CN114606316A

CN114606316A - Construction method of model for early diagnosis and prognosis prediction of NK/T cell lymphoma

Info

Publication number: CN114606316A
Application number: CN202210244353.0A
Authority: CN
Inventors: 蔡清清; 田小朋; 张宇辰; 王金妮; 马淑云; 邹琪华; 蔡君
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2022-03-12
Filing date: 2022-03-12
Publication date: 2022-06-10

Abstract

The invention specifically discloses a construction method of an NK/T cell lymphoma early diagnosis and prognosis prediction model, which comprises the following steps: step S1: screening differential ctDNA methylation sites; step S2: screening a ctDNA methylation probe; step S3: constructing and verifying a ctDNA methylation diagnosis model of NKTCL; step S4: and (3) constructing and verifying a ctDNA methylation prognosis model of NKTCL. The ctDNA methylation detection has the advantages of non-invasive, comprehensive, dynamic and sensitive properties, only needs to collect blood samples of a subject for detection, can solve the defects that a molecular model based on genome sequencing, single nucleotide polymorphism and the like at present needs a large number of tissue slices, is expensive, has poor clinical application performance and the like, and can solve the defect that tissue biopsy is influenced by the type and the part of material to be obtained; the methylation detection sensitivity and the dynamic monitoring value are higher; the establishment of the ctDNA methylation model is very important for the formulation of an individual accurate NKTCL treatment scheme and the improvement of layered treatment bases.

Description

Construction method of model for early diagnosis and prognosis prediction of NK/T cell lymphoma

Technical Field

The invention relates to the technical field of cancer risk assessment, in particular to a construction method of an NK/T cell lymphoma early diagnosis and prognosis prediction model.

Background

NK/T cell lymphoma (NKTCL) is a subtype of non-Hodgkin lymphoma, and the incidence rate of the NK/T cell lymphoma is highest in China worldwide. The 5-year survival rate of the early NKTCL patient can reach 80 percent and is obviously superior to that of the late patient, so that the death rate of the NKTCL can be greatly reduced by early diagnosis and early treatment. NKTCL has strong heterogeneity, the current prognosis evaluation system is based on clinical and pathological features, and patients with the same clinical stage often show obvious prognosis difference. The development of an effective and simple detection means is important for improving the prognosis of patients, guiding accurate treatment to save medical resources, reducing the burden of patients, reducing the side effects of treatment of patients and the like.

At present, research reports on NKTCL molecular models based on genome sequencing and single nucleotide polymorphism, but the NKTCL molecular models respectively have the defects of large tissue slice requirement, high price, incapability of reflecting tumor cell mutation and the like; moreover, due to obvious tissue necrosis of the pathological part of the NKTCL, certain difficulty is brought to the definite diagnosis of the NKTCL.

Circulating tumor DNA (ctDNA) detection is used for sampling and analyzing tumor-associated DNA of non-solid tissues (mainly blood) in a non-invasive/minimally invasive mode, and has the advantages of non-invasiveness, completeness, dynamics and sensitivity. Compared with the traditional ctDNA somatic mutation spectrum detection, the methylation level detection of ctDNA has the following advantages: the methylation detection sensitivity and the dynamic monitoring value are higher; multiple CpG methylation sites per target gene region can be detected; ctDNA methylation changes from the same type of tumor cells are relatively stable. Therefore, ctDNA methylation level is a more ideal indicator for early diagnosis of tumors.

1. At present, NKTCL molecular models based on genome sequencing, single nucleotide polymorphism and the like have the defects of large tissue section requirement, high price, incapability of reflecting tumor cell mutation and the like.

2. The traditional detection aiming at ctDNA somatic mutation spectrum has insufficient sensitivity and dynamic monitoring value, can not detect information of a plurality of sites of a single target gene, and has larger heterogeneity of ctDNA methylation change from the same type of tumor cells.

