RU2656182C1 - Method of diagnostics and monitoring of course of cerebral glyomas - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a method for diagnosing and monitoring the course of cerebral gliomas, including determining the expression levels of the microRNA genes. At the same time, the expression levels of the miRNA-21, -128 and -342 genes in the saliva and the increase in the expression of the miRNA-21 gene and a decrease in the expression of the microRNA-128 and -342 genes in comparison with the reference values, diagnose cerebral glioma, and the degree of change in gene expression compared to the reference values being corresponding to the degree of glioma progression.

EFFECT: invention makes it possible to perform a comprehensive assessment of the state of the pathological process in cerebral gliomas effectively, including diagnosis, activity, sensitivity to chemotherapy, monitoring of patients on the background of ongoing therapy and evaluation of the effect of surgery performed by non-invasive drugs.

3 cl, 2 ex, 2 tbl

Description

The invention relates to medicine, namely to oncology, and can be used in the diagnosis and monitoring of cerebral gliomas.

Known methods for diagnosing cerebral gliomas using neuroimaging techniques - magnetic resonance imaging (MRI) of the brain with contrast enhancement and positron emission tomography (PET) using carbon [ ¹¹ C] (Brandsma, D. Pseudoprogression and pseudoresponse in the the contrast treatment of gliomas / D. Brandsma, MJ van den Bent // Curr. Opin. Neurol. - 2009. - Vol. 22. - P. 633-638). However, the use of radiation techniques has a large number of contraindications: pregnancy, the presence of implants, pacemakers, etc., their effectiveness can be reduced by various postoperative changes, radiation necrosis, hemorrhages, inflammation, etc. Due to the increased radiation load on the patient’s body, such a control method is not always available for regular dynamic observation of patients during their complex treatment.

A known method for the diagnosis of cerebral gliomas by detecting mutations in the genes PTEN (10q23.3), p53, EGFR, p16 (Ohgaki N., Kleihues P. Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas / / J Neuropathol Exp Neurol. 2005 Jun; 64 (6): 479-89; Kita D., Yonekawa Y., PIK3CA alterations in primary (de novo) and secondary glioblastomas // Acta Neuropathol. 2007 Mar; 113 (3): 295-302. Epub 2007 Jan 18). However, these mutations are rare in patients, in less than 30% of cases, which reduces their diagnostic significance.

Studies are known of the levels of expression of various microRNA genes in the blood for the diagnosis of cerebral gliomas (Roth P. et al. A specific miRNA signature in the peripheral blood of glioblastoma patients // J Neurochem. 2011 Aug; 118 (3): 449-57), estimates stages of the pathological process (Visani M, et al. Expression of 19 microRNAs in glioblastoma and comparison with other brain neoplasia of grades I-III // Mol Oncol. 2014 Mar; 8 (2): 417-30), effects on specific therapy ( Zhang KL, et al. Blockage of a miR-21 / EGFR regulatory feedback loop augments anti-EGFR therapy in glioblastomas Cancer Lett. 2014 Jan 1; 342 (1): 139-49), etc.

However, most studies reflect only some aspects of the significance of various miRNAs, without giving an overall clinical characteristic of cerebral gliomas. The results of the work have a pronounced ethnic character: Asian and Central European populations. The use of the blood of patients as the test material indicates the invasiveness of the techniques.

The technical result of the invention is a comprehensive assessment of the state of the pathological process in cerebral gliomas, including diagnosis, activity, sensitivity to chemotherapy, monitoring of patients during therapy and evaluation of the effect of surgical intervention, conducted by non-invasive means.

The technical result is achieved in a method for diagnosing and monitoring the course of cerebral gliomas (CG), including determining the expression levels of miRNA genes, in which the expression levels of miRNA-21, -128 and -342 genes in saliva are determined and with increasing miRNA-21 gene expression and decreasing expression miRNA-128 and -342 genes compared with reference values diagnose CG, and the degree of change in gene expression compared with reference values corresponds to the degree of glioma progression.

It is advisable to use the primer sequence for reverse transcription GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAGAG in the reaction for the preparation of copy DNA of the miRNA-128 gene, and GTCGTATCCAGTGCAGGGGATGTGTCGCT for the microRNA-342 gene.

