RU2523899C1 - Kit of reagents for simultaneous or separate immunohistochemical detection of oestrogen, progesterone and her2 receptors in diagnosing breast cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: kit of reagents contains a solution for providing an antigen bioavailability, a blocking solution of horseradish peroxidase, a protein blocking solution, a washing solution, a solution of primary HER2 receptor antibodies, a solution of primary oestrogen receptor antibodies, a solution of primary progesterone receptor antibodies, a solution of anti-species secondary antibodies to primary antibodies, a solution of streptavidin - horseradish peroxidase, diamine benzidine, a substrate solution, a negative reference sample, three positive reference samples of breast cancer for detecting HER2, oestrogen receptors, progesterone receptors. The kit of reagents according to the invention possesses higher sensitivity and specificity. The invention may be used in medical laboratories and research establishments.

EFFECT: possibility to detect oestrogen and progesterone receptors, as well as HER2 receptors either simultaneously, or separately.

3 ex, 4 tbl

Description

The invention relates to the field of laboratory diagnosis of breast cancer and can be used in medical laboratories and research institutes.

The main method used to determine the level of expression of HER2, estrogen and progesterone markers is currently immunohistochemical analysis (IHC) [1, 2, 3]. The histological material obtained by a biopsy of breast cancer (breast cancer) is examined. An immunohistochemical study determines the markers in the cells of a breast tumor using a special stain, visible during the light-optical study of a histological section. The more receptors the breast cancer test sample contains, the stronger the staining.

The current prototype for immunohistochemical diagnosis of HER2 receptor overexpression using polyclonal affinity purified antibodies to the synthetic HER2 receptor fragment for the diagnosis of breast cancer is the Hercep test TM reagent kit (product No. K520421 Dako).

The reagent kit includes:

1. Blocking solution for peroxidase, includes 3% hydrogen peroxide and sodium azide.

2. Rabbit antibodies against human HER2 protein, affinity-purified, ready to use, are contained in Tris-HCl buffer solution, include sodium chloride, sodium azide, stabilizing protein. An immunogen is the C-terminal fragment (intracytoplasmic portion) of the HER2 receptor. The protein was affinity purified on the HER2 peptide.

3. A visualizing reagent - a polymer dextran conjugated with horseradish peroxidase and with affinity purified goat serum against rabbit immunoglobulins, is contained in Tris-HCl buffer solution, includes an antibacterial agent and a stabilizing protein.

4. Negative control reagent - fraction of normal rabbit serum immunoglobulins, protein concentration is equivalent to antibodies to HER2 protein, is contained in Tris-HCl buffer solution, includes sodium chloride, sodium azide, stabilizing protein.

5. The substrate for the DAB chromogen, contained in the buffer solution, includes less than 0.1% hydrogen peroxide, stabilizers, enhancers and an antibacterial agent.

6. DAB chromogen. A solution of 5% 3,3-diaminobenzidine tetrahydrochloride.

7. A solution of increasing the bioavailability of the epitope, a 10-fold concentrate, is contained in a citrate buffer solution, includes a detergent.

8. Wash buffer solution, 10-fold concentrate, is contained in Tris-HCl buffer solution, includes detergent and antibacterial agent.

9. 5 glasses with positive control samples. Each glass bears sections with 3 formalin-fixed breast fragments in paraffin blocks, representing three levels of immunohistochemical color (0), (1+), (3+).

The procedure for applying the kit includes the following stages [1].

Stage 1. The discovery of epitopes

Fill containers with a solution to ensure bioavailability of epitopes. Place containers in a water bath. Heat the water bath and containers to a temperature of 95-99 ° C. Place slices with test and control samples in containers. Heat the water bath and containers to a temperature of 95-99 ° C. Incubate 40 (± 1) minutes at 95-99 ° C. Remove the containers from the bath. Maintain containers for 20 (± 1) minutes at room temperature. Wash sections with diluted wash buffer.

Stage 2. Blocking endogenous peroxidase

Remove moisture from sections. Apply 3 drops (100 μl) of peroxidase blocking solution to the sample. Incubate 5 (± 1) minutes. Rinse gently with distilled or deionized water or wash buffer. Place in fresh buffer.

