KR101922761B1 - A Real-time Quantitative PCR Analysis for the c-Kit mutant detection to Diagnose an Acute Myeloid Leukaemia and the Use thereof - Google Patents

A Real-time Quantitative PCR Analysis for the c-Kit mutant detection to Diagnose an Acute Myeloid Leukaemia and the Use thereof Download PDF

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KR101922761B1
KR101922761B1 KR1020150161493A KR20150161493A KR101922761B1 KR 101922761 B1 KR101922761 B1 KR 101922761B1 KR 1020150161493 A KR1020150161493 A KR 1020150161493A KR 20150161493 A KR20150161493 A KR 20150161493A KR 101922761 B1 KR101922761 B1 KR 101922761B1
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김용구
김명신
장우리
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가톨릭대학교 산학협력단
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Abstract

The present invention relates to a method for detecting c-Kit mutation using a quantitative real-time PCR (qRT-PCR) method for the diagnosis of acute myelogenous leukemia, and more particularly to a method for detecting mutation of c-Kit mutant D816V, D816Y, D816H, N822K mutation The present invention also relates to a method for tracking a prognosis and a micro residual disease by a real-time quantitative PCR method for detecting an acute myelogenous leukemia.

Description

[0001] The present invention relates to a c-Kit mutant real-time quantitative PCR assay for the diagnosis of acute myelogenous leukemia,

The present invention relates to a method for detecting a c-Kit mutation using a real-time quantitative PCR (RQ-PCR) method for the diagnosis of acute myelogenous leukemia, and more particularly to a method for detecting a c-Kit mutation using the D816V, D816Y, D816H, N822K mutation The present invention relates to a method for diagnosing acute myelogenous leukemia (AML), prognostic evaluation, and tracking a micro residual disease by RQ-PCR.

Leukemia (leukemia) is a generic term for the positive proliferation of leukemia. Leukemia is divided into myeloid leukemia and lymphoid leukemia according to leukocyte origin, and acute leukemia and chronic leukemia according to progression rate. The clinical manifestations of leukemia vary according to the type of disease and the nature of the affected cells. Lymphocytic leukemia is caused by lymphocytic hematopoietic cells, myeloid leukemia by myeloid hematopoietic cells, and chronic myelogenous leukemia by mature cells, and acute myelogenous leukemia is a bone marrow Resulting from disorder of the germ cell

Among these, acute myeloid leukemia (AML) is a blood cancer in which abnormal cells that interfere with the production of normal white blood cells are produced and accumulated in red bone marrow. It usually occurs in adults, and the older the disease, the higher the incidence.

Symptoms of AML include the fact that the number of blood cells (red blood cells, platelets, normal white blood cells) is reduced as normal bone marrow is filled with leukemia cells. Major symptoms include fatigue, mild breathing, frequent bruising or bleeding, and frequent infections. There are a number of things that are suspected of causing AML, but the exact cause of AML is unknown.

The c-kit gene mutation has been reported in patients with acute myeloid leukemia (AML), which is found in 12.7-48.1% of adult patients with acute myelogenous leukemia (AML) Mutations are found at the positions of exons 8 and 10, but point mutations are found in codon D816 and N822 of exon 17, which is a kinase domain mainly. Therefore, c-kit

By examining the exon 9, 11, 13, and 17 mutations of the gene, the prognostic factors of gastrointestinal stromal cancer or acute myelogenous leukemia can be evaluated and used for treatment and follow-up. Thus, expression of c-KIT protein is known to be involved in tumor invasion or metastasis and is associated with prognosis (Longley BJ et al., Leuk Res, 25 (7): 571-576, 2001). Therefore, the presence or absence of these mutations in the c-kit gene is very important for, for example, diagnosis of the above-mentioned diseases and selection of a more effective treatment method for diseases.

In particular, the frequency of c-KIT mutations in patients with central-binding-factor-related acute myelogenous leukemia (CBF AML) differed according to the method of examination, indicating that about 20% In the test, only 46.2% have been reported.

The differences in these tests have also been influenced by clinical evaluation. Recently, various methods have been applied to quantify c-KIT mutations and it has been reported that mutation allele burden is associated with poor prognosis.

However, in the case of c-KIT mutant DNA detection methods of conventional AML, especially CBF AML patients, sensitivity and specificity are not high enough to exhibit satisfactory results in terms of detection limit, detection range, and quantitation limit.

The present inventors have developed a quantitative assay for detecting the D816V, D816Y, D816H, N822K mutations of c- KIT gene by using a real-time quantitative PCR (RQ-PCR) method new, and c- KIT gene mutation devise a quantitative numerical calculation By setting the cut-off value that affects the prognosis, various information for AML, especially CBF AML diagnosis, can be verified and evaluated, the prognosis of such a disease and the possibility of its use as a trace index of residual disease are confirmed, Completed.

1. Korean Patent Publication No. 10-2010-0035404 2. Korean Patent Publication No. 10-2008-0010597 3. Korean Patent Publication No. 10-2014-0138885

1. Hinchey J, Chaves C, Appignani B, Breen J, Pao L, Wange, et al. A reversible posterior leukoencephalopathy syndrome. N Engl J Med 1996; 334: 494-500. 2. Shin RK, Stern JW, Janss AJ, Hunter JV, Liu GT. Reversible posterior leukoencephalopathy during the treatment of acute lymphoblastic leukemia. Neurology 2001; 56: 388-391.

It is an object of the present invention to provide a c-kit mutation detection method using real-time quantitative PCR analysis for the diagnosis of acute myelogenous leukemia.

Another object of the present invention is to provide a c-kit mutation detecting composition for real-time quantitative PCR analysis for the diagnosis of acute myelogenous leukemia and a kit containing the same.

