dNTPs; And a primer set having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

The present invention also provides a microarray for detecting a FLT3 gene mutation comprising a primer having a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.

In the microarray of the present invention, the primer is used as a hybridizable array element and is immobilized on a substrate. Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. The hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by a chemical bonding method or a covalent binding method such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine).

On the other hand, when the sample to be applied to the microarray of the present invention is a nucleic acid can be labeled (labeled), it can be hybridized with the array element on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

Furthermore, the present invention can provide a method for providing information for predicting the prognosis of acute myeloid leukemia, that is, PCR amplifying a DNA sample to be analyzed using a FLT3 specific primer; Capillary electrophoresis of the PCR amplification product; And it provides a method for providing information for predicting the prognosis of acute myeloid leukemia, including the step of quantitative analysis of FLT3 gene mutation by fragment analysis.

In the information providing method according to the present invention, when the quantitative analysis of the FLT3 gene mutation according to the present invention, the quantitative value of the FLT3 gene mutation contained in the DNA sample to be analyzed is 50% or more or the mutation length is 70bp or more. The risk of recurrence of leukemia can be judged to be high. On the other hand, if the quantitative value of the FLT3 gene mutation is less than 50% and the mutation length is less than 70 bp, it can be determined that the risk of relapse is low.

Such information may be useful for predicting survival and recurrence rate of patients, and may be used for predicting survival and prognosis after hematopoietic stem cell transplantation.

Hereinafter, an embodiment is provided to help understanding of the present invention. However, the following examples are merely to help understanding of the present invention, the present invention is not limited by the following examples.

<Example 1>

Target and how

<1-1> Test subject

From April 2009 to March 2014, 182 specimens of FLT3-ITD-positive PCR were tested out of 4235 cases where the FLT3 gene mutation test was submitted to the Seoul National St. Mary's Hospital.

<1-2> DNA extraction

Bone marrow samples at the time of diagnosis were separated from the leukocyte layer by centrifugation, and then extracted with DNA using the QIAamp DNA blood kit (QIAGEN, Germany) according to the instructions of the reagents. Extracted DNA was diluted with 1X TBE buffer to 50 ~ 100ng per 1ul. Primer for quantification of FLT3-ITD attached a fluorescent dye to 5 and the base sequence of the primer is shown in Table 1 below.

Figure 112015110356174-pat00001

<1-3> PCR  Mixture manufacturing

Reaction preparations for PCR amplification were: 10ul reaction buffer with MgCl 2 2.5ul, 200umol dNTPs 0.5ul, 5U Hotstar Taq polymerase (QIAGEN) 0.25ul, 10pmol F Primer 1.5ul, 10pmol R Primer 1.5ul, and DW 13.75 ul was mixed. 20ul of the mixture was added to each PCR tube, and 5ul of diluted DNA was added to prepare a final reaction solution of 25ul.

<1-4> PCR reaction

The PCR instrument used a C-1000 thermal cycler (Bio-Rad, USA).

It was performed once at 95 ° C. for 5 minutes, 30 seconds at 95 ° C., 1 minute at 56 ° C., and 35 times at 72 ° C. for 2 minutes, and then reacted at 60 ° C. for 30 minutes.

After the reaction, the PCR product was electrophoresed using 2% agarose gel to confirm amplification, and the PCR product was diluted 10-fold with distilled water. 1 ul of diluted PCR product was added to a reaction mixture of 10 ul of HiDi formamide and 0.2 ul of size standard, and reacted at 95 ° C. for 2 minutes in a PCR device. The reaction solution was electrophoresed using a 3130xl sequencer (ABI, USA) and analyzed using GeneMapper Software v4.1.

<Example 2>

FLT3-ITD Quantitative Analysis

Quantitative analysis of FLT3-ITD was calculated as the percentage of FLT3-ITD mutants divided by the total FLT3 (sum of wild type and mutant). {Mutant peak height or area / (wild type) peak height or area + mutant peak height or area) x 100}

As a result of analyzing the normal sample by the FLT3-ITD quantitative assay, as shown in Table 2, all 50 samples showed a negative result, confirming that the test method of the present invention can specifically amplify only the mutant, All of the 182 positive samples were detected by the FLT3-ITD assay (median 32.3%, 2.8-94.7%).

Figure 112015110356174-pat00002

As shown in Table 3 below, the minimum detected DNA copy number of each of the wild type and the mutant type was 10.

Figure 112015110356174-pat00003

< Example  3>

Validation of FLT3-ITD Quantitative Methods

In order to determine the specificity of the FLT3-ITD quantitative analysis method of the present invention, the normal bone marrow donor 50 samples were analyzed, and the FLT3 gene was cloned by TOPO TA cloning kit (Invitrogen, USA) to see the detection sensitivity, reproducibility and linearity. The wild type and mutant plasmid DNAs were extracted, respectively. Using the extracted DNA, samples were prepared by mixing and diluting FLT3 mutant alleles from 100% to 0% for comparative analysis. The new FLT3 according to the present invention was tested on 182 specimens which showed FLT3-ITD positive by PCR test among 4235 cases where the FLT3 gene mutation test was submitted to the Catholic University of Seoul St. Mary's Hospital between April 2009 and March 2014. The clinical significance of the -ITD assay was evaluated.