Stage 3. The introduction of primary antibodies or negative control reagent

Remove moisture from sections. Apply 3 drops (100 μl) of anti-HEK2 protein or negative control reagent to the sample. Incubate for 30 (± 1) minutes.

Rinse gently with wash buffer. Place in fresh buffer.

Stage 4. The introduction of a visualizing reagent

Remove moisture from sections. Apply 3 drops (100 μl) of the imaging reagent to the sample. Incubate for 30 (± 1) minutes. Rinse gently with wash buffer. Place in fresh buffer.

Stage 5. Staining with a solution of DAB

Remove moisture from sections. Apply 3 drops (100 μl) of the DAB solution to the sample.

Incubate 10 (± 1) minutes. Rinse gently with distilled or deionized water.

Then, add sections with hematoxylin and enclose under a coverslip.

The disadvantage of this method is the inability to determine the hormonal status of the tumor in one analysis, as well as the use of a system for enhancing specific immunohistochemical staining based on conjugate of dextran with peroxidase and secondary antibodies, which has a lower sensitivity compared to modern conjugates of secondary antibodies with biotin and subsequent use streptavidin peroxidase complex.

A known method for the diagnosis of HER2 receptor using biotinylated humanized monoclonal antibodies, which are a modified version of the antibodies that make up the drug Herceptin. This method was also used for immunohistochemical detection of the localization of extracellular domains of HER2 receptors [4].

The disadvantage of this method is the inability to determine the hormonal status of the tumor in one statement of the analysis, as well as the absence of secondary antibodies in the analysis protocol, which can lead to a decrease in the specificity of the analysis and the appearance of false positive results.

The current prototype for the diagnosis of estrogen and progesterone is the ER / PR pharmDx ™ Kit. (product No. K407111 Dako). The reagent kit includes:

1. The solution of increasing the bioavailability of the epitope, a 10-fold concentrate, includes an antimicrobial component in a nitrate buffer solution.

2. A blocking solution for peroxidase containing 0.5% hydrogen peroxide, detergents, enzyme inhibitors and preservatives.

3. A mixture of primary antibodies to the estrogen receptor. Mouse monoclonal antibodies (IgG1 and IgG2a), against the human estrogen receptor, are contained in Tris-HCl buffer solution, include sodium azide and a stabilizing protein.

4. Primary antibodies to the progesterone receptor. Mouse monoclonal antibodies, against the human progesterone receptor, are contained in Tris-HCl buffer solution, include sodium azide and a stabilizing protein.

5. Negative control reagent. Mouse monoclonal antibodies (IgG1 and IgG2a), contained in Tris-HCl buffer solution, include sodium azide and a stabilizing protein.

6. Visualizing reagent - a polymer of dextran conjugated with horseradish peroxidase and affinity purified goat serum against mouse immunoglobulins, is contained in Tris-HCl buffer solution, includes an antibacterial agent and a stabilizing protein.

7. The substrate for the DAB chromogen, contained in the buffer solution, includes less than 0.1% hydrogen peroxide, stabilizers, enhancers and an antibacterial agent.

8. DAB chromogen. A solution of 5% 3,3-diaminobenzidinate tetrahydrochloride.

9. Wash buffer solution, 10-fold concentrate, is contained in Tris-HCl buffer solution, includes detergent and antibacterial agent.

10. 20 glasses with control samples. Each glass carries sections with 2 formalin-fixed breast fragments in paraffin blocks, representing a section with an average level of expression of estrogen or progesterone, as well as a section from a negative cell line. The procedure for using this reagent kit is similar to the Hercep test TM kit [5].

The disadvantage of this method is the inability to determine the status relative to overexpression of the HER2 tumor receptor in one analysis, as well as the use of a specific immunohistochemical staining amplification system based on dextran conjugate with peroxidase and secondary antibodies, which has lower sensitivity compared to modern secondary antibodies conjugates with biotin and subsequent use of the streptavidin-peroxidase complex.

The technical result of the invention is the creation of a set of reagents for immunohistochemical diagnosis of breast cancer, which has the following advantages over prototypes:

1. The reagent kit allows the simultaneous or separate detection of both estrogen and progesterone receptors and HER2 receptors.

2. The kit has increased sensitivity and specificity.

The specified technical result is achieved:

According to claim 1. - the use of 3 variants of primary antibodies to target receptors, the titers of which are selected so that all procedures are unified.