The claimed invention provides unique primer sequences for reverse transcription of microRNA-128 and -342 genes. The selected thermodynamic characteristics of these primers fully correspond to the characteristics of primers for reverse transcription of the miRNA-21 gene and the U6 reference gene. This allows one to obtain copy DNA and perform PCR under the same temperature conditions, which is extremely important for standardization of the study.

To develop the method, clinical studies were conducted. In the studies, the main group consisted of 90 patients (46 men and 44 women) aged 24 to 75 years (average age 49.5 years) with suspected progression of central nervous system during complex treatment.

At the initial stage, all patients of the main group underwent surgical intervention as part of the standard for the integrated treatment of CG. According to operational protocols, 51.6% performed total and subtotal removal of neoplasm tissue (at least 75% of the volume of tumor tissue), and 30.4% performed partial (at least 50% of the tumor volume). Stereotactic biopsy was used in 12% of patients. Stereotactic biopsy and selective cryodestruction - in 6%. In 2 cases, tumors of subtentorial localization were observed, and in the remaining 88, supratentorial tumors.

Clinical data, EEG, PET and neuroimaging studies, expression levels of miRNA-21, -128, -342 during the adjuvant treatment of the disease were studied in all groups of patients.

The control group consisted of 30 people, of which: 25 potentially healthy volunteers and 5 neurosurgical patients with non-glial tumors: with cavernous angioma (N = 1), colloid cyst of the third ventricle of the brain (N = 1), basal meningiomas (N-2) , meningomatosis (N-1). All patients in the control group also studied the level of expression of miRNA-21, -128, -342.

PET studies were carried out on a Scanditronix PC 2048 positron emission tomograph (Sweden) using L- [methyl- ¹¹ C] -methionine ( ¹¹ C-methionine) radiopharmaceutical (RP). The obtained PET images were examined for the presence of a lesion (s) of increased accumulation of ¹¹ C-methionine, respectively, of the tumor localization.

Neuroimaging was performed using the MRT Achieva 3T instrument.

EEG studies were performed using a standard 10-20 electrode arrangement using mono- and bipolar recordings (Neurotravel computer electroencephalograph and Mitsar 21-channel electroencephalograph), during which functional samples were used to refine the localization of pathological lesions, and visual EEG analysis method, and if necessary - spectral and frequency.

The study of the relative expression of miRNA-21, -128, and -342 was carried out according to the semi-quantitative protocol, using small U6 RNA as the reference gene.

The studied groups of patients were statistically comparable. The information obtained during the study was processed using the STATISTICA for Windows computer software system (version 5.5). The array of initial data for most subjects contained: clinical, genetic, neuroimaging characteristics and PET data with ¹¹ С methionine. Frequency and quality indicators were evaluated using nonparametric methods, χ ² , Yates correction (for small groups), and Fisher's criterion. Comparison of quantitative parameters in the studied groups was carried out according to the criteria of Mann-Whitney, Wald, median χ ² and the ANOVA module. The criterion of statistical reliability of the conclusions was considered to be the generally accepted value in medicine p <0.05.

In the control group, the expression of miRNA-21 in plasma was 0.055 ± 0.041 relative units of expression of the miRNA gene (OE), in saliva - 0.050 ± 0.039 OE. The expression level of miRNA-128 in the blood was 2,330 ± 0,033 OE, in saliva - 0,100 ± 0,014 OE; miRNA-342 in the blood was 1.32 ± 0.02 OE, in saliva - 0.090 ± 0.018 OE. EEG, neuroimaging, and PET scores were normal.

Based on the results of the control group study, reference levels of miRNA gene expression were determined:

- microRNA-21 in the blood <0.05 OE, in saliva <0.05 OE;

- microRNA-128 in the blood> 2.33 OE, in saliva> 0.10 OE;

- miRNA-342 in blood> 1.32 OE, in saliva> 0.09 OE.

In the main group (90 people), the expression level of miRNA-21 exceeded the referent value and was 3.56 ± 0.27 OE in the blood and 2.45 ± 0.17 OE in saliva. Expression of miRNA-128 and -342 was reduced in patients with confirmed disease progression (in 32% of cases). The expression level of miRNA-128 in the blood was 0.0080 ± 0.0004 OE and 0.040 ± 0.004 OE in saliva, and for miRNA-342 in the blood, 0.0080 ± 0.0004 OE and 0.04 ± 0.004 OE in saliva.

