CN111088360A

CN111088360A - Application of PD1-CTLA4 and/or PDL2-CTLA4 in preparation of AML prognosis prediction kit

Info

Publication number: CN111088360A
Application number: CN202010055211.0A
Authority: CN
Inventors: 李扬秋; 陈存特; 梁朝峰; 曾成武; 陈少华; 王彩霞
Original assignee: Jinan University
Current assignee: Jinan University; University of Jinan
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-01

Abstract

The invention provides application of PD1-CTLA4 and/or PDL2-CTLA4 in preparing a kit for predicting AML prognosis. Based on the first discovery by the inventor that the expression of PD1 and CTLA4, and PDL2 and CTLA4 in the bone marrow of AML patients are related to the prognosis of AML patients. When the expression ratio of PD1 and CTLA4 and/or PDL2 and CTLA4 is high, the possibility of poor clinical prognosis of AML patients is shown to be high. The combination of PD1 and CTLA4 and/or the combination expression ratio of PDL2 and CTLA4 has important guiding significance for prognosis judgment of AML patients and formulation of clinical treatment schemes, can provide more clinical prognosis research data for combined application of immune checkpoint inhibitors PD1-CTLA4 and PDL2-CTLA4 of AML patients, and has wide application prospect.

Description

Application of PD1-CTLA4 and/or PDL2-CTLA4 in preparation of AML prognosis prediction kit

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of PD1-CTLA4 and/or PDL2-CTLA4 in preparation of an AML prognosis prediction kit.

Background

Acute Myeloid Leukemia (AML) is the most common type of adult leukemia, with a rate of incidence of 3.7/100000. The prognosis of AML patients deteriorates with age, with 5-year survival rates of less than 10% in patients over 60 years of age. AML is caused by a variety of genetic factors, environmental changes, and their complex interactions. According to the cytogenetics and molecular biology characteristics of patients, the patients can be divided into three categories, namely low-risk, medium-risk and high-risk. Although different classes of AML patients benefit from chemotherapy and hematopoietic stem cell transplantation, the prognosis for these patients varies widely.

Different prognoses of AML patients suggest that other key factors (e.g. cellular immunity) may also affect the therapeutic efficacy. It is well known that immune evasion of cancer and abnormal immune monitoring play a crucial role in the development and progression of cancer. The study of programmed cell death 1 (PD-1), programmed cell death 1ligand 1 (PD-L1) and programmed cell death 1ligand 2 (PD-L2) in the immune escape of cancer cells has made it one of the most promising cell therapy methods in recent years. As Immune Checkpoints (ICs), PD-1 and PD-L1 inhibitors have shown significant effects in a variety of solid tumors, but the role of IC in leukemia is poorly understood. Few reports have shown that, by analyzing the survival curve, AML patients with high PD-L1 expression and concomitant mutations in nuclear phosphosin 1 (NPM 1) and fms-related receptor tyrosine kinase 3 (FLT 3) have a poor prognosis, while high PD-1 expression and T-cell immunoglobulin 3 positive (T-cell immunoglobulin 3, TIM3) T-cells are closely associated with the recurrence of AML patients after hematopoietic stem cell transplantation. In addition, PD-L1 was highly expressed in patients with tumor protein p53(tumor protein p53, TP53) mutations and recurrent mutations, and was positively correlated with risk stratification, based on RNA sequencing data of AML ribonucleic acid (RNA) in The Cancer Genome map (The Cancer Genome Atlas, TCGA) database. These findings suggest that PD-1 and PD-L1 inhibitors may be novel therapeutic strategies for AML. At present, PD-1 and PD-L1 inhibitors are also being clinically tested in AML. Although studies have analyzed the prognostic role of PD-L1 in AML patients with certain types of mutations via survival curves, it is well known that IC may be co-expressed between and involved in the immunosuppressive network. There is still a lack of systematic analysis of different IC expression patterns and combinations of ICs, and the prognosis of AML patients cannot be fully assessed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of PD1-CTLA4 and/or PDL2-CTLA4 in preparing a kit for predicting AML prognosis. When high-expression PD1-CTLA4 and/or PDL2-CTLA4 is detected, the probability of poor prognosis of AML patients is high, and the detection method can be used as an index for predicting the prognosis evaluation of the AML patients.

