RU2800864C1 - Method of diagnostics of remote metastasis in breast cancer patients with her2/neu hyperexpression - Google Patents

Abstract

FIELD: medicine, oncology.

SUBSTANCE: invention can be used for diagnosing distant metastases in breast cancer patients with HER2/neu overexpression. The method includes scintigraphic studies and intravenous administration of a radiopharmaceuticals. The patient is injected with a radiopharmaceutical based on technetium-99m labeled recombinant targeted molecules DARPinG3. The radiopharmaceutical medicinal product is prepared immediately before administration under aseptic conditions: 1,000 µl of ^99mTcO^4- eluate are added to the kit for the preparation of tricarbonyl technetium [^99mTc(CO)₃(H₂O)₃]⁺ and incubated for 30 minutes at a temperature of 100°C, after incubation, 400 µl of tricarbonyl technetium is added to 3,000 µg DARPinG3 and incubated at 60°C for 60 minutes. The resulting compound is purified from protein impurities and DARPinG3 molecules not bound to technetium using purification columns, radiochemical yield and purity are determined using thin-layer radiochromatography (TLX), chromatograms are analyzed using a Hitachi Chromaster HPLC systems chromatograph with a radioactive detector. The medicinal product obtained after purification with a radioactivity of 200–500 MBC is diluted in 10 ml of a sterile 0.9% NaCl solution, taken through a sterilizing filter and slowly administered to the patient at a dose of 3,000 μg. 4 hours after administration, the patient is subjected to single-photon emission computed tomography of the chest organs and distant organs on a two-detector gamma camera, and if areas of medicinal product hyperfixation are detected, a tumor lesion of distant organs and tissues is diagnosed.

EFFECT: use of the invention makes it possible to increase the accuracy, informativeness and availability of diagnostics of tumor lesions of distant organs and tissues in patients with breast cancer with HER2/neu overexpression.

1 cl, 3 dwg, 1 ex

Description

SUBSTANCE: invention relates to medicine, oncology and can be used for diagnosing distant metastases in breast cancer patients with HER2/neu overexpression.

One of the most studied molecular targets is the type 2 epidermal growth receptor (HER2/neu), the expression of which is detected on the surface of tumor cells in cancers of the lung, ovaries, stomach, prostate, etc. [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; 235: 177-182]. At the same time, its high expression is most often found in patients with breast cancer (BC), the frequency of occurrence in which is up to 20% and is usually associated with an unfavorable prognosis and an aggressive course of the disease.

Currently, methods for detecting HER2/neu expression in the tumor tissue of patients with breast cancer, according to the ASCO/CAP recommendations of 2018, are immunohistochemical study (IHC) and fluorescent in situ hybridization (FISH). However, they have a number of significant drawbacks, which include the impossibility of simultaneously determining the extent of the tumor process and determining the molecular characteristics of the detected foci without performing additional invasive interventions. Currently, there is more and more talk about the discrepancy between the status of the epidermal growth receptor HER2 / neu between the primary tumor and metastatic foci in distant organs and tissues, while in most cases, performing a “pistol” biopsy of the tumor node is technically impossible or carries the risk of complications [Schrijver W.A.M. Receptor Conversion in Distant Breast Cancer Metastases: A Systematic Review and Meta-analysis / W.A.M. Schrijver, K.P. Suijkerbuijk, C.H. van Gils [et al.] // JNCI J. Natl. Cancer Inst. - 2018. - Vol. 110, No 6. - P. 568-580.] All this dictates the need to search for additional diagnostic methods to determine the status of type 2 epidermal growth factor receptor.

Currently, for the diagnosis of malignant tumors, targeted radionuclide methods are becoming more widespread, which have a high specificity for various molecular targets located on the surface of tumor cell membranes and allow visualization of foci of various sizes (the main tumor node and metastatic foci) [Chernov V.I., Medvedeva A.A., Sinilkin I.G., and co-authors. Nuclear medicine in the diagnosis and targeted therapy of malignant neoplasms. Bulletin of Siberian medicine. 2018; 17(1):220-231]. Currently, for these purposes, as a “targeting” module, a new class of target molecules is being studied, called “alternative scaffold proteins” or “scaffolds”, which have optimal characteristics for transferring various radioisotopes to target targets [Pluckthun A. Designed ankyrin repeat proteins (DARPins ): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol. 2015; 55: 489-511].

One of the representatives of recombinant targeting molecules are DARPins (DARPin, Design Ankyrin Repeat Protein), which are repeats of the ankyrin protein, which consists of repeating domains 33 a.a. long. and having a stable structure of a B-turn and two Typically, proteins are represented by 4-6 modules with a total molecular weight of 14 to 21 kDa (molecular weight of each 3.5 kDa) [Hanenberg M., McAfoose J., Kulic L. Amyloid-β peptide-specific DARPins as a novel class of potential therapeutics for Alzheimer's disease. J Biol Chem. 2014; 26: 27080-27089]. The main advantages of these molecules are small size, stable structure, high specificity and affinity for the antigen, as well as a significantly lower production cost due to their expression in bacterial media [Kramer L., Renko M., et.al Non-invasive in vivo imaging of tumor-associated cathepsin B by a highly selective inhibitory DARPin. Theranostics. 2017; 8: 2806-2821].

