CN110646557A - Urine metabolic marker of glioblastoma patient carrying IDH gene mutation and application thereof - Google Patents

Abstract

The invention relates to a urine metabolic marker of a glioblastoma patient carrying IDH gene mutation and application thereof. In particular, the present invention relates to the use of an agent for determining the expression level of a metabolite in the urine of a patient with glioblastoma for the preparation of a kit or chip for determining whether said patient carries an IDH gene R132H point mutation, wherein the metabolite in the urine is selected from one or more of the following: N-acetyl-L-glutamic acid, alginic acid, L-glutamic semiuronic acid, cytokinin, jasmone isoleucine, and pyrogallol.

Description

Urine metabolic marker of glioblastoma patient carrying IDH gene mutation and application thereof

Technical Field

The invention relates to the field of biotechnology, and more specifically relates to urine metabolic markers of glioblastoma patients carrying IDH gene mutation and application thereof in prognosis and treatment selection of glioblastoma patients.

Background

The latest yearbook of the Ministry of health in 2013 shows that more than 30 ten thousand patients die of brain tumors every year in China. Glioblastoma, the most common Primary malignancy of the Brain, accounts for 47.6% of all Primary Brain malignancies (CBTRUS reported by the american Brain tumor registration center in 2018), see q.t. ostrom, h.gitleman, g.truitt, a.boscia, c.kruchko, j.s.barholtz-Sloan, CBTRUS Statistical Report: Primary Brain and other Central Nervous System Tumors clustered diagnosis in the United States in 2011-2015, Neuro Oncol 20(2018) IV1-IV86, which is the most malignant, WHO graded to the most malignant IV grade, and poor therapeutic effect. Median survival of patients after standard surgery, radiation and chemotherapy is only 14.6 months, see r.stupp, w.p.mason, m.j.van den Bent, m.weller, b.fisher, m.j.tapho, k.belanger, a.a.brandes, c.marosi, u.bogdahn, j.currchmann, r.c.janzer, s.k.ludwin, t.gorlia, a.allgecomier, d.lacombe, j.g.cairnross, e.eisenhauer, r.o.mirrimoff, radiotheraprosis conduran and juvatomoglomide for glabrato.n.n.2005 engglj 987-. This not only causes great pain to the patient's body, but also causes a psychological heavy burden to the patient and his family members. In addition, the damage of glioblastoma itself to brain function and the disability rate caused by surgical treatment are also high. Therefore, the disease brings great burden to national medical resources and social security systems.

Isocitrate Dehydrogenase (IDH) is a key enzyme in the tricarboxylic acid cycle, and IDH gene mutations, which specifically alter the catalytic activity of the enzyme and are thought to be involved in the development of tumors, have been discovered in recent years to be early and key mutations in glioma.

There is a significant difference between patients with IDH mutation and wild-type glioblastoma in prognosis, and patients with glioblastoma having IDH gene R132H point mutation in tumor tissue have a significantly better survival prognosis than patients with IDH wild-type glioblastoma, see P.J.Killela, C.J.Pirozzi, P.Healy, Z.J.Reitman, E.Lipp, B.A.Rasheed, R.Yang, B.H.Displas, Z.Wang, P.K.Greer, H.Zhu, C.Y.Wang, A.B.Carpenter, H.Friedman, A.H.Friedman, S.T.Keir, J.He, Y.He, R.E.McLendon, J.E.Herndon,2nd, H.Yan, D.D.Bigni, Mutatain, H.15132 about 5 Onclear, III, D.E.E.Cheng. Cheng. Herndon,2nd, H.Yan, D.D.D.D.D.6332, Mutingin, H.32, H.Onclear, III, E.5 Onclearnt, III, E.5 Onclear, Onclear. Therefore, it is important to distinguish whether patients with glioblastoma carry the IDH gene R132H point mutation or not for the prognosis and treatment mode selection of patients with glioblastoma.