It is another object of the present invention to provide a kit for predicting the prognosis of AML.

The purpose of the invention is realized by the following technical scheme:

the application of PD1-CTLA4 and/or PDL2-CTLA4 in preparing a kit for predicting AML prognosis is based on the first discovery by the inventor of the invention that the expression of PD1-CTLA4 and PDL2-CTLA4 in the bone marrow of AML patients is related to the prognosis of the AML patients. The PD1-CTLA4 refers to the combination of PD1 and CTLA4 genes; the PDL2-CTLA4 refers to the combination of PDL2 and CTLA4 genes.

In the application, the expression level of the gene is obtained by detecting the expression level of one or two of PD1 gene and PDL2 gene and the expression level of CTLA4 gene in bone marrow of a clinical patient and combining the expression level of an internal reference gene for statistical analysis, thereby predicting the AML prognosis.

The reference gene is preferably 18 SrRNA.

The statistical analysis method is preferably a delta CT method; the expression level is 2^ (-Delta CT), Delta CT is Delta CT_{Clinical patients}–ΔCT_{Normal person}，ΔCT_{Clinical patients}＝(CT_Gene-CT_{Internal reference})，ΔCT_{Normal person}＝(CT_Gene-CT_{Internal reference})，CT_GeneAnd CT_{Internal reference}The fluorescent probe is obtained by detecting through a real-time fluorescent quantitative PCR method.

The prediction specifically refers to:

① when the expression level of PD1 is more than or equal to 1.1 and the expression level of CTLA4 is more than or equal to 0.5, and/or when the expression level of PDL2 is more than or equal to 0.4 and the expression level of CTLA4 is more than or equal to 0.5, the AML patient has a high possibility of poor clinical prognosis;

② when the expression level of PD1 is more than or equal to 1.1 or the expression level of CTLA4 is more than or equal to 0.5, and/or when the expression level of PDL2 is more than or equal to 0.4 or the expression level of CTLA4 is more than or equal to 0.5, the possibility of poor clinical prognosis of AML patients is relatively high;

③ when the expression level of PD1 <1.1 and CTLA4 <0.5, and/or when the expression level of PDL2 <0.4 and CTLA4 <0.5, there is a greater likelihood that the clinical prognosis of AML patients will be good.

A kit for predicting AML prognosis comprising one or both of a primer for amplifying PD1 cDNA and a primer for amplifying PDL2 cDNA, and a primer for amplifying CTLA4 cDNA.

The primers for amplifying PD1 cDNA are as follows:

PD1(F):5'-CTCAGGGTGACAGAGAGAAG-3'；

PD1(R):5'-GACACCAACCACCAGGGTTT-3'；

the primers used for amplifying the PDL2 cDNA were as follows:

PDL2(F):5'-ACAGTGCTATCTGAACCTGTGG-3'；

PDL2(R):5'-CTGCAGGCCACCGAATTCTT-3'；

the primers used for amplifying CTLA4 cDNA are as follows:

CTLA4(F):5'-GCCCTGCACTCTCCTGTTTTT-3'；

CTLA4(R):5'-GGTTGCCGCACAGACTTCA-3'。

the kit also comprises primers for amplifying the internal reference, and the primers are as follows:

18SrRNA(F):5'-CGGCGGCTTTGGTGACTCTAGA-3'；

18SrRNA(R):5'-CCTGCTGCCTTCCTTGGATGTG-3'。

the kit also comprises any one or at least two of a reagent for separating bone marrow mononuclear cells, a reagent for extracting total RNA, a reagent for Reverse transcription polymerase chain reaction (RT-PCR) and a reagent for real-time quantitative PCR (qRT-PCR).

The reagent for separating bone marrow mononuclear cells is preferably lymphocyte separation liquid (Ficoll).

The reagent for extracting the total RNA is preferably TRIZOL.

The kit for predicting AML prognosis is applied to non-diagnostic detection of PD1-CTLA4 and/or PDL2-CTLA4 expression.

