RU2797698C1 - Method of determining the conversion of the molecular biological subtype of the totality of circulating tumor cells relative to the molecular biological subtype of the primary tumor in patients with breast cancer - Google Patents

Abstract

FIELD: oncology.

SUBSTANCE: invention can be used to determine the conversion of the molecular biological subtype (MBP) of a set of circulating tumor cells (CTC) relative to the MBP of the primary tumor in patients with breast cancer. The MBP of the primary tumor is determined according to the regulations of the clinical recommendations of RUSSCO and the MBP of the CSC combination, for this, in the venous blood fraction enriched with leukocytes after erythrocyte sedimentation, the expression in nucleated cells of the common leukocyte marker CD45, EpCam, as well as MBP markers ER, PR, is determined by flow cytometry. HER2 and Ki67, the obtained values are compared with those in the main tumor node, and if a discrepancy between them is detected, the MBP conversion of the CTC set relative to the MBP of the primary tumor is determined.

EFFECT: proposed method of determining the MBP conversion of the CTC population in patients with breast cancer makes it possible to predict the effectiveness of the chemotherapy regimen used.

1 cl, 3 dwg, 2 ex

Description

SUBSTANCE: invention relates to medicine, namely to oncology, and can be used to determine the change (conversion) of the molecular biological subtype (MBP) of a set of circulating tumor cells (CTCs) relative to the MBP of the primary tumor in patients with breast cancer (BC).

Determining the molecular biological subtype of the primary tumor is an important step in assessing the prognosis of the clinical course of the disease and determining the tactics of treating patients with breast cancer.

The method for determining the molecular biological subtype of a primary breast cancer tumor is strictly regulated by the RUSSCO clinical guidelines and is based on an integral assessment of the expression of markers of estrogen receptors (ERα,) and progesterone (PR), a marker of proliferative activity of Ki67 cells and human epidermal growth factor receptor (HER2) in collection of tumor cells. Taking into account the integral assessment of the expression of these markers, 5 main molecular biological subtypes of breast cancer are distinguished:

- luminal A positive expression of ERα and/or PR; Ki67 less than 20%, no overexpression of the HER2 gene);

- luminal B, HER2-negative (positive expression of ERα and/or PR; Ki67 over 20%, no overexpression of the HER2 gene);

- luminal B, HER2-positive (positive expression of ERα and/or PR, the presence of overexpression of the HER2 gene);

- HER2-positive (negative expression of ERα and PR, the presence of overexpression of the HER2 gene);

- triple negative (negative expression of ERα, PR, HER2).

The appointment of adequate hormonal therapy, as well as conventional chemotherapy and targeted anti-HER2 therapy for breast cancer, directly depends on the molecular biological subtype of the primary tumor. It is known that the expression of individual markers of the molecular biological subtype differs in the primary tumor, metastatic node and circulating tumor cells (CTC) (Aktas B. et al. Comparison of the HER2, estrogen and progesterone receptor expression profile of primary tumor, metastases and circulating tumor cells in metastatic breast cancer patients // BMC cancer. - 2016. - T. 16. - No. 1. - C. 1-8.). Adjuvant therapy is recommended to be chosen taking into account the expression of molecular markers by tumor cells of the metastatic focus. However, metastases are not always available for research. CTCs are a heterogeneous collection of tumor cells in circulation, and include cells with aggressive properties, some of which become disseminated, "dormant" and, as a result, are a source of distant metastases.

Thus, it became necessary to introduce a definition of the molecular biological subtype of the CTC population, compare it with the subtype of the primary tumor, and identify its change (conversion) relative to the MBP of the primary tumor in order to significantly expand the diagnostic, prognostic, and therapeutic possibilities in breast cancer.

To date, methods are known that allow determining the expression of individual CTC markers and detecting changes in their expression relative to the primary tumor, but not the conversion of the molecular biological subtype as a whole.

