RU2635628C1 - Method for tumour process prediction in brain - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method includes obtaining of a paraffin block from tumour biopsy, the resulting paraffin block is microtomized, the resulting cut is stained, scanned and compared with the paraffin block from which the tumour tissue is then isolated, then it is dewaxed, hydrotreated, its cell membrane is enzymatically destroyed until a cell nuclei suspension is obtained, the suspension is stained with a fluorescent dye and DNA cytometry is performed. If the number of aneuploid nuclei in the resulting suspension is less than or equal to 21%, a favourable course of the tumour process is predicted, if it is above 21% - unfavourable.

EFFECT: increased accuracy of prediction of the tumour process course in the brain by DNA cytometry of a precision-isolated tumour from the paraffin block.

2 ex, 2 dwg

Description

The invention relates to medicine, namely to Oncology, pathomorphology, and may find application in assessing the degree of cell proliferation in order to predict the course of the disease.

Malignant brain tumors remain one of the most difficult problems in modern oncology. Primary brain tumors account for about 2% of all identified cases of malignant neoplasms. In pediatric oncology, they take second place after leukemia and are the most common form of tumor disease. According to the US National Cancer Institute, the absolute number of new cases of the disease increased from 16,500 in 2005 to 23,130 in 2013, of which glial tumors - gliomas account for about 80% of all malignant tumors of the central nervous system (CNS), of which about 40% make up glioblastomas. According to the Moscow Scientific Research Institute P.A. Herzen, in Russia the annual increase in the incidence of brain tumors is 4.2%.

Currently, there is no doubt that the application of the clinical characteristics of a neurooncological disease in the form of the prevalence of the process, the histological structure of the tumor, the molecular profile of surface antigens is insufficient to predict the prognosis of the disease and the possible effectiveness of the prescribed therapy. Progress in understanding the molecular biology of the cell, the mechanisms of control of cell division and apoptosis, and signal transmission pathways from receptors to the nucleus have led to the realization of new individualization schemes in therapy.

With the modern approach to choosing an individual program for treating patients with malignant gliomas and predicting the course of the disease, the most promising is the study of molecular biological markers of tumor tissue.

Proliferative activity is one of the main morphological characteristics of progression and allows to predict the effectiveness of treatment, the development of relapses, as well as metastasis along the central nervous system.

One of the fundamental processes in biology is the cell cycle - the period of the cell’s existence from the moment of its formation by dividing the mother cell to its own division or death. All body cells, with the exception of sex cells, have a diploid set of chromosomes and cell replication precedes cell division.

The passage of the entire cell cycle follows an ordered scheme, ensuring that DNA replication occurs only once during the cell cycle and precedes the cleavage of chromosomes, which, in turn, precedes cytokinesis. Throughout the cell cycle, built-in mechanisms in the form of control points guarantee the complete and correct transfer of genetic information to daughter cells. The detection of irreparable genetic damage at these control points leads to the activation of apoptosis or programmed cell death. Cells that do not have effective control points reflect genomic instability, defective DNA replication, or abnormal separation.

A quantitative assessment of the DNA content in a cell is determined by flow cytometry, which is widely known and used both in oncology and basic biological research. Due to the ongoing life processes in the cell, the DNA content is normally not constant and depends on the phase of the cell cycle of the cell.

In pathology, the amount of DNA in a non-dividing cell can be either normal — diploid, or deviant — aneuploidy in the form of a decrease — hypoploidy or an increase — hyperploidy. A multiple increase in the amount of DNA of hyperploidy in the form of triploidy and tetraploidy is most often characteristic of tumor growth. When registering the amount of DNA, not only ploidy is taken into account, but also its amount in dividing cells. This is due to the fact that in pathology the presence of normal populations in the same study is most likely - diploid and pathological aneuploid cells.

It is known that using flow-DNA cytometry with a high degree of certainty, one can determine the ploidy of a tumor and evaluate its cell structure and its distribution over the phases of the cell cycle. Diploid (with normal DNA content) solid tumors have a more favorable prognosis than aneuploid (with higher DNA content). Tumors with a high content of cells in the cell cycle synthesis phase are usually characterized by rapid growth and high metastatic activity.

The known method of flowing DNA cytometry, described in cancers of various localizations [Medical science and the formation of the Urals. - 2011. - No. 2. - P.89-91., Bulletin of the Russian Research Center named after N.N. Blokhina RAMS. - 2006. - T. 17, No. 1. - S. 29-36.], It allows you to determine the ploidy of pathological cells, estimate their number, separately count the aneuploid cells in the tumor and distribute them according to the phases of the cell cycle with the determination of the proliferation index. This method of flow cytometric DNA was developed for the study of ascites fluid in ovarian tumors and is not suitable for the study of brain biopsy in patients with central nervous system tumors.