The application comprises the following steps:

(1) taking a bone marrow sample to be detected, and separating to obtain mononuclear cells;

(2) adding a reagent for extracting total RNA into the mononuclear cells obtained in the step (1), and uniformly mixing;

(3) adding chloroform, mixing, and centrifuging;

(4) sucking the upper layer liquid, adding isopropanol, and centrifuging to remove the supernatant;

(5) washing with ethanol, and drying to obtain an RNA sample;

(6) reverse transcribing the RNA sample obtained in step (5) into cDNA;

(7) and (4) detecting the expression quantity of the target gene by using the cDNA obtained in the step (6) by using the kit.

The proportion of the mononuclear cells, the total RNA extraction reagent and the chloroform is preferably 5-10 multiplied by 10⁶And (2) cell: 1 ml: 0.1-0.3 ml.

The reagent for extracting total RNA in the step (2) is preferably TRIZOL.

The conditions for the centrifugation in step (3) are preferably: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 15-30 minutes.

The amount of the isopropyl alcohol in the step (4) is preferably as follows: calculating the volume ratio of isopropanol to isopropanol being 1-2: 1-2; preferably calculated as 1: 1.

The conditions for the centrifugation in the step (4) are preferably: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 10-20 minutes;

the number of ethanol washes described in step (5) is preferably two; more preferably, the first time is preferably washed by 75% ethanol at the temperature of minus 20 ℃, the second time is preferably washed by 100% ethanol at the temperature of minus 20 ℃, after each time of uniform mixing for 30s, the mixture is centrifuged for 5 to 10 minutes at the temperature of 2 to 8 ℃ and 8000 to 12000 g.

The drying in the step (5) is preferably vacuum centrifuge drying; the conditions are preferably 2-8 ℃ and 5-10 minutes.

The reverse transcription described in the step (6) is carried out by a reagent for reverse transcription PCR.

The detection of the expression level of the target gene in the step (7) is realized by a reagent for real-time quantitative PCR.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention adopts a real-time quantitative polymerase chain reaction (qRT-PCR) to detect the expression conditions of PD1, CTLA4 and PDL2 in the bone marrow of AML patients, and firstly discovers that the combination of PD1 and CTLA4 or PDL2 and CTLA4 better predicts the prognosis of the AML patients than the individual PD1 or PDL 2. When PD1 and CTLA4 or PDL2 and CTLA4 are highly expressed at the same time, the prognosis of AML patients is poor. The expression ratio of the PD1 and CTLA4 or the PDL2 and CTLA4 has important guiding significance for the prognosis judgment of AML patients and the establishment of clinical treatment schemes.

2. The invention can provide more clinical prognosis research data for combined application of the immune checkpoint inhibitors PD1-CTLA4 and PDL2-CTLA4 to AML patients, and has wide application prospect in predicting the prognosis evaluation of the AML patients and combining the immune checkpoint inhibitors.

3. The invention uses qRT-PCR method to express PD1, CTLA4 and PDL2 in AML patient marrow, the method is simple and easy, and the method is stable. When high-expression PD1-CTLA4 or PDL2-CTLA4 is detected, the probability of poor prognosis of AML patients is high, and the detection method can be used as an index for predicting the prognosis evaluation of the AML patients.

Drawings

FIG. 1 is a graph showing the effect of expression levels of PD1, CTLA4 and PDL2 on the prognosis of AML naive patients; wherein, panel a is the prognosis of PD1 for AML naive patients, panel b is the prognosis of CTLA4 for AML naive patients, and panel c is the prognosis of PDL2 for AML naive patients.

FIG. 2 is a graph of the effect of PD1 in combination with CTLA4 and PDL2 in combination with CTLA4 on the prognosis of AML naive patients; wherein, graph a is the comparison of low expression PD1 with CTLA4, high expression PD1 or high expression CTLA4, high expression PD1 with CTLA4, graph b is the comparison of high expression PD1 with low expression CTLA4, high expression PD1 with CTLA4, graph c is the comparison of low expression PDL2 with CTLA4, high expression PDL2 or high expression CTLA4, high expression PDL2 with CTLA4, and graph d is the comparison of high expression PDL2 with low expression CTLA4, high expression PD1 with CTLA 4.

