RU2739107C1 - Diagnostic technique for stomach cancer with her2/neu overexpression - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology, and can be used for radionuclide diagnosis of gastric cancer with Her2/neu overexpression. Method involves administering a radiopharmaceutical and performing single-photon emission computed tomography. Radiopharmaceutical used is a radiopharmaceutical based on technetium-99m-labeled recombinant address molecules DARPin9_29, which is prepared immediately before administration according to the developed laboratory regulations: in aseptic conditions 1 ml eluate ^99mTcO^4- 7 GBq is added by syringe into kit for preparation of tricarbonyl technetium and incubated at temperature 100 °C for 30 minutes, after incubation 1000 mcl of technetium tricarbonyl is added to 334 mcl of DARPin9_29 with concentration of basic substance solution of 3.6 mg/l and incubated at temperature of 40 °C for 60 minutes, method includes purifying the obtained compound from protein impurities and non-technetium-bound DARPin9_29 molecules using purification columns, preparation obtained after purification is 500 MBq diluted in 10 ml of normal saline and slowly introduced into a patient through a sterilizing filter. Examination is performed 2 hours after the intravenous introduction; the obtained images are subjected to post-processing treatment and if the accumulation of the preparation increases in the projection of the stomach more than 8 times in relation to the "background" area, a hereditary cancer of Her2/neu expressing expression is diagnosed.

EFFECT: use of the invention enables higher specificity and information value, reduced number of complications and more accessible diagnostic technique.

1 cl, 1 dwg, 1 ex

Description

The invention relates to medicine, namely to oncology, and can be used for radionuclide diagnosis of gastric cancer with overexpression of Her2 / neu.

Stomach cancer (GC) currently occupies a leading position in terms of morbidity, both among the female and male population. In recent years, for the treatment of gastric cancer, along with chemotherapy, the targeted drug trastuzumab (Herceptin) is widely used, which is a humanized monoclonal antibody specific to the human epidermal growth factor receptor 2 (HER2) [Slamon D.J., Clark G.M., Wong S.G. et.al. Human breast cancer: correlation of relapse a survival with amplification of the Her-2 / neu oncogenes // Science. 1987. Vol. 235. P. 177-182]. The open international randomized controlled trial of phase III (TOGA), which included 122 research centers from 24 countries, showed that gastric cancer with a positive HER2 status occurs in 16-22% of cases, and the use of trastuzumab in the complex therapy of patients with overexpression of HER2 in combination with capecitabine and fluorouracil for locally advanced gastric cancer leads to a significant increase in the life expectancy of patients. The median survival rate in the group of patients receiving Herceptin in combination with chemotherapy was 13.8 months, and in the group of patients receiving only chemotherapy - 11.1 months.

Currently, several methods are actively used to determine the Her2 / neu status: immunohistochemical study (IHC), fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) [[Chernov V.I., Bragina O.D., Sinilkin I.G. et al. Radioimmunotherapy: current state of the problem // Problems of Oncology. 2016. T. 62. No. 1. S. 24-26]. However, with the undoubted advantages of the methods used, they have a number of significant disadvantages, which include the impossibility of conducting in vivo studies to assess the prevalence of the tumor process (primary tumor node, the state of regional lymph nodes, assessment of distant organs and tissues), tumor heterogeneity, and material inaccessibility. -technical equipment in most laboratories in the Russian Federation.

One of the directions of molecular imaging is the use of non-invasive radionuclide methods. For targeted delivery of a radionuclide, it is necessary to use a structure with high affinity, specificity of binding directly to the target, localized in both normal and pathologically altered tissues, low immunogenicity, and moreover, high stability, manifested by the ability to withstand various chemical environments in the process tagging. Moreover, in the process of medical imaging, high-affinity targeted molecules must quickly find a target in the patient's body, unbound components must be quickly removed from the body, thus facilitating tumor visualization and shortening the time between drug administration and examination. Recently, a new class of binding proteins, called "alternative scaffold proteins", has been actively studied, one of which is DARPin (Design Ankyrin Repeat Protein), which meets all the necessary requirements for creating an effective radiochemical compound [Nicholes N., Date A., Beaujean P. et.al. Modular protein switches derived from antibody mimetic proteins // Protein Engineering, Design and Selection. 2016. Vol. 29. P. 77-85].

The most frequently used radionuclide for diagnostic studies both in the Russian Federation and in the world is the short-lived (T _1/2 = 6.02 h) technetium-99m ( ^99m Tc). As a rule, technetium radiopharmaceuticals (RP) are manufactured in the form of standard reagent kits (lyophilisates) for the technetium-99m generator, which are mixtures prepared by freeze drying at low temperatures [Lykov AB Freeze drying // In the book: Theory of drying. - M., Energy. -1968. - S. 334 - 362]. When mixed with technetium-99m eluate (sodium pertechnetate solution, ^99m Tc) isolated from the generator, a ready-made RPF with desired properties is obtained. The shelf life of lyophilisates is usually 1 year.

The closest method for diagnosing gastric cancer with overexpression of Her2 / neu to the proposed method is a method using indium-111 labeled alternative framework proteins affibody ( ¹¹¹ In-ABY-025). The radiopharmaceutical used in the known method is specific and is fixed on the surface of the membranes of tumor cells expressing Her2 / neu, which allows them to visualize both the main tumor node and metastatic foci [Sorensen J., Sandberg D., Sandstrom M., et.al ... First-in-Human Molecular Imaging of HER2 Expression in Breast Cancer Metastases Using the ¹¹¹ In-ABY-025 Affibody Molecule // Tee Journal of Nuclear Medicine. 2014. Vol. 55 (5). P. 730-735]. The main disadvantage of this method is the non-optimal radiological characteristics of the drug associated with a long half-life (T _1/2 = 2.8 days) and, therefore, a high dose of radiation per patient, high cost of cyclotron production, as well as the presence of high-energy gamma quanta in its irradiation spectrum. preventing high-quality scintigraphic images. A significant drawback is also the inaccessibility of obtaining affibody on the territory of the Russian Federation.

The new technical result is an increase in specificity and information content, a decrease in complications and an increase in the availability of a diagnostic method.

To achieve a new technical result in a method for diagnosing gastric cancer with overexpression of Her2 / neu, including the introduction of a radiopharmaceutical and single-photon emission computed tomography (SPECT). As the radiopharmaceutical used radiopharmaceuticals based on technetium-99m-labeled recombinant molecules targeted DARPin9_29, a radiopharmaceutical is prepared immediately prior to administration according to the rules developed by the authors laboratory: aseptically 1 ml eluate ^99m TcO ^4- 7 GBq added via syringe to a kit for preparing technetium tricarbonyl and incubated at a temperature of 100 ° C for 30 minutes, after incubation, 1000 μl of technetium tricarbonyl is added to 334 μl of DARPin9_29 at a concentration of a basic substance solution of 3.6 mg / l and incubated at a temperature of 40 ^° C for 60 minutes, the resulting compound is purified from protein impurities and DARPin9_29 molecules not bound to technetium using purification columns, the drug obtained after purification in a dose of 500 MBq is diluted in 10 ml of saline and slowly injected through a sterilizing filter to the patient. The study is performed 2 hours after intravenous administration, the obtained images are subjected to post-processing processing, and with an increase in the accumulation of the drug in the projection of the stomach more than 8 times in relation to the "background" site, a gastric malignant tumor expressing Her2 / neu is diagnosed.

FIG. 1 shows the results of single-photon emission computed tomography with " ^99m Tc-DARPin9_29" 2 hours after administration in a patient diagnosed with gastric body cancer (T3N0M0).

The method is carried out as follows

Patients administered radiopharmaceutical based on technetium-99m-labeled recombinant molecules targeted DARPin9_29, which is prepared immediately prior to administration according to the developed laboratory regulations: aseptically 1 ml eluate ^99m TcO ^4- 7 GBq added via syringe to a kit for preparing technetium tricarbonyl and incubated at temperature of 100 ° C for 30 minutes, after incubation 1000 μl of technetium tricarbonyl is added to 334 μl of DARPin9_29 at a concentration of the basic substance solution of 3.6 mg / l and incubated at 40 ° C for 60 minutes, the resulting compound is purified from protein impurities and DARPin9_29 molecules not bound to technetium using purification columns, the preparation obtained after purification at a dose of 500 MBq is diluted in 10 ml of saline and slowly injected through a sterilizing filter to the patient. The study is performed 2 hours after intravenous administration of the drug. Single-photon emission computed tomography is performed on a two-detector gamma camera; during the study, the patient is placed on the table of the gamma camera in the "supine" position with his hands thrown back behind his head. The projection of the chest and abdomen must be in the field of view of the gamma camera detectors. They record 32 frames (64 projections) for 30 seconds per frame in a 64x64 pixel matrix without hardware zoom according to standard protocols. The resulting images are post-processed using a specialized software package. When visualizing areas of drug hyperfixation in the projection of the stomach more than 8 times the accumulation in the "background" area, a malignant tumor is diagnosed.

The method is based on the analysis of the results of experimental clinical studies, in order to confirm the effectiveness of which in the detection of malignant tumors of the stomach with overexpression of Her2 / neu, a study of the characteristics of the accumulation of the radiopharmaceutical preparation "^99mTc-DARPin9_29 ". For this purpose, a group of 6 patients with a verified diagnosis of gastric cancer was formed T_1-2N_0-3M₀... All patients were injected intravenously with a radiopharmaceutical based on technetium-99m labeled recombinant targeting molecules DARPin9_29 at a dose of 500 MBq. The radiopharmaceutical preparation was prepared immediately before administration according to the laboratory regulations developed by the authors: for the preparation of technetium tricarbonyl [^99mTc (CO)₃(H₂O)₃]⁺ used the kit "CRS Isolink" (Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland). Eluate^99mTcO^4- (400–500 µl, 7 GBq) was added to the kit followed by incubation at 100 ° C for 30 minutes. After incubation, 1000 μl technetium tricarbonyl was added to 1200 μg DARPin9_29 (concentration 3.6 mg / l) in 130 μl PBS (sodium phosphate buffer) and incubated at 40 ° C for 60 minutes (laboratory regulations receiving RFP LR-02069303-0217 dated 03.02.2017). Radiochemical yield and purity were determined by thin layer radiochromatography (TCPX), purification was performed using NAP-5 columns (GE Healthcare, Sweden). Chromatogram analysis was performed using a Hitachi Chromaster HPLC systems chromatograph with a radioactive detector.

2 hours after the intravenous administration of the drug, SPECT is performed on a two-detector gamma camera E.CAM manufactured by SIEMENS in the standard mode, 64 projections are recorded in a 64x64 pixel matrix using low-energy collimators with an energy of 140 keV. The differential discriminator window is set to 20%, no hardware zoom is used.

The images (scintigrams) obtained during the study were post-processed using the E. Soft proprietary software package (SIEMENS, Germany). Asymmetric areas of increased drug accumulation in the stomach projection were considered pathological (Fig. 1). In addition, during post-processing processing, the "tumor / background" index was calculated, reflecting the ratio of drug accumulation in tumor tissue and in healthy tissue (mediastinal projection). The results of the study demonstrated 100% sensitivity of the method in the diagnosis of gastric cancer with overexpression of Her2 / neu, that is, using the indicated radiopharmaceutical DARPin9_29, it was possible to detect a tumor in all 6 patients included in the study. The calculations showed that the average values of the "tumor / background" index were 8.35 ± 0.54.

Clinical example.

Patient I., 53 years old: Ds .: Cancer of the body of the stomach (T3N0M0).

Histological and immunohistochemical study No. хххх-93/20 from 04/27/2020: Low-grade gastric adenocarcinoma. Her2 / neu 3+.

In terms of the survey, additionally performed single-photon emission computed tomography with DARPin9_29 according to the proposed method.

The results of the study are shown in FIG. 1. Metabolic hyperfixation of the drug is visualized in the projection of the body of the stomach.

Thus, the proposed method for diagnosing gastric cancer with overexpression of Her2 / neu using a radiopharmaceutical based on technetium-99m labeled recombinant targeting molecules DARPin9_29 makes it possible to clearly visualize malignant gastric tumors at the metabolic level, and the degree of accumulation of the presented radiopharmaceutical in the tumor makes it possible to obtain scintigraphic images of proper quality. Thus, the use of a new radiopharmaceutical based on technetium-99m labeled recombinant targeting molecules DARPin9_29 will increase the efficiency and specificity of radionuclide diagnostics of gastric cancer with overexpression of Her2 / neu.

Claims (1)

A method for diagnosing gastric cancer with overexpression of Her2 / neu, including the introduction of a radiopharmaceutical and single-photon emission computed tomography, characterized in that a radiopharmaceutical based on technetium-99m labeled recombinant targeting molecules DARPin9_29 is used as a radiopharmaceutical, according to the regulation, which is prepared immediately before the introduction aseptically 1 ml eluate ^99m TcO ^4- 7 GBq added via syringe to a kit for preparing technetium tricarbonyl and incubated at 100 ° C for 30 minutes, after incubation 1000 .mu.l tricarbonyl technetium added to 334 .mu.l DARPin9_29 at a concentration of the basic substance solution 3.6 mg / L and incubated at 40 ° C for 60 minutes, the resulting compound is purified from protein impurities and DARPin9_29 molecules not bound to technetium using purification columns, the preparation obtained after purification at a dose of 500 MBq diluted in 10 ml of physiological solution and slowly injected through a sterilizing filter to the patient, the study is performed 2 hours after intravenous administration, the resulting images are subjected to post-processing processing and with an increase in the accumulation of the drug in the projection of the stomach more than 8 times in relation to the "background" area, a malignant gastric tumor expressing Her2 / neu.

Images