According to claim 2, by using a mixture of secondary antibodies conjugated with biotin, and the introduction of streptavilin-peroxidase complex to enhance the color of a specific reaction.

The composition of the proposed set of reagents includes the following components:

1. A solution to ensure the bioavailability of the antigen (ROB), containing reagents (primarily detergents and denaturants) that reduce the number of non-specific interactions, using the mechanism to prevent their formation or destroying already formed non-specific interactions before starting the main stages of the analysis.

2. Blocking solution of horseradish peroxidase (a key staining enzyme during the course of the reaction) (PDP). This component is necessary to maximize the reduction of the amount of active peroxidase in the sections of the studied tissue and to reduce the nonspecific signal arising under the influence of the tissue enzyme.

3. Protein blocking solution (BBR). A component containing protein compounds in a buffer solution that, when bound to polypeptides, reduces the number of non-specific interactions.

4. Wash solution (PR), is a buffer solution and is intended for washing glasses with control and test samples between stages.

5. A solution of primary antibodies No. 1 (ATP-1) to the HER2 receptor in a stabilizer designed for long-term storage of antibodies. At the first stage of IHC, the antibodies contained in the solution form the “HER2 receptor - primary antibody” complex. The solution is ready to use.

6. A solution of primary antibodies No. 2 (ATP-2) to the estrogen receptor in a stabilizer designed for long-term storage of antibodies. At the first stage of IHC, the antibodies contained in the solution form the complex “estrogen receptor - primary antibody”. The solution is ready to use.

7. A solution of primary antibodies No. 3 (ATP-3) to the progesterone receptor in a stabilizer designed for long-term storage of antibodies. At the first stage of the IHC reaction, the antibodies contained in the solution form the complex “progesterone receptor - primary antibody”. The solution is ready to use.

8. A solution of antispecies secondary antibodies to primary antibodies (ATV) conjugated with biotin in a stabilizer designed for long-term storage of antibodies. At the second stage of the IHC reaction, the antibodies contained in the solution form the “receptor-primary antibody – secondary antibody – biotin” complex. The solution is ready to use.

9. A solution of streptavidin-horseradish peroxidase (SPH) in a stabilizer, designed to detect biotin contained in the above complexes.

10. Diaminbenzidine (DAB), a concentrate intended for the preparation of a working solution of diaminbenzidine, which provides color staining under the influence of the enzyme.

11. The substrate solution (SR) used to prepare the working solution of diaminbenzidine.

12. A negative control sample (K-), which is a non-immunogenic serum, introduced in parallel with primary antibodies and designed to control non-specific staining.

13. A positive control sample No. 1 (K + 1), which is a glass slide with sections mounted on it from paraffin blocks containing breast tissue. Positive control samples are placed in the same statement of analysis as the test samples and serve to verify the adequacy of the analysis procedures and compare the results. It is used to check the adequacy of the procedures and to compare the color during detection of the HER2 receptor.

14. Positive control sample No. 2 (K + 2), similar to the previous one. Serves to verify the adequacy of the procedures and to compare the color during the detection of estrogen receptor.

15. Positive control sample No. 3 (K + 3), similar to the previous one. Serves to verify the adequacy of the procedures and to compare the color during detection of the progesterone receptor.

Below are the options for the volume of solutions in the vial and the number of components in the kit with a kit option designed to perform studies of up to 100 test and control samples in ten independent analyzes.

The antigen bioavailability solution contains guanidine hydrochloride, urea, and Tween 20 in standard phosphate buffered saline. (The standard phosphate-salt buffer necessary for the preparation of some of the following solutions contains sodium phosphate disubstituted 12-aqueous, sodium phosphate monosubstituted 2-aqueous, sodium chloride, purified water according to FS 42-2619-97.) After preparing the solution, the pH is adjusted to 7, 2-7.4. The solution is a 10-fold concentrate and is prepared before use. The solution is poured into 250 ml vials. The kit contains 1 bottle.

The blocking solution for horseradish peroxidase contains a 3% solution of hydrogen peroxide and sodium azide in purified water according to FS 42-2619-97. After preparing the solution, the pH is not adjusted. The solution is ready to use. The solution is poured into 20 ml vials. The kit contains 1 bottle.

The protein blocking solution contains a solution of bovine serum albumin and tween 20 in standard phosphate buffered saline. After preparing the solution, the pH is adjusted to 7.2-7.4. The solution is ready to use. The solution is poured into 20 ml vials. The kit contains 1 bottle.

The glass washing solution with the samples contains Tween 20 in standard phosphate buffered saline. After preparing the solution, the pH is not adjusted. The solution is a 10-fold concentrate and is prepared before use. The solution is poured into 250 ml vials. The kit contains 1 bottle.

The primary antibody solution No. 1 contains monoclonal antibodies to C-erB-2 oncoprotein (internal domain) in a titer of 1: 5000 in the stabilizer. Composition of the stabilizer: bovine serum albumin, Triton X-100 and ProClin 300 in standard phosphate-buffered saline. After preparing the solution, the pH is adjusted to 7.2-7.4. The solution is ready to use. The solution is poured into 10 ml vials. The kit contains 1 bottle.

The primary antibody solution No. 2 contains monoclonal mouse antibodies (IgG1 isotype) to the human estrogen receptor and in the titer 1: 8000 in the stabilizer. Composition of the stabilizer: bovine serum albumin, Triton X-100 and ProClin 300 in standard phosphate-buffered saline. After preparing the solution, the pH is adjusted to 7.2-7.4. The solution is ready to use. The solution is poured into 10 ml vials. The kit contains 1 bottle.

The primary antibody solution No. 3 contains monoclonal mouse antibodies (Kappa IgG1 isotype) to the human progesterone receptor (both A-form and B-form) in a titer of 1: 150 in the stabilizer. Composition of the stabilizer: bovine serum albumin, Triton X-100 and ProClin 300 in standard phosphate-buffered saline. After preparing the solution, the pH is adjusted to 7.2-7.4. The solution is ready to use. The solution is poured into 10 ml vials. The kit contains 1 bottle.

The secondary antibody solution contains a mixture of antibodies against mouse IgG, mouse IgM, and rabbit IgG conjugated with biotin in a titer of 1: 10000 in a stabilizer. Composition of the stabilizer: bovine serum albumin, Triton X-100 and ProClin 300 in standard phosphate-buffered saline. After preparing the solution, the pH is adjusted to 7.2-7.4. The solution is ready to use. The solution is poured into 10 ml vials. The kit contains 1 bottle.

The horseradish streptavidin peroxidase solution contains horseradish streptavidin peroxidase in a titer of 1: 5000. The solution is ready to use. The solution is poured into 10 ml vials. The kit contains 1 bottle.

Diaminbenzidine, the concentrate contains 3,3'-diamenbenzidine-tetrochloride in purified water according to FS 42-2619-97. After preparing the solution, the pH is not adjusted. The solution is intended for the preparation of a working solution of DAB by mixing with a substrate solution for subsequent use. The solution is poured into 1.5 ml vials. The kit contains 1 bottle.

Substrate solution. The solution is intended for mixing with diaminbenzidine for subsequent use as a working solution of DBA. The solution is poured into 10 ml vials. The kit contains 2 bottles.

The negative control sample is a non-immunogenic serum filtered through a nitrocellulose membrane with a pore diameter of 5 μm and containing ProClin 300. After preparing the solution, the pH is not adjusted. The solution is ready to use. The solution is poured into 10 ml vials. The kit contains 1 bottle.

The positive control sample No. 1 is a slide of size 76 × 26 × 1 with mounted on it slices from paraffin blocks containing breast tissue. Two sections were mounted on each glass (0 and 3+ with respect to the HER2 receptor for breast cancer). The set contains 10 glasses with positive control samples No. 1.

Positive control sample No. 2 is a slide of size 76 × 26 × 1 mm with mounted on it sections from paraffin blocks containing breast tissue. Three sections are mounted on each glass (0, 1+ and 3+ with respect to staining of the breast cancer estrogen receptors). The set contains 10 glasses with positive control samples No. 2.

Positive control sample No. 3 is a slide of size 76 × 26 × 1 mm with mounted on it sections from paraffin blocks containing tissue breast. Two sections were mounted on each glass (0 and 3+ relative to staining of the breast cancer progesterone receptors). The set contains 10 glasses with positive control samples No. 3.