Thus, differences in miRNA expression between the control and the main group were statistically significant: p <0.001.

The distribution of miRNA-21 expression values correlated with the degree of glioma malignancy: r = + 0.97, and micro RNA-128 and -342: r = + 0.65. In cerebral gliomas with a high degree of malignancy (Grade II-III, III and IV), the correlation of miRNA-21 expression with the Karnowski index reached a significant level: r = + 0.89, and miRNA-128 expression, -342: r = + 0, 38.

It should be noted that the progression of gliomas correlated with the degree of expression of miRNA-21, -128, and -342 (r = + 0.89).

The above microRNA-21 indices significantly correlated with PET data with [ ¹¹ C] methionine. So, with convincing remission of the tumor, the expression of miRNA-21, -128, and -342 did not go beyond the reference values, they were highly correlated with stabilization of glioma growth (r = + 0.77) and with its progression (r = + 0.96).

Thus, the development of cerebral gliomas is accompanied by a convincing increase in miRNA-21 gene expression and a decrease in miRNA-128 and -342 gene expression. The availability of a method for determining the dynamics of microRNAs during monitoring of patients with CG allows substantiating indications for PET studies.

The proposed method allows epigenetic diagnosis and monitoring of the course of cerebral gliomas at the time of diagnosis and at any stage of tumor development, including chemotherapy. The lack of normalization of miRNA-21 gene expression during therapy indicates the lack of effectiveness of the latter.

The method consists in determining the relative number of copies of microRNA in saliva, so the study is non-invasive and has no contraindications.

The method is carried out, for example, as follows.

The study of the relative expression of miRNA-21, -128, and -342 is carried out according to the semi-quantitative protocol, using small U6 RNA as the reference gene. Total RNA is isolated from saliva using the phenol reagent Trireagent-LS, followed by extraction with chloroform.

Copy DNA (cDNA) is obtained using the StemLoop technology with specific primers, separately for each miRNA using the OT-1 reverse transcription kit (Synthol, Moscow) according to the instructions. Reverse transcription primer sequences are shown in Table 1.

Amplification is carried out in RealTime mode on a TLite device (Russia) according to the standard two-primer scheme according to a semi-quantitative protocol using a 2.5x reaction mixture for PCR-RV in the presence of EVA Green dye (Synthol, Moscow). PCR mixtures for miRNA-21, -128, 342 and U6 contain one common primer and specific primers according to table 2.

Amplification program: 95 ° C - 5 minutes, then 45 cycles (60 ° C - 30 seconds and 95 ° C - 1 minute). After amplification, the device automatically calculates the sample exit cycle to the logarithmic curve (Ct). The relative level of expression of the miRNA-21 gene is determined by the ΔΔCt protocol and calculated by the formula 2 ^{(- (AB))} , where A is the Ct of the miRNA gene and B is the Ct of the U6 gene (Rao X, et al. An improvement of the 2 ^{^} (-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis // Biostat Bioinforma Biomath. 2013 Aug; 3 (3): 71-85). Calculation of relative levels of gene expression of miRNA-128 and -342 is carried out in a similar way.

With increasing miRNA-21 gene expression and decreasing miRNA-128 and -342 gene expression compared with reference values, cerebral glioma is diagnosed, and the degree of change in gene expression compared with reference values corresponds to the degree of glioma progression.

The method is confirmed by clinical examples.

1. Patient D., 36 years old, came to the clinic with complaints of periodic intense diffuse headaches, general weakness coming. The state of moderate severity, mucous membranes and skin dry, pulse up to 68 beats. min, A / D up to 115/70 mm Hg, temperature 36.7 ° С, respiration 16 min. The manifestation of the disease 2 months before hospitalization with somatomotor irritative syndrome. In neurological status: consciousness is clear, asthenic, no aphatic disorders. The movements of the eyeballs are in full, there is no anisocoria, there is left-side reflex hemiparesis, abdominal reflexes are sluggish with a slight accent on the right, coordination is preserved, no loss of sensitivity and pathological stop signs. On the fundus of stagnation is not observed, the field of view - without loss.

