WO2017086589A1 - C-kit mutation quantitative real-time pcr analysis method for diagnosing acute myeloid leukemia, and use thereof - Google Patents

Abstract

The present invention relates to a method for detecting c-Kit mutations by using a quantitative real-time PCR (qRT-PCR) method in order to diagnose acute myeloid leukemia and, more specifically, to a method for diagnosing acute myeloid leukemia, evaluating the prognosis thereof, and performing a follow-up on minimal residual disease by using a qRT-PCR method for detecting D816V, D816Y, D816H and N822K mutations of a c-KIT gene.

Description

Real-time quantitative PCR assay and its use of c-mutant mutations for the diagnosis of acute myeloid leukemia

The present invention relates to a method for detecting c-mutant mutations using real-time quantitative PCR (RQ-PCR) for the diagnosis of acute myeloid leukemia, and more specifically, the D816V, D816Y, D816H, and N822K mutations of the c-KIT gene. The present invention relates to a method for diagnosing acute myeloid leukemia, evaluating prognosis, and tracking microresistive diseases by the RQ-PCR method for detecting.

Leukemia refers to a disease in which leukocytes proliferate on a tumor basis. The leukemia is divided into myeloid leukemia and lymphocytic leukemia according to leukemia from which leukemia originates, and is classified into acute leukemia and chronic leukemia according to the progress rate. The clinical manifestations of leukemia vary depending on the type of disease and the nature of the cells involved. Lymphocytic leukemia is caused by mutations in lymphoid blood cells, myeloid leukemia in myeloid blood cells, and chronic myeloid leukemia in mature cells. Acute myeloid leukemia is a bone marrow that begins to differentiate in a relatively early stage of hematopoiesis. Is caused by the failure of the stem cells

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

Symptoms of AML are caused by a sharp drop in the number of blood cells (erythrocytes, platelets, normal white blood cells) as normal bone marrow is filled with leukemia cells. The main symptoms are fatigue, shortness of breath, easy bruising or bleeding, and frequent infections.There are a number of alleged causes of AML, but the exact cause of AML is unknown.

C-kit gene mutations have been reported in patients with acute myeloid leukemia (AML), which are found in 12.7-48.1% of adult patients with acute myeloid leukemia (AML). Although mutations may occur at the positions of exons 8 and 10, point mutations are mainly found in codons D816 and N822 of exon 17, a kinase domain. Thus c-kit

By examining exon 9, 11, 13, 17 mutations in genes, the prognostic factors of gastrointestinal stromal cancer or acute myeloid leukemia can be evaluated, used for treatment and follow-up. Thus, expression of c-KIT protein is known to be involved in tumor invasion or metastasis and related to 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 example, in the diagnosis of the above-described diseases, the selection of more effective treatments for diseases, and the like.

In particular, the frequency of c-KIT mutations in patients with Core Binding Factor acute myeloid leukemia (CBF AML) differed according to the test method. Only 46.2% of the tests are reported.

The differences in these methods also influence the clinical significance. Recently, various methods have been applied to quantitatively measure c-KIT mutations, and it has been reported to be associated with poor prognosis when the mutation allele burden is high.

However, in the conventional detection method of c-KIT mutant DNA of AML, in particular, CBF AML patients, the sensitivity and specificity of the detection limit, the detection range, and the limit of quantification were not high.

The present inventors have developed a quantitative assay for detecting the D816V, D816Y, D816H, N822K mutation of the c-KIT gene by using a real-time quantitative PCR (RQ-PCR) method and new, c -KIT mutation devise a quantitative numerical calculation By setting cut-off values affecting prognosis, various information for diagnosing AML, particularly CBF AML, can be validated and evaluated, as well as the prognostic assessment of these diseases and their applicability as follow-up indicators of micro-residual disease and the present invention. Completed.

An object of the present invention is to provide a c-kit mutation detection method using a real-time quantitative PCR assay for the diagnosis of acute myeloid leukemia.

Another object of the present invention is to provide a c-kit mutation detection composition and kit comprising the same for performing a real-time quantitative PCR assay for the diagnosis of acute myeloid leukemia.

Still another object of the present invention is to provide information necessary for diagnosing and prognostic acute myeloid leukemia by c-kit mutation detection using real-time quantitative PCR analysis.

In order to solve the above control, the present invention

For samples obtained from patients with Acute Myeloid Leukaemia (AML), the D816V, D816Y, D816H, D816F, N822K (A) and N822K of c-KIT genes using real-time quantitative PCR (RQ-PCR). A method of providing information for diagnosing acute myeloid leukemia, comprising measuring at least one mutation expression level selected from the group consisting of (G) and comparing the expressed levels with those expressed for normal individuals, wherein the comparison is standard Or it provides a method for providing information for diagnosing or diagnosing acute myeloid leukemia, characterized in that performed by a cutoff value.