Reagent Kit Application

Preliminary preparation of solutions

The solution to ensure the bioavailability of the antigen is diluted in a ratio of 1:10 with distilled or deionized water in the amount necessary for the analysis (40 ml per test glass). The solution is prepared before analysis. The prepared solution is stored for 2 weeks at a temperature of 2-8 ° C.

The solution for washing glasses with samples is diluted in a ratio of 1:10 with distilled or deionized water in the amount necessary for the analysis (40 ml per test glass). The solution is prepared before analysis. The prepared solution is stored for 2 weeks at a temperature of 2-8 ° C.

A working solution of diaminobenzidine is prepared as follows. 1 ml of the substrate solution is transferred to a clean container. 1 drop (23-30 μl) of DAB concentrate is added to this container. Stir the solution thoroughly. A working solution of DAB is prepared in the amount necessary for the analysis (0.2 ml per test glass). The solution is prepared immediately before the analysis. The prepared solution is stored for 3 days at a temperature of 2-8 ° C. Before use, the stored solution must be mixed.

Statement of analysis

When setting up the analysis, in parallel with the studied samples, control (s) positive (s) sample (s) are put at the rate of one glass per set, depending on the determined target markers.

At the first stage of the analysis, pour 20-30 drops (500-750 μl) of the ROB on the samples, cover with a clean container and place in the microwave for 8-10 minutes at a power of 120 watts. Do not allow the sections to completely dry when heated. After this, the ROB should be drained from the surface of the glass into a container with a disinfectant liquid, the remaining moisture should be removed by gently touching the filter paper to the edge of the liquid, without touching the sections themselves. Next, pour 20-30 drops (500-750 μl) of cold ROB on the glass with samples and hold for 10 minutes.

At the end of the first stage, rinse the samples. The protocol for washing glasses with samples is uniform for any washing procedure during the analysis and is carried out according to the following scheme. The liquid located on the glass should be drained from the glass surface into a container with a disinfectant liquid, pour 20-30 drops (500-750 μl) of the glass washing solution on top of the slices, stand for 2-3 minutes and repeat the washing procedure one more time. OL merge and perform the next step.

In the second stage, apply 2-3 drops (50-75 μl) to each of the test or control glasses on a section of a blocking solution for horseradish peroxidase. Maintain PDU for 15 minutes at a temperature of 18-25 ° C in a wet chamber (it is permissible to use a resealable container with pieces of wet filter paper located under the glasses but not touching them instead of the wet chamber) and rinse the glass according to the above diagram.

The third stage consists of pouring 2-3 drops (50-75 μl) of protein blocking solution onto the sections and keeping it for 15 minutes at a temperature of 18-25 ° C in a humid chamber. After performing this stage, the RBD merges with the glasses, but do not rinse the glass.

At the fourth stage, add 2-3 drops (50-75 μl) of the solution of primary antibodies to the glass, their choice is determined by the purpose of the analysis (determination of receptors for HER2, estrogen or progesterone). It is acceptable to define different receptors in one formulation without changing the formulation protocol. Additionally, on one or more glasses containing sections from the same tissues as the test samples (it is acceptable to use one of the positive control glasses), instead of the solution of primary antibodies, add a negative control sample in the same volume. Incubate sections at a temperature of 18-25 ° C in a humid chamber for 30 minutes and rinse according to the procedure described above.

The fifth stage is that 2-3 drops (50-75 μl) of a solution of secondary antibodies are applied to each of the test or control sections, the sections are kept for 18 minutes at a temperature of 18-25 ° C in a humid chamber. After that, the samples are washed according to the above procedure.

When performing the sixth stage, 2-3 drops (50-75 μl) of the SPC solution are applied to each of the sections. Samples are incubated for 20 minutes at a temperature of 18-25 ° C in a humid chamber. After that, the samples are washed according to the scheme described above.

At the seventh stage, a DBA working solution of 2-3 drops (50-75 μl) is applied to the glass. The solution is kept for 20 minutes at a temperature of 18-25 ° C in a humid chamber. Slices are washed according to the above scheme with distilled or deionized water, stained with hemotoxylin and enclosed in polystyrene under a cover glass.