The short-lived (T _1/2 = 6.02 h) technetium-99m ( ^99m Tc) remains the most commonly used radionuclide for diagnostic studies both in the Russian Federation and in the world, the preparations of which are manufactured in the form of standard reagent kits (lyophilizates ) to the technetium-99m generator [Lykov A.V. Freeze drying // In the book: Drying theory. - M., Energy. - 1968. - S. 334-362].

A known method of radionuclide diagnosis of breast cancer with overexpression of Her2/neu" Bragina O.D. et al. (Patent for invention 2720801 C1, May 13, 2020. Application No. 2019103792 dated February 11, 2019.) using technetium-99m-labeled DARPinG3 molecules (“ ^99m Tc-DARPinG3”). An obvious drawback of this work is the lack of information on the possibility of using this compound in the diagnosis of damage to distant organs and tissues and the assessment of their molecular characteristics.

The short-lived (T _1/2 = 6.02 h) technetium-99m ( ^99m Tc) remains the most commonly used radionuclide for diagnostic studies both in the Russian Federation and in the world, the preparations of which are manufactured in the form of standard reagent kits (lyophilizates ) to the technetium-99m generator [Lykov A.V. Freeze drying // In the book: Drying theory. - M., Energy. - 1968. - S. 334-362].

However, the method is not sufficiently accurate and informative.

A new technical result is an increase in the accuracy, information content and accessibility of diagnosing tumor lesions of distant organs and tissues in patients with breast cancer with HER2/neu overexpression.

To achieve a new technical result in a method for diagnosing distant metastases in patients with breast cancer with HER2/neu overexpression, including intravenous administration of a radiopharmaceutical and scintigraphic studies, a radiopharmaceutical based on technetium-99m labeled recombinant targeted DARPinG3 molecules at a dosage of 3000 μg is administered to the patient, the drug is prepared immediately before administration the radiopharmaceutical preparation is prepared immediately before administration under aseptic conditions: in the CRS Isolink kit (Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland) for the preparation of tricarbonyl technetium [ ^99m Tc(CO) ₃ (H ₂ O ) ₃ ] ⁺ add 1000 µl (2-4 GBq) of ^99m TcO ^4- eluate and incubate for 30 minutes at 100°C, after incubation 400 µl tricarbonyl technetium is added to 3000 µg DARPinG3 and incubate at 60°C for 60 minutes, the resulting compound is purified from protein impurities and DARPinG3 molecules not bound to technetium using Sephadex G-25 M purification columns (GE Healthcare, Sweden), radiochemical yield and purity are determined using thin-layer radiochromatography (TSRH), chromatograms are analyzed using chromatograph Hitachi Chromaster HPLC systems with a radioactive detector, obtained after purification, the drug at a dose of 200-500 MBC is diluted in 10 ml of a sterile 0.9% NaCl solution, taken through a sterilizing filter and slowly injected into the patient, 4 hours after administration, the patient is subjected to single-photon emission computer tomography of the chest organs and distant organs on a two-detector gamma camera and, if areas of drug hyperfixation are detected, a tumor lesion of distant organs and tissues is diagnosed.

The method is carried out as follows, the radiopharmaceutical preparation is prepared immediately before administration under aseptic conditions: in the CRS Isolink kit (Center for Radiopharmaceutical Science, Paul Scherrer Institute, Villigen, Switzerland) for the preparation of tricarbonyl technetium [ ^99m Tc(CO) ₃ (H ₂ O ) ₃ ] ⁺ add 1000 μl (2-4 GBq) of eluate ^99m TcO ^4- and incubate for 30 minutes at 100°C. After incubation, 400 μl tricarbonyl technetium is added to 3000 μg DARPinG3 and incubated at 60° C. for 60 minutes. Subsequently, the resulting compound is purified from protein impurities and DARPinG3 molecules not bound to technetium using Sephadex G-25 M purification columns (GE Healthcare, Sweden). Radiochemical yield and purity are determined by thin-layer radiochromatography (TLPC). Chromatogram analysis is carried out using a Hitachi Chromaster HPLC systems chromatograph with a radioactive detector. The drug obtained after purification in a dose of 200-500 MBC is diluted in 10 ml of a sterile 0.9% NaCl solution, taken through a sterilizing filter and slowly injected into the patient. 4 hours after administration, the patient is subjected to single-photon emission computed tomography of the chest and upper abdomen on a two-detector gamma camera, the results are evaluated, and if areas of drug hyperfixation are detected, a tumor lesion of distant organs and tissues is diagnosed.

The method is based on the analysis of the results of clinical studies, to confirm the effectiveness of which in the detection of metastatic lymph nodes with HER2/neu overexpression, the diagnostic efficacy of " ^99m Tc-DARPinG3" at a dosage of 3000 μg was studied. The study included 28 breast cancer patients with (T _1-4 N _1-3 M _0-1 ) with different expression of HER2/neu. In two patients during the study, metastatic lesions of the liver and bones of the skeleton were detected.

All patients underwent histological and immunohistochemical examination of the biopsy material of the primary tumor with the determination of the status of HER2/neu according to standard methods. FISH analysis was performed at HER2/neu 2+ value.

Visualization of metastatic foci in distant organs and tissues was noted in all cases of using the drug " ^99m Tc-DARPinG3" at a dosage of 3000 μg and was comparable with the data of standard instrumental, morphological and immunohistochemical studies.

Clinical example 1.

Patient L., aged 48: Ds.: Cancer of the right breast, edematous-infiltrative form, stage IV (T4N3M1), metastatic lesions of the axillary, subclavian, supraclavicular lymph nodes, liver and bones of the skeleton. Histological and immunohistochemical examination: Non-specific invasive carcinoma of the 2nd degree of malignancy. RE+, RP+, HER2/neu 3+.

In terms of examination, single-photon emission computed tomography of the chest, head and neck, and abdominal organs was additionally performed with the drug " ^99m Tc-DARPinG3" at a dose of 3000 μg. On the scintigraphic picture, all tumor foci identified by standard diagnostic studies are clearly visualized, Fig. 1-3 Applications:

On FIG. Figure 1 shows SPECT of the head and neck, chest and upper abdomen with " ^99m Tc-DARPinG3" 4 hours after injection in a patient diagnosed with right breast cancer, edematous-infiltrative form, stage IV (T4N3M1), metastatic lesion of the axillary , subclavian, supraclavicular lymph nodes, liver and bones of the skeleton (overview scintigram).

On FIG. 2 - SPECT of the upper floor of the abdominal cavity with " ^99m Tc-DARPinG3" 4 hours after injection in a patient diagnosed with Cancer of the right breast, edematous-infiltrative form, stage IV (T4N3M1), metastatic lesions of the axillary, subclavian, supraclavicular lymph nodes, liver and bones of the skeleton. Arrows indicate metastatic foci in the liver.

On FIG. 3 - SPECT of the head and neck organs with " ^99m Tc-DARPinG3" 4 hours after injection in a patient diagnosed with Cancer of the right breast, edematous-infiltrative form, stage IV (T4N3M1), metastatic lesions of the axillary, subclavian, supraclavicular lymph nodes, liver and bones of the skeleton. The arrow indicates a metastatic lesion of the skull bones.

Thus, the proposed method for diagnosing distant metastases in breast cancer with HER2/neu overexpression using a radiopharmaceutical based on technetium-99m-labeled recombinant target molecules DARPinG3 at a dose of 3000 μg and performing SPECT makes it possible to clearly assess the prevalence of the tumor process at the metabolic level, and the degree The accumulation of the presented radiopharmaceutical in the tumor tissue makes it possible to obtain scintigraphic images of adequate quality, which improves the diagnostic efficiency, information content and availability of radionuclide diagnostics of distant breast cancer metastases with HER2/neu overexpression.

Claims (1)

A method for diagnosing distant metastases in patients with breast cancer with HER2/neu overexpression, including intravenous administration of a radiopharmaceutical and scintigraphic studies, characterized in that the patient is administered a radiopharmaceutical based on technetium-99m-labeled recombinant targeted DARPinG3 molecules, the radiopharmaceutical is prepared immediately before administration under aseptic conditions: add 1000 µl of ^99m TcO ^4- eluate to the kit for the preparation of tricarbonyl technetium [ ^99m Tc(CO) ₃ (H ₂ O) ₃ ] ⁺ and incubate for 30 minutes at 100°C, after incubation with 400 µl of tricarbonyl technetium is added to 3000 μg of DARPinG3 and incubated at 60°C for 60 minutes, the resulting compound is purified from protein impurities and DARPinG3 molecules that have not bound to technetium using purification columns, radiochemical yield and purity are determined using thin layer radiochromatography (TSPX), analysis chromatograms are carried out using a Hitachi Chromaster HPLC systems chromatograph with a radioactive detector, the preparation obtained after purification with a radioactivity of 200-500 MBC is diluted in 10 ml of a sterile 0.9% NaCl solution, taken through a sterilizing filter and slowly administered to the patient at a dose of 3000 μg, after 4 hours after administration to the patient, single-photon emission computed tomography of the chest organs and distant organs is performed on a two-detector gamma camera, and if areas of drug hyperfixation are detected, a tumor lesion of distant organs and tissues is diagnosed.