CN111424083A - Leukemia marker and application thereof in leukemia prognosis evaluation - Google Patents
Leukemia marker and application thereof in leukemia prognosis evaluation Download PDFInfo
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Abstract
The invention discloses a leukemia marker, which is a gene shown in SEQ ID No.1 or a gene with 85% -99% homology with the gene, and is used for preparing a product for predicting and diagnosing whether a patient is a patient with refractory myeloid leukemia or selecting a patient with refractory myeloid leukemia, predicting and diagnosing whether a patient is a patient with myeloid leukemia which has a recurrence risk after being treated to achieve complete remission of the marrow, or selecting a patient with myeloid leukemia which has a recurrence risk after being treated to achieve complete remission of the marrow, and predicting or evaluating the total survival period of the patient with myeloid leukemia. The marker can be detected before chemotherapy to judge the responsiveness, recurrence and prognosis survival of patients, can well distinguish patients sensitive to chemotherapy, difficult to treat and recurrence, overcomes the defect of the existing risk stratification, achieves the aim of further finely layering the patients, and effectively guides subsequent chemotherapy, transplantation and other treatment schemes.
Description
Technical Field
The invention relates to the field of in vitro diagnosis, in particular to a leukemia marker and application thereof in leukemia prognosis evaluation.
Background
The Acute myeloid leukemia (AM L) is a highly heterogeneous disease, and is the most common type of adult, and the elderly patients are more, AM L has poor prognosis, the five-year survival rate is less than 27%, 70-80% of patients relapse into refractory types even after chemotherapy completely relieves, the treatment of AM L clinically determines the induction treatment scheme, the chemotherapy scheme and the long and short treatment course of the patients according to the risk degree stratification of the patients, and whether hematopoietic stem cell transplantation should be actively carried out after the patients achieve complete relief.
At present, NCCN (National Comprehensive cancer network) risk stratification is mainly adopted globally, and E L N (European leukemia network) risk stratification is also adopted in part of European countries.
However, clinically 40-50% of patients have normal chromosomes and fusion genes are negative; in addition, some patients can not obtain the mutant gene information because the sequencing is not carried out, and the patients can not be layered accurately according to the existing scheme and can only be classified into the middle-risk group in a fuzzy manner. In addition, current stratification has a role in indicating the natural history and prognosis of a patient's disease across a population, but often indicates deviations among individuals, particularly in indicating refractory relapse situations and frequent clinical outcomes for patients. And the clear outcome of patient refractory and relapsed before chemotherapy is one of the most valuable guidelines for treatment and prognosis.
The limitations of current stratified diagnostic and therapeutic decisions are as follows:
the risk stratification of NCCN and E L N, which are the same basically, is composed of abnormal chromosome of each type of patient, several fusion gene with very definite clinical significance and mutation gene, the mutation gene for evaluating chemotherapy sensitivity is obtained from scattered patient sequencing, each mutation frequency is only found in about 10% of patients, and the survival period of the fusion gene and chromosome evaluation patients is more long, which causes the problems of the existing stratification that 1) about 40-50% of patients have normal chromosome clinically, fusion gene is negative, mutation gene is not detected, the existing scheme can not be stratified, and only can be classified into middle-risk/middle-high-risk group, 2) the ability of predicting the chemotherapy reactivity of patients in advance is insufficient, the indication of primary drug resistance and difficult treatment and recurrent clinical outcome of the patients is unclear, and the common and clinical outcome is different, the influence on the elderly is particularly obvious because the old people have poor tolerance, 3) the effect of indicating the disease severity and prognosis on the population, but the deviation of indicating individuals is not constant.
Therefore, there is a need to find a marker for accurately predicting the risk of refractory and relapsing of patients with myeloid leukemia before chemotherapy to solve the above-mentioned deficiencies in the prior art.
Disclosure of Invention
One aspect of the invention provides a leukemia marker aiming at the defects that the risk degree of a patient with myeloid leukemia is not clearly layered and whether the patient has the risks of treatment difficulty and relapse cannot be clearly indicated in the prior art.
The technical scheme provided by the invention is as follows:
a leukemia marker is a gene shown in SEQ ID No.1 or a gene which has 85% -99% homology with the gene shown in SEQ ID No.1, and the marker is used for preparing products for predicting and diagnosing whether a patient is a refractory myeloid leukemia patient or sorting the refractory myeloid leukemia patient.