A known method for determining the molecular biological subtype of the primary tumor and individual markers of the molecular biological subtype in the CTC by flow cytometry. RU patent No. 2757590 describes a method for determining the expression of Ki67, PR and HER2 markers in non-fixed tumor tissue obtained by core biopsy using flow cytometry. The undoubted advantages of this method include the validation of the obtained data by immunohistochemistry.

In the work of Paoletti et al., expression of the estrogen receptor in the CTC is determined using the CellSearch® system (Paoletti C. et al. Circulating tumor cell number and endocrine therapy index in ER positive metastatic breast cancer patients //NPJ breast cancer. - 2021. - T 7. - No. 1. - C. 1-9.). A similar system is used by Beije et al. to determine the expression of HER2 in the CTC (Beije N. et al. Prognostic impact of HER2 and ER status of circulating tumor cells in metastatic breast cancer patients with a HER2-negative primary tumor //Neoplasia. - 2016. - T. 18. - No. 11. - C. 647-653.).

In the work of Kalinsky et al. to determine the expression of estrogen and progesterone receptors, CTCs are isolated by the microfluidic method using the OncoCEE™ platform and further immunocytochemical staining (Kalinsky K. et al. Correlation of hormone receptor status between circulating tumor cells, primary tumor, and metastasis in breast cancer patients // Clinical and Translational Oncology - 2015. - V. 17. - No. 7. - C. 539-546.).

A known method for determining the expression of estrogen receptor and Ki67 in the CTC using the CellSearch® system (Paoletti C. et al. Circulating Biomarkers and Resistance to Endocrine Therapy in Metastatic Breast Cancers: Correlative Results from AZD9496 Oral SERD Phase I TrialLiquid Biopsies, Metastatic Breast Cancer, Oral SERD AZD9496 // Clinical Cancer Research - 2018. - T. 24. - No. 23. - C. 5860-5872.).

A known method for determining the molecular biological subtype of the primary tumor by flow cytometry (Wopereis S. et al. Evaluation of ER, PR and HER2 markers by flow cytometry for breast cancer diagnosis and prognosis //Clinica Chimica Acta. - 2021. - T. 523. - C. 504-512.). The disadvantage of this method is the inconsistency of the developed approach for determining the overexpression of the HER2 marker to generally accepted clinical guidelines. The authors interpret intermediate values of HER2 expression "+" and "++" as the presence of overexpression of the HER2 marker, while positive expression is generally considered to be only staining for "+++".

Thus, none of the above analogues is suitable for solving the problem. A common disadvantage of the methods under consideration is the determination of individual markers without a conclusion about the molecular biological subtype of the CTC population. At the same time, the method proposed by Wopereis S. et al. makes it possible to determine the molecular biological subtype by flow cytometry, but in the primary tumor, and not in the aggregate of CTCs.

A new technical result is the creation of a method for determining the conversion of the molecular biological subtype of the CTC population in patients with breast cancer in order to predict the effectiveness of the chemotherapy regimen used.

To achieve a new technical result in the method for determining the conversion of the molecular biological subtype (MBP) of the population of circulating tumor cells relative to the molecular biological subtype of the primary tumor in patients with breast cancer, the MBP of the primary tumor is determined according to the regulations of the RUSSCO clinical guidelines, and the MBP of the CTC population is also determined, for this in the leukocyte-enriched venous blood fraction obtained after erythrocyte sedimentation, the expression in nucleated cells of the common leukocyte marker CD45, EpCam, as well as the expression of markers of the molecular biological subtype ER, PR, HER2 and Ki67, respectively, are determined by flow cytometry, the obtained values are compared with those in the main tumor node and if a discrepancy between them is detected, the conversion of the molecular biological subtype in the aggregate of CTCs is determined.