The acute myeloid leukemia is most preferably a core binding factor acute myeloid leukemia (CBF AML).

The diagnosis also includes determining the progression, recurrence, metastasis, prognosis, and follow-up of microscopic residual disease in acute myeloid leukemia.

At this time, in the real-time quantitative PCR (RQ-PCR) method, the c-KIT gene mutation quantitative value is characterized in that it is determined by the number of mutation gene copies per 100 copies of the reference gene, in one embodiment, the c-KIT For gene mutation quantitative values, the median mutant allele level was determined to be 0.3 and the cutoff value to 10. The D816 mutation quantification has a higher mutation quantification than the N822K mutation.

In addition, the prognostic evaluation can be performed by the long-term survival rate (OS) and accident-free survival rate (EFS) value, the three-year survival rate is 35.6% in patients with high c-KIT gene mutation quantification level (> 10), quantitative mutation It is statistically significantly lower than patients with low levels (<10) and without mutations.

In addition, whether or not the trace of the micro-remaining disease is characterized by recurrence and micro-remaining disease when the quantitative quantification of the D816V and D816Y mutation of the c-KIT gene is amplified.

The c-KIT mutation quantitative assay using the real-time quantitative PCR (RQ-PCR) of the present invention can be easily and effectively quantitated D816V, D816Y, D816H, N822K mutations due to its excellent sensitivity and specificity. In addition, it is possible to accurately evaluate the prognosis of acute myeloid leukemia using cut-off values affecting prognosis by the c-KIT gene mutation quantitative numerical calculation method of the present invention. The use of the disease can be traced back to it.

1 and 2 illustrate one program of a holding stage and a cycling stage for performing the RQ-PCR method of the present invention.

3 is a graph of the linearity evaluation results of each mutation of c-KIT .

Figure 4 shows the results of analyzing the c-KIT mutation level using the melting curve analysis (melting curve analysis).

5 is a quantitative numerical calculation and cut-off value of c-KIT gene mutations calculated by calculating the number of mutant gene copies per 100 copies of the reference gene.

6 is a graph showing the combination of mutations in a total of 111 CBF AML patients.

Figure 7 shows the genetic variation quantitative values for the D816 mutation and the N822K mutation.

FIG. 8 shows a comparison of mutant quantification values of t (8; 21) AML patients and inv (16) AML patients.

9 shows a comparison of mutational quantitative values of patients with two or more mutations and patients with a single mutation.

10 is a graph showing long-term survival (OS) and accident-free survival (EFS) for the prognosis of acute myeloid leukemia.

Figure 11 is a result of observing the quantitative value of the genetic variation according to the recurrence of the genetic mutation in order to trace the micro residual disease of acute myeloid leukemia.

Definitions of terms used in the present invention are as follows.

"Diagnosis" means identifying the presence or characteristic of a pathological condition. In particular, the present invention is useful for diagnosing acute myeloid leukemia, especially core binding factor acute myeloid leukemia (CBF AML). The present invention includes predicting recurrence progression and metastasis of acute myeloid leukemia. Therefore, the indicators that can accurately predict the recurrence and progression of acute myeloid leukemia are very important, and factors that can predict the response of treatment while complementing clinical indicators such as tissue differentiation and stage are needed, c-KIT mutation of the present invention The real-time quantitative PCR analysis of the combination functions as an indicator and can be used as a diagnostic factor for acute myeloid leukemia. That is, measurement of expression characteristics of these c-KIT mutant genes by real-time quantitative PCR analysis can be used as an indicator (diagnosis marker) useful for predicting the degree of differentiation, stage, and progression of acute myeloid leukemia. The term "diagnosis" in the present invention encompasses prognostic assessment and traceability of microresidual disease.

"Diagnostic marker or diagnostic marker (diagnosis marker) is a substance that can diagnose acute myeloid leukemia cells and distinguish them from normal cells, polypeptides or nucleic acids (eg. mRNA, etc.), lipids, glycolipids, glycoproteins, organic biomolecules such as sugars (monosaccharides, disaccharides, polysaccharides, etc.) and the like.

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included are any subjects who participated in clinical research trials showing no disease clinical findings or subjects who participated in epidemiologic studies or who used as controls.

"Tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and / or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; Cells at any time of pregnancy or development in the subject. Tissue samples may also be primary or cultured cells or cell lines.

"Nucleic acid" is meant to include any DNA or RNA, eg, chromosomes, mitochondria, viruses, and / or bacterial nucleic acids present in tissue samples. One or both strands of a double stranded nucleic acid molecule and any fragment or portion of an intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. The gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities in exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to genes.

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 a cell. In that sense, gene expression includes not only transcriptional and translational processes, but also posttranscriptional and posttranslational processes that can affect the biological activity of a gene or gene product. The processes include, but are not limited to, RNA synthesis, processing and transport, as well as post-translational modifications of polypeptide synthesis, transport and polypeptide. In the present invention, aspects of gene expression include all cases of methylation of gene promoter, mRNA expression and protein expression.

An “primer” is an oligonucleotide sequence that hybridizes to complementary RNA or DNA target polynucleotides and functions as a starting point for the stepwise synthesis of polynucleotides from mononucleotides, for example by the action of nucleotidyltransferases that occur in polymerase chain reactions. Means.

"Probe" refers to a nucleic acid fragment, such as RNA or DNA, that corresponds to a few bases to hundreds of bases that can achieve specific binding with mRNA. Probes can be made in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes and the like. In the present invention, after hybridization is performed using a probe complementary to a gene to be detected or quantified, absolute quantification of microorganisms in a sample may be predicted through hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

A “protein” is also to include fragments, analogs and derivatives of a protein that possess essentially the same biological activity or function as the reference protein.

By "label" or "label" is meant a compound or composition that directly or indirectly facilitates the detection of a reagent conjugated to, fused, conjugated or fused to a reagent, eg, a nucleic acid probe or antibody. The label may itself be detected (eg, a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze the chemical modification of the detectable substrate compound or composition.

The expression "up-regulation" refers to a significant increase in the expression level of a particular gene into mRNA or protein by intracellular transcription or translation compared to normal tissue cells. it means.

"Prognosis" means the prediction of the course and outcome of the disease, in the present invention, the development and progression of acute myeloid leukemia (AML), preferably the central binding factor acute myeloid leukemia (CBF AML) And predicting microscopic residual diseases.

"Treatment" means an approach to obtain beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (ie, not worsening), delay or slowing of disease progression, disease state Improvement or temporary mitigation and alleviation (partially or wholly), detectable or not detected. "Treatment" may also mean increasing survival compared to expected survival when untreated. Treatment refers to both therapeutic treatment and prophylactic or preventive measures. Such treatments include the treatments required for the disorders that have already occurred as well as the disorders to be prevented. "Palliating" a disease may reduce the extent of the disease state and / or undesirable clinical signs and / or slow or lengthen the time course of progression as compared to untreated treatment. It means losing.

"Kit" means 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 is suitable as an analytical method as well as an artificial sequencing including one or more primers and / or probe sites specific for a gene to be detected or quantified as a standard sample for calibration of real-time quantitative PCR. One or more other component compositions, solutions, or devices may be included.

"About" means 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4 for reference quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, quantities, weights, or lengths. , Amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length, varying by about 3, 2 or 1%.

Throughout this specification, the terms “comprises” and “comprising”, unless otherwise indicated in the context, include a given step or element, or group of steps or elements, but any other step or element, or step or element It should be understood that this group is not excluded.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

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

Core binding factor acute myeloid leukemia (CBF AML) is a chromosomal abnormality of (8; 21) (q22; q22) or inv (16) (p13.1q22). Accounted for 15%.

The acute myeloid leukemia of the present invention is characterized by ① M0 (minimally differentiated acute myeloid leukemia) by France-American-British (FAB) classification according to morphological and cytochemical findings on an optical microscope. , minimally differentiated acute myeloblastic leukemia, ② M1 (acute myeloblastic leukemia, without maturation), ③ M2 (acute myeloblastic leukemia, with granulocytic maturation), ④ M3 (acute promyelocytic leukemia), ⑤ M4 (acute myelomonocytic leukemia), ⑥ All cases are subdivided into eight subtypes, such as acute monoblastic leukemia or acute monocytic leukemia, ⑦ M6 (acute erythroid leukemias), and ⑧ M7 (acute megakaryoblastic leukemia).

According to the 2014 National Comprehensive Cancer Network (NCCN) guidelines, CBF AML is classified as a prognostic group with a better prognosis compared to other subtypes, but with a c-KIT mutation, but overall survival is significant. It has been reported that the recurrence rate is very short and the recurrence rate is significantly high. Therefore, the molecular genetic risk is classified as a medium risk group. In particular, mutations occur frequently at the D816 and N822 positions of exon 17, which is associated with poor prognosis of AML.

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

In particular, the present inventors have newly developed a quantitative assay that detects the combination of D816V, D816Y, D816H, and N822K mutations of the c-KIT gene using real-time quantitative (qRT) PCR methods, which are used for the diagnosis and prognostic evaluation of acute myeloid leukemia The first use of the residual disease as a tracking indicator was identified.

Accordingly, the present invention relates to a diagnosis of acute myeloid leukemia using a specific mutation combination of the c-KIT gene and a diagnostic method using the RQ-PCR method. Myeloid leukemia (CBF AML) can be determined and diagnosed. Progression, recurrence and metastasis of the acute myeloid 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 a diagnostic marker of the D816V, D816Y, D816H, N822K combination.

That is, by analyzing the expression level of one or more combinations selected from the group consisting of specific mutations D816V, D816Y, D816H, and N822K of the c-KIT gene, the diagnosis and prognosis of acute myeloid leukemia, furthermore, the use as a tracing indicator of micro residual disease and It is about use thereof.

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

The selection and application of significant diagnostic markers determines the reliability of the diagnostic results. Significant diagnostic markers mean markers of high reliability such that the results obtained by diagnosis are accurate, have high validity, and show consistent results in repeated measurements. The diagnostic marker of the present invention shows the same result in repeated experiments with genes whose expression changes by direct or indirect factors with the onset of acute myeloid leukemia, especially central binding factor acute myeloid leukemia (CBF AML). The differences are very high compared to the control group and are highly reliable markers with little chance of producing false results. Therefore, the result of diagnosis based on the result obtained by 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 phosphoprotein that is a transmembrane receptor for a KIT ligand called stem cell factor (SCF) (Yarden Y. et al. , EMBO J, 6 (11): 3341-3351,1987). The kit gene is a tyrosine kinase receptor subclass III family including platelet-derived growth factor (PDGF), macrophage-colony stimulating factor, and flt3. family (Claesson-Welsh L. et al., Proc Natl Acad Sci USA, 86: 4917-4921, 1989; Yarden Y. et al., Nature, 323: 226-232, 1986; Coussens L. et al. , Nature, 320: 277-280, 1986; Rosnet O. et al., Blood, 82: 1110-1119, 1993; Small D. et al., Proc Natl Acad Sci USA, 91: 459-463, 1994). When a stem cell factor is attached to KIT, a tyrosine kinase receptor, KIT becomes auto-phosphorylation and dimerizes to participate in AKT activation through PI3K, which is involved in cell survival. It also regulates MAPK pathways and is involved in cell growth and transcription.

However, when c-kit mutations occur, phosphorylation occurs in the absence of ligands such as stem cell factors, and KIT is activated. Cell lines become factor-independent growth from factor-dependent growth, resulting in malignant cell hyperdivision (Hashimoto K. et al., Am J). Pathol, 148 (1): 189-200, 1996; Tsujimura T. et al., Blood, 93 (4): 1319-1329, 1993; Jacobs H. et al., Seminars in immunology, 12 (5): 487 -502, 2000).

The c-kit mutants D816V, D816Y, D816H, N822K variants of the invention include those produced by nucleotide substitutions, deletions or additions. The addition, deletion or replacement may be associated with one or more nucleotides. The variants may be altered at the coding site, the non-coding site, or both. The alteration at the coding site can produce conservative or non-conservative amino acid substitutions, deletions or additions.

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

Nucleotide sequences that are at least 90% identical, more preferably at least 95%, 96%, 97%, 98% or 99% identical to the mutant sequences of the present invention; Or isolated polynucleotides comprising nucleotides complementary to such exemplary sequences.

As such, the present invention enables diagnosis of acute myeloid leukemia and diagnosis of microscopic residual disease through quantitative real-time PCR analysis of a combination of c-KIT mutant genes. The c-KIT mutations can be used in particular in combination and diagnosed by comparison of standard or cutoff values.

<Method of execution>

As the c-kit mutation detection method of the present invention, any known method capable of measuring the expression level of the c-kit mutation can be used.

As a c-kit mutation detection method, a mutation detection method using a sequencing method after a polymerase chain reaction (PCR) and a 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 ( ELISA) (Wiesner et al., Neoplasia. 10 (9): 996-1003, 2008) and immunofluorescence staining (Immunohistochemistry method) (Noack F. et al., Leuk Lymphoma. 45 (11): 2295-302, 2004 ) And Pyrosequencing (Sundstrom M., Immunology. 108 (1): 89-97. 2003), which can complement low sensitivity detection.

Among them, however, in the present invention, it is particularly preferable to use Real-time Quantitative PCR (RQ-PCR).

RQ-PCR quantitatively accumulates amplified cumulative genes at each cycle in real time and analyzes and displays them by amplification cycle, thereby eliminating the method of analyzing amplification products after the amplification of a fixed cycle of conventional PCR methods. By displaying the amount amplified each time, you can measure the exact amount.

A particularly important parameter is the CP (CT) value, which is the amplification cycle in which the amount of fluorescence generated by the separation of the probe reaches a certain reference value above the default value. This is done through values and standard curves. The present invention can be analyzed using known software in which production of such CP values, preparation of standard curves, and initial gene amounts are supported together.

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

The real-time PCR method is divided into absolute quantification and relative quantification analysis. For this purpose, it is essential to prepare a calibration curve of a standard sample. A standard curve and an unknown sample are simultaneously subjected to quantitative real-time PCR to derive a calibration curve from the standard sample. From this calibration curve, the amount of target DNA in the unknown sample is quantified. The calibration curve from the standard sample (x-axis, Log10 (standard sample); y-axis, number of threshold periods) is ideal as the correlation index (R ² ) is close to one. In an embodiment of the present invention, the R ² coefficients of crystallization were 0.998 or more in the linearity evaluation of D816V, D816H, D816Y and N822K.

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 one or more primer pairs and probes selected from the group consisting of primer pairs and probes capable of amplifying c-kit mutant markers according to the present application and said amplification And comparing the amount to the amount expressed for a normal person.

The step of comparing the amplified amount with the amount amplified for a normal person is characterized by a standard or cutoff value.

At this time, the expression amount refers to the value of ΔCt, and confirms the Ct values of D816V, D816Y, D816H, N822K, and IC.The threshold value of D816V, D816Y, D816H, N822K, and IC channels is set to 0.1, and then Ct. Check the value.

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

A method for separating totally used full RNA and synthesizing cDNA therefrom can be carried out through 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 the like can be incorporated as a reference of the present invention.

Important probes can be labeled for detection and can be labeled, for example, with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. Proper labeling of such probes is a technique well known in the art and can be carried out by conventional methods.

Diagnostic Compositions and Kits

In another aspect, the present invention also provides acute myeloid leukemia, preferably, a central binding factor acute myeloid leukemia (CBF AML) comprising at least one c-KIT mutation combination selected from the group consisting of D816V, D816Y, D816H, and N822K. ) Diagnostic marker composition.

At this time, the gene expression level is measured to determine whether overexpression of the D816V, D816Y, D816H, and N822K mutant genes. The 'gene expression level measurement' includes both measuring the mRNA or its protein expression level.

According to one aspect of the invention, the present invention is to measure the expression level at the mRNA level via QR-PCR. To this end, novel primer pairs and fluorescently labeled probes that specifically bind to the mutant genes are required. In the present invention, the primers and probes specified by specific nucleotide sequences may be used, but are not limited thereto. As long as it can perform RQ-PCR by specifically binding to provide a detectable signal, it can be used without limitation. In one embodiment of the present invention FAM and VIC means a fluorescent dye.

In a similar aspect the present invention relates to a kit or system for the diagnosis and prognostic assessment of acute myeloid leukemia, in particular for the diagnosis or prognostic analysis of central binding factor acute myeloid leukemia (CBF AML).

Detection reagents and methods in which such reagents are used are described above. Reagents capable of detecting such markers of the present application may be separately dispensed in a compartment in which the compartment is divided, and in this sense, the present application also relates to an apparatus / apparatus comprising compartmentally containing the marker detection reagent of the present application.

In other embodiments, the detection reagent may be provided in the form of an array or chip comprising a microarray. Detection reagents may be attached to the surface of a substrate 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. Nos. 5,599,695, 5,556,752 or 5,631,734. The detection reagent can be labeled directly or indirectly in the form of a sandwich for detection. In the case of the direct labeling method, serum samples used for arrays and the like are labeled with fluorescent labels such as Cy3 and Cy5. Fluorescence luminosity can be used with scanning confocal microscopy, for example Affymetrix, Inc. Or Agilent Technologies, Inc.

Kits herein may further include one or more additional ingredients required for binding assays, and may further include, for example, binding buffers, reagents for sample preparation, blood sampling syringes or negative and / or positive controls. .

For example, when the kit of the present invention is subjected 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.

Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above.