Prior to hospitalization, a microRNA study was performed:

During hospitalization. EEG: the presence of slow pathological and epileptiform activity in the leads from the right hemisphere of the brain, dysfunction of the median stem structures, indirect signs of intracranial hypertension are determined.

MRI scan of the brain - a volumetric process of the right frontal lobe with a zone of perifocal edema was revealed. In a series of PE tomograms in a study with ¹¹ C-methionine in the cortex of the anterior half of the right frontal lobe and subcortically, a large lesion of pathologically increased heterogeneous radiopharmaceutical accumulation (IN = 3.35) measuring 49 × 45 × 43 mm, corresponding to the localization of the lesion identified on MRI Drain foci of maximum accumulation of ¹¹ C-methionine occupy more than half of the total volume of education.

In a planned manner, the patient is prepared for surgery. Under endotracheal anesthesia, osteoplastic trepanation was performed in the right frontotemporal region, subtotal tumor removal (according to histology, Grade III anaplastic oligodendroglioma).

In a series of control postoperative PE tomograms in the study with ¹¹ C-methionine in the middle third of the right frontal lobe, an ametabolic lesion measuring 25 × 27 × 21 mm due to the postoperative cavity is determined. Below and medial to the described cavity, a small focus of pathologically increased accumulation of radiopharmaceuticals (IN = 1.65) with dimensions of 15 × 15 × 16 mm is recorded. The postoperative cavity is surrounded by a discontinuous border of increased accumulation of the radiopharmaceutical (IN = 1.2) up to 8 mm thick. The accumulation of radiopharmaceuticals in the zone of interest in the early vascular phase of the study is not visualized. Deformation of the anterior horn of the right lateral ventricle decreased. PET data were treated as a condition after subtotal removal of anaplastic oligodendroglioma of the right frontal lobe.

By the 10th and 14th days of the postoperative period, the patient retained asthenic and cephalgic syndrome, decreased appetite, nausea, dry skin and mucous membranes, subfebrile condition. Neurologically: contact, adequate in behavior, however lethargic, inhibited, left-sided hemiparesis up to 4 points.

MicroRNA analysis after surgery:

Thus, an increase in the expression of miRNA-21 and a decrease in the expression of miRNA-128 and -342 at the stage of a neuroimaging examination indicated the existence of a tumor and was confirmed instrumentally. Normalization of parameters after surgical treatment of the tumor reflects the effect of surgical removal of the tumor.

2. Patient S., 27 years old, was admitted to the hospital with complaints of intense diffuse headaches, general weakness, loss of appetite, nausea and vomiting. Upon admission - the patient's condition is severe, mucous membranes and skin are dry, pulse up to 62 beats. in min, A / D up to 115/70 mm Hg, low-grade fever 37.1 ° C, respiratory rate up to 18-19 per min. The manifestation of the disease 2 months before admission to the clinic.

In neurological status: consciousness is narrowed to light stunning, speech is monosyllabic intermittent, without distinct aphasic disorders, moderate upward gaze restriction, no anisocoria, there is reflex and power (up to 3 points) right hemiparesis, sensitivity is preserved, bilateral pathological stop signs. Severe edema of the optic disc.

With EEG: pathological slow activity in the leads from the left hemisphere of the brain, dysfunction of the stem structures, signs of intracranial hypertension.

With ultrasound dopplerography - a decrease in cerebral perfusion, mainly in the basin of the left midbrain artery, left-sided venous dyshemia in the basal, orbital and jugular collectors.

With echoencephalography - dislocation of the middle structures from left to right up to 8 mm.

An MRI and PET scan of the brain revealed a three-dimensional process of the deep parts of the left temporal lobe of the brain with lateral dislocation of the middle structures from left to right. In a series of PE tomograms, in a study with ¹¹ C-methionine in the deep part of the left cerebral hemisphere, a focus area of pathologically increased inhomogeneous radiopharmaceutical accumulation (ID = 3.2) 40 × 30 × 40 mm in size was found paraventricularly corresponding to the posterior part of the hippocampus, optic tubercle and parietal lobe. The drain center of the maximum accumulation of the radiopharmaceutical occupies more than half of the total volume of education and is localized in its upper-posterior part. The tumor is located in intimate proximity with the temporal horn, triangle and posterior part of the body of the left lateral ventricle.