What is new is that the MBP of the primary tumor is determined according to the regulations of the RUSSCO clinical guidelines, the MBP of the CTC set is also determined, for this, in the leukocyte-rich blood fraction obtained after the precipitation of venous blood erythrocytes, the expression in nucleated cells of the common leukocyte marker CD45, EpCam, is determined by flow cytometry. , as well as the expression of markers of the molecular biological subtype ER, PR, HER2 and Ki67, respectively, the obtained values are compared with those in the main tumor node and, if a discrepancy between them is detected, the conversion of the molecular biological subtype in the CTC set is determined.

The method is carried out as follows. After surgical removal of the tumor, an immunohistochemical study of tumor tissue samples is performed to determine the molecular biological subtype of the main tumor node. Expression of ERα, PR, HER2 and Ki67 in tumor tissue is assessed using a Leica Bond-Max automatic immunostainer (Leica, Bannockburn, IL). Staining is done with a Leica Refine kit according to an automated protocol. Antigen unmasking for PR was performed in Bond Solution #1 (Leica Biosystems, equivalent to citrate buffer, pH 6.0), for ERα, HER2 and Ki67 in Bond Solution #2 (Leica Biosystems, equivalent to EDTA buffer, pH 9.0) at 100°С for 20 min . Primary antibodies against ERα (RTU, clone 6F11, mouse, Leica Biosystems, Germany), PR (RTU, clone 16, mouse, Leica Biosystems, Germany), HER2 (1:1000, polyclonal, rabbit, Dako, USA) and Ki67 ( 1:250, clone SP6, rabbit, Sigma-Aldrich, USA) were incubated at room temperature for 25 min. Primary antibody detection was performed using a Bond Polymer Refine Detection kit (Leica Biosystems, Germany) and diaminobenzidine (DAB) at room temperature for 5 min. The samples were then counterstained with hematoxylin. The results are calculated in accordance with the recommendations of RUSSCO, for ERα and PR more than 1% of tumor cells with expression, for Ki67 more than 20% of tumor cells, for HER2 more than 10% of tumor cells with pronounced and moderate circular expression or a positive result with fluorescent hybridization in situ . A small fragment of tumor tissue (0.5×0.5 cm) is frozen at -80°C for further analysis by flow cytometry.

Determination of HER2 expression in tumor cells by flow cytometry

To determine the status of HER2, it is necessary to separate normal expression from overexpression, which is an indication for targeted therapy, it is necessary to set a threshold value of fluorescence intensity, determined by flow cytometry, which corresponds to "+++" when determining HER2 expression in a tumor by immunohistochemistry or a positive reaction by fluorescence hybridization in situ.

To determine the level of fluorescence intensity corresponding to the expression of HER2 at "+++", samples of the primary tumor with different expression of HER2 (0, "+", "++" "+++") were selected for analysis by flow cytometry. Samples were thawed at room temperature, then homogenized using MediMachine (BD Biosciences, USA) in 1 ml DMEM containing 5% FBS. The resulting cell suspension was filtered through a cell sieve (Corning Falcon™ Cell Strainer) with a pore size of 70 μm and washed twice in PBS (pH=7.4) at 400×g for 10 min. Next, the samples were stained with antibodies against CD45, EpCam and HER2. Cell viability was assessed by 7AAD staining.

According to the results of our study, the expression of HER2 at "+++" in the tumor corresponded to the expression of HER2 in more than 0.5% of cells with a fluorescence level corresponding to the 5th decade of the logarithmic scale of the Novocyte 3000 flow cytometer (ACEA Bioscience, USA).

Note In a medical institution where the proposed method will be used, it is necessary to determine once, using the above method, the percentage of CTCs and the threshold of the HER2 fluorescence level, which correspond to the expression of HER2 in the tumor at "+++".

Re-determination of the level of HER2 fluorescence intensity by cells of the primary tumor is performed only when the technical characteristics of the instrument change or when using a new lot of antibodies for flow cytometry.

Determination of ERα, PR, HER2 and Ki67 receptors of the CTC population

A sample of EDTA-stabilized venous blood in a volume of 9 ml is taken in the morning on an empty stomach, the first ml of blood is not taken into the study to exclude skin epithelial cells from entering the sample. Next, the samples are incubated in a thermostat at 37°C for 90 minutes. After sedimentation of the erythrocyte fraction, the leukocyte-enriched fraction located at the interface between plasma and erythrocytes is taken. The selected cell concentrate was washed in 2 ml of Cell Wash buffer (BD Biosciences, USA) and centrifuged at 800 × g for 15 min. The supernatant is removed, the precipitate is resuspended in 150 µl of sterile phosphate-buffered saline (pH=7.4). For further research, the sample is divided into two parts: 50 µl - unstained control, 100 µl - stained sample. CTC staining for subsequent detection by flow cytometry is carried out in two stages, the first stage stains surface markers (CD45, EpCam, HER2), and the second stage stains intracellular markers (EpCam, ERα, PR, Ki67).

Samples are incubated with Fc Receptor Blocking Solution to block non-specific antibody binding (Sony Biotechnology, USA) at room temperature for 10 min. Then add 5 µl of monoclonal antibodies: BV570-anti-CD45 (clone HI30, mouse IgGl, Sony Biotechnology, USA), BV650-anti-EpCam (clone 9C4, mouse IgG2b, Sony Biotechnology, USA), AF750-anti-HER2 ( clone 24D2, mouse IgGl, BioLegend, USA), samples were incubated in the dark at room temperature for 20 min. After incubation, erythrocytes are lysed in 250 μL of OptiLyse buffer (Beckman Coulter, France) in the dark at room temperature for 10 min, then the samples are washed in 2 mL of Cell Wash buffer (BD Biosciences, USA) and centrifuged at 800 × g for 6 min.

For intracellular staining, samples are permeabilized in 250 µl of BD Cytofix/Cytoperm (BD Biosciences, USA) and incubated in the dark at 4°C for 30 min, then washed in 1 ml of Cell Wash buffer (BD Biosciences, USA) and centrifuged at 800 × g for 6 min . Then, resuspend in 50 µl BD Perm/Wash buffer (BD Biosciences, USA) and add 5 µl Fc Receptor Blocking Solution (Human TruStain FcX, Sony Biotechnology, USA) samples are incubated in the dark at 4°C for 10 min. Then add 5 µl of monoclonal antibodies: BV650-anti-EpCam (clone 9C4, mouse IgG2b, Sony Biotechnology, USA), AF488-anti-ERα (clone AER314, mouse IgGl, Novus Biologicals, USA), APC-anti-PR ( clone PR484, mouse IgGl, Novus Biologicals, USA), BV421-anti-Ki67 (clone 16A8, mouse IgG2a, Sony Biotechnology, USA), samples were incubated in the dark at 4°C for 20 min. After incubation, the samples are washed in 1 ml Cell Wash buffer (BD Biosciences, USA) and centrifuged at 800 × g for 6 min. Samples are resuspended in 100 µl of sterile phosphate-buffered saline (pH=7.4) and analyzed on a Novocyte 3000 flow cytometer (ACEA Biosciences, USA).

Analysis of the expression of ERα and PR receptors is carried out as follows. As a negative control for the expression of ERα and PR receptors, lymphocytes are used, which, as is known from the literature data, do not express ERα and PR. Samples in which more than 10% of CTCs have a fluorescence intensity higher than lymphocytes are considered positive for ERα and PR expression. When evaluating the expression of Ki67, the percentage of CTCs with a fluorescence intensity higher than the unstained control is determined. A CTC sample was considered HER2-positive if ≥0.5% of the total CTC population was characterized by an intensity of HER2 expression at the level of the 5th decade of the logarithmic scale.

Given the expression of ERα, PR, HER2 and Ki67 in the CTC, the molecular biological subtype of the CTC population is determined (Fig. 1, Determination of fluorescence intensity levels by flow cytometry, corresponding to different expression of HER2 (0, "+", "++" "+++" ).

The method is based on the results of experimental clinical studies. Determining the molecular biological subtype of the primary tumor is an important step in the treatment of breast cancer. In clinical practice, the molecular biological subtype of the residual tumor in breast cancer, as well as metastases, is determined (Dergunova Yu.A. et al. Correlation of the molecular biological subtype of the primary tumor and regional metastases in patients with breast cancer // Scientific and practical peer-reviewed journal Khailenko V.A., Kozlov N.A. Clinical significance of changes in the receptor status in recurrent and metastatic tumors in cancer patients Clinical and experimental morphology. breast.). However, metastasis material is not always available. The molecular biological classification of breast cancer is a surrogate one, that is, belonging to a subtype is based on the analysis of the expression of ERα, PR, HER2, Ki67 markers. In this regard, it is advisable to use generally accepted methods for analyzing the expression of markers of the molecular biological subtype of the CTC population.

The method is based on the analysis of the results of laboratory and clinical studies. In the analysis of 43 cases of breast cancer, stage T1-4N0-3M0, in addition to determining the molecular biological subtype of the main tumor node in accordance with the regulations of the RUSSCO clinical guidelines using the method described above, the molecular biological subtype (MBP) of the CTC set was determined using the above described proposed method. When comparing the molecular biological subtype of the CTC with the molecular biological subtype of the primary tumor, a conversion (change) of the MBP was detected, which was expressed in the fact that in 88.4% of cases (38/43) the MBP changed. And in 11.6% (5/43) cases, the MBP of the CTC aggregate did not change compared to the MBP of the primary tumor.

Based on the data obtained, it was concluded that a change (conversion) of the molecular biological subtype of the CTC population relative to the MBP of the primary tumor is likely to be the basis for making recommendations for clarifying the MBP and, as a result, changing the chemotherapy regimen to a more adequate one, which will promote more effective anticancer therapy.

The essence of the proposed method is illustrated by the following examples.

Example 1. Patient S., aged 39, diagnosed with invasive ductal carcinoma of a nonspecific type of breast. Stage IIB disease. The state of menstrual function - saved. The degree of malignancy - II. The size of the primary tumor node is 2.5 cm. In order to select therapy, the MBP of the primary tumor was determined according to the RUSSCO clinical guidelines. Received: ERα+, PR+, HER2 0, Ki67 - 50%. The molecular biological subtype of the primary tumor was defined as luminal B HER2-negative.

The determination of the MBP of the totality of CTCs isolated from venous blood and its conversion relative to the MBP of the primary tumor was carried out according to the proposed method. It turned out that more than 10% of circulating tumor cells had a fluorescence intensity above the threshold for ER and PR markers. There were no tumor cells with HER2 expression above the threshold (≥0.5%) fluorescence intensity value. Ki67 expression had a level above the threshold level of fluorescence intensity in 5% of tumor cells. Thus, the molecular biological subtype of the population of circulating tumor cells was defined as luminal B HER2-negative. It was confirmed that there was no change in the MBP of the population of circulating tumor cells, relative to the MBP of the primary tumor subtype (Fig. 2 The histograms show the average fluorescence intensity (MFI) of the expression of markers of the molecular biological subtype ER, PR, HER2 and Ki67 in the population of CD45-EpCam + circulating tumor cells in sick S.).

Example 2. Patient G., aged 39, diagnosed with invasive ductal carcinoma of a nonspecific type of breast. Disease stage IA. The state of menstrual function - saved. The degree of malignancy - II. The size of the primary tumor node is 2 cm. In order to select therapy, the MBP of the primary tumor was determined according to the RUSSCO clinical guidelines. Received: ERα+, PR+, HER2 0, Ki67 - 24%. The molecular biological subtype of the primary tumor was defined as luminal B HER2-negative.

The determination of the MBP of the totality of CTCs isolated from venous blood and its conversion relative to the MBP of the primary tumor was carried out according to the proposed method. More than 10% of circulating tumor cells had a fluorescence intensity above the threshold for ERα and PR markers. HER2 expression was above the threshold value of fluorescence intensity in 46.9% of tumor cells. The expression of Ki67 was above the threshold level of fluorescence intensity in 5% of tumor cells. Thus, the molecular biological subtype of the population of circulating tumor cells is defined as luminal B HER2-positive. There was a conversion (change) of the MBP of the totality of circulating tumor cells, which was confirmed by the presence of a difference from the MBP of the primary tumor (Figure 3. The histograms show the average fluorescence intensity (MFI) of the expression of markers of the molecular biological subtype ER, PR, HER2 and Ki67 in the CD45- EpCam+ circulating tumor cells in patient K.)

Based on the results obtained - the presence of a change in the MBP of the CTC population relative to the MBP of the primary tumor, it became necessary to recommend a change in the chemotherapy regimen.

Thus, the proposed method allows to determine the molecular biological subtype of the CTC population, to compare it with the subtype of the primary tumor to detect the conversion of the molecular biological subtype, which can significantly expand the diagnostic, prognostic and therapeutic possibilities in breast cancer. The method has a number of advantages. For the first time, all markers of the molecular biological subtype (ERα, PR, HER2, Ki67) are determined in the aggregate of CTCs by flow cytometry. Due to the focus on modern clinical guidelines for determining the molecular biological subtype of the primary tumor, the method can be easily integrated into clinical practice. This method is cost-effective, does not require the purchase of additional expensive equipment and can be performed not only in research, but also in medical institutions, including oncology dispensaries.

Sources of information taken into account when compiling the description:

1. Description of the invention according to the application RU 2757590 C1.

2. Aktas B. et al. Comparison of the HER2, estrogen and progesterone receptor expression profile of primary tumor, metastases and circulating tumor cells in metastatic breast cancer patients //BMC cancer. - 2016. - T. 16. - no. 1. - C. 1-8.).

3. Beije N. et al. Prognostic impact of HER2 and ER status of circulating tumor cells in metastatic breast cancer patients with a HER2-negative primary tumor //Neoplasia. - 2016. - T. 18. - no. 11. - C. 647-653.

4. Kalinsky K. et al. Correlation of hormone receptor status between circulating tumor cells, primary tumor, and metastasis in breast cancer patients //Clinical and Translational Oncology. - 2015. - T. 17. - No. 7. - C. 539-546.

5. Paoletti C. et al. Circulating Biomarkers and Resistance to Endocrine Therapy in Metastatic Breast Cancers: Correlative Results from AZD9496 Oral SERD Phase I TrialLiquid Biopsies, Metastatic Breast Cancer, Oral SERD AZD9496 //Clinical Cancer Research. - 2018. - T. 24. - No. 23. - C. 5860-5872.

6. Paoletti C. et al. Circulating tumor cell number and endocrine therapy index in ER positive metastatic breast cancer patients //NPJ breast cancer. - 2021. - T. 7. - no. 1. - C. 1-9.

7. Wopereis S. et al. Evaluation of ER, PR and HER2 markers by flow cytometry for breast cancer diagnosis and prognosis //Clinica Chimica Acta. - 2021. - T. 523. - C. 504-512.

Claims (1)

A method for determining the conversion of the molecular biological subtype of the population of circulating tumor cells relative to the molecular biological subtype of the primary tumor in patients with breast cancer, characterized in that the molecular biological subtype of the primary tumor is determined according to the regulations of the RUSSCO clinical guidelines and the molecular biological subtype of the CTC population, for this in the leukocyte-enriched venous blood fraction obtained after erythrocyte sedimentation, the expression in nucleated cells of the common leukocyte marker CD45, EpCam, as well as the expression of markers of the molecular biological subtype ER, PR, HER2 and Ki67, respectively, are determined by flow cytometry, the obtained values are compared with those in the main tumor node and if a discrepancy between them is detected, the conversion of the molecular biological subtype of the CTC population relative to the molecular biological subtype of the primary tumor is determined.

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