Another object of the present invention is to provide information necessary for the diagnosis and prognostic evaluation of acute myelogenous leukemia by c-kit mutation detection using real-time quantitative PCR analysis.

In order to solve the above control,

D816V, D816Y, D816H, D816F, N822K (A), and N822K (A) of the c-KIT gene were amplified using a real-time quantitative PCR (RQ-PCR) method on samples obtained from patients suspected of having acute myeloid leukemia (G), and comparing said expressed level with that expressed in normal individuals, said method comprising the steps of: comparing said expression level with that of a normal person, Or a cut-off value. The present invention also provides an information providing method for diagnosing or diagnosing acute myelogenous leukemia.

Most preferably, the acute myelogenous leukemia is a central binding factor acute myeloid leukemia (CBF AML).

In addition, the diagnosis includes progression, recurrence, metastasis, prognosis evaluation, and whether or not micro-remnant disease is traced, of acute myelogenous leukemia.

In this case, in the above-described real-time quantitative PCR (RQ-PCR) method, c- KIT gene mutation quantitative levels in characterized in that to determine the copy number mutant gene per 100 copies reference gene, in one embodiment, the c-KIT In the gene mutation quantification, the median mutant level was determined as 0.3 and the cutoff value was 10. The D816 mutation quantification value has a higher mutation quantification value than the N822K mutation.

The 3-year survival rate was 35.6% in patients with a high c- KIT gene mutation level (> 10), a mutation level Statistically significantly lower than patients with low (<10) and non-mutated patients.

In addition, whether or not the micro residual disease is traced is characterized in that when the D816V and D816Y mutation quantitative values of the c-KIT gene are amplified, it is determined that there is recurrence and micro residual disease.

The c-KIT mutation assay using the real-time quantitative PCR (RQ-PCR) of the present invention is excellent in sensitivity and specificity, so that mutations of D816V, D816Y, D816H and N822K can be quantitatively measured easily and effectively. In addition, the cut-off value that affects the prognosis can be used to estimate the prognosis of acute myelogenous leukemia using the quantitative calculation method of mutation of c-KIT gene of the present invention. Based on this, the diagnosis of acute myelogenous leukemia, The disease can be traced and the utilization will be very high.

FIG. 1 and FIG. 2 show one program of a holding stage and a cycling stage for carrying out RQ-PCR of the present invention.
Fig. 3 is a graph of the linearity evaluation result of each mutation of c- KIT .
FIG. 4 is a graph showing the relationship between c- KIT This is the result of analysis of mutation level.
FIG. 5 is a numerical value calculation and cut-off value of the c- KIT gene mutation obtained by calculating the number of mutation gene copies per 100 copies of the reference gene.
FIG. 6 is a graph showing the combination of mutations in a total of 111 patients with CBF AML.
Fig. 7 shows the gene mutation quantification values for the D816 mutation and the N822K mutation.
Figure 8 shows comparison values of mutation quantification values in t (8; 21) AML patients and inv (16) AML patients.
Figure 9 shows a comparison of mutation quantities in patients with more than two mutations and patients with single mutations.
FIG. 10 is a graph showing the long-term survival rate (OS) and the unsafe survival rate (EFS) for predicting the prognosis of acute myelogenous leukemia.
FIG. 11 shows the result of observing the numerical value of gene mutation according to the recurrence of the gene mutation in order to trace the micro residual disease of acute myelogenous leukemia.

The terms used in the present invention are defined as follows.

&Quot; Diagnosis " means identifying the presence or characteristic of a pathological condition. In particular, the present invention is useful for the diagnosis of acute myelogenous leukemia, particularly, a central binding factor acute myelogenous leukemia (CBF AML). The present invention includes prediction of recurrence progression and metastasis of acute myelogenous leukemia. Therefore, it is very important to accurately predict the recurrence and progression of acute myelogenous leukemia. Factors that can predict the response of the treatment while complementing the clinical indications such as the degree of differentiation and stage of the disease are required. The c-KIT mutation Real-time quantitative PCR analysis of the combination can be used as a diagnostic tool for acute myelogenous leukemia since it has such an indicator function. That is, the measurement of the expression characteristics of these c-KIT mutant genes by the real-time quantitative PCR analysis method can be used as an indicator (diagnostic marker) for predicting the differentiation, stage and progression of acute myelogenous leukemia. In the present invention, the term " diagnosis " includes prognostic evaluation and ability to follow up residual microdisease.

"Diagnostic markers or diagnostic markers are substances that can distinguish acute myelogenous leukemia cells from normal cells and include polypeptides or nucleic acids that show increased patterns in cells with acute myelogenous leukemia compared to normal cells, mRNA, etc.), lipids, glycolipids, glycoproteins, sugar (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

&Quot; Subject " or " patient " means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.

&Quot; Tissue or cell sample " refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.

&Quot; Nucleic acid " is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

&Quot; Gene " means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term " gene expression " generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide. The present invention encompasses all cases of methylation, mRNA expression, and protein expression of a gene promoter as a mode of gene expression.

&Quot; Primer " refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .

&Quot; Probe " means a nucleic acid fragment such as RNA or DNA corresponding to several bases or several hundreds of bases in a short time, which can achieve specific binding with mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. In the present invention, it is possible to predict the absolute amount of microorganisms in the sample through hybridization after hybridization using a complementary probe to a gene to be detected or quantified. Selection of suitable probes and hybridization conditions may be modified based on those known in the art.

&Quot; Protein " also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein

&Quot; Label " or " label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

The term " up-regulation " refers to the expression of a specific gene into mRNA or a marked increase in the amount of protein expressed by gene transcription or gene translation, as compared to normal tissue cells it means.

"Prognosis" refers to the progression of disease and the prediction of outcome. In the present invention, acute myeloid leukemia (AML), preferably central oncogenic factor, acute myelogenous leukemia (CBF AML) , Microscopic residual disease, and the like.

&Quot; Treatment " means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, " treatment " may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented. &Quot; Palliating " a disease may reduce the extent of the disease state and / or undesirable clinical symptoms and / or delay or slow the time course of the progression, It means to lose.

The term " kit " refers to a tool that enables absolute quantification of a target microorganism by confirming the number of copies of a gene to be detected or quantified in a sample by real-time quantitative PCR. The kit for quantification according to the present invention can be used not only as an artificial base sequence containing at least one primer and / or probe site specific to a gene to be detected or quantified as a standard sample for preparing a check for real-time quantitative PCR, One or more other component compositions, solutions, or devices may be included.

&Quot; about " means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising " and " comprising &quot;, unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The disease to be diagnosed in the present invention is an acute myelogenous leukemia, more preferably a disease associated with a central binding factor acute myeloid leukemia (CBF AML).

The central binding factor acute myeloid leukemia (CBF AML) has chromosomal abnormalities of (8; 21) (q22; q22) or inv (16) (p13.1q22) And 15%.

The acute myelogenous leukemia of the present invention was classified according to the French-American-British (FAB) classification as revised in 1985 according to the morphological findings and cytochemical findings on the optical microscope. (1) M0 (minimal differentiated acute myelogenous leukemia , M3 (acute myelomonocytic leukemia), M4 (acute myeloblastic leukemia), M3 (acute myeloblastic leukemia), M3 (acute myeloblastic leukemia) acute monoblastic leukemia or acute monocytic leukemia), ⑦ M6 (acute erythroid leukemias), and ⑧ M7 (acute megakaryoblastic leukemia).

According to the National Comprehensive Cancer Network (NCCN) guidelines in 2014, CBF AML is classified as a prognostic group with a better prognosis than the other subtypes and with a high risk of molecular genetics. However, in the case of c-KIT mutation, And the risk of recurrence was significantly higher than that of the control group. In particular, mutations occur frequently at the D816 and N822 positions of the 17th exon, which is known to be associated with a poor prognosis of AML.

The present invention relates to a method for evaluating the diagnosis and prognosis of acute myelogenous leukemia using the mutation combination at the D816 and N822 positions.

In particular, the present inventors have newly developed a quantitative assay for detecting the combination of the mutations of D816V, D816Y, D816H, and N822K of the c- KIT gene using a real-time quantitative (qRT) PCR method and confirmed this by a diagnosis of acute myelogenous leukemia, And the first use as a follow-up index of residual disease.

Accordingly, the present invention relates to a diagnostic method using an acute myelogenous leukemia marker and a RQ-PCR method using a specific mutation combination of the c- KIT gene. Through the identification of the specific mutant combination, acute myelogenous leukemia, (CBF AML) can be identified and diagnosed. Progression, recurrence and metastasis of said acute myelogenous leukemia.

<c-kit mutation combination>

Therefore, the present invention relates in one aspect to the use of certain mutant combinations of the c- KIT gene, in particular as diagnostic markers of D816V, D816Y, D816H, N822K combinations.

Specifically, the expression level of at least one selected from the group consisting of the specific mutations D816V, D816Y, D816H and N822K of the c- KIT gene is analyzed to evaluate the diagnosis and prognosis of acute myelogenous leukemia. Further, And the use thereof.

The c-KIT protein can be obtained from known, for example, known human-derived sequences from known DBs, including, but not limited to, functional equivalents thereof. Preferably, the c-KIT protein of the present invention may comprise the amino acid sequence of SEQ ID NO: 1.

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. Significant diagnostic markers are those markers that are highly reliable with high validity and consistency in repeated measurements. The diagnostic markers of the present invention show the same results in repeated experiments with genes whose expression changes either directly or indirectly as well as the onset of acute myelogenous leukemia, particularly central connective factor acute myelogenous leukemia (CBF AML) These are highly reliable markers that are very large when compared to controls and have little chance of producing false results. Therefore, the diagnosis result based on the result of measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

The c-KIT protein expressed by the c-kit gene is a 145-kDa phosphorus protein and is a light-cell membrane receptor for a KIT ligand called stem cell factor (SCF) (Yarden Y. et al. , EMBO J., 6 (11): 3341-3351, 1987). kit gene is a member of the tyrosine kinase subclass III family including platelet-derived growth factor (PDGF), macrophage-colony stimulating factor, and flt3 Yarden et al., Nature, 323: 226-232, 1986; Coussens L. et al., Proc Natl Acad Sci USA, 86: 4917-4921, Et al., Proc Natl Acad Sci USA, 91: 459-463,1994). &Lt; / RTI &gt; When the stem cell factor is attached to the receptor tyrosine kinase (KIT), KIT is auto-phosphorylated and dimerizes to participate in the activation of AKT through PI3K, thereby participating in cell survival. It also regulates the MAPK pathway and is involved in cell growth and transcription.

However, when the c-kit gene mutation occurs, phosphorylation (ligand-independent phosphorylation) occurs in the absence of a ligand such as stem cell factor, and KIT is activated. Cell lines undergo factor-independent growth in factor-dependent growth, resulting in malignant cell hypertrophy (Hashimoto K. et al., Am J Seminars in Immunology, 12 (5): 487 (1986), pp. 183-180, 1996; Tsujimura T. et al., Blood, 93 (4): 1319-1329, 1993; -502, 2000).

The c-kit mutants D816V, D816Y, D816H, N822K mutants of the present invention include those produced by nucleoside replacement, deletion or addition. The addition, deletion or substitution may be associated with one or more nucleoside. The variants may be altered at the coding site, the non-coding site, or both. Changes at the coding site may result in conservative or non-conservative amino acid substitutions, deletions or additions.

The c-kit mutants D816V, D816Y, D816H, and N822K are variants in which the 816th D in the c-kit amino acid sequence of SEQ ID NO: 1 is mutated to V, Y, and H, respectively, and N822K is an amino acid sequence of SEQ ID NO: And the 822nd N is K.

At least 90% identical, more preferably at least 95%, 96%, 97%, 98% or 99% identical nucleotide sequence to the mutant sequence of the invention; Or isolated polynucleotides comprising nucleoside complementary to such exemplary sequences.

Thus, in the present invention, the quantitative real-time PCR analysis of the combination of the c-KIT mutant genes enables diagnosis of acute myelogenous leukemia and microscopic residual disease diagnosis. The c-KIT mutations can be used in particular in combination and can be diagnosed through comparison of standard or cut-off values.

<How to Perform>

The c-kit mutation detection method of the present invention may employ any known method capable of measuring the expression level of the c-kit mutation.

For the c-kit mutation detection method, polymerase chain reaction (PCR) followed by sequencing, mutation detection, fluorescence melting curve analysis (Satzger I. et al., British Journal of Cancer 99, 2065-2069, 2008), single-strand conformation polymorphism analysis (RL Paquette et al., Blood Cells 31: 159-168, 1996), enzyme linked immunosorbent assay Immunohistochemistry (Noack F. et al., Leuk Lymphoma. 45 (11): 2295-302, 2004) and ELISA (Wiesner et al., Neoplasia. 10 (9): 996-1003, 2008) ) And Pyrosequencing (Sundstrom M., Immunology. 108 (1): 89-97, 2003), which can complement low sensitivity detection.

However, in the present invention, it is most preferable to use Real-time Quantitative PCR (RQ-PCR).

The quantification of RQ-PCR is performed by measuring the gene amplified and accumulated in each cycle in real time and analyzing it by the amplification cycle. By this method, the conventional PCR method is used to analyze the amplification product after the completion of the fixed cycle, So that the accurate amount can be measured.

The most important parameter is CP (CT) value. This means that the amount of fluorescence generated by the separation of the probe reaches a constant reference value above the basal value, and the amount of the unknown gene is determined by the CP Values and standard curves. The present invention can be analyzed using known software that supports the production of such CP values, the preparation of standard curves, and the amount of initial genes.

In one embodiment, it may be carried out in the following manner.

The real-time PCR method is classified into absolute quantification and relative quantification. For this purpose, calibration of the standard sample is indispensable. Quantitative real-time PCR is performed simultaneously on standard and unknown samples to derive a calibration curve from the standard sample, and quantify the amount of target DNA in the unknown sample from this calibration curve. At this time, the calibration curve (x-axis, log10 (standard sample), y-axis, threshold frequency number from the standard sample) is ideal as the correlation index (R 2 ) approaches 1. In the embodiment of the present invention, the R 2 crystal coefficient of the D816V, D816H, D816Y, and N822K was superior to the R 2 coefficient of 0.998 or more.

Primers / probes that can be used in the quantitative real-time PCR method can be easily synthesized through the base sequence of the target DNA to be detected. .

In one embodiment, the method comprises performing RQ-PCR using at least one primer pair and a probe selected from the group consisting of a primer pair capable of amplifying c-kit mutation markers according to the present invention and a probe, To the amount expressed for a normal person.

Comparing the amplified amount with an amplified amount for a normal person is performed by a standard or a cutoff value.

The threshold value of D816V, D816Y, D816H, N822K and IC channels is set to 0.1, and the Ct value of C8 Check the value.

The c-KIT gene mutation quantification value in the present invention uses a reference gene (c-KIT exon 2) and a mutation gene copy number calculated by a known software in comparison with a quantitative standard graph.

A method for separating a total RNA (total RNA) to be used and a method for synthesizing cDNA from the same can be carried out by a known method. For a detailed description of this process, see Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); And Noonan, K.F. And can be inserted as a reference of the present invention.

Important probes can be labeled for detection and can be labeled, for example, as radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. It is well known in the art to appropriately label such a probe, and can be carried out by a conventional method.

&Lt; Diagnostic composition and kit &

The present invention also provides, in a different aspect, an acute myelogenous leukemia, preferably a myeloid leukemia, comprising one or more c-KIT mutant combinations selected from the group consisting of D816V, D816Y, D816H, and N822K, ) Diagnostic marker composition.

At this time, overexpression of the D816V, D816Y, D816H, and N822K mutant genes is confirmed by measuring gene expression levels. The above-mentioned 'gene expression level measurement' includes measuring the mRNA or its protein expression level.

According to one aspect of the present invention, the present invention measures the level of expression at the mRNA level through QR-PCR. For this purpose, a novel primer pair specifically binding to the mutant gene and a probe labeled with fluorescence are required. In the present invention, corresponding primers and probes specified by the specific nucleotide sequence may be used, but the present invention is not limited thereto. Can be used without limitation as long as it can specifically perform RQ-PCR by providing a detectable signal. In one embodiment of the present invention, FAM and VIC refer to fluorescent dyes.

From a similar point of view, the present invention relates to a kit or system for the diagnosis and prognosis evaluation of acute myelogenous leukemia, particularly for diagnosis or prognosis of central connective factor acute myelogenous leukemia (CBF AML).

Detection reagents and methods in which these reagents are used are as described above. Reagents capable of detecting such markers of the present invention can be present separately in divided containers, and in this sense, the present invention also relates to a device / apparatus that includes the marker detection reagent of the present invention in a divided manner.

In other embodiments, the detection reagent may be provided in the form of an array or chip comprising a microarray. Detection reagents can be attached to the surface of substrates such as glass or nitrocellulose, and array fabrication techniques are described, for example, in Schena et al., 1996, Proc Natl Acad Sci USA. 93 (20): 10614-9; Schena et al., 1995, Science 270 (5235): 467-70; And U.S. Pat. Pat. Nos. 5,599,695, 5,556,752, or 5,631,734. Detection reagents may be labeled indirectly, either directly or in sandwich form for detection. In the case of direct labeling methods, serum samples used in arrays and the like are labeled with fluorescent labels such as Cy3 and Cy5. Fluorescence intensity can be measured using a scanning confocal microscope, for example Affymetrix, Inc. Or from Agilent Technologies, Inc., and the like.

The kit of the present invention may further comprise one or more additional components required for binding analysis, for example, a binding buffer, reagents for sample preparation, a blood sampling syringe or a negative and / or positive control .

For example, if the kit of the present invention is applied to a PCR amplification procedure, the kit of the present invention may optionally comprise reagents necessary for PCR amplification, such as buffers, DNA polymerase (e.g., Thermus aquaticus (Taq), Thermus thermophilus Thermostable DNA polymerases obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs.

The kit of the present invention may be made from a number of separate packaging or compartments containing the above reagent components.

<Diagnosis and Information Delivery Method>

In another aspect, the present invention provides a method for diagnosing or diagnosing acute myelogenous leukemia, comprising the step of measuring the level of expression of a specific mutation of the c-kit gene using RQ-PCR.

In yet another embodiment, the invention provides a method for identifying a nucleic acid or protein level of one or more biomarkers selected from the group consisting of D816V, D816Y, D816H, and N822K from a biological sample of a subject to provide information necessary for the diagnosis or prognosis of acute myelogenous leukemia, Or a nucleic acid or a protein in a sample,

The method also provides information useful as a prognostic indicator, including a recurrence of acute myelogenous leukemia, particularly central connective factor acute myelogenous leukemia (CBF AML), and as a tracing index of micro-residual disease.

In one embodiment,

For acute myeloid leukemia (AML) samples from suspected patients

One or more mutant expression levels selected from the group consisting of D816V, D816Y, D816H, D816F, N822K (A) and N822K (G) of the c-KIT gene were measured using real-time quantitative PCR (RQ- Wherein the comparison is performed by a standard or cut-off value, wherein the comparison is performed by a standard or cut-off value. The method of claim 1, wherein the comparison is for a diagnosis or diagnosis of acute myelogenous leukemia. Provide information providing method. The diagnosis includes the progression, recurrence, metastasis, prognosis assessment, and the follow-up of the micro residual disease in acute myelogenous leukemia.

In this case, the sample is preferably a bone marrow. In the real-time quantitative PCR (RQ-PCR) method, the c-KIT gene mutation quantification value is determined as a mutation gene copy number per 100 copies of the reference gene.

If the mutated expression of the c-kit gene in the biological sample isolated from the patient is over-expressed in the normal control sample, it is judged to be acute myelogenous leukemia.

In one embodiment, in the c-KIT gene mutation quantification value, the median mutant level was determined as 0.3 and the cutoff value was determined as 10. The D816 mutation quantification value has a higher mutation quantification value than the N822K mutation.

The 3-year survival rate was 35.6% in patients with a high c-KIT gene mutation level (> 10), a mutation level Statistically significantly lower than patients with low (<10) and non-mutated patients.

Whether or not the micro residual disease is traced is characterized in that when the D816V and D816Y mutation quantitative values of the c-KIT gene are amplified, it is determined that there is recurrence and micro residual disease.

As such, the method is directed to investigating the expression characteristics of a particular marker according to acute myelogenous leukemia, and the methods disclosed herein are useful for obtaining data and information useful in assessing appropriate or effective therapies for the treatment of patients with acute myelogenous leukemia Efficient, and cost-effective means. &Lt; RTI ID = 0.0 &gt;

In another aspect, the present invention relates to a method for screening a substance for treating acute myelogenous leukemia, which inhibits the mutation expression of the c-kit gene based on the facts described above.

A change in expression of a marker according to the present invention in an acute myelogenous leukemia tissue or cell can be selected as a candidate agent for acute myelogenous leukemia therapy.

In one embodiment, the method comprises the steps of: providing cells comprising at least one mutation selected from the group consisting of D816V, D816Y, D816H, D816F, N822K (A) and N822K (G); Contacting the cell with the test substance;

Measuring the degree of expression of the nucleic acid sequence at an mRNA or protein level; And when the amount of expression of the at least one nucleic acid sequence is decreased in the cells to which the test substance has been treated as compared with the control group in which the test substance is not treated as a result of the measurement, the candidate substance is selected.

The present invention extends to a genetic approach to the upregulation or downregulation of c-kit mutant expression and gene expression.

The test substance used in the method of the present invention is a substance expected to regulate the expression of the marker gene according to the present invention. For example, for screening purpose of a drug, a compound having a therapeutic effect of a low molecular weight may be used.

For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such a library of compounds can be prepared by reacting peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles, and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto.

Thus, in the present invention, the RQ-PCR profile characteristics of acute myelogenous leukemia, in particular, the c-KIT mutation combination, were found to increase the sensitivity and specificity of the diagnosis and prognostic evaluation of acute myelogenous leukemia, Suggesting that the use of the gene as a diagnostic marker can be utilized in various ways.

<Examples>

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples. Unless otherwise indicated, nucleic acids are recorded in a 5 'to 3' orientation from left to right. The numerical ranges recited in the specification include numerals defining the ranges and include each integer or any non-integral fraction within a defined range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.

Object and method

1. Target

Between April 2009 and July 2013, 111 adult patients diagnosed with CBF AML at Seoul St. Mary's Hospital, Catholic University were used. Bone marrow specimens and 100 normal bone marrow donors were used to test specificity.

2. Method

Bone marrow specimens at the time of diagnosis were centrifuged to separate leukocyte layers, and DNA was extracted according to the guidelines of reagents using QIAamp DNA blood mini kit (QIAGEN, Germany). The extracted DNA was diluted with 1 × TBE buffer to 50 to 100 ng per 1 μl.

Screening  inspection

(1) RQ-PCR master mixture for reaction was prepared.

(Unit: μl) edifice Volume 2X PCR reaction mixture 12.5 C-kit Probe & Primer mix 3 IC Probe & Primer mix 3 DW 3.5 Total 22

(2) Mix RQ-PCR master mixture well and dispense 22 μl into 96-well plate or optical tube.

(3) Sample DNA and positive DNA were dispensed into each well at 3 μl.

(4) To confirm contamination of the PCR reaction, 3 μl of water was added to the negative control well.

(5) After mounting the tube on ABI PRISM 7500, the experiment was performed using the program stored in the procedure of FIG.

(6) Analysis of inspection

* Positive control

The threshold values of D816V, D816Y, D816H, N822K and IC channels were set to 0.1, and the Ct values of D816V, D816Y, D816H, N822K and IC amplified by positive control were confirmed. The range of D816V, D816Y, D816H, N822K and IC values is shown in Table 2. If the range is out of range, the test is repeated again. When no signal appeared in the negative control and amplification of either FAM or VIC was confirmed, the PCR reaction was performed again.

C- kit  ( VIC ) Ct IC
( FAM ) Ct Result Comment Positive control 27 ± 3 28 ± 3 Positive Valid Negative control Neg Neg Negative Valid

* Interpretation of results

① Set the threshold value of D816V, D816Y, D816H, N822K and IC channel to 0.1 and check the Ct value.

② Be sure to check the raw data of each sample to confirm whether it is amplified normally.

③ Analyze the results according to the table below.

C- kit  ( VIC ) Ct IC  ( FAM ) Ct Result Comment ≤38 ≤30 mutant > 38 or neg ≤ 30 Wild neg ≤ 30 Invalid Re-examine

Genotyping  inspection

Screening tests were performed to examine the mutant samples.

(1) RQ-PCR master mixtures of D816V, D816Y, D816H and N822K were prepared for each reaction.

(Unit: μl) edifice Volume 2X PCR reaction mixture 12.5 Probe & Primer mix
(D816V, D816Y, D816H or N822K) 3 IC Probe & Primer mix 3 DW 3.5 Total 22

(2) Mix RQ-PCR master mixture well and dispense 22 μl into 96-well plate or optical tube.

(3) Sample DNA and positive DNA were dispensed into each well by 3 μl.

(4) To confirm contamination of the PCR reaction, 3 μl of water was added to the negative control well.

(5) After mounting the tube on ABI PRISM 7500, the experiment was performed using the program stored in the procedure of FIG.

(6) Analysis of inspection

* Positive control

The threshold value of D816V, D816Y, D816H, N822K and IC channel was set to 0.1, and Ct value of D816V, D816Y, D816H, N822K IC amplified by positive control was confirmed. The range of D816V, D816Y, D816H, N822K, and IC values is shown in Table 5. If the range is out of range, the test is re-examined. When no signal was detected in the negative control and amplification of either FAM or VIC was confirmed, the PCR reaction was performed again.

D816V , D816Y , D816H ,
N822K  ( VIC ) Ct IC
( FAM ) Ct Result Comment Positive control 27 ± 3 28 ± 3 Positive Valid Negative control Neg Neg Negative Valid

* Interpretation of results

① Set the threshold value of D816V, D816Y, D816H, N822K and IC channel to 0.1 and check the Ct value.

② Be sure to check the raw data of each sample to confirm whether it is amplified normally.

③ The results were analyzed according to the contents of the table below.

VIC Ct FAM Ct Result Comment D816V ≤38 ≤30 D816V mutant > 38 or neg ≤30 Wild type D816Y ≤38 ≤30 D816V mutant > 38 or neg ≤30 Wild type D816H ≤38 ≤30 D816V mutant > 38 or neg ≤30 Wild type N822K ≤38 ≤30 N822K mutant > 38 or neg ≤30 Wild type Neg Neg Invalid Re-examine

④ Quantification of c- KIT gene mutation was performed using the reference gene ( c- KIT exon 2) and mutant gene number automatically calculated by software in comparison with the quantitative standard graph.

Example  1: Specificity and sensitivity evaluation

1-1 Verification of Specificity Using Normal Specimens

Analysis of normal specimens by real-time quantitative PCR showed that the test with negative results in all 100 specimens could specifically amplify only the mutant

1-2 c- KIT mutation quantitation test Minimum detection plasmid DNA copy number

The minimum detection DNA copy number of D816V, D816H, D816Y and N822K was less than 200 copies at 43.4, 13.8, 168.9 and 1.7 copies, respectively.

type D816V D816H D816Y N822K Number of copies (copy /?) 43.4 13.8 168.9 1.7

1-3 c- KIT Mutation Quantification Linearity Verification

In the D816V, D816H, D816Y, N822K linearity evaluation of the measurement using the mutant plasmid DNA of 5 different concentrations determined coefficient R 2 were superior in both 0.998 or more.

Comparative Example  1: Comparison with other methods

We compared the results of real - time quantitative PCR and melting curve analysis in 108 patients. The concordance rate between the two tests was 74.1%, which was the same in 80 patients. All patients who were positive in melting curve analysis showed positive results in real-time quantitative PCR. Twenty-eight patients who were negative in Melting curve analysis showed positive results in real-time quantitative PCR. As a result of quantitative analysis, they were all patients with low mutant allele burden, and they were positive by other highly sensitive test (pyrosequencing or MEMO-PCR) Respectively. Therefore, the real-time quantitative PCR test showed a higher sensitivity than the melting curve analysis (FIG. 4).


Example  2: Evaluation of clinical usefulness: calculation of quantitative value and prognosis cut - off  Set

D816V, D816H, D816Y and N822K mutations of the c-KIT gene were performed by real-time quantitative PCR in 111 patients with CBF AML (male 65, female 46, age range 18-72 years).

2-1. c- KIT gene mutation quantitative calculation and cut-off

Using the reference gene ( c- KIT exon 2) analyzed in the quantitative standard graph and the number of mutant gene copies, the quantification value of c-KIT gene mutation was calculated by calculating the number of mutant gene copies per 100 copies of the reference gene (mutation gene copy number / Reference gene 100 copies). When one person has multiple mutations (30/69, 43.5%), the quantification of all mutations is added. The distribution of mutant allele levels showed a median value of 0.3 (range 0.002-532.7) and a cut-off value of 10. This is shown in Fig.

2-2. Diagnosis of acute myeloid leukemia

Of the total 111 patients with CBF AML, 69 (62.2%) had D816 and N822 mutations in c- KIT gene, 14 (20.3%) were D816V, 7 were D816Y and 10 were D816H and D816H, respectively 5.8%), N822K (14.3%), and 30 patients (43.5%) had more than two mutations (Figure 6).

As shown in Figure 7, D816 mutant showed a higher value than quantitative genetic variation;; (range, median 0.006 to 43.53, 0.1) (P = 0.016) ( median, 0.7, 0.002 to 532.7) for N822K mutation .

Range, 0.002-532.7) and inv (16) AML patients with t (8; 21) AML patients, as shown in Figure 8, 293.0) showed no statistically significant difference ( P = 0.243).

And, as shown in Figure 9, the mutation quantitative comparison of patients with two or more mutations (median, 0.2; range, 0.002-490.9) and patients with single mutation (median, 2.2; range, 0.002-532.7) There was no statistically significant difference between the two groups ( P = 0.077).

Example  3: Of acute myelogenous leukemia  Prognosis prediction

c - KIT quantitation showed a significant effect on long-term survival (OS) and unresponsive survival (EFS) when the variable quantification level was divided into the high and low groups based on the quantitative value of c- KIT gene mutation 10 10)

In patients with high c- KIT gene mutation levels (> 10), the 3-year survival rate was 35.6%, patients with low mutation levels (<10) (3-year survival rate, 72.5% Year survival rate, 72.5%). There was no significant difference in the 3-year survival rate between the low-dose group and the non-mutated group ( P = 0.943). The 3-year unresponsive survival rate was also lowest in the high-dose group (28.9%, P = 0.0252) and the 3-year unresponsive survival rate was not significantly different between the low-dose group and the non-mutation group (61.2% vs. 63.9%, P = 0.963).

Example  4: Of acute myelogenous leukemia  Tracking micro residual disease

After treatment, 27 patients out of c- KIT gene mutations were detected in the samples at diagnosis was the result of both hematological negative results underwent c- KIT gene mutation test in real-time quantitative PCR method in remission. In 6 of the relapsed patients, the c- KIT gene mutation observed at the time of diagnosis was also observed at relapse (Fig. 11).

In particular, two mutations were observed in each of the two patients, and it was confirmed that a small amount of gene mutation, which can be detected only by a highly sensitive test at the time of diagnosis, was amplified upon recurrence 0.2 in recurrence, 40.9 in diagnosis, 0.01 in recurrence and 4.9 in recurrence).

These results indicate that the c- KIT mutation using real-time quantitative PCR of the present invention The quantitative method is superior in sensitivity and specificity, and it can be seen that the D816V, D816Y, D816H, and N822K mutations can be quantitatively measured relatively easily and effectively.

In addition, when the cut-off value of the present invention is used as a reference, the prognosis is analyzed using the quantification value of the c- KIT gene mutation. Based on these results, utilization in the future of AML patients will be high.

<110> Industry Academic Cooperation Foundation of Catholic University <120> A Real-time Quantitative PCR Analysis for the c-Kit mutant          detection to Diagnose an Acute Myeloid Leukaemia and the Use          the <130> PN1508-245 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 976 <212> PRT <213> c-kit animoacid sequence <400> 1 Met Arg Gly Ala Arg Gly Ala Trp Asp Phe Leu Cys Val Leu Leu Leu   1 5 10 15 Leu Leu Arg Val Gln Thr Gly Ser Ser Gln Pro Ser Val Ser Pro Gly              20 25 30 Glu Pro Ser Pro Pro Ser Ile His Pro Gly Lys Ser Asp Leu Ile Val          35 40 45 Arg Val Gly Asp Glu Ile Arg Leu Leu Cys Thr Asp Pro Gly Phe Val      50 55 60 Lys Trp Thr Phe Glu Ile Leu Asp Glu Thr Asn Glu Asn Lys Gln Asn  65 70 75 80 Glu Trp Ile Thr Glu Lys Ala Glu Ala Thr Asn Thr Gly Lys Tyr Thr                  85 90 95 Cys Thr Asn Lys His Gly Leu Ser Asn Ser Ile Tyr Val Phe Val Arg             100 105 110 Asp Pro Ala Lys Leu Phe Leu Val Asp Arg Ser Leu Tyr Gly Lys Glu         115 120 125 Asp Asn Asp Thr Leu Val Arg Cys Pro Leu Thr Asp Pro Glu Val Thr     130 135 140 Asn Tyr Ser Leu Lys Gly Cys Gln Gly Lys Pro Leu Pro Lys Asp Leu 145 150 155 160 Arg Phe Ile Pro Asp Pro Lys Ala Gly Ile Met Ile Lys Ser Val Lys                 165 170 175 Arg Ala Tyr His Arg Leu Cys Leu His Cys Ser Val Asp Gln Glu Gly             180 185 190 Lys Ser Val Leu Ser Glu Lys Phe Ile Leu Lys Val Arg Pro Ala Phe         195 200 205 Lys Ala Val Pro Val Val Ser Val Ser Lys Ala Ser Tyr Leu Leu Arg     210 215 220 Glu Gly Glu Glu Phe Thr Val Thr Cys Thr Ile Lys Asp Val Ser Ser 225 230 235 240 Ser Val Tyr Ser Thr Trp Lys Arg Glu Asn Ser Gln Thr Lys Leu Gln                 245 250 255 Glu Lys Tyr Asn Ser Trp His His Gly Asp Phe Asn Tyr Glu Arg Gln             260 265 270 Ala Thr Leu Thr Ile Ser Ser Ala Arg Val Asn Asp Ser Gly Val Phe         275 280 285 Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val Thr Thr Thr     290 295 300 Leu Glu Val Val Asp Lys Gly Phe Ile Asn Ile Phe Pro Met Ile Asn 305 310 315 320 Thr Thr Val Phe Val Asn Asp Gly Glu Asn Val Asp Leu Ile Val Glu                 325 330 335 Tyr Glu Ala Phe Pro Lys Pro Glu His Gln Gln Trp Ile Tyr Met Asn             340 345 350 Arg Thr Phe Thr Asp Lys Trp Glu Asp Tyr Pro Lys Ser Glu Asn Glu         355 360 365 Ser Asn Ile Arg Tyr Val Ser Glu Leu His Leu Thr Arg Leu Lys Gly     370 375 380 Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser Asp Val Asn 385 390 395 400 Ala Ala Ile Ala Phe Asn Val Tyr Val Asn Thr Lys Pro Glu Ile Leu                 405 410 415 Thr Tyr Asp Arg Leu Val Asn Gly Met Leu Gln Cys Val Ala Ala Gly             420 425 430 Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Pro Gly Thr Glu Gln         435 440 445 Arg Cys Ser Ala Ser Val Leu Pro Val Asp Val Gln Thr Leu Asn Ser     450 455 460 Ser Gly Pro Pro Phe Gly Lys Leu Val Val Gln Ser Ser Ile Asp Ser 465 470 475 480 Ser Ala Phe Lys His Asn Gly Thr Val Glu Cys Lys Ala Tyr Asn Asp                 485 490 495 Val Gly Lys Thr Ser Ala Tyr Phe Asn Phe Ala Phe Lys Gly Asn Asn             500 505 510 Lys Glu Gln Ile His Pro His Thr Leu Phe Thr Pro Leu Leu Ile Gly         515 520 525 Phe Val Ile Val Ala Gly Met Met Cys Ile Val Met Ile Leu Thr     530 535 540 Tyr Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val 545 550 555 560 Glu Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu                 565 570 575 Pro Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly             580 585 590 Lys Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala         595 600 605 Tyr Gly Leu Ile Lys Ser Asp Ala Met Thr Val Ala Val Lys Met     610 615 620 Leu Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu 625 630 635 640 Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu                 645 650 655 Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr             660 665 670 Cys Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser         675 680 685 Phe Ile Cys Ser Lys Gln Glu Asp His Ala Glu Ala Ala Leu Tyr Lys     690 695 700 Asn Leu Leu His Ser Lys Glu Ser Ser Cys Ser Asp Ser Thr Asn Glu 705 710 715 720 Tyr Met Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Ala                 725 730 735 Asp Lys Arg Arg Ser Val Arg Ile Gly Ser Tyr Ile Glu Arg Asp Val             740 745 750 Thr Pro Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Glu Asp         755 760 765 Leu Leu Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala     770 775 780 Ser Lys Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu 785 790 795 800 Thr His Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp                 805 810 815 Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro             820 825 830 Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Cys Val Tyr Thr Phe         835 840 845 Glu Ser Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser     850 855 860 Leu Gly Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr 865 870 875 880 Lys Met Ile Lys Glu Gly Phe Arg Met Leu Ser Pro Glu His Ala Pro                 885 890 895 Ala Glu Met Tyr Asp Ile Met Lys Thr Cys Trp Asp Ala Asp Pro Leu             900 905 910 Lys Arg Pro Thr Phe Lys Gln Ile Val Gln Leu Ile Glu Lys Gln Ile         915 920 925 Ser Glu Ser Thr Asn His Ile Tyr Ser Asn Leu Ala Asn Cys Ser Pro     930 935 940 Asn Arg Gln Lys Pro Val Val Asp His Ser Val Arg Ile Asn Ser Val 945 950 955 960 Gly Ser Thr Ala Ser Ser Gln Pro Leu Leu Val His Asp Asp Val                 965 970 975

Claims (8)

(1) Central binding factor N-linked immunoglobulin (A) of c-KIT gene was amplified by real-time quantitative PCR (RQ-PCR) in in vitro samples obtained from suspected patients with acute myeloid leukemia (CBF AML) Measuring a quantitative value of the mutation; And
(2) The above quantitative values were compared with the N822K (A) mutant quantitative value and And diagnosing and prognosing a disease associated with acute myelogenous leukemia when the cut-off value is 10; and providing information for diagnosis and prognosis of acute myelogenous leukemia. delete The method according to claim 1,
Wherein said diagnosis and prognosis includes progression, recurrence and metastasis of a central connective factor acute myelogenous leukemia. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
In the real-time quantitative PCR (RQ-PCR) method,
wherein the N822K (A) mutation quantification value of the c-KIT gene is determined by the number of mutation gene copies per 100 copies of the reference gene, and a method for providing information for diagnosis and prognosis of acute myelogenous leukemia. delete delete The method according to claim 1,
Wherein said prognosis is evaluated by performing a long-term survival rate (OS) and an unsafe survival rate (EFS) on the basis of the N822K (A) mutation quantitative value. delete
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