3. The NKTCL lesion part has obvious tissue necrosis, and the deviation of the material quality, the material part and the tumor heterogeneity can not be eliminated;

4. at present, no noninvasive early diagnosis mode aiming at NKTCL exists; furthermore, PINK-E, IPI and other prognosis evaluation systems based on clinical and pathological characteristics are mainly used for prognosis judgment, and the root cause of differential prognosis cannot be explained.

Disclosure of Invention

The invention provides a construction method of an NK/T cell lymphoma early diagnosis and prognosis prediction model, which is used for solving at least one technical problem in the background technology.

A construction method of an NK/T cell lymphoma early diagnosis and prognosis prediction model comprises the following steps:

step S1: screening differential ctDNA methylation sites;

step S2: screening a ctDNA methylation probe;

step S3: constructing and verifying a ctDNA methylation diagnosis model of NKTCL;

step S4: and (3) constructing and verifying a ctDNA methylation prognosis model of NKTCL.

Preferably, in step S1, the step of screening for differential ctDNA methylation sites comprises:

s11: collecting plasma samples of 250-350 NKTCL patients and 250-350 normal persons;

s12: extracting free DNA by using a cfDNA extraction kit, carrying out whole genome ctDNA methylation level detection by high-throughput whole genome methylation sequencing, and establishing a methylation library with about 300 ten thousand sites;

s13: methylation sites with p less than 0.05 and methylation level difference multiple of 2000 are obtained by screening through methylation level difference analysis and are used for subsequent queue research;

s14: corresponding methylation probes are designed according to the 2000 differential methylation sites, and methylation sites capable of generating positive and specific PCR amplification signals are screened through PCR verification.

Preferably, in step S12, the step of extracting free DNA using the cfDNA extraction kit includes:

s121: putting 200 μ l of blood into a 1.5ml centrifuge tube, adding 400 μ l of cell lysate CL, reversing and mixing uniformly, centrifuging at 10000rpm for 1min, discarding supernatant, adding 200 μ l of buffer GS into cell nucleus precipitate, shaking to thoroughly mix uniformly;

s122: adding 200 μ l buffer solution GB and 20 μ l premixed solution of protease K, fully inverting and mixing, standing at 56 deg.C for 10min until the solution becomes clear;

s123: after standing at room temperature for 2-5min, 350. mu.l of buffer BD was added, and the mixture was thoroughly mixed by inversion.

Preferably, the step S12 further includes:

s124: adding the solution obtained in the step S123 into an adsorption column CG2, centrifuging at 12000rpm for 30S, and pouring out waste liquid in the collection tube;

s125: adding 600 μ l buffer GDB into adsorption column CG2, centrifuging at 12000rpm for 30s, and removing waste liquid in the collection tube;

s126: adding 500 μ l of rinsing solution PWB into adsorption column CG2, centrifuging at 12000rpm for 30s, pouring off waste liquid in the collection tube, and repeating the steps; centrifuging at 12000rpm for 2min, pouring off waste liquid in the collecting tube, standing adsorption column CG2 at room temperature for 2min, and completely air drying residual rinsing liquid in the adsorption material;

s127: transferring the adsorption column CG2 into a 1.5ml centrifuge tube, suspending 80 μ l of elution buffer TB to the middle position of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, collecting the solution in the centrifuge tube, and storing at-20 deg.C for later use.

Preferably, in step S2, the step of screening the ctDNA methylation probe comprises:

s21: collecting 100 cases of NKTCL tumor tissue specimens and corresponding plasma specimens before treatment, extracting tumor tissue DNA and plasma ctDNA by using a tissue DNA and plasma ctDNA kit, wherein the extraction steps are the same as S12;

s22: detecting the methylation level of the candidate methylation sites of the tumor tissue DNA and the corresponding matched plasma ctDNA by using targeted bisulfite sequencing by taking the candidate methylation sites as difference sites;

s23: and comparing the methylation level consistency of the DNA of the tissue sample and the plasma ctDNA, and selecting the candidate methylation sites with better consistency and higher detection abundance for subsequent modeling.

Preferably, in step S22, the bisulfite sequencing step of detecting the DNA methylation level of tumor tissue is:

s221: the bisulfite treated product was centrifuged and transferred to a 1.5ml EP tube;

s222: adding 310 mu l B mu ffer BL, shaking and mixing uniformly, and adding carrier RNA if the DNA content is lower than 100 ng; adding 250 μ l ethanol (concentration 90-100%), shaking for 15s, and centrifuging;

s223: adding the solution obtained in the previous step into an adsorption column MinEl μ te DNA Spin Col μmes, centrifuging at 12000rpm for 1min, and pouring off the waste liquid in the collection tube;

s224: adding 500 mu l B mu ffer BW into an adsorption column MinEl mu te DNA Spin Col mu mes, centrifuging at 12000rpm for 30s, and pouring waste liquid in a collecting tube;

s225: adding 500 μ l B μ ffer BD to adsorption column MinEl μ te DNA Spin Col μmes, and cooling for 15min at room temperature; centrifuging at 12000rpm for 1min, and pouring off waste liquid in the collecting pipe;

s226: adding 500 mu l B mu ffer BW into the adsorption column MinEl mu te DNA Spin Col mu mes, centrifuging at 12000rpm for 1min, pouring waste liquid in the collection tube, and repeating the step;

s227: adding 250 μ l of anhydrous ethanol into MinEl μ te DNA Spin Col μmes adsorption column, centrifuging at 12000rpm for 1min, and pouring off waste liquid in the collection tube;

s228: centrifuging at 12000rpm for 2min, pouring out waste liquid in the collecting tube, standing adsorption column MinEl μ te DNA Spin Col μmes at room temperature for 2-5min, and air drying residual rinsing liquid in the adsorption material;

s229: transferring the adsorption column MinEl μ te DNA Spin Col μmes into a 1.5ml centrifuge tube, suspending 80 μ l of elution buffer solution B μ ffer EB into the middle position of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12000rpm for 2min, collecting the solution into the centrifuge tube, and storing at-20 ℃ for later use.

Preferably, in step S3, the step of constructing and verifying the ctDNA methylation diagnostic model of NKTCL comprises:

s31: collecting 650 cases of NKTCL patients before treatment and 550 cases of normal human plasma samples before treatment internally, randomly distributing the samples to a training set and an internal verification set according to the proportion of 7:3, and performing targeted bisulfite sequencing by taking the differential methylation sites as the background of the queue research; collecting plasma samples of about 100 and 200 cases of pre-treatment NKTCL patients and 100 and 200 cases of normal persons as an external verification set;

s32: respectively using LASSO (last Absol μ te Shrinkage and Selection Operator) regression model and Random Forest (Random Forest) regression model method to construct diagnosis model, selecting methylation locus most relevant to disease diagnosis as binary classification model construction variable.

Preferably, in step S3, the step of constructing and verifying the ctDNA methylation diagnostic model of NKTCL further comprises:

s33: calculating to obtain a methylation label diagnosis score by using a logistic regression (logistic regression) model and integrating a regression coefficient and a methylation level expression matrix of the locus;

s34: the prediction performance of the model was evaluated in the training set, the internal validation set and the external validation set by means of Receiver Operating Characteristic (ROC) curve, Area under curve (Area m der C μ rve, a m C), confusion matrix (conf μ fusion table).

Preferably, in step S4, the step of constructing and verifying the ctDNA methylation prognosis model of NKTCL comprises:

s41: randomly distributing 550-650 NKTCL patients collected internally to a training set and an internal verification set according to a ratio of 7: 3; externally collected 100-200 pre-treatment NKTCL patient plasma samples are used as an external verification set;

s42: taking a progression-free (PFS) survival period of a patient as an outcome index, successively using a single-factor Cox proportional risk regression model, a LASSO-Cox method and a multi-factor Cox regression model to construct a prognosis model, screening methylation sites closely related to prognosis as construction variables of the patient prognosis model of NKTCL, and calculating to obtain a methylation label prognosis score.