Results Analysis

In the developed set of reagents, the immunohistochemical reaction to all three receptor variants is supposed to be evaluated according to the generally accepted point system approved by the FDA [6, 7]. The severity of color is evaluated on a scale from 0 to 3+.

0 - complete absence of IHC staining or when detecting it on the membranes of less than 10% of invasive cancer cells:

- when determining HER2 overexpression - negative breast cancer, treatment with herceptin is not indicated;

- in determining hormonal status regarding estrogen and progesterone receptors - negative breast cancer;

1+ - weak staining of membranes of more than 10% of invasive cancer cells:

- when determining HER2 overexpression - negative breast cancer, it is possible to conduct additional research to recheck HER2 status;

- when determining hormonal status relative to estrogen and progesterone receptors - negative breast cancer, it is possible to conduct additional research to check the hormonal status;

2+ - moderate staining of membranes of more than 10% of invasive cancer cells:

- when determining HER2 overexpression - the boundary value, an unclear case, it is mandatory to recheck HER2 status with the help of additional research;

- when determining hormonal status relative to estrogen and progesterone receptors - borderline value, unclear case, mandatory re-examination of hormonal status with the help of additional research;

3+ - bright staining of the entire cell membrane of most invasive cancer cells:

- when determining HER2 overexpression - positive breast cancer, treatment with herceptin is indicated;

- in determining hormonal status regarding estrogen and progesterone receptors - positive breast cancer, hormone therapy is indicated.

In the light-optical study of sections, the results of the analysis should be taken into account only if sections with a negative control sample contain staining at level 0 on a scale for the degree of immunohistochemical staining of breast cancer estrogen receptors, and glass with a positive control sample has staining at levels 0, 1+, and 3 + depending on the control slice.

The use of the reagent kit we developed can be illustrated by the following examples.

Example 1. We studied the "Working panel of HEK2-positive and HER2-negative breast cancer samples" (RP (+/-) HER2 breast cancer), consisting of the following samples:

Samples No. 1-6 had a score of 0 (-). Samples No. 7-10 had a score of 1 + (+). Samples No. 11-14 had a score of 2 + (++). Samples No. 15-20 had a rating of 3 + (+++).

The results of the verification of three series of the test system “A set of reagents for simultaneous or separate immunohistochemical detection of estrogen, progesterone and HER2 receptors” on the working panel of RP (+/-) HER2 breast cancer samples are presented in table 1:

Table 1 Test results C1, C2 and C3 on the RP panel (+/-) HER2 breast cancer Sample Number HER2 receptor content C1 C2 C3 No. 1 0 (-) 0 (-) 0 (-) 0 (-) Number 2 0 (-) 0 (-) 0 (-) 0 (-) Number 3 0 (-) 0 (-) 0 (-) 0 (-) Number 4 0 (-) 0 (-) 0 (-) 0 (-) Number 5 0 (-) 0 (-) 0 (-) 0 (-) Number 6 0 (-) 0 (-) 0 (-) 0 (-) Number 7 1 + (+) 2 + (++) 2 + (++) 1 + (+) Number 8 1 + (+) 1 + (+) 1 + (+) 1 + (+) Number 9 1 + (+) 1 + (+) 2 + (++) 1 + (+) Number 10 1 + (+) 1 + (+) 1 + (+) 1 + (+) Number 11 2 + (++) 1 + (+) 2 + (++) 2 + (++) Number 12 2 + (++) 2 + (++) 2 + (++) 2 + (++) Number 13 2 + (++) 2 + (++) 2 + (++) 2 + (++) Number 14 2 + (++) 2 + (++) 1 + (+) 1 + (+) Number 15 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) Number 16 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) Number 17 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) Number 18 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) Number 19 3+ (+++) 3+ (+++) 2 + (++) 3+ (+++) Number 20 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++)

According to the results of the analysis, it is evident that, according to the criteria, sensitivity and specificity (by definition of 0 and 3+ samples), the experimental series fully correspond to the control panel data. For samples requiring additional verification (1 + and 2+), the results obtained in the experimental series are very close to those in the control.

Example 2. We studied the general panel of samples "The working panel of estrogen- and progesterone-positive and estrogen- and progesterone-negative samples of breast cancer" (RP (+/-) ER / PR breast cancer). Characteristics of the samples are shown in table 2.

table 2 Characteristics of samples of the panel RP (+/-) ER / PR breast cancer Sample Number Estrogen receptor levels Progesterone Receptor Content No. 1 0 (-) 0 (-) Number 2 0 (-) 0 (-) Number 3 0 (-) 0 (-) Number 4 0 (-) 0 (-) Number 5 1 + (+) 0 (-) Number 6 3+ (+++) 0 (-) Number 7 0 (-) 1 + (+) Number 8 0 (-) 2 + (++) Number 9 0 (-) 3+ (+++) Number 10 1 + (+) 1 + (+) Number 11 1 + (+) 2 + (++) Number 12 1 + (+) 3+ (+++) Number 13 2 + (++) 1 + (+) Number 14 2 + (++) 2 + (++) Number 15 3+ (+++) 1 + (+) Number 16 3+ (+++) 2 + (++) Number 17 3+ (+++) 3+ (+++)

The results of the verification of three series of the test system “Set of reagents for simultaneous or separate immunohistochemical detection of estrogen, progesterone and HER2 receptors” (RP (+/-) ER / PR breast cancer) are presented in table 3.

Table 3 Results of checking C1, C2 and C3 on the RP panel (+/-) ER / PR breast cancer Sample Number determination of estrogen receptors determination of progesterone receptors Control values C1 C2 C3 Control values C1 C2 C3 No. 1 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) Number 2 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) Number 3 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) Number 4 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) 0 (-) Number 5 1 + (+) 1 + (+) 1 + (+) 1 + (+) 0 (-) 0 (-) 0 (-) 0 (-) Number 6 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) 0 (-) 0 (-) 0 (-) 0 (-) Number 7 0 (-) 0 (-) 1 + (+) (-) 1 + (+) 1 + (+) 1 + (+) 1 + (+) Number 8 0 (-) 0 (-) 0 (-) 0 (-) 2 + (++) 3+ (+++) 2 + (++) 2 + (++) Number 9 0 (-) 0 (-) 0 (-) 0 (-) 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) Number 10 1 + (+) 1 + (+) 1 + (+) 1 + (+) 1 + (+) 1 + (+) 1 + (+) 2 + (++) Number 11 1 + (+) 1 + (+) 2 + (++) 1 + (+) 2 + (++) 2 + (++) 2 + (++) 2 + (++) Number 12 1 + (+) 2 + (++) 1 + (+) 1 + (+) 3+ (+++) 3+ (+++) 3+ (+++) 2 + (++) Number 13 2 + (++) 2 + (++) 2 + (++) 2 + (++) 1 + (+) 1 + (+) 1 + (+) 1 + (+) Number 14 2 + (++) 2 + (++) 2 + (++) 3+ (+++) 2 + (++) 2 + (++) 3+ (+++) 2 + (++) Number 15 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) 1 + (+) 1 + (+) 1 + (+) 1 + (+) Number 16 3+ (+++) 2 + (++) 3+ (+++) 3+ (+++) 2 + (++) 2 + (++) 2 + (++) 2 + (++) Number 17 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++) 3+ (+++)

According to the results of the analysis, it is evident that, according to the criteria of sensitivity and specificity (by definition of 0 and 3+ samples), the experimental series of test systems for determining hormonal status almost completely correspond to the control panel data. For samples requiring additional verification (1+ and 2+), the results obtained in the experimental series are very close to those in the control.

Example 3. To check the experimental sets of reagents on a random sample of clinical material, an array of breast tumor tissue samples was collected, collected from surgical material and samples after biopsy (171 samples). For each of the samples, a clinical diagnosis was available regarding the hormonal status of the tumor and the presence of HER2 markers in them.

For the experimental set, a screening analysis of an array of random samples of breast cancer material was carried out to determine the markers HER2, estrogen and progesterone. The results are presented in table 4.