As a prototype we took the work published in J. Neurooncol. - 2005. - T. 71. - No. 2. - P. 85-89, in which a quantitative analysis of DNA from a biopsy specimen of glial tumors was performed. The ploidy of biopsy cells was evaluated in 64 patients with a confirmed diagnosis of an astrocytic tumor and a direct correlation was established with the degree of anaplasia. In this work, no reliable correlation dependencies of patient survival and ploidy of the biopsy specimen of glial tumors were obtained. In our opinion, this is due to the fact that the entire paraffin block was used for the study, containing not only tumor, but also connective tissue, sections of blood vessels and zones of necrosis, which in turn gives a diagnostic “noise” and changes statistical parameters, not allowing you to make an accurate conclusion about the prognosis of the course of the disease.

The technical result of the present invention consists in increasing the accuracy of predicting the course of a tumor process in the brain due to the use of a precision isolated tumor from a paraffin block in the DNA cytometry method.

This result is achieved in that in the known method for predicting the course of a tumor process in the brain to obtain a paraffin block from a tumor biopsy, according to the invention, the obtained paraffin block is microtomated, the obtained section is stained, scanned and compared with the paraffin block, from which tumor tissue is then isolated. after which it is dewaxed, hydrated, enzymatically destroy the cell membrane in it until a suspension of cell nuclei is obtained, the suspension is stained with a fluorescent dye DNA cytometry was performed and if the number of aneuploid cell nuclei in the resulting suspension is less than or equal to 21%, predict favorable course of tumor process, higher than 21% - unfavorable.

Microtomination of the paraffin block, its staining and scanning of the obtained histological section allows you to analyze it, identify the tumor area and select it without areas of necrosis, blood vessels and connective tissue components (“punch”).

Punch dewaxing, its hydration and enzymatic removal of cell membranes from it allows a suspension to be obtained from tumor cell nuclei, and subsequent staining with a fluorescent dye allows DNA cytometry to evaluate the distribution of nuclei over the cell cycle.

Our conclusion about the favorable or unfavorable course of the tumor process according to DNA cytometry data of a suspension of cell nuclei was made by us on the basis of a preliminary analysis of the results of DNA cytometry of a suspension of cell nuclei in 30 patients with traced actual survival. Based on a statistical analysis of aneuploidy, it was found that the content of aneuploid cell nuclei of 21% or less corresponded to a favorable course of the tumor process, and above 21% to an unfavorable one.

For a better understanding of the essence of the proposed method, we present an algorithm of the actions performed.

The patient takes the tumor material with the subsequent manufacture of a paraffin block from it, which is microtominated and the resulting section is stained with hematoxylin-eosin. The stained section is scanned (Fig. 1), tumor zones without areas of necrosis, blood vessels and connective tissue components (1) are revealed. Compare the resulting image with a paraffin block (Fig. 2) with the allocation of the zone of interest - "punch". Punch is dewaxed, hydrated, the cell walls in it are destroyed by trypsin to obtain a suspension of cell nuclei, which is stained with propidium iodide and DNA cytometry is performed. When the suspension contains aneuploid nuclei in an amount of 21% or less, a favorable course of the tumor process is predicted, and more than 21% - an unfavorable one.

The essence of the method is illustrated by a clinical example.

Example 1

Patient B., 60 years old, medical history No. 1858/13. He was admitted to the RSCRHT clinic on May 20, 2013 with a diagnosis of Glioblastoma of the right parietal-temporal region, condition after osteoplastic craniotomy from March 25, 2013.

From the anamnesis: he became ill in March 2013, when headaches of a hypertensive nature arose and began to increase. According to MRI from 03.25.2013 a volumetric formation of the right parietal-temporal region was revealed. He was hospitalized at the Research Institute of Emergency Medicine. I.I. Janelidze, 03/25/2013 performed osteoplastic trepanation of the skull, subtotal removal of the tumor. Then, from the obtained biopsy material, a paraffin block was made for histological examination and the patient was sent to the Federal State Budget Scientific Center of the Russian Center for Science and Technology for diagnosis and treatment.

At admission: a previously established diagnosis was confirmed.

The resulting paraffin block was microtominated, after which the section was stained with hemotoxylin-eosin. The micropreparation thus obtained from the tumor was scanned and the tumor zone was revealed without areas of necrosis, blood vessels and connective tissue components. Then, the resulting image was compared with the paraffin block, followed by the isolation of a “punch”. Punch was dewaxed, hydrated, and cell membranes were removed using the trypsin enzyme. Cell nuclei obtained as a suspension were stained with propidium iodide and DNA cytometry was performed. A group of aneuploid nuclei was detected by DNA cytometry - 30%, which corresponds to an unfavorable prognosis. In this regard, the patient is subject to immediate chemoradiation treatment.