The current diagnosis of IDH gene mutations in glioblastoma tumor tissues is mainly obtained by genetic sequencing of surgical specimens. The IDH gene mutation is detected by a non-invasive method, and the method has important significance for early diagnosis, prognosis judgment and treatment selection of glioblastoma. Current IDH mutation noninvasive diagnostic studies are currently mainly focused in two directions: firstly, the characteristics of IDH mutant glioma image group are judged by an imaging method based on nuclear magnetic resonance, see B.Zhang, K.Chang, S.Ramkesson, S.Tanguturi, W.L.Bi, D.A.Reardon, K.L.Ligon, B.M.Alexander, P.Y.Wen, R.Y.Huang, Multimodal MRI defects later and gene expression specific metabolite 2-HG, see A.Tiet, C.Choi, B.Mickey, E.A.Maher, B.Parm Uli, R.gigi, Y.Lassen-Rad, S.Lucoco, L.Lu.Bi, D.A.Reardon, K.L.Ligon, B.M.Alexander, P.Y.Wen, R.Y.Huang, M.Huang, Mumodal MRI defects and D.M.M.D.A.G.G.H.J.D.M.M.M.J.D.M.M.M.S.S.C.C.C.Choi, B.M.M.M.S.S.S.S.S.S.S.M.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.A.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S; second, 2-HG in the body fluid of the patient is detected based on mass spectrometry, see A.T.Fathi, B.V.NaHed, S.A.wander, A.J.Iafrate, D.R.Borger, R.Hu, A.Thabet, D.P.Cahill, A.M.Perry, C.P.Joseph, A.Muzikansky, A.S.Chi, Elevation of Urriny 2-hydrogluterate in IDH-Mutant Glioma.Oncologrist 21(2016) 214-219.

The metabonomics method is a research method for finding a whole set of small molecule metabolites in a biological sample by a nuclear magnetic resonance spectroscopy method and a mass spectrometry method. The research discovers a group of brand-new metabolic markers capable of well distinguishing IDH gene R132H point mutation from wild glioblastoma multiforme in the urine of a glioma patient by a metabonomics method based on a mass spectrum technology.

Disclosure of Invention

In view of the above-mentioned needs in the art, according to some embodiments of the present disclosure, there is provided a use of an agent for determining an expression level of a metabolite in urine of a glioblastoma patient, wherein the metabolite in urine is selected from one or more of the following:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

jasmone isoleucine, and

and (3) pyrogallol.

Preferably, the subject is a human, more preferably, the glioblastoma patient carries an IDH gene R132H point mutation or a wild-type IDH gene in the tumor tissue.

In a further specific embodiment, the kit or chip is used for prognosis or selection of treatment regimen for a patient with glioblastoma.

In specific embodiments, a glioblastoma patient having changes in one or more of the following metabolites in the urine compared to a control sample indicates that the glioblastoma patient carries the IDH gene R132H point mutation: increased cytokinin content, decreased N-acetyl-L-glutamic acid content, decreased alginic acid content, decreased L-glutamic semialdehyde acid content, decreased jasmone isoleucine content, and decreased pyroglutamic glycol content, wherein the control sample is derived from a glioblastoma patient carrying a wild-type IDH gene.

In a further specific embodiment, the kit is used in a method of mass spectrometric identification. The simple identification is used under the condition of mass spectrum full-scan mode combined target analysis. The mass spectrum full-scanning mode is to simultaneously acquire primary information of all small molecules within a mass range of 100m/z to 1000m/z, screen differential metabolites through multivariate statistical analysis, further perform targeted secondary fragmentation on the differential metabolites, and finally determine the differential molecules by combining a database secondary spectrogram.

In particular embodiments, the expression level is the relative amount of metabolite; preferably, the expression level is relative to the amount of metabolite in the urine of a glioblastoma patient carrying the wild-type IDH gene.

In specific embodiments, the subject is a human.

Another aspect of the present invention provides a kit or chip for determining whether a glioblastoma patient carries an IDH gene R132H point mutation, said kit or chip comprising a detection reagent for a metabolite selected from the group consisting of:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

jasmone isoleucine, and

pyrogallol; preferably, the kit or chip comprises a control sample derived from a glioblastoma patient carrying the wild-type IDH gene.

In a specific embodiment, the kit or chip is used for mass spectrometric identification, in particular, the mass spectrometric identification is used in a full scan mode in combination with a targeted identification mode.

In specific embodiments, the kit or chip is used for prognosis of glioblastoma or selection of treatment regimen.

Another aspect of the present invention provides a method for determining whether a subject carries an IDH gene R132H point mutation, comprising the steps of:

1) a urine sample is obtained from the subject,

2) optionally, extracting metabolites from the urine sample,

3) determining the level of one or more metabolites selected from the group consisting of:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

the jasmone-isoleucine is added into the mixture,

pyrogallol and combinations thereof.

In a specific embodiment, the content level is determined using mass spectrometry methods.

When mass spectrometry is used to determine metabolite levels, metabolite extraction, protein removal, may also be included after the step of obtaining a urine sample. In a specific embodiment, the metabolites in the urine sample are extracted with 2 volumes of acetonitrile while removing the proteins.

In specific embodiments, the mass spectrometry method is a primary full scan mode in combination with targeted secondary analysis. Specifically, the urine metabolome is detected through primary full scanning, a potential marker is screened out through multivariate statistical analysis, targeting secondary fragmentation is carried out on the potential marker, and the potential marker is determined by combining a database secondary spectrogram. Quantification was performed using the first-order spectral peak area of the marker.

Specifically, detection:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

the jasmone-isoleucine is added into the mixture,

pyrogallol and combinations thereof.

The method for quantitatively detecting the six metabolites based on urine can be used for establishing a baseline of the six metabolites in a human population by using a control sample derived from the urine of a glioblastoma patient carrying a wild type IDH gene, and judging whether the patient carries an IDH gene R132H point mutation or the wild type IDH gene based on the content range of the baseline and the obtained expression level of each metabolite in the urine of the glioblastoma patient.

Drawings

FIG. 1: glioblastoma IDH gene R132H point mutation and glioblastoma control group urine metabolism profile PCA classification map carrying the wild-type IDH gene.

FIG. 2: glioblastoma IDH gene R132H point mutation and glioblastoma control group urine metabolic profile OPLS-DA classification map carrying wild type IDH gene; wherein the control group refers to patients with glioblastoma having no mutation in IDH gene.

FIG. 3: the combination of six metabolites predicted the ROC curve for the R132H point mutation of the IDH gene in glioblastoma patients.

Detailed Description

The invention is further illustrated by the following examples, but not by way of limitation, in connection with the accompanying drawings. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the present invention, and that materials that are the same as or similar to the type, model, quality, nature, or function of the following reagents and instruments may be used in the practice of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

We used liquid chromatography-high resolution mass spectrometry (LC-MS) to detect metabolites in urine by full scan mode and screened metabolites associated with glioblastoma IDH gene mutation by multivariate statistical analysis. The identification of the marker is carried out by matching or resolving the secondary fragments by using a secondary targeting analysis method.

Materials and reagents

1) The instrument comprises the following steps: waters H-class liquid chromatograph (Waters Corp.) LTQ-Orbitrapvelos pro mass spectrometer (Thermofeisher Scientific).

2) The main reagents are as follows: acetonitrile (Thermofisher Scientific); c18 reverse phase chromatography column (3.0 mm. times.100 mm, C18,1.7 μm, Waters Co.).

3) Sample preparation: urine from 15 patients with glioblastoma and carrying the IDH gene R132H point mutation and urine from 20 glioblastoma patients (as a control group) carrying the wild-type IDH gene, from beijing tiantan hospital. Wherein, the IDH gene state is determined after gene sequencing of the taken-down tumor tissue after operation. The R132H point mutation of IDH1 gene is the most common type of IDH mutation, and is clearly related to the prognosis of patients, and the prognosis of patients with the mutation is obviously better.