The gene shown in SEQ ID No.1 in the invention is an essential gene for cell survival, and plays an important role in the most basic biological processes of cells, such as pre-mRNA shearing, RNA nuclear retention, cell transcription, DNA repair and the like. The gene shown in SEQ ID No.1 of the invention is expressed in every bone marrow and is a gene which is continuously expressed in cells. At present, few reports about the gene in the blood diseases exist, no report related to the difficulty in treating and relapse of the myeloid leukemia exists, and no report exists that the gene can be used as a new prognosis evaluation and a new marker for the difficulty in treating and relapse and can be included in the risk degree stratification of the myeloid leukemia.
The gene shown in SEQ ID No.1 comprises an mRNA single-chain sequence and a complementary sequence shown in the gene. Meanwhile, the marker also comprises a cDNA single-chain sequence obtained by reverse transcription of the gene shown in SEQ ID No.1 and a sequence obtained by other transcription forms.
In the present invention, the gene represented by SEQ ID No.1 may be derived from nucleated cells in peripheral blood and/or bone marrow of a patient.
In another aspect of the invention, a leukemia marker is provided, the leukemia marker is a gene shown in SEQ ID No.1 or a gene with 85% -99% homology with the gene shown in SEQ ID No.1, and the marker is used for preparing products for predicting and diagnosing whether a patient is a myeloid leukemia patient who has a recurrence risk after being treated to achieve complete remission of bone marrow, or sorting the myeloid leukemia patient who has the recurrence risk after being treated to achieve complete remission of bone marrow.
The Complete Remission (CR) is based on the complete remission of bone marrow morphology according to the international cooperative group standard, and specifically comprises: the patient satisfied a morphological leukemic state, dropped from transfusion, < 5% of primary cells in the bone marrow aspiration smear (at least 200 nucleated cells counted), absence of primary cells with Auer bodies or absence of extramedullary leukemia persists. Patient's neutrophil count in peripheral blood > 1.0x109/L, platelets > 100X109/L。
In the present invention, the gene represented by SEQ ID No.1 may be derived from nucleated cells in the peripheral blood and/or bone marrow of a patient.
The term "treated" as used herein means that the patient with myeloid leukemia is treated with a conventional clinical treatment, such as chemotherapy (including induction remission, consolidation, maintenance) or supportive treatment, after being subjected to risk stratification (risk stratification in NCCN-2016 and E L N-2016).
In another aspect of the invention, a leukemia marker is provided, the leukemia marker is a gene shown in SEQ ID No.1 or a gene with 85% -99% homology with the gene shown in SEQ ID No.1, and the marker is used for preparing a product for predicting or evaluating the prognosis of patients with myeloid leukemia.
The above-mentioned gene having 85% -99% homology with the gene shown by SEQ ID No.1 may be a gene having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the gene shown by SEQ ID No. 1.
In another aspect of the invention, a marker composition is provided, wherein the marker composition comprises the above marker, and the marker composition is used for preparing products for predicting and diagnosing whether patients are refractory and/or have a recurrence risk after being treated to achieve complete remission of bone marrow, or sorting refractory and/or treated patients have a recurrence risk after being treated to achieve complete remission of bone marrow, or predicting and evaluating the overall survival of patients with myeloid leukemia.
The marker compositions described above may be derived from a sample of peripheral blood and/or bone marrow cells. Preferably, however, in one embodiment of the present invention, the gene is derived from nucleated cells in bone marrow of the patient.
Preferably, the marker composition also comprises one or more selected from a mutant gene, a fusion gene or an abnormal chromosome related to leukemia onset and/or prognosis evaluation, more preferably, the marker composition is one or more selected from a fusion gene, a mutant gene or an abnormal chromosome related to leukemia onset and/or prognosis evaluation in WHO and E L N risk degree stratification, more preferably, the marker composition is one or more selected from a PM L-RAR α or AM L1-ETO fusion gene, RUNX1 or ASX L1 mutant gene, and a-5 or-7 abnormal chromosome.
Preferably, the level of any of the above biomarkers is determined by mass spectrometry coupled chromatography, such as gas chromatography mass spectrometry (GC-MS) or liquid chromatography mass spectrometry (L C-MS).
Also, the present invention may utilize any suitable method for determining the level of any of the above biomarkers. Preferably, in another embodiment of the present invention, the level of the biomarker is determined by a method of real-time fluorescent quantitative PCR.
In the present invention, the gene markers represented by SEQ ID No.1 and the marker compositions can be used for predicting patients with myeloid leukemia, such as acute myeloid leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome-transformed acute myeloid leukemia. Preferably, in one embodiment of the present invention, the gene marker represented by SEQ ID No.1 and the marker composition are used for predicting patients with acute myeloid leukemia.
In another aspect of the invention, a kit is provided, which comprises a product for detecting the gene marker shown in the SEQ ID No.1 and the marker composition.