Preferably, in step S4, the step of constructing and verifying the ctDNA methylation prognosis model of NKTCL further comprises:

s43: dividing patients into a high-risk group and a low-risk group based on methylation label prognosis scores, evaluating the prediction efficiency of a model in a training set, an internal verification set and an external verification set through an ROC curve and A [ mu ] m C, and exploring the early prediction value of a methylation label prognosis score system on the P-GEMOX (pemetrexed, gemcitabine and oxaliplatin) scheme chemotherapy curative effect;

s44: the ctDNA methylation label constructed by the method is compared with IPI (International pathological index), KPI (Korean pathological index) and PINK (pathological index of nat mu ral killer cell lymphoma) prognosis models in sensitivity and specificity to evaluate the prognostic prediction value of the methylation label prognosis score.

Compared with the prior art, the invention has the following beneficial effects:

(1) the method is based on a methylation chip sequencing technology, and utilizes a Lasso-cox and random forest high-dimensional modeling method to screen out the differential ctDNA methylation sites of NKTCL patients and normal people, so as to realize the early diagnosis of NKTCL; screening ctDNA methylation sites related to NKTCL patient prognostic survival, constructing a prognostic label, individually evaluating the patient progress risk, and then exploring the curative effect prediction value of the patient;

(2) the use value of the plasma ctDNA methylation label as an NKTCL early diagnosis marker is determined, the early diagnosis of high risk groups is realized, scientific evidence is provided for the early diagnosis of the NKTCL, and the long-term survival of NKTCL patients is further promoted;

(3) the plasma ctDNA methylation label is used as the prediction value of the NKTCL patient survival prognosis, the prognosis evaluation of the NKTCL patient is realized, and the plasma ctDNA methylation label is used as a biomarker for potential immune chemotherapy curative effect prediction and early relapse detection, so that a basis is provided for accurate prognosis layering and individualized treatment scheme selection of the NKTCL, and a design thought is provided for future clinical research;

(4) the ctDNA methylation detection has the advantages of non-invasive, comprehensive, dynamic and sensitive properties, only blood samples of a subject need to be collected for detection, so that the defects that a large number of tissue slices are needed, the price is high, the clinical application performance is poor and the like in the molecular model based on genome sequencing, single nucleotide polymorphism and the like at present can be overcome, and the defect that tissue biopsy is influenced by the material drawing type and the material drawing part can be overcome;

(5) compared with the traditional ctDNA somatic mutation spectrum detection, the methylation level detection of ctDNA has the following advantages: the methylation detection sensitivity and the dynamic monitoring value are higher; multiple CpG methylation sites per target gene region can be detected; ctDNA methylation changes from the same type of tumor cells are relatively stable; the establishment of the ctDNA methylation model is very important for the formulation of an individual accurate NKTCL treatment scheme and the improvement of layered treatment bases.

Drawings

FIG. 1 is a schematic diagram of the steps of the present invention.

Fig. 2 is a flow chart demonstrating the principle of the present invention.

FIGS. 3-7 are schematic diagrams showing the different results of QUMA software according to the present invention.

Detailed Description

The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic views illustrating only the basic structure of the present invention in a schematic manner, and thus show only the constitution related to the present invention.

Example 1

This example 1 provides a method for constructing model for early diagnosis and prognosis of NK/T cell lymphoma, which comprises the following steps:

step S1: screening differential ctDNA methylation sites;

step S2: screening a ctDNA methylation probe;

By adopting the scheme, the ctDNA methylation detection has the advantages of non-invasiveness, comprehensiveness, dynamics and sensitivity, only blood samples of a subject need to be collected for detection, so that the defects that a large number of tissue slices are needed, the price is high, the clinical application line is poor and the like in the molecular model based on genome sequencing, single nucleotide polymorphism and the like at present can be overcome, and the defect that tissue biopsy is influenced by the type and the part of the obtained materials can be overcome.