On May 23, 2013, combined radiation therapy was started (isoeffective total radiation dose of 60 Gy) with simultaneous chemotherapy with nitrosourea preparations (Kartmustin 150 mg, Vincristine 3 mg). The treatment was satisfactory.

06/14/2013 was discharged in satisfactory condition under the supervision of a district oncologist with a turnout at the Federal State Budget Scientific Center of the Russian Center for Science and Technology, after 1 month for a follow-up examination.

07/15/2013 the patient was hospitalized for a maintenance course of chemotherapy. The state of moderate severity. A course (2 days) of chemotherapy was carried out (Kartmustin 200 mg, Vincristine 1 mg).

07/18/2013 the patient was discharged. His condition remained stable.

08/21/2013 relatives reported that the patient died. Actual survival from the day of surgery was 149 days.

Example 2

Patient B., 66 years old, medical history No. 2997/13. He was admitted to the RSCRHT clinic on September 16, 2013 with a diagnosis of Glioblastoma of the left temporal region, condition after bone-plastic skull trepanation from 08/28/2013.

From the anamnesis: he became acutely ill when, against the background of general well-being, motor aphasia occurred, he was hospitalized with a suspected stroke. According to MPT dated 08/18/2013, a volumetric formation of the left temporal lobe with hemorrhagic impregnation was detected.

On August 28, 2013, an operation was performed - osteoplastic trepanation of the skull, subtotal removal of the tumor. Then, a paraffin block for histological examination was made from the obtained biopsy material.

The patient was referred to the Federal State Budget Scientific Center of the Russian Center for Science and Technology for specific treatment.

At admission: a previously established diagnosis was confirmed. The resulting paraffin block was microtominated, after which the section was stained with hemotoxylin-eosin. The micropreparation thus obtained from the tumor was scanned and the tumor zone was revealed without areas of necrosis, blood vessels and connective tissue components. Then, the resulting image was compared with the paraffin block, followed by the isolation of a “punch”. Punch was dewaxed, hydrated, and cell membranes were removed with the help of the trypsin enzyme. Cell nuclei obtained as a suspension were stained with propidium iodide and DNA cytometry was performed. A group of aneuploid nuclei was detected by DNA cytometry - 17%, which corresponded to a favorable prognosis.

09/25/2013 in the radiotherapy department No. 4 of the Federal State Budget Scientific Center of the Russian Center for Science and Technology, combined radiation therapy (isoeffective total radiation dose of 60 Gy) was carried out with simultaneous chemotherapy with nitrosourea preparations (Kartmustin 150 mg, Vincristine 3 mg).

On October 17, 2013, the patient was discharged in satisfactory condition under the supervision of a district oncologist with an appearance at the Federal State Budget Scientific Center of the Russian Center for Science and Technology, after 1 month for a follow-up examination.

Subsequent adjuvant chemotherapy courses (Kartmustin 200 mg, Vincristine 1 mg) were carried out with an interval of 1 month. The patient after three courses of chemotherapy was in satisfactory condition.

Since 2014, a general deterioration was noted in the form of an increase in general cerebral symptoms. The patient refused supportive chemotherapy courses.

07/12/2014 the patient died. Actual survival from the day of surgery was 474 days.

To date, the proposed method has been predicted in 20 operated patients with glioblastomas of the brain. All patients underwent combined chemoradiotherapy in the radiological department No. 4 of the Federal State Budget Scientific Center of the Russian Center for Science and Technology. In 15 patients, the prognosis was poor - life expectancy ranged from 112 to 197 days. In 5 patients, the prognosis was favorable - life expectancy ranged from 332 to 645 days.

The proposed method for the first time on the basis of the proposed forecasting allows you to optimize the tactics of chemoradiation treatment.

The method was developed in the radiological department No. 4 of the Federal State Budget Scientific Center of the Russian Center for Scientific and Technical Research and passed clinical testing in 20 patients with a positive result.

Claims (1)

A method for predicting the course of a tumor process in the brain, including obtaining a paraffin block from a tumor biopsy, characterized in that the obtained paraffin block is microtomated, the obtained section is stained, scanned and compared with the paraffin block, from which tumor tissue is then isolated, after which it is dewaxed, they hydrate, enzymatically destroy the cell membrane in it until a suspension of cell nuclei is obtained, the suspension is stained with a fluorescent dye and DNA cytometry is performed and, if GUT aneuploid nuclei in the resulting suspension is less than or equal to 21%, predict favorable course of tumor process, higher than 21% - unfavorable.

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