Prior to hospitalization, a microRNA study was performed:

In connection with a gradual deterioration of the condition in the form of an increase in cerebral and focal neurological symptoms, the patient was operated on according to emergency indications. Under endotracheal anesthesia, osteoplastic trepanation was made in the left frontoparietal-temporal region, partial removal of the neoplasm (according to histological examination - glioblastoma, Grade IV) of the deep parts of the left temporal lobe of the brain. By the 5th day of the postoperative period: the patient's condition is severe, tachypical up to 22 per min, hyperthermia up to 38.8 ° C, dry mucous membranes and skin. Level of consciousness - stunning, elements of psychomotor agitation. In cerebrospinal fluid analyzes: cytosis within 5/3, protein 0.63 g / l.

The patient was given a course of infusion-detoxification therapy. After the above treatment, the patient's condition improved: by the 10th day of the postoperative period, the patient is in a compensated state, clear consciousness, contact and adequate, notes satisfactory subjective well-being. In addition, there is a regression of cerebral and focal symptoms. With an ophthalmic examination, the fundus and visual fields are normal.

In a series of control postoperative PE tomograms during the study with ¹¹ C-methionine in the left temporal lobe, an ametabolic lesion measuring 38 × 27 × 23 mm due to the postoperative cavity was recorded. At the upper medial wall of the described cavity in the left optic tubercle, a locus of pathologically increased accumulation of radiopharmaceuticals (IN = 2.8) with dimensions 26 × 26 × 27 mm corresponding to the upper part of the primary glioma massif is determined. The focus of maximum accumulation of ¹¹ C-methionine occupies half the volume of the glioma residue. At the lower medial wall of the cavity there is a small focus of pathologically increased accumulation of radiopharmaceuticals (IN = 1.59) with a diameter of 7 mm, corresponding to a small glioma residue in the para-hippocampal gyrus. According to the PET control, the state is after partial removal of glioblastoma of the deep part of the left cerebral hemisphere.

Clinical, biochemical and immunological parameters of blood and urine are normal. The course of adjuvant therapy - MXT with Temodal has begun.

Conducted 6 courses of the Moscow Art Theater Temodal. After 6 months with MRI with contrast enhancement, the size of the remaining part of the tumor was not significantly changed, and with a PET study of IN 2.7, this indicated a relative stabilization of the neoplastic process (taking into account the histological diagnosis).

MicroRNA study:

This observation demonstrates the positive dynamics of changes in miRNA expression levels up to normalization (plasma miRNA-342 level) after surgery and during therapy, which makes it possible to use the method to monitor the state of the tumor. The fact that not all indicators returned to normal is confirmed by the presence of a residual focus of CG with a diameter of 7 mm (IN = 1.59).

Using the claimed invention allows a comprehensive assessment of the state of the pathological process in cerebral gliomas, including diagnosis, activity, sensitivity to chemotherapy, monitoring of patients during therapy and assessment of the effect of surgical intervention, non-invasive means.

Claims (3)

1. A method for diagnosing and monitoring the course of cerebral gliomas, including determining the expression levels of miRNA genes, characterized in that the expression levels of miRNA-21, -128 and -342 genes are determined in saliva and with an increase in the expression of the miRNA-21 gene and a decrease in the expression of miRNA-genes 128 and -342, compared with reference values, diagnose cerebral glioma, and the degree of change in gene expression compared with reference values corresponds to the degree of glioma progression. 2. The method according to p. 1, characterized in that for the detection of the expression level of the miRNA-128 gene, two interdependent primers are used: for the preparation of the copy DNA of the miRNA-128 gene, the primer GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAGAG and for the amplification reactionCGTCTCGCCCCGCC. 3. The method according to p. 1, characterized in that for the detection of the expression level of the miRNA-342 gene, two interdependent primers are used: for the preparation of copy DNA of the miRNA-342 gene, the primer GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACGGGT and for the amplification reaction GCCA TCGCACCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGA.