Method for assessing radiosensitivity of airway cancer. RU patent 2505817.

FIELD: medicine.

SUBSTANCE: what is found is a pre-therapeutic rate of haemopoietic stem cells with immunophenotype CD34+CD45 low among peripheral lymphocytes at the stages T3 or T4 to be compared to its discrimination level of 6.0×10^-4. If the value occurs to exceed 6.0×10^-4, a high radiosensitivity of the tumour is predicted, while the value of 6.0×10^-4 or less enables predicting a low radiosensitivity of the tumour.

EFFECT: using the presented method enables predicting the radiosensitivity of the malignant growths to irradiation for the purpose of indicating for the radiation therapy in the patients with the stage T3 or T4 of airway cancer.

2 ex

The invention relates to medicine, more precisely, to the Oncology-related immunological analysis linking blood cells with antibodies labeled .

It is known that the radiosensitivity of cancer of the upper respiratory tract (var) (and other locations) varies greatly on the individual level, at the same clinical and morphological tumor process (anatomic region, stage, histological type, differentiation degree of tumor cells). This fact makes inefficient use of radiotherapy patients and determines the need to identify such patients before the start of treatment with the purpose of its optimization. Among the reasons for wide individual variability of radiosensitivity should specify the well-known biological factors, differences in the oxygenation of tumors, proliferative activity of tumor cells etc [1]. In recent years being investigated molecular-biological indicators (profile of gene expression, different mutations, the presence of a papilloma virus infection), also affecting the radiosensitivity tumors [2-7].

Known method for determining the radiosensitivity of tumors of the mucous membranes of the oral cavity (RF patent №2387472, IPC A61N 5/067 publ. 2008). Method is to determine the prognostic indicators baseline level antigen expression of Ki-67 in the tumor and the ratio of intensities of fluorescence at certain wavelengths (420 nm, 460 nm, 520 nm, 560 nm, 608 nm) in the tumor and the intact tissue after exposure to laser radiation (370 nm and 532 nm) on the tumor and the surrounding fabric. Definition of the radiosensitivity of conduct on the sum of all criteria of the forecast.

The disadvantage of this method are quite laborious manipulation using laser radiation and the use of invasive methods of obtaining biological material (samples of tumor tissue).

There is a method of forecasting of individual radiosensitivity in hypoxia (RF patent №2075078, IPC A61N 5/00 G01N 33/48 publ. 1997). The method consists in the hypoxic samples with double capture of skin biopsy before and after samples. Perform a histological processing biopsies to detect tissue basophiles by dye, and then count any structural-metabolic forms of tissue basophiles and carry out the forecast calculations.

The disadvantage of this method is the need for the use of invasive methods of obtaining biological material and labor-intensive manipulation of the most used only in experimental studies, and unsuitable for wide practical application.

There are methods for identification and quantification of hematopoietic stem cells (HSC) in human peripheral blood using flow cytometry in Oncohematology and autotransplantation. Methods based on the use of monoclonal antibodies () to protein markers cluster of differentiation (cluster of differentiation, CD), characteristic for GSK. In this case the best antibodies to identify the HSC rights recognized to CD34 class III [8, 9]. For flow cytometry use to CD34 labelled , often phycoerythrin (PE) (US patent # 4520110, the European patent №76695, Canadian patent №1179942). Add in the additional sample antibody labeled with other , most often (FITZ) or , allows to increase the specificity and accuracy of identification GSK [10].

The closest technical solution is to use a commercial kit company Becton Dickinson (USA) called ProCOUNT. Set includes labelled and DNA-binding dye, as well as procedures for the preparation and analysis of blood samples (instruction of the manufacturer is attached to a set). In particular, the Protocol ProCOUNT includes the use of a pair to CD34-Feh and CD45-, that provides identification of GSK on intensive binding to CD34 (CD34 + - high expression marker CD34) and weak binding of to CD45 (CD45 low - low marker expression of CD45). The Protocol also includes the use of DNA-binding dye and analysis of the number of CD34 + CD45 low cells with low lateral dispersion, that allows to exclude out debris (damaged cells).

Disadvantages prototype is no data on the prognostic value of the number of GSK in human blood as a determinant of radioresistant malignant tumors of the person.

None of the known methods for the identification and quantification of GSK is not used as a method for predicting the radiosensitivity of malignant neoplasms. Experience of Russian and foreign research suggests that GSK are able to differentiate into endothelial cells, stimulate angiogenesis and thereby maintain blood circulation and oxygenation of tumor, which is one of the main factors of its radiosensitivity [11-14].

The technical result of the claimed invention consists in increasing the accuracy of the assessment of the indications for the radiation therapy of cancer patients TTP in stages T3 or T4 on the basis of a new principle of radiosensitivity of malignant neoplasms.

Summary of the invention consists in that the patient has cancer of the var on the stage T3 or T4 to treatment with labeled determine the frequency of the HSC circulating in the peripheral blood, and compare them with the discriminatory level - 6,0·10 -4 . Frequency GSK predict the nature of the reaction of growths on radiation therapy for cancer patients VDP. If the frequency of GSK in a patient's blood before treatment is more than 6.0·10 -4 , predict a high radiosensitivity of the tumor. If the frequency of the GCS less than or equal to 6,0·10 -4 , predict low radioresistant tumor.

List of figures:

Figure 1 - Distribution of cells in intensity of direct and lateral light scattering. R1 (study area) is a region of cells with low lateral dispersion.

Figure 2 - Distribution of cells by direct and colouring of a DNA-binding dye 33342 (R2 - region of nucleus-containing cells).

Figure 3 - Distribution of cells taken from .R1 (figure 1)and R2 (figure 2), the intensity of binding to CD45 and to CD34. R3 - region of cells with CD34 + CD45 low (study area).

Procedure of implementation of the method:

I stage includes collection of samples and staining cells in peripheral blood of patients with cancer of the var on the stage T3 or T4 to treatment with monoclonal antibodies, DNA-binding dye. Collection and staining blood samples carry out as follows:

1. A sample of blood volume not less than 0.5 ml is taken from the cubital vein in the tube with geparinom at a final concentration of 25 IU/ml of blood. Staining sample begin not later than 24 hours after a vein;

2. In the labeled test tube bring 100 MKL of blood, 20 ml labelled FITZ, CD45 and 20 ml labelled Feh, CD34 (Becton Dickinson - BD, USA);

3. A mixture of blood, to CD45 and to CD34 carefully mix and incubate at room temperature in the dark for 20 minutes, then are lysed RBCs. For this concentrated solution FACS-Lysing (BD, USA) was diluted with distilled water 10 times, then 1 ml of this solution is contributed into a test tube with a mixture of blood and these , mixed and incubated in the dark for 10-12 minutes;

4. Precipitated leukocytes by centrifugation at 300 g for 5 minutes;

5. Produce double laundering blood cells in 1 ml of 0.01 M phosphate buffer salt, pH 7,2 (FSB), contains 0.15 M NaCl, with the help tsentrifugirovanija (300g within 5 minutes);

6. In the test tube with the sediment make 200 cells FSB and 5 MKL DNA-binding dye Hoechst 33342 concentration of 250 mg/ml (Sigma, USA). The mixture is stirred and incubated for 15 minutes.

Stage II includes data acquisition using flow cytometry:

1. Sample, prepared as described at the first stage, analyze the flow cytometer FACS Vantage (BD, USA), equipped with lasers with wavelength 364 and 488 nm, or other flow-through with the specified characteristics;

2. To measure fluorescence FITZ, and use narrowband filters 530/30 nm, for PV - 575/30 nm, for höchst 33342 - 424/20 nm;

3. In each test, analyze 200 thousand cells. Information on the intensity of direct and lateral light scattering, fluorescence , FE, höchst 33342 save a file. The obtained results are recorded in digital form;

4. The saved data is treated using a program CellQuestPro (BD, USA) or other software with the necessary characteristics.

Stage III is the treatment of the data collected using flow cytometry, in accordance with the algorithm of identification of GSK on the following criteria:

- strong expression marker CD34 on the surface of cells (CD34 + );

- weak expression token CD45 (CD45 low ).;

- low lateral light scattering, characteristic of lymphoid populations;

- intense coloration 33342, which allows us to differentiate cells from debris, conglomerates, platelets and other non-specific events and, ultimately, ensure high precision quantitative analysis of GSK.

In particular, the analysis data to identify and determine the frequency of GSK in peripheral blood include:

1. the construction of the point graph of the distribution of cells in direct and lateral (figure 1). In the distribution emit region lymphoid cells (R1) low-lateral dispersion and determine the number of cells in R1;

2. the construction of the point graph of cells by direct and the intensity of the fluorescence höchst 33342 (figure 2). On the chart emit region of nucleus-containing cells (R2), a high fluorescence;

3. the construction of the point graph of cell fluorescence intensity to CD34 and CD45 (figure 3). Schedule are built only for cells, satisfying the conditions of R1 and R2. On the chart emit region cells (R3) with CD34 + CD45 low and determine the number of cells in R3;

4. calculation of the relative number (frequency) GSK by dividing the number of cells in R3 on the number of cells in R1.

Stage IV: Forecasting of the radiosensitivity of tumors:

1. The frequency of GSK, calculated according to item 4 of III stage, compared with discriminatory level, the rationale for which is shown in example # 1.

2. If the frequency of GSK in a patient's blood before treatment more than 6.0·10 -4 , the probability of high tumor regression in response to radiation exposure is 74%is expected to be high radiosensitivity of the tumor. If the frequency of the GCS less than or equal to 6,0·10 -4 , projected low radioresistant tumor.

Example # 1. The degree of regression of malignant tumors of water users (stage T3 or T4) after I stage of radiation therapy in groups of patients with different number of circulating GSK before treatment

Frequency HSC cells defined in the peripheral blood 65 cancer patients VDP before treatment in accordance with the methodology described above. Patients were admitted for treatment at the Department of radiotherapy and surgical treatment of diseases of the upper respiratory tract FSBO MRRC health Ministry of Russia. Stage T3 diagnosed in 45 patients, T4 in 20 of the patient. Regional lymph nodes were involved in tumor process in 31 patients in the other 34 patients signs of damage to the lymph nodes were noted. All the patients were under M0. Treatment was started with a course of radiation therapy, total focal dose (SOD) 35-40 Gr followed by a 2 week break, after which endoscopically evaluated the degree of regression of the tumor. While the volume growths of more than 50% of the tumor regarded as and conservative treatment continued until the doses of 60 to 65 Gr. With the decrease of the tumor 50% and less than surgical treatment was carried out in the necessary volume.

Frequency GSK in the blood of patients before treatment varied from 1.7·10 -4 to 57.5·10 -4 . The average value of GSK in blood was 10,2·10 -4 . The degree of regression of primary tumor in the groups of patients with cancer of the var on the stages of T3 or T4 with varying frequency GSK in peripheral blood before treatment is shown in table 1. The results of calculations according to the criterion ^ 2 show a high radiosensitivity of tumors in the group of patients with high frequency circulating GSK compared with that in the low amount of GSK (p=0.001). So, the regression of primary focus of more than 50% was reported in 74% of patients with a high frequency of GSK (more than 6.0·10 -4 ) and only 31% of patients with a low frequency of such cells. Established discriminatory level of frequency GSK (6,0·10 -4 ) is optimal, because other discriminatory frequency levels GSK had a lower prognostic value.

The degree of regression of primary tumor in the groups of patients with cancer of the var on the stages of T3 or T4 with varying frequency GSK in peripheral blood before treatment

Frequency of GSK's x 10 -4

Regression of primary lesion

More than 50%

Less than 50%

29/39 (74%)

10/39(26%)

8/26(31%)

18/26(69%)

*In the table indicates the number of patients (in brackets the percentage of patients in the corresponding subgroup).

Example # 1 proves the prognostic value of the frequency of circulating GSK to treatment on the direct response of the tumor to radiation and justifies the use of discriminatory frequency of these cells 6,0·10 -4 as a criterion for classification of tumors in their radiosensitivity. This approach is informative to the assessment of the indications for the radiation therapy of patients with cancer of the upper respiratory tract in stage T3 or T4.

Example # 2. The degree of regression of malignant tumors of water users (stage T1 or T2) after I stage of radiation therapy in groups of patients with different number of circulating GSK before treatment

Frequency HSC cells defined in the peripheral blood of 27 patients with cancer of the SDT to treatment in accordance with the methodology described above. Patients were admitted for treatment at the Department of radiotherapy and surgical treatment of diseases of the upper respiratory tract FSBO MRRC health Ministry of Russia. Phase T1 was observed in 8, T2 in 19 patients. Regional lymph nodes were involved in tumor process in 9 patients in the other 18 patients signs of damage to the lymph nodes were noted. All the patients were under MO. Treatment was started with a course of radiation therapy to SOD 35-40 Gr followed by a 2 week break, after which endoscopically evaluated the degree of regression of the tumor. While the volume growths of more than 50% of conservative treatment continued until the doses of 60 to 65 Gr. With the decrease of the tumor 50% and less than surgical treatment was carried out in the necessary volume.

Frequency GSK in the blood of patients before treatment ranged from 3.6·10 -4 to 60.5·10 -4 . The average value of GSK in blood was 11,7·10 -4 . The degree of regression of tumors did not differ in the groups of patients with different frequency circulating GSK to treatment (table 2). The results of calculations according to the criterion ^ 2 show similar radiosensitivity of tumors in the group of patients with low and high frequency circulating GSK (p>0.05). So, the regression of primary focus of more than 50% was reported in 68% of patients with a high frequency of GSK (more than 6.0·10 -4 ) and 62% of patients with a low frequency of such cells.

The degree of regression of primary tumor in the groups of patients with cancer of the var on the stages of T1 and T2 with varying frequency GSK in peripheral blood before treatment

Frequency of GSK's x 10 -4

Regression of primary lesion

More than 50%

Less than 50%

13/19 (68%)

6/19(32%)

5/8 (62%)

3/8 (38%)

*In the table indicates the number of patients (in brackets the percentage of patients in the respective subgroup.

As shown by figure 1, the new principle allows to predict the radiosensitivity of cancer var stages T3 or T4 with high statistical significance (p=0.001) prior to treatment. Information about the individual radiosensitivity of malignant neoplasms improves the accuracy of estimates of the indications for the radiation therapy of cancer patients var, and, thus, improves treatment planning. Moreover, the identification of patients with malignant tumors resistant to the standard options of radiation exposure, before the start of treatment helps you develop the optimal strategy for their treatment, including the use of special modes dose fractionation radiation, the use of hadron therapy, combined methods of treatment, etc. It should also be noted that for forecasting the radiosensitivity of cancer VDP used a relatively non-invasive method of obtaining biological material (peripheral blood) and well-known method of preparation of a material to the analysis, which is of essential importance for the wide distribution of this method for the assessment of the radiosensitivity of cancer VDP.

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The method of evaluation of the radiosensitivity of cancer of the upper respiratory tract, including the identification of the frequency of hematopoietic stem cells CD34+CD45low among peripheral blood lymphocytes, wherein the frequency of hematopoietic stem cells CD34+CD45low on stages of cancer T3 or T4 to treatment compared with its discriminatory level of 6.0·10 -4 and for the values of more than 6.0·10 -4 predict a high radiosensitivity of the tumor and if the values are less than or equal to 6,0·10 -4 predict low tumor.