Example 2

The present embodiment 2 includes all the technical features of the embodiment 1, and the differences are that:

in step S1, the step of screening for differential ctDNA methylation sites comprises:

In step S12, the step of extracting free DNA using the cfDNA extraction kit includes:

s123: standing at room temperature for 2-5min, adding 350 μ l buffer BD, and fully reversing and mixing;

s124: adding the solution obtained in the step S123 into an adsorption column CG2, centrifuging at 12000rpm for 30S, and pouring out waste liquid in the collection pipe;

Wherein, using a kit: genomic DNA extraction kit (DP 320): blood (DP 348), soil (DP 336), feces (DP 328).

Wherein, the high-throughput genome-wide methylation sequencing comprises the following steps:

I. PCR amplification

50 mu L system

Reaction procedure

Thirdly, adding all the amplified PCR products into agarose gel pores, and then carrying out electrophoresis.

Agarose gel electrophoresis

Preparation of a 1% agarose gel: weighing 0.5 g of agarose, placing in a conical flask, adding 50ml of 1 XTAE, heating and boiling in a microwave oven until the agarose is completely melted, and shaking up to obtain 1.0% agarose gel solution;

preparing a rubber plate: and (5) washing the glass plate and the glue making groove and drying. And (3) putting the glass plate into a glue making groove, placing the glass plate in a horizontal position, and placing a comb in a fixed position. The agarose gel cooled to about 65 ℃ is mixed evenly and carefully poured onto a glass plate, and the gel solution is slowly spread until a uniform gel layer is formed on the whole surface of the glass plate. Standing at room temperature until the gel is completely solidified, slightly pulling the comb vertically, taking off the adhesive tape, and putting the gel into an electrophoresis tank. Adding 1 XTAE electrophoresis buffer solution until the gel plate is submerged;

adding sample: mixing the DNA sample and the sample buffer solution on the spotting plate or the disposable glove, and adding the sample into the sample cell of the gel plate by using a 10 mu l micropipettor without damaging the gel surface around the sample hole during sample addition;

and fourthly, electrophoresis: electrifying the gel plate after sample adding for electrophoresis immediately, carrying out electrophoresis at 100V for 10 minutes, and stopping electrophoresis when the bromophenol blue moves to a position about 1cm away from the lower edge of the gel plate;

taking out the gel after electrophoresis, observing under an ultraviolet lamp, and taking a picture by using a gel imaging system for storage.

Purification of PCR products

After the electrophoresis is completed, the gel containing the target fragment is cut out from the agarose gel, and the weight is estimated or weighed accurately.

② adding 100 mul Binding Sol mu into each 100mg agarose gel, carrying out water bath at 50-60 ℃ for 3-5 min, and intermittently slightly reversing and mixing evenly every 2-3 min until the gel block is completely melted.

Note: for convenient operation, 400 μ l Binding solution μ tion can be uniformly added.

Thirdly, the mixed solution is transferred into an adsorption column GC-2 mu sleeved with a 2ml collecting pipe, placed for 2min at room temperature, centrifuged for 1min at 6000rpm at room temperature, taken out of the adsorption column GC-2 mu, and waste liquid in the collecting pipe is poured out.

And fourthly, putting the adsorption column GC-2 mu back into the collecting pipe again, adding 500 mul WA Sol mu, centrifuging at 12000rpm for 1min at room temperature, and pouring out the waste liquid in the collecting pipe.

Fifthly, the adsorption column GC-2 mu is put back into the collecting pipe again, 500 mu l of Wash Sol mu is added, the mixture is centrifuged for 1min at 12000rpm, and the waste liquid in the collecting pipe is poured out.

Sixthly, repeating the step 5 once.

Seventhly, putting the adsorption column GC-2 mu back into the collecting pipe again, centrifuging at 12000rpm for 1min at room temperature, then opening the cover of the adsorption column GC-2 mu, and standing at room temperature for 5-10 min or 50 ℃ for 3-5 min to completely remove the Wash Sol mu.