1.1 Collection of human urine samples

Collecting fasting morning urine, centrifuging at 5000g for 30min, and removing precipitate.

1.2 metabolite extraction

200ul of urine supernatant is taken, 200ul of acetonitrile is added, vortex is carried out, standing is carried out for 30min at 4 ℃,14000g is centrifuged for 10min, the supernatant is taken, centrifugal concentration is carried out, 200ul of acetonitrile with 2 percent of acetonitrile is used for redissolving, 14000g is centrifuged for 10min, 10ul of sample is taken after passing through a 10kD filter membrane.

1.3 liquid phase analysis

Waters H-class

A chromatographic column: waters BEH C18(3.0X100mm,1.7um), column temperature 50 ℃; the mobile phase A is 0.1 percent formic acid water, and the mobile phase is acetonitrile; the analytical gradient was: 0-1min, 2% B; 1-8min, 2% B-98% B; 8-8.1min, 98% B-100% B; 8.1-12min, 100% B; 12-12.1min, 100% -2% B; 12.1-17min, 2% B; the flow rate is 0.5 ml/min; the injection volume was 10 ul.

1.4 Mass Spectrometry

UPLC Mass Spectroscopy tandem LTQ-Orbitrapvelos (Thermo Fisher Scientific, SanJose, Calif., USA) Mass Spectroscopy, using electrospray ion source positive ion mode; the sheath gas is nitrogen and auxiliary gas, and the flow rates are respectively 45arbitraryunits and 10 arbitraryunits; the mass spectrum scanning range is 100-1000 m/z; the spray volts is set to 4.2 KV; the ion transfer tube temperature was 350 ℃. The data is obtained by adopting a high-resolution Fourier Transform (FT) mode, and the first-level resolution is 60000; the secondary resolution is 15000.

1.5 Mass spectrometric data analysis

Raw data obtained from UPLC-LTQ orbitrap were processed using the genomic analysis software progenesis QI (Version 2.0, Nonlinear Dynamics, UK) from Waters. The software can automatically complete pretreatment procedures such as peak alignment, peak identification, peak correction and the like, and finally output a three-dimensional matrix, namely a spectrum peak index variable consisting of retention time and accurate mass-to-charge ratio, a sample name and peak intensity/area. The obtained data matrix is imported into multivariate statistical software SIMCA-P software 14.0(Umetrics AB, Umea, Sweden) for PCA analysis, and the change trend among groups is visualized. And (3) screening the difference variable between groups by using a VIP value obtained by an OPLS-DA model, wherein the VIP value is more than 1, and the variable with the non-reference test p value less than 0.05 is considered as the significant difference variable between groups and is screened as the early potential marker of IDH gene mutation of the glioblastoma multiforme. And (3) performing secondary fragmentation on the screened differential variables, and selecting 20,40 and 60eV energy according to specific metabolites by adopting an HCD (high dilution fragmentation) fragmentation mode. Deconvoluting the secondary fragment by using progenesis QI software, searching HMDB (human METABOLOME DATABASE) database, and determining the structure of the differential metabolite.

Urine metabolome for distinguishing glioblastoma IDH gene mutation from control group

The unsupervised PCA score shows (fig. 1) that the glioblastoma IDH gene mutation group and the control group exhibited a certain degree of discrimination. And a supervised OPLS-DA model is further adopted to construct a model, so that the two groups of discrimination are more obvious (figure 2). 20 different metabolites were selected. Research shows that the combination application of a plurality of metabolites can better predict the occurrence of diseases, model optimization is carried out on 20 different metabolites by adopting a logistic regression calculation method, and finally, the combination application of 6 metabolites, namely N-acetyl-L-glutamic acid, alginic acid, L-glutamic semialdehyde acid, cytokinin, jasmone isoleucine and pyroglutamine can achieve a better prediction effect, the AUC value predicted for the sample group is 0.956, the AUC value of ten-fold cross validation is 0.827 (figure 3), and the sensitivity and the specificity are shown in table 1. The prediction effect is good.

TABLE 1 combination of six metabolites predicting sensitivity and specificity of glioblastoma IDH gene mutations

	AUC	Sensitivity of the probe	Specificity of
				Training/discovery	0.956(0.933-0.978)	0.900(0.856-0.944)	0.881(0.827-0.936)
10-fold cross validation	0.827(0.670-0.983)	0.850(0.850-1.000)	0.867(0.695-1.000)

TABLE 2 early diagnosis of glioblastoma IDH gene R132H point mutation by six metabolites alone

Database ID	Name (R)	Area under curve	P value	Multiple of change
					HMDB01138	N-acetyl-L-glutamic acid	0.83667	0.00057134	-0.10438
HMDB03148	Alginic acid	0.83667	0.0019526	-0.073509
					HMDB02104	L-glutamic acid semialdehyde acid	0.83	0.00086454	-0.084668
HMDB39238	Cytokinins	0.80667	0.0021461	0.6017
					HMDB29391	Jasmone isoleucine	0.77667	0.0046908	-0.33698
HMDB30858	Pyroglycol	0.76667	0.0039395	-0.11487

Claims (10)

1. Use of an agent for determining the level of expression of a metabolite in urine of a glioblastoma patient, wherein the metabolite in urine is selected from one or more of the following:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

jasmone isoleucine, and

and (3) pyrogallol.

2. The use of claim 1, wherein the glioblastoma patient is a human.

3. The use of claim 2, wherein the glioblastoma patient carries an IDH gene R132H point mutation or a wild-type IDH gene in tumor tissue.

4. The use of any one of claims 1-3, wherein a change in one or more of the following metabolites in the urine of a glioblastoma patient, as compared to a control sample, indicates that the glioblastoma patient carries the IDH gene R132H point mutation: increased cytokinin content, decreased N-acetyl-L-glutamic acid content, decreased alginic acid content, decreased L-glutamic semialdehyde acid content, decreased jasmone isoleucine content, and decreased pyroglutamic glycol content, wherein the control sample is derived from a glioblastoma patient carrying a wild-type IDH gene.

5. Use of an agent for determining the level of expression of a metabolite in urine of a glioblastoma patient, wherein the metabolite in the urine is selected from one or more of the following:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

jasmone isoleucine, and

and (3) pyrogallol.

6. The use of claim 5, wherein a change in one or more of the following metabolites in the urine of the glioblastoma patient, as compared to a control sample, is indicative of a good prognosis for the glioblastoma patient: increased cytokinin content, decreased N-acetyl-L-glutamic acid content, decreased alginic acid content, decreased L-glutamic semialdehyde acid content, decreased jasmone isoleucine content, and decreased pyroglutamic glycol content, wherein the control sample is derived from a glioblastoma patient carrying a wild-type IDH gene.

7. The use of any one of claims 1-3 or 5, wherein the kit or chip is used in a method of mass spectrometric identification.

8. The use of any one of claims 1-3 or 5, wherein the level of metabolite expression in the urine is the relative content of metabolite compared to a control sample, wherein the control sample is derived from a glioblastoma patient carrying a wild-type IDH gene.

9. A kit or chip for determining whether a glioblastoma patient carries an IDH gene R132H point mutation, said kit or chip comprising detection reagents for the following metabolites:

N-acetyl-L-glutamic acid,

the acid-resistant agent is prepared from alginic acid,

l-glutamic acid semialdehyde acid is used,

(ii) a cytokinin, wherein the cytokinin is,

jasmone isoleucine, and

pyrogallol;

preferably, the kit or chip comprises a control sample derived from a glioblastoma patient carrying the wild-type IDH gene.

10. The kit or chip of claim 9, for use in prognosis or selection of treatment regimen for a glioblastoma patient.

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