FIG. 3 is a graph of the impact of the combination of PD1 and PDL2 on the prognosis of AML naive patients; wherein, graph a is a comparison of low-expression PD1 with PDL2, high-expression PD1 or high-expression PDL2, high-expression PD1 with PDL2, graph b is a comparison of high-expression PD1 with low-expression PDL2, high-expression PD1 with PDL2, and graph c is a comparison of high-expression PDL2 with low-expression PD1, high-expression PDL2 with PD 1.

FIG. 4 is a graph of the impact of PD1 in combination with PDL2 and CTLA4 on the prognosis of AML naive patients; wherein, the graph a is the comparison of low-expression PD1 and PDL2 with CTLA4, high-expression PD1 or high-expression PDL2 or high-expression CTLA4, high-expression PD1 and PDL2 with CTLA4, the graph b is the comparison of high-expression PD1 and low-expression PDL2 with low-expression CTLA4, high-expression PD1 with high-expression PDL2 or high-expression CTLA4, high-expression PD1 with high-expression PDL2 with high-expression CTLA4, the graph c is the comparison of high-expression PDL2 and low-expression PD1 with low-expression CTLA4, high-expression PDL2 and high-expression PD1 or high-expression CTLA4, and the comparison of high-expression PDL2 with high-expression PD1 with high-expression CTLA 4.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The reagent information used in the examples is specifically as follows:

lymphocyte isolate Ficoll (available from GE Healthcare Life Sciences);

TRIZOL (available from Invitrogen);

reverse transcription PCR kit (purchased from Promega);

real-time quantitative PCR kit (purchased from TIANGEN);

example 1

(1) Bone marrow was collected with patient informed consent. 62 primary AML samples and 12 healthy adult bone marrow samples were collected from the first human hospital in Guangzhou, the hematological department of the department of research, all of which were anticoagulated with heparin from the time of admission, and which were approved by the ethical Committee of the department. Clinical data, such as survival time and survival status of AML patients, were also collected (as shown in table 1).

(2) Mononuclear cells from bone marrow were isolated. 4mL of lymphocyte separation medium (Ficoll, density 1.077) is added into a 15mL centrifuge tube, the diluted anticoagulated bone marrow specimen suspension is laid on the separation medium, and the suspension is centrifuged for 15 minutes at 1500rpm by a horizontal centrifuge. Sucking the middle single core layer, transferring to another 15mL centrifuge tube, adding appropriate amount of 1 XPhosphate buffer saline (PBS), gently blowing, centrifuging at 1000rpm for 10 minutes, discarding supernatant, adding 1 XPBS solution to 2mL, mixing, counting, washing twice with the same method, and washing with 5-10 × 10⁶1 ml of TRIZOL reagent was added to each mononuclear cell to prepare a single cell suspension for use.

(3) Total RNA extraction

3.1 placing the single cell suspension on ice, adding 0.2 ml of chloroform after 5 minutes, fully and uniformly mixing (15 seconds), placing on ice for 2-3 minutes, and centrifuging at low temperature and high speed (2-8 ℃, 12000g) for 15-30 minutes;

3.2 carefully sucking the supernatant (about 50% of the total volume) and transferring to a 1.5 ml new tube, adding 0.5 ml isopropanol and mixing uniformly, standing on ice for 10 minutes, and centrifuging at low temperature and high speed (2-8 ℃, 12000g) for 10-20 minutes;

3.3 removing the supernatant and washing twice with 1 ml of 75% ethanol (-20 ℃), mixing evenly for 30 seconds each time, and centrifuging for 5-10 minutes (2-8 ℃, 10000 g);

3.4 removing the supernatant, centrifuging again, and removing the residual ethanol;

3.5 drying in a vacuum centrifuge at the low temperature of 2-8 ℃ for 5-10 minutes;

3.6 Add ultra-clean water (Biotecx BL-5700) 50. mu.l (optionally increased or decreased) and mix well to obtain RNA sample.