Table 4 Test system test results on an array of breast tissue samples Clinical diagnosis and number of samples in relation to HER2 Data obtained using an experimental test system for the determination of HER2 Clinical diagnosis and number of samples in relation to estrogen receptors Data obtained using an experimental estrogen receptor test system Clinical diagnosis and progesterone receptor count Data obtained using an experimental test system for determining progesterone receptors 3+ (26 samples) 3+ (20 samples) 3+ (88 samples) 3+ (76 samples) 3+ (78 samples) 3+ (70 samples) 2+ (4 samples) 2+ (2 samples) 2+ (8 samples) 1 + (-) 1+ (5 samples) 1 + (-) 0 (2 samples) 0 (5 samples) 0 (-) 2+ (12 samples) 3+ (1 sample) 2+ (27 samples) 3+ (4 samples) 2+ (18 samples) 3+ (3 samples) samples) 2+ (6 samples) 2+ (22 samples) 2+ (13 samples) 1+ (3 samples) 1+ (1 sample) 1+ (1 sample) 0 (2 samples) 0 (-) 0 (1 sample) 1+ (24 samples) 3+ (5 samples) 1+ (6 samples) 3+ (4 samples) 1+ (14 samples 3+ (1 sample) 2+ (2 samples) 2 + (-) 2+ (1 sample) 1+ (11 samples) 1+ (2 samples) 1+ (9 samples) 0 (6 samples) 0 (-) 0 (3 samples) 0 (80 samples) 3 + (-) 0 (31 samples) 3+ (1 sample) 0 (37 samples) 3+ (2 samples) 2+ (4 samples) 2+ (3 samples) 2+ (3 samples) 1+ (4 samples) 1+ (2 samples) 1+ (3 samples) 0 (72 samples) 0 (25 samples) 0 (29 samples) Undefined status 3+ (5 samples) Undefined status 3+ (14 samples) Undefined status 3+ (12 samples) (29 samples) 2+ (2 samples) 19 (samples) 2 + (-) (24 samples) 2+ (2 samples) 1+ (1 sample) 1+ (2 samples) 1+ (5 samples) 0 (21 samples) 0 (3 samples) 0 (5 samples) Total total total 3+ (31 samples) 3+ (99 samples) 3+ (88 samples) 2+ (18 samples) 2+ (27 samples) 2+ (27 samples) 1+ (19 samples) 1+ (12 samples) 1+ (18 samples) 0 (103 samples) 0 (33 samples) 0 (38 samples)

The analysis of the data shows that when testing the test system “A set of reagents for simultaneous or separate immunohistochemical detection of estrogen, progesterone and HER2 receptors”, the analysis results obtained using it adequately correspond to the clinical data and the percentage distribution of samples in the point system is consistent with the statistical results investigation of arbitrary sample arrays [6, 7].

Bibliography

1. Hercept test TM, 16 Edition, Manual.

2. Walker RA, Harris AL, Balslev E. Immunohistochemistry and Breast Cancer. Diagnosis, Therapy and Prognosis. Corporate Headquartes Denmark. 2004, 23p.

3. Zafrani B, Aubriot MH, Mouret E et al. High sensitivity and specificity of immunohistochemistry for the detection of hormone receptors in breast carcinoma: comparison with biochemical determination in a prospective study of 793 cases. Histopathology 2000; 37 (6): 536-545.

4. European Patent Application EP1357132.

5. Dako ER / PR pharmDx ™ Kit For the Dako Autostainer English Code K4071 Edition 10/11

6. O'Malley F. P, Parkers R, Latta E et al. Am J ClinPathol 2001; 115 (4): 504-11.

7. Zavalishina L.E., Frank G.A. Morphological testing of HER2 status. Methodology and atlas. M .: Mediamedica, 2006.

Claims (1)

A set of reagents for immunohistochemical separate or simultaneous detection of overexpression of HER2 receptors and hormonal status with respect to estrogen and progesterone receptors for breast cancer, including a solution for ensuring the bioavailability of antigen, blocking solution of horseradish peroxidase, protein blocking solution, washing solution, solution of primary antibodies No. 1, solution of primary antibodies antibodies No. 2, a solution of primary antibodies No. 3, a solution of antispecies secondary antibodies to primary antibodies, a solution of streptavidin-p horseradish oxidase, diaminobenzidine, substrate solution, negative control sample (K-), positive control sample No. 1, positive control sample No. 2, positive control sample No. 3, characterized in that the set contains three variants of primary antibodies simultaneously (to estrogen receptors , progesterone and HER2), a mixture of secondary antibodies conjugated with biotin, and introduced a unified protocol for the use of all three variants of primary antibodies.