<Diagnosis and Information Provision Method>

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

In another aspect the invention provides nucleic acid or protein levels of at least one biomarker selected from the group consisting of D816V, D816Y, D816H and N822K, from a biological sample of a subject to provide information necessary for diagnosing or prognostic acute myeloid leukemia, Or it relates to a method for detecting acute myeloid leukemia markers for detecting the presence of nucleic acids or proteins.

The method also provides useful information as a prognostic assessment and tracer indicator of microresistive disease, including recurrence of acute myeloid leukemia, particularly central binding factor acute myeloid leukemia (CBF AML), and the like.

In one embodiment, the present invention

For samples obtained from patients with suspected Acute Myeloid Leukaemia (AML)

Real-time quantitative PCR (RQ-PCR) method is used to measure the expression level of one or more mutations selected from the group consisting of D816V, D816Y, D816H, D816F, N822K (A) and N822K (G) of the c-KIT gene, and expressing the expression A method of providing information for diagnosing acute myeloid leukemia, comprising comparing the level of the level to that expressed in normal subjects, wherein the comparison is performed by a standard or cutoff value for diagnosing or diagnosing acute myeloid leukemia. Provide information The diagnosis includes progression, recurrence, metastasis, prognostic evaluation, and follow-up of micro-resistance disease in acute myeloid leukemia.

At this time, the sample is preferably bone marrow, in the real-time quantitative PCR (RQ-PCR) method, c-KIT gene mutation quantitative value is characterized in that the mutant gene copy number per 100 copies of the reference gene is determined.

If the c-kit gene mutant expression in a biological sample isolated from the patient is overexpressed than the normal control sample, it is determined to be acute myeloid leukemia.

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

In addition, the prognostic evaluation can be performed by the long-term survival rate (OS) and accident-free survival rate (EFS) value, the three-year survival rate is 35.6% in patients with high c-KIT gene mutation quantification level (> 10), quantitative mutation It is statistically significantly lower than patients with low levels (<10) and without mutations.

Whether or not the trace of the micro-remaining disease is characterized by recurrence and micro-remaining disease when the quantitative quantification of the D816V and D816Y mutation of the c-KIT gene is amplified.

As such, the method relates to investigating the expression characteristics of specific markers according to acute myeloid leukemia, and the methods disclosed herein are useful for obtaining useful data and information when evaluating appropriate or effective therapies for treating patients with acute myeloid leukemia. It may provide a convenient, efficient and cost effective means.

On the other hand, in another aspect, the present invention relates to a method for screening a substance for treating acute myeloid leukemia that suppresses the expression of mutations in the c-kit gene based on the above-described fact.

Bringing changes in the expression of a marker according to the invention in acute myeloid leukemia tissues or cells can be selected as a candidate drug for treating acute myeloid leukemia.

In one embodiment the method comprises providing a cell comprising one or more mutations selected from the group consisting of D816V, D816Y, D816H, D816F, N822K (A) and N822K (G); Contacting the cell with a test substance;

Measuring the expression level of the nucleic acid sequence at the mRNA or protein level; And when the measurement result, the expression level of the one or more nucleic acid sequences in the cells treated with the test material is reduced compared to the control group not treated, comprising the step of selecting the candidate material.

The present invention extends to genetic approaches to c-kit mutant expression and up-regulation or down-regulation of gene expression.

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

For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely illustrative. It is not limited to this.

As such, in the present invention, the factors that increase the sensitivity and specificity of acute myeloid leukemia, especially the central binding factor acute myeloid leukemia diagnosis and prognostic evaluation, showed the characteristics of RQ-PCR profile according to c-KIT mutation combination. It suggests that the use of the gene as a diagnostic marker can be variously utilized.

<Example>

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples. Unless otherwise indicated, nucleic acids are recorded in a 5 '→ 3' orientation from left to right. The numerical ranges listed in the specification include numbers defining the ranges and include each integer or any non-integer fraction within the defined ranges.

Unless defined otherwise, 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 for testing the invention, preferred materials and methods are described herein.

Target and how

1. Target

Bone marrow samples from 111 adult patients first diagnosed with CBF AML at the Catholic University of Korea St. Mary's Hospital from April 2009 to July 2013 were used.

2. How to

The bone marrow specimens at the time of diagnosis were centrifuged to separate the leukocyte layer, and then DNA was extracted using the QIAamp DNA blood mini kit (QIAGEN, Germany) according to the instructions of the reagents. Extracted DNA was diluted with 1X TBE buffer to 50 ~ 100ng per 1ul.

Screening check

(1) Prepare RQ-PCR master mixture for each reaction.

(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) After mixing the RQ-PCR master mixture well, 22 μl was dispensed into 96-well plates or optical tubes.