In the present invention, any suitable product or method for detecting the above genes can be used, which is considered to be within the scope of the present invention, for example, Polymerase Chain Reaction (PCR) such as qPCR, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (FISH), Transcription Mediated Amplification (TMA), ligase chain reaction (L CR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA), microarray, Southern or Northern blot, high throughput sequencing methods.
Preferably, in one embodiment of the present invention, the product is a primer or probe for detecting the leukemia marker or the marker composition.
More preferably, the product is a primer for detecting the leukemia marker or the marker composition. In one embodiment of the invention, the target gene is detected using a method of qPCR.
Preferably, the primers in the kit comprise an amplification primer pair of internal reference GAPDH and an amplification primer pair of the gene marker shown in SEQ ID No. 1. More preferably, the amplification primer pair of the internal reference GAPDH is in the forward direction: 5'-CAACTACATGGTTTACATGTTC-3', respectively; and (3) reversing: 5'-GCCAGTGGACTCCACGAC-3', respectively; the amplification primer pair of the gene marker shown in SEQ ID No.1 is a forward direction: 5'-GGATCCACGGGAAAGAGACA-3', respectively; and (3) reversing: 5'-CACTCATGGTTGCTGGTGG-3' are provided. As a preferenceThe kit also comprises PCR reaction liquid and kit instructions. The PCR reaction solution comprises dNTP and Mg2+The kit comprises Taq enzyme, a fluorescent dye and buffer solution, wherein the fluorescent dye is SYBR GreenII, and the Taq enzyme is hot-start enzyme.
In one embodiment of the present invention, the predicting the risk of patients with myeloid leukemia comprises the following steps:
step 1) determining the level Ct of each of the above leukemia markers, above biomarker compositions, in a sample from said patient;
step 2) detecting the GAPDH level of the sample according to △ Ct ═ Ct-CtGAPDH△ Ct value is calculated, △ Ct is more than or equal to 4.913, the patient is a sensitive type patient, △ Ct is less than 4.913, and the patient is a difficult-to-cure type patient.
Preferably, the sample of the patient is bone marrow nucleated cells of the patient.
The invention has the beneficial effects that:
1) the marker of the invention is expressed in bone marrow nucleated cells of everyone, so the marker is suitable for all types and all age groups of AM L patients;
2) the marker can judge the responsiveness of the patient to chemotherapy before chemotherapy, and can well distinguish patients who are sensitive to chemotherapy and patients who are difficult to treat and relapse; therefore, the aim of further finely layering the patients under the existing risk stratification can be achieved, and the chemoresponse is directly pointed out.
3) The marker layering scheme is added under the existing risk layering, so that the transplantation of the hidden refractory and relapsing patients in a low-risk group can be prompted as early as possible; the patients with high risk, especially high risk and old age are known to be refractory before chemotherapy, or can directly adopt supportive therapy or demethylation therapy, so as to avoid the side effect of ineffective chemotherapy and reduce the infection rate; these are necessary to win survival time, extend survival time, and improve quality of life for the patient.
4) The detection of the marker can be completed only by using the intermediate product cDNA of the essential examination item of the patient and the gene detection process, and the sample does not need to be additionally processed and prepared, so that the invasive sample collection and sample consumption of the patient can not be increased, and the medical cost of the patient is saved.
5) The work is not limited to the application significance in the field of diagnosis, and has important guiding effects on later-stage clinical reasonable medication, the relieving treatment of the malignant diseases of the old patients and the suggestion that the transplantation scheme is more objective (quantitative by using detection indexes).
Drawings
FIG. 1 is a flow chart of a clinical study design protocol in an example of the present invention;
FIG. 2 is a graph showing the results of differential analyses of markers of the invention among patients with different types of AM L;
FIG. 3 is a graph of the results of inter-patient variability analysis of markers of the invention under risk stratification at E L N2018;
FIG. 4 is a graph showing the results of differential analyses of markers of the present invention among susceptible, refractory and relapsing patients;
FIG. 5 is a graph of the results of a differential analysis between patients further subdivided by markers of the invention under the current risk stratification of E L N2018;
FIG. 6 is a graph of the performance characteristics of subjects with markers of the present invention;
FIG. 7 is a graph of the level of a marker of the present invention versus overall survival of a patient.
DESCRIPTION OF THE SEQUENCES
SEQ ID No.1 is a nucleotide sequence of the leukemia marker of the invention.
Detailed Description
The invention discloses a leukemia marker and application thereof in leukemia prognosis evaluation. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Some of the pertinent terms appearing in the present invention are explained below.
The acute myeloid leukemia is classified into M0, M1, M2, M3, M4, M5, M6 and M7 types according to the AM L morphological classification of FAB.
The term "marker", also referred to as "biological marker", refers to a measurable indicator of a biological state or condition of a subject. Such biomarkers can be any substance in the subject, such as nucleic acid markers (e.g., DNA), protein markers, cytokine markers, chemokine markers, carbohydrate markers, antigen markers, antibody markers, species markers (species/genus markers), functional markers (KO/OG markers), and the like, so long as they are associated with a particular biological state or condition (such as a disease) of the subject. Detection and assessment of biomarkers is often used to examine normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions and is useful in many scientific fields.
The terms "a", "an" and "the" are not intended to refer to only a single entity, but include the general class of which a specific example may be used for illustration.
The English full name of the term "QPCR" is Real-time Quantitative PCR detection System. Namely a real-time fluorescence quantitative nucleic acid amplification detection system, also called a real-time quantitative gene amplification fluorescence detection system, which is called QPCR for short.
The term "polymerase chain reaction", PCR, is a molecular biological technique for amplifying and amplifying a specific DNA fragment, which utilizes the principle that DNA becomes single-stranded when denatured at a high temperature of 95 ℃ in vitro, primers are combined with single-stranded at a low temperature (usually about 60 ℃) according to the principle of base complementary pairing, and then the temperature is adjusted to the optimum reaction temperature (about 72 ℃) of DNA polymerase, and the DNA polymerase synthesizes a complementary strand along the direction from phosphate to pentose (5 '-3').
The term "susceptible" means that clinically Complete Remission (CR) is achieved within two standard treatment regimens, with no recurrence within 18 months after CR, and no two or more relapses after CR.
The term "refractory" refers to treatment of 2 treatment-ineffective primary cases via standard protocols; 2 or more relapsers; extramedullary leukemia persists.
The term "relapse" refers to a relapse within 6 months, 12 months, or 18 months after CR with consolidation and strengthening therapy. Recurrence is based on recurrence of leukemic cells in peripheral blood after CR or > 5% of blasts in bone marrow (except for consolidation of other reasons such as bone marrow regeneration after chemotherapy) or leukemic cell infiltration outside the marrow.
It will be understood by those skilled in the art that the terminology herein is provided for the purpose of promoting a better understanding of the invention and is not intended to be limiting of the invention except as outlined in the claims.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
The clinical scheme of the invention comprises the steps of carrying out conventional clinical examination after a patient retains a specimen, carrying out gene detection on the marker, confirming diagnosis and typing, carrying out risk degree layering on the patient, carrying out clinical treatment according to guidelines, grouping the patient according to the outcome of clinical exposure (sensitive, refractory and recurrent) after the treatment is finished, analyzing the relation between the expression level of the marker and the outcome of treatment-sensitive, refractory and recurrent of the patient, and the flow of the clinical research design scheme is shown in figure 1. the risk degree layering of the patient adopts NCCN-2016 and E L N-2016 type risk degree layering, and the sensitive, refractory and recurrent patients are grouped according to the recurrent/refractory AM L Chinese diagnosis and treatment guidelines (2017).
Example 1: patient compliance and sample retention
Patients with acute myelogenous leukemia of various types and ages without chemotherapy are initially diagnosed. Excluding 1) patients with various infections; 2) patients with bleeding tendency, failure to fight infection, inability to undergo chemotherapy for various reasons; 3) the traditional patients with solid tumor, blood tumor and aplastic anemia have a history. The patient undergoes bone marrow puncture, while the necessary clinical routine examination is performed, a bone marrow specimen is kept for each patient, the total RNA of the bone marrow nucleated cells is extracted, and is inverted into cDNA, and the cDNA is frozen and stored at-20 ℃.
Example 2: separation of bone marrow nucleated cells, total RNA extraction and reverse transcription
1. Separating nucleated cells:
standing a bone marrow sample of a patient, layering bone marrow plasma and cell components, gently washing the bone marrow plasma, adding the cell components and erythrocyte lysate into the lysate according to the volume ratio of 1:4, reversing and uniformly mixing, standing at room temperature for 8-10 minutes, reversing and uniformly mixing for a plurality of times, and centrifuging to collect cells when the solution is clear and transparent. Lysis of 5X10 by 400 cell clear TRIzol6TRIzol lysate (ThermoFisher) is added to the ratio of cells, and RNA is extracted immediately or frozen at-80 ℃ for subsequent concentrated extraction.
2. Total RNA extraction:
the TRIzol containing the nucleated cells is mixed and shaken violently by reversing, chloroform with the volume of 1/5 is added, the mixture is mixed evenly by reversing for 1 minute, the mixture is kept still for 5 minutes at the room temperature and is centrifuged for 15 minutes at 13000rpm, the centrifuged supernatant is carefully transferred into a new centrifugal tube, isopropanol with the same volume is added, the mixture is mixed evenly by shaking gently, the mixture is kept still for 10 minutes at the room temperature and is centrifuged for 15 minutes at 13000rpm at 4 ℃ and 13000rpm, the supernatant is discarded, the precipitate is reserved, 1m L70% of absolute ethyl alcohol is added into the precipitate, the mixture is mixed evenly by blowing, the mixture is centrifuged for 15 minutes at 4 ℃ and 13000rpm, the supernatant is sucked off gently, a proper amount of DEPC water is added after the precipitate is dried in the air at the room temperature, the precipitate is fully dissolved, the RNA is.
3. Reverse transcription:
RNA was denatured at 70 ℃ for 5 minutes in an ice bath for 5 minutes, and then reverse transcribed using a reverse transcriptase manufactured by Promega corporation in an ice bath system in the system shown in Table 1. The prepared reaction system carries out reverse transcription on a PCR instrument, and the reaction conditions are as follows: 5 minutes at 25 ℃, 1 hour at 42 ℃ and 15 minutes at 70 ℃. After the reaction is finished, the cDNA is frozen and stored at the temperature of-20 ℃ or immediately subjected to real-time fluorescent quantitative PCR identification.
TABLE 1 reverse transcription System
Example 3: real-time fluorescent quantitative PCR detection of target gene
1. Primer sequences
GAPDH (product length: 185bp)
Forward：5’-CAACTACATGGTTTACATGTTC-3’ (22nt)
Backward：5’-GCCAGTGGACTCCACGAC-3’ (18nt)
The marker of the present invention (product length: 148bp)
Forward：5’-GGATCCACGGGAAAGAGACA-3’ (20nt)
Backward：5’-CACTCATGGTTGCTGGTGG-3’ (19nt)
2. Amplifying a target gene by real-time fluorescent quantitative PCR:
the reaction adopts a Bio-rad real-time fluorescence quantitative PCR instrument and the reagent adopts
qPCR Master Mix (Promega), the expression level of the marker of the invention and GAPDH gene in each patient bone marrow specimen was measured according to the system shown in Table 2, while negative control without template was set, the reaction conditions were pre-denaturation at 95 ℃ for two minutes, denaturation at 95 ℃ for 15 seconds, annealing at 60 ℃ for one minute, 40 cycles, and finally, the reaction was terminated at 95 ℃ for 15 seconds, 60 ℃ for one minute, and 95 ℃ for 15 seconds, and the total reaction system was 25. mu. L.
TABLE 2 PCR System
3. And (3) result processing and statistical analysis:
ct values of the genes of interest (markers of the invention) were corrected using the Ct value of the GAPDH gene as an internal reference according to the qPCR results, and the expression level of the marker comparable to each patient was represented by △ dar, △ ═ Ct-CtGAPDH. Patients were according to acute myeloid leukemia WHO-2008 classification, dividing patients into 8 groups, gr: AM with t (15; 17)/PM RAR, gr: AM 0 with t (8; 21)/AM 11-ETO, gr: AM 2 with inv (16)/CBF-MYH, gr: AM 3 with minimal differentiation and with out-regulation (FAB AM 4-M, M), gr: AM 5 with regulation (FAB AM 6-M), gr: AM 8 with intracellular myogenic leukemia (FAB AM 9-M), gr: AM with intracellular myogenic leukemia/monomeric myelogenic leukemia (FAB AM-M), gr: other types of AM (AM myelodysplastic syndrome) using one way ANOVA to analyze patient target genes (markers of the invention) 7, results are shown in FIG. 2. results of the present invention are indicative of the stratification of high or low risk among the groups using the current markers of the present invention, and the present invention is indicative of high risk of the present invention, low risk of the stratification of the present invention using the one way ANOVA-2016.
The Chinese diagnosis and treatment guideline of 2017 version AM L for relapse/refractory disease is divided into three groups of sensitive, refractory and relapsing groups, and the grouping standard is shown in Table 3.
TABLE 3 grouping criteria for sensitive, refractory, relapsing patients
The difference of the target gene (marker of the present invention) between the two groups was analyzed by t-test, and the results are shown in FIG. 4. The transcription level of the marker of the invention has very obvious difference between the groups of treatment difficulty of sensitive vs and relapse of sensitive vs, and P is less than 0.0001; can well distinguish sensitive and refractory relapsing patients. The patients are further divided into two layers of low-risk (low-risk and medium-low-risk) and high-risk (high-risk and medium-high-risk) under the existing layered system, the transcription level of the marker can still well distinguish the sensitive and refractory relapse conditions of patients in more layers, the groups have very obvious difference, P is less than 0.0001, and the result is shown in figure 5. The transcription level of the marker can further refine and stratify the patient under the current stratification, and identify the responsiveness of the patient to chemotherapy before chemotherapy, thereby being an important supplement of the current stratification.
Example 4: diagnostic efficacy determination
The marker value detected by a patient with complete follow-up data (n is 166) is used for making a receiver operating characteristic curve (ROC curve), R software is used for drawing the curve by a Youden method and calculating a cutoff value, the specificity and the sensitivity corresponding to a critical point are obtained according to coordinate values, the result is shown in figure 6, the sensitivity is 83.3 percent, the specificity is 82.9 percent, the cutoff value of the marker △ Ct is 4.913, and the area AUC under the curve is 0.88.
Example 5: transcription level of target gene and overall survival time (OS) of patient
The survival curve chart is made for patients (n is 166) with complete follow-up data, the three-place survival curve is made according to the detected marker value of the invention, GraphPad Prism software is adopted to draw the survival curve, and L og-rank (Mantel-Cox) method is used for statistical analysis, the result is shown in figure 7.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Beijing Hospital
<120> leukemia marker and application thereof in leukemia prognosis evaluation
<130>None
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>3740
<212>DNA/RNA
<213>Human
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gcctgtgtca tccgccattt tgtgagaagc aaggtggcct ccacgtttcc tgagcgtctt 60
cttcgctttt gcctcgaccg ccccttgacc acagacatgt ctcgggatcg gttccggagt 120
cgtggcggtg gcggtggtgg cttccacagg cgtggaggag gcggcggccg cggcggcctc 180
cacgacttcc gttctccgcc gcccggcatg ggcctcaatc agaatcgcgg ccccatgggt 240
cctggcccgg gccagagcgg ccctaagcct ccgatcccgc caccgcctcc acaccaacag 300
cagcaacagc caccaccgca gcagccaccg ccgcagcagc cgccaccgca tcagccgccg 360
ccgcatccac agccgcatca gcagcagcag ccgccgccac cgccgcagga ctcttccaag 420
cccgtcgttg ctcagggacc cggccccgct cccggagtag gcagcgcacc accagcctcc 480
agctcggccc cgcccgccac tccaccaacc tcgggggccc cgccagggtc cgggccaggc 540
ccgactccga ccccgccgcc tgcagtcacc tcggcccctc ccggggcgcc gccacccacc 600
ccgccaagca gcggggtccc taccacacct cctcaggccg gaggcccgcc gcctccgccc 660
gcggcagtcc cgggcccggg tccagggcct aagcagggcc caggtccggg tggtcccaaa 720
ggcggcaaaa tgcctggcgg gccgaagcca ggtggcggcc cgggcctaag tacgcctggc 780
ggccacccca agccgccgca tcgaggcggc ggggagcccc gcgggggccg ccagcaccac 840
ccgccctacc accagcagca tcaccagggg cccccgcccg gcgggcccgg cggccgcagc 900
gaggagaaga tctcggactc ggaggggttt aaagccaatt tgtctctctt gaggaggcct 960
ggagagaaaa cttacacaca gcgatgtcgg ttgtttgttg ggaatctacc tgctgatatc 1020
acggaggatg aattcaaaag actatttgct aaatatggag aaccaggaga agtttttatc 1080
aacaaaggca aaggattcgg atttattaag cttgaatcta gagctttggc tgaaattgcc 1140
aaagccgaac tggatgatac acccatgaga ggtagacagc ttcgagttcg ctttgccaca 1200
catgctgctg ccctttctgt tcgtaatctt tcaccttatg tttccaatga actgttggaa 1260
gaagccttta gccaatttgg tcctattgaa agggctgttg taatagtgga tgatcgtgga 1320
agatctacag ggaaaggcat tgttgaattt gcttctaagc cagcagcaag aaaggcattt 1380
gaacgatgca gtgaaggtgt tttcttactg acgacaactc ctcgtccagt cattgtggaa 1440
ccacttgaac aactagatga tgaagatggt cttcctgaaa aacttgccca gaagaatcca 1500
atgtatcaaa aggagagaga aacccctcct cgttttgccc agcatggcac gtttgagtac 1560
gaatattctc agcgatggaa gtctttggat gaaatggaaa aacagcaaag ggaacaagtt 1620
gaaaaaaaca tgaaagatgc aaaagacaaa ttggaaagtg aaatggaaga tgcctatcat 1680