<3-1> Detection Sensitivity and Reproducibility Verification Results

As shown in Table 4 below, the lowest detectable concentration measured through wild-type and mutant mixed dilution experiments showed a sensitivity of 10% in the conventional PCR method, while the FLT3-ITD quantitative method of the present invention showed a sensitivity of 3%. It was shown to show higher sensitivity compared to the conventional PCR method. Therefore, it can be seen that the method proposed in the present invention can quantitatively analyze FLT3-ITD with higher accuracy and higher sensitivity.

Figure 112015110356174-pat00004

<3-2> Linearity  Verification result

As shown in Fig. 1 and Fig. 2, in the linearity evaluation, the r value was 0.991, and the value obtained by using the height of the peak and the area of the peak when calculating the quantitative percentage was calculated as y = 1.0297x (y: Height, x: area).

In addition, 75 patients (male 41, female 34, age distribution 18-79 years, median age 49) who were asked for FLT3-ITD tests at the initial diagnosis of AML were found to have a median 35.2% (9.3-69.2%). (See FIG. 3), and the variation ratio of subtypes according to AML diagnostic criteria is shown in Table 5 below.

Figure 112015110356174-pat00005

<3-3> Analysis result of association with therapeutic outcome

Furthermore, the present inventors conducted the following experiments to confirm whether the method of the present invention was actually applied to the patient, and whether or not it was associated with therapeutic results. The duration of the patient's survival from the time point was compared. The survival rate between the FLT3-ITD-positive group over 50% and less than 50% was compared by Kaplan-Meier survival analysis. Disease free survival refers to the length of time the disease survived without recurrence of bone marrow morphology and molecular genetics. The survival rate was compared between FLT3-ITD positive and> 50% groups by Kaplan-Meier survival analysis.

As shown in FIG. 4 and FIG. 5, the results of quantitative analysis of FLT3-ITD have a significant effect on overall survival, disease free survival, and survival after hematopoietic stem cell transplantation. In other words, according to the quantitative analysis of FLT3-ITD, the patient group was divided into more than 50% and less than 50% of the FLT3-ITD, and as a result, the percentage of leukemia cells in the peripheral blood was high in the group of 50% or more. (Groups with an average of 63% vs. 50% or more appear as 81%).

In addition, the rate of reaching complete remission (a state in which little leukemia cells can be observed) after 2 cycles of chemotherapy was found to be lower in the group with 50% or more of FLT3-ITD (54.5% in the group of 50% or more). vs. groups below 50% appear to be 71.2%). The relapse rate after complete remission was 50.0% in the group with 50% or more of FLT3-ITD quantitative analysis, and 27.0% in the group with less than 50%. I could see that it can be used. In addition, overall survival and disease-free survival rate was also shorter than the group with less than 50% of FLT3-ITD, and it can be seen that survival and recurrence rate can be predicted by the method of the present invention, especially in patients with hematopoietic stem cell transplantation. (See FIGS. 4 and 5).

<Example 4>

Additional clinical trial subjects and methods

FLT3 mutation was detected in 363 patients diagnosed as acute myelogenous leukemia at the Catholic University of Seoul, Seoul. The results were as follows: FLT3-ITD mutation was detected in 73 (20.1%) patients. In the experiment, the PCR test used the same method as in Example 1.

Example 5

Association between FLT3-ITD Quantitative Analysis and Acute Myeloid Leukemia Treatment Results

The present inventors conducted the following experiments to confirm whether the method of the present invention is actually associated with therapeutic results when applied to a patient, and the long-term survival rate is determined from the time of diagnosis, even if the disease is relapsed or only partially treated. The length of time the patient has survived The survival rate of the group with FLT3-ITD positive content more than 50% and less than 50%, and the mutation length more than 70bp and less than 70bp was compared by Kaplan-Meier survival analysis.

Uncomplicated survival rate refers to the recurrence of acute myeloid leukemia symptoms after treatment, the duration of survival without developing a new bone marrow-related disease, and the survival rate of the group with a FLT3-ITD positive content of 50% or more and less than 50% and a mutation length of 70bp or more. Survival rates of the group less than 70bp were compared by Kaplan-Meier survival analysis.

As shown in FIGS. 6 and 7, the results of quantitative analysis of FLT3-ITD were found to have a significant effect on overall survival, disease free survival, and survival after hematopoietic stem cell transplantation. In other words, according to the quantitative analysis of FLT3-ITD, the patient group was analyzed by dividing the FLT3-ITD content by 50% or more, or 70 bp or more in mutation length, the FLT3-ITD content by less than 50%, and mutation length less than 70 bp. In the group with 50% or more of FLT3-ITD and 70bp or more of mutation length, the leukemia (blast) ratio of peripheral blood was high.