(3) 3 μl of sample DNA and positive DNA were dispensed into each well.

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

(5) After the tube was mounted on the ABI PRISM 7500, the experiment was performed using the program stored in the procedure of FIG. 1.

(6) Inspection Analysis

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, which were amplified in a positive control, were confirmed. The ranges of D816V, D816Y, D816H, N822K, and IC values are shown in Table 2, and when the ranges were out of range, re-examination was performed. When the signal was not displayed in the negative control (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 The threshold values of D816V, D816Y, D816H, N822K and IC channels were set to 0.1 and the Ct values were checked.

② Always check the raw data of each sample to confirm normal amplification.

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	Conduct a review

Genotyping inspection

Screening test resulted in mutant specimens.

(1) RQ-PCR master mixtures of D816V, D816Y, D816H and N822K were prepared for the 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) After mixing the RQ-PCR master mixture well, 22 μl was dispensed into 96-well plate or optical tube.

(3) 3 μl of sample DNA and positive DNA were dispensed into each well.

(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 the ABI PRISM 7500 was tested using the program stored in the process of FIG.

(6) Inspection Analysis

Positive control

The threshold values of the D816V, D816Y, D816H, N822K and IC channels were set to 0.1 and the Ct values of the D816V, D816Y, D816H, and N822K ICs amplified by the positive control were confirmed. The ranges of D816V, D816Y, D816H, N822K, and IC values are shown in Table 5, and when the ranges were out of range, they were reviewed again. When no signal was detected in the negative control and amplification was confirmed in either FAM or VIC, 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 The threshold values of D816V, D816Y, D816H, N822K and IC channels were set to 0.1 and the Ct values were checked.

② Check the raw data of each sample to confirm normal amplification.

③ The results were analyzed according to 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	Conduct a review

④ c-KIT gene mutation quantitative value was used for the reference gene ( c-KIT exon 2) and mutant gene copy number automatically calculated by the software compared to the quantitative standard graph.

Example 1: Evaluation of Specificity and Sensitivity

1-1 Specificity Verification Using Normal Specimens

As a result of analyzing normal samples by real-time quantitative PCR method, all 100 samples showed negative results, which confirmed that this test method can specifically amplify only mutants.

1-2 c-KIT mutation quantification minimum detection plasmid DNA copy number

By real-time quantitative PCR, the minimum number of detected DNA copies of D816V, D816H, D816Y, and N822K was less than 200 copies with 43.4, 13.8, 168.9, and 1.7 copies, respectively.

type	D816V	D816H	D816Y	N822K
Copy number (copy / μL)	43.4	13.8	168.9	1.7

1-3 c-KIT Mutation Quantitative Linearity Verification

In the linearity evaluation of D816V, D816H, D816Y, and N822K measured using five concentrations of mutant plasmid DNA, the R ² crystal coefficients were all superior to 0.998.

Comparative example  1: comparison with other methods

In 108 patients, the results of real-time quantitative PCR and melting curve analysis were compared. The concordance rate of the two tests was 74.1%, which was the same in 80 patients, and the positive results in the melting curve analysis were also positive in real time quantitative PCR. Twenty-eight patients who were negative in the Melting curve analysis showed positive results in real-time quantitative PCR, which were all patients with low mutant allele burden and were additionally positive by other highly sensitive tests (pyrosequencing or MEMO-PCR). It was confirmed that. Therefore, real-time quantitative PCR test showed a high sensitivity compared to the melting curve analysis (Fig. 4).

Example 2 Evaluation of Clinical Usefulness: Calculation of Quantitative Values and Prognostic Related Cut-Off Settings

A total of 111 CBF AML patients (65 male, 46 female, age 18-72 years old) were tested for the D816V, D816H, D816Y, and N822K mutations of the c-KIT gene by real-time quantitative PCR.

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

Quantification of c-KIT gene mutations using the reference gene ( c-KIT exon 2) and the number of mutant gene copies analyzed in the quantitative standard graph was obtained by calculating the number of mutant gene copies per 100 copies of the reference gene (mutant gene copy number). / 100 copies of reference gene). If a person had several mutations (30/69, 43.5%), the quantitative values of all mutations were summed. The distribution of mutant allele levels showed a median of 0.3 (range 0.002-532.7) with a cut-off of 10. This is illustrated in FIG. 5.

2-2. Diagnosis of Acute Myeloid Leukemia

Of the 111 patients with CBF AML, 69 patients (62.2%) had D816 and N822 mutations in the c-KIT gene, 14 patients (20.3%) with D816V, 7 patients (10.1%) with D816Y, and 4 patients with D816H ( 5.8%), N822K had 14 patients (20.3%) and 30 patients (43.5%) had two or more mutations (FIG. 6).