gaacatcagg caaatctttt gcgccaagat ctgatgagac gacaggaaga attaagacgc 1740
atggaagaac ttcacaatca agaaatgcag aaacgtaaag aaatgcaatt gaggcaagag 1800
gaggaacgac gtagaagaga ggaagagatg atgattcgtc aacgtgagat ggaagaacaa 1860
atgaggcgcc aaagagagga aagttacagc cgaatgggct acatggatcc acgggaaaga 1920
gacatgcgaa tgggtggcgg aggagcaatg aacatgggag atccctatgg ttcaggaggc 1980
cagaaatttc cacctctagg aggtggtggt ggcataggtt atgaagctaa tcctggcgtt 2040
ccaccagcaa ccatgagtgg ttccatgatg ggaagtgaca tgcgtactga gcgctttggg 2100
cagggaggtg cggggcctgt gggtggacag ggtcctagag gaatggggcc tggaactcca 2160
gcaggatatg gtagagggag agaagagtac gaaggcccaa acaaaaaacc ccgattttag 2220
atgtgatatt taggctttca ttccagtttg ttttgttttt ttgtttagat accaatcttt 2280
taaattcttg cattttagta agaaagctat ctttttatgg atgttagcag tttattgacc 2340
taatatttgt aaatggtctg tttgggcagg taaaattatg taatgcagtg tttggaacag 2400
gagaattttt ttttcctttt tatttcttta ttttttcttt tttactgtat aatgtccctc 2460
aagtttatgg cagtgtacct tgtgccactg aatttccaaa gtgtaccaat tttttttttt 2520
ttactgtgct tcaaataaat agaaaaatag ttataatatt gatcttcaac tttgccattc 2580
atgcttctat gcatattagg ctacgtattc cacattgaaa gcatgagagt gtctaggcct 2640
ttgaatggca tatgccattt ctgggaaatg catctggagg ctaagtattg ctttctacaa 2700
ataattgccc cctttgtttt aaaaagaaga aatgcatatt gaagtagttt gatgatttgt 2760
ttggcatata ggaagcacgc tggtgctaag tattttttaa atggttatgt aagcaaagct 2820
gaactgtaaa tcttcaggaa tatgtattaa gattgtggaa tgggtgtaag acaattggta 2880
gggggtgaaa gtgggtttga ttaaatggat cttttatggc cctatgatct atcctttact 2940
tgaaagcttt tgaaaagtgg aaaggtcatt ttgttgcatt tccccatttc ttgtttttaa 3000
aagaccaaca aatctcaagc cctataaatg gcttgtattg aacttttaca tttgaattaa 3060
agatgttaaa catgaagtct tttcatgtat tcaagtgatt tataaagatg gggtgtagtc 3120
taaaaaacaa aacttgaaag taagaaggtt tagtaactgg gccgggcgcg gtggctcacg 3180
cctgtaatcc cagcactttg ggaggctgag gtgggcaaat catgagatca ggagttcaag 3240
accagcctgg ccaacatggt gaaaccccgt ctctactaaa aatacaaaaa attagctggg 3300
cgtggtggca ggcgcctgta atcccagcta ctcgggaggc tgagccagga gaattgcttg 3360
aacctggaga tggaggttgc agtgagctga gactgccact gcactccagc ctggacaaca 3420
gagcgagact ctgtctcaaa aaaaaaaaaa ggtttagtac tgaagtaggt ttagttatat 3480
atacatttgt actctgtaac ttttacatct ccttgtgcta cttctgttgg gcaaaatcta 3540
taggactata aaaatctatt ggttctcttc taagagctag tggtgattgt taacaaatag 3600
ggttgcagat aatcattctt attgtaaatt taacttgttt tcacatagtt tcgttttcat 3660
gtagcactaa atctggagaa agttgctcat gctacacagt acaaatatcc cttcctccca 3720
ccataaaggc ttggaatgtt 3740
Claims (10)
1. The leukemia marker is a gene shown in SEQ ID No.1 or a gene which has 85% -99% homology with the gene shown in SEQ ID No.1, and is used for preparing products for predicting and diagnosing whether a patient is a refractory myeloid leukemia patient or sorting the refractory myeloid leukemia patients.
2. The leukemia marker is a gene shown in SEQ ID No.1 or a gene which has 85% -99% homology with the gene shown in SEQ ID No.1, and is used for preparing products for predicting and diagnosing whether patients are myeloid leukemia patients with recurrence risk after the patients are treated to achieve complete remission of bone marrow or sorting the myeloid leukemia patients with recurrence risk after the patients are treated to achieve complete remission of bone marrow.