3.7 taking 2 microliter RNA sample and diluting to 400 microliter with ultra-clean water, detecting the optical density of the sample at A260nm wavelength in an ultraviolet spectrophotometer, and estimating the purity and content (1 OD)_A260nm40 μ g/ml), total amount of RNA OD × 400 μ g;

3.8RNA samples were stored at-70 ℃ for future use after addition of 0.1 volume (5. mu.l) of 3mol/L NaAC (sodium acetate) and 2 volumes (100. mu.l) of ethanol.

(4)RT-PCR

4.1 500ng of RNA, 0.5. mu.l of oligo (dT) (0.5. mu.g/reaction), 0.5. mu.l of random primer (0.5. mu.g/reaction) and double distilled water (ddH) without RNase₂O) (up to 5 μ l) were added together and incubated at 70 ℃ for 5 minutes followed by rapid cooling on ice for 5 minutes;

4.2 Add 4.0. mu.l of GoScript^TM5 Xreaction buffer, 1.7. mu.l MgCl₂(final concentration 2.0mmol/L), 1.0. mu.l 0.5mmol/L dNTP, 0.3. mu.l RNase inhibitor (20U), 1. mu.l reverse transcriptase, ddH₂O is complemented to 15 mu l;

4.3 after mixing, 20 microliter of the sample was incubated at 42 ℃ for 60 minutes and then inactivated at 70 ℃ for 15 minutes;

4.4 addition of 80. mu.l of ddH₂And (4) diluting with oxygen.

(5)qRT-PCR

5.1qRT-PCR 20 microliter system was formulated as: 10. mu.l Mix, 0.5. mu.l forward primer (10. mu. mol/L), 0.5. mu.l reverse primer (10. mu. mol/L), 1. mu.l cDNA, ddH₂O is added to 8 mu l;

wherein, the sequences of the primers are as follows:

PD1(F):5'-CTCAGGGTGACAGAGAGAAG-3'；

PD1(R):5'-GACACCAACCACCAGGGTTT-3'；

PDL2(F):5'-ACAGTGCTATCTGAACCTGTGG-3'；

PDL2(R):5'-CTGCAGGCCACCGAATTCTT-3'；

CTLA4(F):5'-GCCCTGCACTCTCCTGTTTTT-3'；

CTLA4(R):5'-GGTTGCCGCACAGACTTCA-3'；

internal reference primers:

18SrRNA(F):5'-CGGCGGCTTTGGTGACTCTAGA-3'；

18SrRNA(R):5'-CCTGCTGCCTTCCTTGGATGTG-3'；

5.2qRT-PCR the program settings were as follows: 15 minutes at 95 ℃; 10s at 95 ℃, 20s at 60 ℃ and 45 cycles;

5.3 Gene expression level is shown to be 2^ (- Δ Δ CT).

Gene expression quantity CT obtained by qRT-PCR determination_PD1、CT_PDL2、CT_CTLA4Further performing statistical analysis by a delta CT method to obtain a gene expression level, wherein:

ΔCT_PD1＝(CT_PD-1-CT_18SrRNA)；

ΔCT_PDL2＝(CT_PDL2-CT_18SrRNA)；

ΔCT_CTLA4＝(CT_CTLA4-CT_18SrRNA)；

ΔΔCT＝ΔCT_{incipient AML patient}–ΔCT_{Healthy adult}；ΔCT_{Healthy adult}Obtained by calculating the average value of 12 healthy adult bone marrow samples;

the gene expression level is 2^ (-Delta CT).

The gene expression profile and clinical prognosis data of AML patients are shown in Table 1.

The expression profiles of PD1, CTLA4 and PDL2 were analyzed in combination with clinical prognosis data of AML patients, and optimal prognostic cut-off values of gene expression of PD1, CTLA4 and PDL2 were obtained in X-tile software (version 3.6.1, yale university) by inputting gene expression levels, survival status and survival time. 1.1, 0.5 and 0.4 respectively. As shown in FIG. 1, the prognosis of AML patients with PD1 ≧ 1.1, CTLA4 ≧ 0.5 and PDL2 ≧ 0.4 was found to be poor. The survival analysis was carried out by combining PD1 with CTLA4 and PDL2 with CTLA4, and the results are shown in fig. 2. The results suggest that the prognosis of AML patients with high expression of PD1 and CTLA4 is the worst (figure 2a), and the analysis of the low expression and high expression of CTLA4 shows that PD1 is used for patients with high expression of PD1^highCTLA4^highShort survival of the patient (fig. 2 b); the results suggest that the prognosis of AML patients with high PDL2 expression and CTLA4 expression was the worst (fig. 2c), and patients with high PDL2 expression were further classified into CTLA4 low-expression and high-expression componentsFound out that PDL2^highCTLA4^highThe survival of the patients was shorter (fig. 2 d). From the above results, it is understood that the combination of PD1 and CTLA4 is more predictive of prognosis of AML patients than PD1 alone, and simultaneously, the combination of PDL2 and CTLA4 is more predictive of prognosis of AML patients than PDL2 alone.

Meanwhile, survival analysis was carried out by combining PD1 with PDL2, PD1 with PDL2 with CTLA4, respectively, and the results suggest that the prediction effect of PD1 with PDL2 (fig. 3) or PD1 with PDL2 with CTLA4 (fig. 4) on the prognosis of AML patients is not different from that of PD1 or PDL2 alone (P > 0.05).

The above experimental results show that the combination of PD1 and CTLA4 or PDL2 and CTLA4 has important significance in the evaluation of AML clinical prognosis prediction, and provides prognostic research data for clinical combined application of immune checkpoint inhibitors.

TABLE 1 AML patients PD1, CTLA4, PDL2 Gene expression profiles and clinical data

Note: 0 in the state represents survival and 1 represents death.

The gene expression level measured as described above, although not directly leading to future diagnosis and health condition, can be used as an intermediate result as one of reference information for the clinical treatment planning of patients.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> PD1-CTLA4 and/or PDL2-CTLA4 in preparation of AML prognosis prediction kit

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PD1 (F)

<400>1

ctcagggtga cagagagaag 20

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PD1 (R)

<400>2

gacaccaacc accagggttt 20

<210>3

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PDL2 (F)

<400>3

acagtgctat ctgaacctgt gg 22

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>PDL2 (R)

<400>4

ctgcaggcca ccgaattctt 20

<210>5

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>CTLA4 (F)

<400>5

gccctgcact ctcctgtttt t 21

<210>6

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>CTLA4 (R)

<400>6

ggttgccgca cagacttca 19

<210>7

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>18SrRNA (F)

<400>7

cggcggcttt ggtgactcta ga 22

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>18SrRNA (R)

<400>8

cctgctgcct tccttggatg tg 22

Claims

Use of PD1-CTLA4 and/or PDL2-CTLA4 in the preparation of a kit for predicting AML prognosis.
2. Use of PD1-CTLA4 and/or PDL2-CTLA4 according to claim 1 for the preparation of a kit for predicting AML prognosis, characterized in that: the expression level of the gene is obtained by detecting the expression level of one or two of PD1 and PDL2 and the expression level of CTLA4 gene in bone marrow of a clinical patient and combining the expression level of an internal reference gene for statistical analysis, thereby predicting the AML prognosis.
3. Use of PD1-CTLA4 and/or PDL2-CTLA4 according to claim 2 for the preparation of a kit for predicting AML prognosis, characterized in that: the internal reference gene is 18 SrRNA; the statistical analysis method is a delta CT method; the expression level is 2^ (-Delta CT), Delta CT is Delta CT_{Clinical patients}–ΔCT_{Normal person}，ΔCT_{Clinical patients}＝(CT_Gene-CT_{Internal reference})，ΔCT_{Normal person}＝(CT_Gene-CT_{Internal reference})；CT_GeneAnd CT_{Internal reference}Detecting by a real-time fluorescent quantitative PCR method; the prediction specifically refers to:

① when the expression level of PD1 is more than or equal to 1.1 and the expression level of CTLA4 is more than or equal to 0.5, and/or when the expression level of PDL2 is more than or equal to 0.4 and the expression level of CTLA4 is more than or equal to 0.5, the AML patient has a high possibility of poor clinical prognosis;

② when the expression level of PD1 is more than or equal to 1.1 or the expression level of CTLA4 is more than or equal to 0.5, and/or when the expression level of PDL2 is more than or equal to 0.4 or the expression level of CTLA4 is more than or equal to 0.5, the possibility of poor clinical prognosis of AML patients is relatively high;

③ when the expression level of PD1 <1.1 and CTLA4 <0.5, and/or when the expression level of PDL2 <0.4 and CTLA4 <0.5, there is a greater likelihood that the clinical prognosis of AML patients will be good.
4. A kit for predicting AML prognosis, comprising: comprises one or two of a primer for amplifying PD1 cDNA and a primer for amplifying PDL2 cDNA, and a primer for amplifying CTLA4 cDNA.
5. The kit for predicting AML prognosis as claimed in claim 4, characterized in that:

the primers for amplifying PD1 cDNA are as follows:

PD1(F):5'-CTCAGGGTGACAGAGAGAAG-3'；

PD1(R):5'-GACACCAACCACCAGGGTTT-3'；

the primers used for amplifying the PDL2 cDNA were as follows:

PDL2(F):5'-ACAGTGCTATCTGAACCTGTGG-3'；

PDL2(R):5'-CTGCAGGCCACCGAATTCTT-3'；

the primers used for amplifying CTLA4 cDNA are as follows:

CTLA4(F):5'-GCCCTGCACTCTCCTGTTTTT-3'；

CTLA4(R):5'-GGTTGCCGCACAGACTTCA-3'。
6. the kit for predicting AML prognosis as claimed in claim 5, characterized in that: the kit also comprises primers for amplifying the internal reference, and the primers are as follows:

18SrRNA(F):5'-CGGCGGCTTTGGTGACTCTAGA-3'；

18SrRNA(R):5'-CCTGCTGCCTTCCTTGGATGTG-3'。
7. kit for predicting the prognosis of AML according to claim 5 or 6, characterized in that: the kit also comprises any one or at least two of a reagent for separating bone marrow mononuclear cells, a reagent for extracting total RNA, a reagent for reverse transcription PCR and a reagent for real-time quantitative PCR.
8. Use of a kit according to any one of claims 4 to 7 for the non-diagnostic detection of PD1-CTLA4 and/or PDL2-CTLA4 expression in AML prognosis.
9. Use according to claim 8, characterized in that: the method comprises the following steps:

(1) taking a bone marrow sample to be detected, and separating to obtain mononuclear cells;

(2) adding a reagent for extracting total RNA into the mononuclear cells obtained in the step (1), and uniformly mixing;

(3) adding chloroform, mixing, and centrifuging;

(4) sucking the upper layer liquid, adding isopropanol, and centrifuging to remove the supernatant;

(5) washing with ethanol, and drying to obtain an RNA sample;

(6) reverse transcribing the RNA sample obtained in step (5) into cDNA;

(7) and (4) detecting the expression quantity of the target gene by using the cDNA obtained in the step (6) by using the kit.
10. Use according to claim 9, characterized in that:

the ratio of the mononuclear cells, the total RNA extraction reagent and chloroform is 5-10 multiplied by 10⁶And (2) cell: 1 ml: calculating 0.1-0.3 ml;

the reagent for extracting the total RNA in the step (2) is TRIZOL;

the centrifugation conditions in the step (3) are as follows: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 15-30 minutes;

the dosage of the isopropanol in the step (4) is as follows: calculating the volume ratio of isopropanol to isopropanol being 1-2: 1-2;

the centrifugation conditions in the step (4) are as follows: the temperature is 2-8 ℃, the rotating speed is 10000-15000 g, and the time is 10-20 minutes;

washing with ethanol twice in the step (5), washing with 75% ethanol at-20 ℃ for the first time, washing with 100% ethanol at-20 ℃ for the second time, uniformly mixing for 30s each time, and centrifuging at 2-8 ℃ and 8000-12000 g for 5-10 minutes;

the drying in the step (5) is vacuum centrifuge drying, and the conditions are that the temperature is 2-8 ℃ and the time is 5-10 minutes;

the reverse transcription in the step (6) is realized by a reagent for reverse transcription PCR;

the detection of the expression level of the target gene in the step (7) is realized by a reagent for real-time quantitative PCR.