In addition, in patients with acute myeloid leukemia showing normal chromosomes, the prognosis was improved by hematopoietic stem cell transplantation when the mutation was quantified within 50% or the mutation length was 70 bp or less. However, if the mutation is more than 50% or the mutation length is 70bp or more, hematopoietic stem cell transplantation does not improve the therapeutic results. Therefore, it is expected that a new therapy, for example, an FLT3 inhibitor or the like will be required.

Furthermore, the rate of reaching complete remission (a state in which little leukemia cells can be observed) after 2 cycles of chemotherapy was found to be lower in the group with more than 50% of the FLT3-ITD content or 70bp of mutation length. . The relapse rate after complete remission was 6.6% in the group with a FLT3-ITD quantitative value of 50% or more and a mutation length of 70bp or more, and 13.5% in a group with a FLT3-ITD quantitative value of less than 50% and a mutation length of 70bp or less. As shown, it can be seen that the method of the present invention can be used as a means for predicting the degree of recurrence after treatment.

In addition, overall survival and disease-free survival were also shorter in the group with FLT3-ITD content of 50% or more than 70 bp in length, compared to the group with FLT3-ITD quantification less than 50% and mutation length of less than 70 bp. In patients with hematopoietic stem cell transplantation, the survival rate and recurrence rate can be predicted by the method of the present invention (see FIGS. 6 to 8).

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Industry-Academic Cooperation Foundation of Catholic University <120> Method for quantitative analysis of FLT3 gene mutation and kit <130> PN1508-226 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FLT3 forward <400> 1 tttaggtatg aaagccagct aca 23 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> FLT3 reverse <400> 2 agcattttga cggcaacct 19

Claims (13)

PCR amplifying a DNA sample of blood to be analyzed using a primer set represented by nucleotide sequences of SEQ ID NOs: 1 and 2 with respect to the FLT3 gene and labeled with 5 'fluorescent material;
Capillary electrophoresis of the PCR amplification product to analyze the length of the FLT3 gene mutation (FLT3-ITD), and quantitatively analyzing the minimum detected DNA copy number to 10 copy numbers;
Acute myeloid leukemia has a poor prognosis when the length of the FLT3 gene mutation (FLT3-ITD) is 70bp or more and the quantitative analysis value is 50% or more. A method for quantitative analysis of FLT3 gene mutations (FLT3-ITD) for prognostic assessment of acute myeloid leukemia, comprising: assessing that the prognosis of myeloid leukemia is good. delete delete The method of claim 1,
The PCR amplification,
Reaction once for 3 to 5 minutes at 90 ~ 100 ℃;
20 to 40 seconds at 90 to 100 ℃, 30 seconds to 1 minute at 55 to 65 ℃ and 1 to 3 minutes at 70 to 75 ℃ repeated for 30 to 40 times the reaction: And
Method for quantitative analysis of FLT3 gene mutation (FLT3-ITD) for prognostic evaluation of acute myeloid leukemia, characterized in that it comprises a reaction for 30 minutes at 60 ℃. delete The method of claim 1,
The quantitative analysis is a method of quantitative analysis of FLT3 gene mutations (FLT3-ITD) for prognostic evaluation of acute myeloid leukemia, characterized in that performed using GeneMapper software. The method of claim 1,
The quantitative analysis is a quantitative analysis method of FLT3 gene mutation (FLT3-ITD) for prognostic evaluation of acute myeloid leukemia, characterized in that calculated by the following equation (1);
[Equation 1]
{Variable peak height or area / (wild peak height or area + variant peak height or area) x 100}. delete PCR buffer solution;
DNA polymerase;
dNTPs; And
Kit for detecting the FLT3 gene mutation for prognostic evaluation of acute myeloid leukemia, comprising a primer set represented by the nucleotide sequence of SEQ ID NO: 1 and 2 and labeled with 5 'fluorescent material. PCR amplifying a DNA sample of blood to be analyzed using a primer set represented by nucleotide sequences of SEQ ID NOs: 1 and 2 with respect to the FLT3 gene and labeled with 5 'fluorescent material;
Capillary electrophoresis of the PCR amplification product to analyze the length of the FLT3 gene mutation (FLT3-ITD), and quantitatively analyzing the minimum detected DNA copy number to 10 copy numbers;
Acute myeloid leukemia has a poor prognosis when the length of the FLT3 gene mutation (FLT3-ITD) is 70bp or more and the quantitative analysis value is 50% or more. Evaluating that the prognosis of myeloid leukemia is good; providing information for prognostic assessment of acute myeloid leukemia according to the FLT3 gene mutation (FLT3-ITD). delete The method of claim 10,
The quantitative analysis is an information providing method for prognostic evaluation of acute myeloid leukemia according to the FLT3 gene mutation (FLT3-ITD), characterized in that calculated by the following equation (1);
[Equation 1]
{Variable peak height or area / (wild peak height or area + variant peak height or area) x 100}.
delete
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