3. The leukemia marker is a gene shown in SEQ ID No.1 or a gene which has 85% -99% homology with the gene shown in SEQ ID No.1, and is used for preparing a product for predicting or evaluating the total survival time of a myeloid leukemia patient.
4. A marker composition comprising the marker of claim 1 or 2 for use in the preparation of a product for predicting, diagnosing whether a patient is refractory and/or is treated to achieve complete myeloid remission before the risk of relapse is present, or for sorting refractory and/or treated to achieve complete myeloid remission before the risk of relapse is present, or for predicting, assessing the overall survival of patients with myeloid leukemia.
5. The marker composition according to claim 3, wherein the marker composition further comprises one or more selected from the group consisting of a mutant gene, a fused gene or an abnormal chromosome involved in the onset and/or prognosis evaluation of leukemia, preferably one or more selected from the group consisting of a fused gene, a mutant gene or an abnormal chromosome involved in the onset and/or prognosis evaluation of leukemia in WHO and E L N risk stratification, more preferably one or more selected from the group consisting of a PM L-RAR α or AM L1-ETO fused gene, RUNX1 or ASX L1 mutant gene, a-5 or-7 abnormal chromosome.
6. The leukemia marker according to any one of claims 1-3, or the marker composition according to claim 4 or 5, wherein the myeloid leukemia is acute myeloid leukemia.
7. A kit comprising a product for detecting a leukemia marker according to any one of claims 1 to 3, or a marker composition according to claim 4 or 5.
8. The kit according to claim 7, wherein the product is a primer or probe for detecting the leukemia marker according to any one of claims 1 to 3, or the marker composition according to claim 4 or 5.
9. The leukemia marker according to any one of claims 1-3, the marker composition according to claim 4 or 5 or the kit according to claim 7, wherein the predicting the risk of a patient with myeloid leukemia comprises the steps of:
step 1) determining the level Ct of each biomarker in the leukemia marker according to any one of claims 1 to 3, the marker composition of claim 4 or 5 in a sample from the patient;
step 2) detecting GAPDH water of the sampleFlat, according to △ Ct ═ CtMarker substance-CtGAPDH△ Ct value is calculated, △ Ct is more than or equal to 4.913, the patient is a sensitive type patient, △ Ct is less than 4.913, and the patient is a difficult-to-cure type patient.
10. The leukemia marker, marker composition or kit of claim 9, wherein the sample is nucleated cells in bone marrow of the patient.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910024579.8A CN111424083A (en) | 2019-01-10 | 2019-01-10 | Leukemia marker and application thereof in leukemia prognosis evaluation |
| PCT/CN2019/130230 WO2020143500A1 (en) | 2019-01-10 | 2019-12-31 | Leukemia marker and use thereof in assessment of leukemia prognosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910024579.8A CN111424083A (en) | 2019-01-10 | 2019-01-10 | Leukemia marker and application thereof in leukemia prognosis evaluation |
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| CN111424083A true CN111424083A (en) | 2020-07-17 |
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| CN201910024579.8A Pending CN111424083A (en) | 2019-01-10 | 2019-01-10 | Leukemia marker and application thereof in leukemia prognosis evaluation |
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| WO (1) | WO2020143500A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891813A (en) * | 2010-04-12 | 2010-11-24 | 中国医学科学院血液学研究所 | Leukemia drug resistant epicyte protein and purpose thereof as drug resistant target protein |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006048263A2 (en) * | 2004-11-04 | 2006-05-11 | Roche Diagnostics Gmbh | Gene expression profiling in acute promyelocytic leukemia |
| AU2017258901A1 (en) * | 2016-12-30 | 2018-07-19 | Allarity Therapeutics Europe ApS | Methods for predicting drug responsiveness in cancer patients |
-
2019
- 2019-01-10 CN CN201910024579.8A patent/CN111424083A/en active Pending
- 2019-12-31 WO PCT/CN2019/130230 patent/WO2020143500A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101891813A (en) * | 2010-04-12 | 2010-11-24 | 中国医学科学院血液学研究所 | Leukemia drug resistant epicyte protein and purpose thereof as drug resistant target protein |
Non-Patent Citations (3)
| Title |
|---|
| ANNA D等: "Commonly altered genomic regions in acute myeloid leukemia are enriched for somatic mutations involved in chromatin remodeling and splicing", 《BLOOD》 * |
| NM_005066.3: "NM_005066.3", 《GENBANK》 * |
| 任思楣等: "细胞膜富含脯氨酸和谷氨酰胺的剪切因子维持急性髓性白血病HL-60细胞对多柔比星的敏感性", 《白血病.淋巴瘤》 * |
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| WO2020143500A1 (en) | 2020-07-16 |
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