As shown in FIG. 7, the D816 mutation (median, 0.7; range, 0.002-532.7) showed higher genetic mutation quantitative value than the N822K mutation (median, 0.1; range, 0.006-43.53) ( P = 0.016). .

As shown in FIG. 8, the quantitative quantification of the mutations in the t (8; 21) AML patients (median, 0.5; range, 0.002-532.7) and the quantification of the inv (16) AML patients (median, 0.1; range, 0.002- 293.0) showed no statistically significant difference ( P = 0.243).

As shown in FIG. 9, the comparison of quantitative mutations between patients with two or more mutations (median, 0.2; range, 0.002-490.9) and patients with a 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: Prediction of Acute Myeloid Leukemia

Based on the c-KIT mutant quantitative value of 10, c-KIT quantitatively showed significant effects on long - term survival (OS) and accident-free survival (EFS). 10)

Three-year survival rate was 35.6% in patients with high c-KIT gene quantification (> 10), low mutation (<10) in patients (3 year survival, 72.5%) and no mutation (3). Yearly survival rate, 72.5%). There was no significant difference in the three-year survival rate between the groups with low quantification and no mutations ( P = 0.943). Three-year accident-free survival was also lowest in the group with high quantitative variability (28.9%, P = 0.0252), and the group with low mutant quantification and no mutation showed no significant difference in the three-year accident free survival (61.2%). vs. 63.9%, P = 0.963).

Example 4 Tracking of Microsurvivors of Acute Myeloid Leukemia

Twenty-seven patients who were detected c-KIT gene mutations at diagnosis were negative in all of the patients who underwent c-KIT mutation mutations by real-time quantitative PCR. In 6 of the patients who relapsed after treatment, c-KIT gene mutations observed at diagnosis were also observed at relapse (FIG. 11).

In particular, two patients were found to have two types of mutations at the time of diagnosis, confirming that a small amount of gene mutations that could only be detected by high-sensitivity tests were amplified at recurrence. 40.9 at relapse at 0.2 and 4.9 at 0.01 at diagnosis).

Based on these results, c-KIT mutation using real-time quantitative PCR of the present invention. The quantitative assay is excellent 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, based on the cut-off value of the present invention, the prognosis is analyzed using the quantitative c-KIT gene mutation quantitative value, and based on these results, the utilization in the future AML patients will be high.

Claims (8)

1. For samples obtained from patients with acute myeloid leukaemia (AML), the D816V, D816Y, D816H, D816F, N822K (A) and N822K of c-KIT genes using real-time quantitative PCR (RQ-PCR). An information providing method for diagnosing acute myeloid leukemia, comprising measuring at least one mutation expression level selected from the group consisting of (G) and comparing the expressed level with that expressed in normal persons,

   The comparison is an information providing method for diagnosing acute myeloid leukemia, characterized in that performed by a standard or cutoff value.
2. The method of claim 1,

   The acute myeloid leukemia is a core binding factor acute myeloid leukemia (Core binding factor acute myeloid leukemia, CBF AML) information providing method for diagnosing acute myeloid leukemia.
3. The method of claim 1,

   The diagnosis is an information providing method for diagnosing acute myeloid leukemia, comprising the progression (progression), recurrence (metastasis), metastasis (prognosis), prognostic evaluation, and tracking the presence of micro residual disease.
4. The method of claim 1,

   In the real-time quantitative PCR (RQ-PCR) method,

   c-KIT gene mutation quantitative value is determined by the number of mutant gene copies per 100 copies of the reference gene information providing method for diagnosing acute myeloid leukemia.
5. The method of claim 4, wherein

   In the c-KIT gene mutation quantitative value,

   A method of providing information for diagnosing acute myeloid leukemia, characterized in that the median mutant allele level is 0.3 and the cutoff value is 10.
6. The method of claim 4, wherein

   The D816 mutant quantitative value is higher than the N822K mutant, characterized in that the information providing method for diagnosing acute myeloid leukemia.
7. The method of claim 3,

   The prognosis evaluation is an information providing method for diagnosing acute myeloid leukemia, characterized in that it is carried out by the value of long-term survival (OS) and accident-free survival (EFS).
8. The method of claim 3,

   Whether the trace of the micro residual disease is

   Method for providing information for diagnosing acute myeloid leukemia, characterized in that the recurrence and micro-residual disease is determined when the quantitative D816V and D816Y mutations of the c-KIT gene are amplified.

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