Eighthly, placing the adsorption column GC-2 mu into a clean 1.5ml collecting tube (provided in a kit), suspending 20 mu l of El mu tion B mu ffer in the center of the membrane, covering the cover, placing for 2min at 37 ℃, centrifuging for 1min at 12000rpm, and obtaining the liquid in the centrifugal tube as the solution containing the target DNA fragment.

Cloning of

The general pTG19-T vector was used and the procedures described in the kit were followed.

And (3) connection reaction: ligation systems were prepared from purified DNA fragments according to the following table

Mixing, centrifuging, and connecting at 22 deg.c for 2 hr;

secondly, conversion: adding 5 μ L or the total amount of ligation product into 100 μ L of competent cells;

③ carrying out ice bath for 30 minutes;

fourthly, after heat shock is carried out for 90 seconds at 42 ℃, the mixture is placed in ice for 2 minutes;

adding 500 mul LB culture medium, shaking and culturing for 60 minutes at 37 ℃;

sixthly, culturing the bacterial strain on an L-agar plate culture medium containing X-Gal, IPTG and Amp to form a single colony. Counting white and blue colonies;

note: this step requires adjustment to the efficiency of competent cells, allowing 100 to 1000 colonies to grow on each plate, for subsequent experimental manipulations.

And seventhly, identifying the insert fragment of the white clone by an enzyme cutting method.

And taking positive plasmid for sequencing.

V. sequencing

Sequencing PCR reaction conditions:

sequencing PCR reaction conditions:

reaction system:

PCRmix 15ul

primers (5 pM) were 1ul each

Stencil- -ul (50 ng)

H2O to 30ul

Purifying and sequencing a sequencing PCR reaction product: the product was recovered directly after amplification and finally eluted at 20. mu.l. Mu.l of the purified product was taken for sequencing.

Sequencing result analysis software: the sequencing instrument ABI3730XL, the sequencing reagent BigDye V3.1 and the sequencing Analysis software Seq μ science Analysis V5.02. the sequencing result can also be viewed and the sequence can be derived by software such as Seq Scan, chromas and the like.

(2) Methylation sites with p <0.05 and the methylation level difference multiple of 2000 are obtained by screening through methylation level difference analysis and are used for subsequent cohort research.

(3) Corresponding methylation probes are designed according to the 2000 differential methylation sites, and methylation sites capable of generating positive and specific PCR amplification signals are screened through PCR verification.

Example 3

The present embodiment 3 includes all the technical features of the embodiment 1, and the differences are that:

in step S2, the step of screening the ctDNA methylation probe is:

In step S22, the bisulfite sequencing step of detecting the DNA methylation level of tumor tissue is:

s225: adding 500 μ l B μ ffer BD to adsorption column MinEl μ te DNA Spin Col μmes, and standing at room temperature for 15 min; centrifuging at 12000rpm for 1min, and pouring off waste liquid in the collecting pipe;

Wherein bisulfite sequencing tumor tissue DNA methylation levels:

reaction system:

DNA 20 ul

Bisulfite Solution 85 ul

DNA protect Buffer 35 ul

reaction procedure:

95℃ 5 min

60℃ 20 min

95℃ 5 min

60℃ 20 min

20℃

example 4

This example 4 includes all the technical features of example 1, with the difference that:

in step S3, the step of constructing and verifying the ctDNA methylation diagnostic model of NKTCL comprises:

s31: collecting 650 cases of NKTCL patients before treatment and 550 cases of normal human plasma samples before treatment internally, randomly distributing the samples to a training set and an internal verification set according to the proportion of 7:3, and performing targeted bisulfite sequencing by taking the differential methylation sites as the background of the queue research; collecting plasma samples of about 100-200 cases of NKTCL patients before treatment and 100-200 cases of normal persons as an external verification set;

s32: a diagnosis model is constructed by respectively using a LASSO (last Absol μ te springage and Selection Operator) regression model and a Random Forest (Random Forest) regression model method, and methylation sites most relevant to disease diagnosis are selected as construction variables of the binary model.

Example 5

This example 5 includes all the technical features of example 1, with the difference that:

in step S4, the step of constructing and verifying the ctDNA methylation prognosis model of NKTCL is:

The kit can determine the use value of the plasma ctDNA methylation label as the NKTCL early diagnosis marker, realize early diagnosis of high risk groups, provide scientific evidence for the early diagnosis of the NKTCL, and further improve the long-term survival of NKTCL patients.

The invention can determine the prediction value of the plasma ctDNA methylation label as the survival prognosis of the NKTCL patient, realize the prognosis evaluation of the NKTCL patient, and serve as a biomarker for the potential immune chemotherapy curative effect prediction and early relapse detection, provide basis for the accurate prognosis stratification and the individualized treatment scheme selection of the NKTCL, and provide a design thought for the future clinical research.

The ctDNA methylation detection has the advantages of non-invasive, comprehensive, dynamic and sensitive properties, only needs to collect blood samples of a subject for detection, can overcome the defects that a molecular model based on genome sequencing, single nucleotide polymorphism and the like at present needs a large number of tissue slices, is high in price, poor in clinical application performance and the like, and can overcome the defect that tissue biopsy is influenced by material taking types and material taking parts.

Compared with the traditional ctDNA somatic mutation spectrum detection, the methylation level detection of ctDNA has the following advantages: the methylation detection sensitivity and the dynamic monitoring value are higher; multiple CpG methylation sites per target gene region can be detected; ctDNA methylation changes from the same type of tumor cells are relatively stable; the establishment of the ctDNA methylation model is very important for the formulation of an individual accurate NKTCL treatment scheme and the improvement of layered treatment bases.

The primer sequences are as follows:

in the description herein, references to the description of the terms "one embodiment," "certain embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. A construction method of an NK/T cell lymphoma early diagnosis and prognosis prediction model is characterized by comprising the following steps:

step S1: screening differential ctDNA methylation sites;

step S2: screening a ctDNA methylation probe;

2. The method of claim 1, wherein the step of screening differential ctDNA methylation sites in step S1 comprises:

3. The method for constructing an NK/T cell lymphoma early diagnosis and prognosis prediction model according to claim 2, wherein in step S12, the step of extracting free DNA using cfDNA extraction kit comprises:

s121: putting 200 μ l of blood into a 1.5ml centrifuge tube, adding 400 μ l of cell lysate CL, reversing and mixing, centrifuging at 10000rpm for 1min, discarding the supernatant, adding 200 μ l of buffer GS into the cell nucleus precipitate, shaking to thoroughly mix;

4. The method for constructing model for early diagnosis and prognosis of NK/T cell lymphoma according to claim 3, wherein said step S12 further comprises:

5. The method for constructing model for early diagnosis and prognosis of NK/T cell lymphoma according to claim 1, wherein in step S2, the step of screening ctDNA methylation probe comprises:

6. The method for constructing model for early diagnosis and prognosis of NK/T cell lymphoma according to claim 5, wherein in step S22, the step of bisulfite sequencing to detect the DNA methylation level of tumor tissue is:

s228: centrifuging at 12000rpm for 2min, pouring out waste liquid in the collecting tube, standing adsorption column MinEl μ te DNA Spin Col μmes at room temperature for 2-5min, and completely air drying residual rinsing liquid in the adsorption material;

7. The method of claim 1, wherein the step of constructing and verifying the ctDNA methylation diagnosis model of NKTCL in step S3 comprises:

8. The method of claim 7, wherein the step of constructing and validating the ctDNA methylation diagnosis model of NKTCL in step S3 further comprises:

9. The method of claim 1, wherein in step S4, the step of constructing and verifying the prognosis model of NKTCL ctDNA methylation comprises:

10. The method of claim 9, wherein the step of constructing and validating the ctDNA methylation prognosis model of NKTCL in step S4 further comprises: