WO2023115171A1 - Method, panel and kit for the diagnosis and treatment monitoring of breast cancer patients - Google Patents

Method, panel and kit for the diagnosis and treatment monitoring of breast cancer patients Download PDF

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WO2023115171A1
WO2023115171A1 PCT/BR2021/050561 BR2021050561W WO2023115171A1 WO 2023115171 A1 WO2023115171 A1 WO 2023115171A1 BR 2021050561 W BR2021050561 W BR 2021050561W WO 2023115171 A1 WO2023115171 A1 WO 2023115171A1
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breast cancer
diagnosis
analysis
epcam
panel
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PCT/BR2021/050561
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French (fr)
Portuguese (pt)
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Paula Philbert Lajolo Canto
Donizeti William Santos
Yara Cristina de Paiva MAIA
Alinne Tatiane Faria Silva
Cláudia Mendonça Rodrigues
Juliana Carvalho Penha Pereira
Luiz Ricardo Goulart Filho
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Universidade Federal de Uberlândia
Imunoscan Engenharia Molecular Ltda
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Priority to PCT/BR2021/050561 priority Critical patent/WO2023115171A1/en
Publication of WO2023115171A1 publication Critical patent/WO2023115171A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • This patent application refers to a method of diagnosing and monitoring the treatment of patients with breast cancer, more particularly in which the method comprises the detection of subpopulations of circulating tumor cells (CTCs), based on the presence or absence of proteins analyzed by flow cytometry in peripheral blood to be used as biomarkers for breast cancer (BC).
  • CTCs circulating tumor cells
  • the present patent application refers to a panel of markers and a kit for rapid diagnostic testing of breast cancer and rapid monitoring of the treatment of patients with breast cancer.
  • the present patent application describes a method for detecting subpopulations of CTCs based on (i) the absence of CD45 and (ii) the presence of vimentin, CD44 and EpCAM.
  • the method, marker panel and detection kit based on the result of CTC analysis by flow cytometry has the potential to be used as a tool complementary to the clinical diagnosis, and can also be used to monitor the treatment. It should also be noted that the methodology proposed here comprises a fast and non-invasive technique.
  • Carcinogenesis is characterized as a multi-step process driven by changes both at the genotype and phenotype levels, and the uncontrolled growth of cells that have undergone such changes is defined as cancer (BARTOSCH et al., 2017; WEIGELT et al. ., 2014).
  • Metastatic progression is an undetermined sequence of events that involves migration of cells across the basement membrane and surrounding tissue, displacement through the bloodstream to distant sites, readaptation to the new tissue environment, and ultimately the formation of clinically detectable metastases. & MASSAGUE, 2013).
  • evidence suggests that tumor cells undergo the transition from an epithelial to mesenchymal phenotype, which will trigger the release and dissemination of these cells into the bloodstream (WERNER et al., 2017).
  • Tissue biopsy is used for diagnosis and provides histological information and allows establishing the immunohistochemical profile of hormone receptors and HER-2 receptor status.
  • it has limitations and can provide information from just one moment and only from the collected tumor tissue (TAY & TAN, 2019).
  • tumors are heterogeneous and can still undergo changes according to the type of treatment performed (GERLINGER et al. al., 2012).
  • CTCs to complement or replace tumor tissue biopsies for diagnosis and therapy makes such a methodology relevant and applicable as "real-time liquid biopsies" (PANTEL & ALIX-PANABIERES, 2010; BARDELLI & PANTEL, 2017).
  • Liquid biopsy can fill both the lack of data in relation to different times and still detect the inherent heterogeneity of the tumor tissue.
  • EDA Food and Drug Administration
  • CellSearch The only method to perform liquid biopsy approved by the Food and Drug Administration (EDA) is CellSearch. This method is based on the expression of epithelial cell adhesion molecules (EpCAM) for selection of CTCs, followed by characterization of selected cells as: i) negative for CD45 and ii) positive for cytokeratins.
  • EpCAM epithelial cell adhesion molecules
  • studies have shown that this technique does not adequately capture CTCs with low EpCAM expression.
  • CTCs that undergo the process of epithelial-mesenchymal transition (EMT) lose their epithelial antigens and are not detected by CellSearch.
  • EMT epithelial-mesenchymal transition
  • these EpCAM-negative CTCs appear to be a highly aggressive and invasive subtype (PUNNOOSE et al., 2010; GEORGES et al., 2012).
  • JP2018079327A, TW201920928A and US20200055048A1 use a device (microfluid chip or microfilters) to capture and/or detect CTCs. Although they also use antibodies, but immobilized on the microchip or filter, there is a need to use post-capture techniques to characterize the target cells, such as immunohistochemistry, flow cytometry, in situ hybridization, analysis of DNA mutations and immunofluorescence .
  • Patent document US20190107542A1 provides methods to detect and isolate CTCs by antibodies linked to magnetic beads and subsequent characterization of the gene expression of these cells.
  • Patent documents EP2948776B, CA2798250A1 and US20140154799A1 select CTCs based on a set of antibodies such as EpCAM, CD45, CD44, CD14, CD24, CD34, Cytokeratins like 8, 18 and 19, EGFR (epidermal growth factor receptor) , ALDH1 (aldehyde dehydrogenase 1), vimentin, among others, and then put them in culture for enrichment, with the main objective of selecting drugs for patient therapy, in addition to being able to characterize the CTCs in culture by imaging.
  • the patent document AU2016228165A1 provides methods to obtain CTCs and the possibility of analyzing a single cell. This identification is made by immunohistochemistry. with aspiration of cells by pipetting and subsequent morphological, genomic, epigenetic or proteomic analysis. Uses epithelial and mesenchymal markers.
  • the patent document JP2006509185A provides a method to detect and discriminate changes in the expression levels of molecular markers, using, among other markers, CD44.
  • the patent document WO2017180499A2 uses a method to capture CTCs from biological fluids that comprises the contact of the biological material with the lectin trapped on the magnetic surface. The separation takes place in a magnetic apparatus. The analysis of CTCs occurs a posteriori by techniques such as immunohistochemistry, genomics and metabolomics, for example. Similarly, patent document CN107428837A uses a method for enrichment and detection of CTCs based on anti-TROP2 antibody, however such analysis is valid only for tumors positive for this marker. Further analyzes include immunofluorescence, FISH and RT-PCR.
  • the patent document US20170038389A1 uses a method of detection of CTCs aiming at their differentiation from circulating endothelial cells and confirms that vimentin is expressed in these last cells, but when analyzed together with other markers (CD45 and CD144) it is possible to separate the populations.
  • the patent document CN108588018A uses an erythrocyte membrane to capture CTCs, being a more effective capture than the available methods .
  • the patent document EP3160654A2 identifies nucleic acids of CTCs using the combination of PCR and FACS (PCR-activated selection), the primers can be EpCAM, CD44 and vimentin, among others.
  • the patent document CN106868101A uses similar markers for detection of CTCs by probe and in situ hybridization, but there were no analyzes of this method for monitoring treatment or diagnosis.
  • Patent documents JP2017207516A, US20130129681A1 and EP2529223B1 use epithelial and mesenchymal markers and detection by flow cytometry, but use enrichment methods such as initial steps, magnetic beads, RosetteSep and CellSearch, respectively.
  • This patent application aimed to solve a problem in the state of the art by developing a method, panel of markers and kit for the diagnosis and monitoring of the treatment of patients with breast cancer, sensitive, specific and simple that can be implemented in the clinical routine in an optimized way.
  • FIGURE 1 a shows the percentage of mesenchymal CTCs. Mann-Whitney test with *p ⁇ 0.05;
  • FIGURE 1 b shows the percentage of intermediate CTCs. Mann-Whitney test with *p ⁇ 0.05;
  • FIGURE 1 c shows the percentage of epithelial CTCs. Mann-Whitney test with *p ⁇ 0.05.
  • the inventors of this patent application through experiments using biological samples of peripheral blood and tissues from patients with benign breast disease or breast cancer submitted or not to chemotherapy, were able to identify that the expression of only four specific markers would be useful in the development of a method, panel of markers and diagnostic and monitoring kit for simplified breast cancer treatment. More particularly, the analysis of the referred markers within the developed method is capable of indicating, through a cut-off defined specifically for the separation of groups, whether the patient has: benign breast disease (BMD), breast cancer not treated by chemotherapy ( BC without CT) or breast cancer with chemotherapy treatment (CM with CT).
  • BMD benign breast disease
  • BC without CT breast cancer not treated by chemotherapy
  • CM with CT breast cancer with chemotherapy treatment
  • the panel of markers proposed by the present invention encompasses the detection of: CD45, a marker for negative selection, defined as a pan-leukocyte marker; Vimentin, a typical marker of the mesenchymal phenotype and with studies demonstrating that higher levels of this protein are associated with greater invasive power of tumor cells in vitro and, consequently, its silencing reduces the formation of metastases (ZELENKO et al., 2017); CD44, established marker for stem cells, being an adhesion molecule, related to cell-to-cell and cell-matrix interactions (KLINGREIL et al., 2010).
  • CD44 promotes the invasion and migration of CTCs (SENBANJO et al., 2017); EpCAM, a transmembrane glycoprotein, which has an adhesion function and is related to cell signaling, proliferation, differentiation and migration (OSTA et al., 2004).
  • the present patent application proposes the use of a panel of markers, comprising labeled antibodies anti-CD45 (for negative selection), anti-CD44, anti-vimentin and anti-EpCAM.
  • This panel of markers is capable of identifying subpopulations of breast cancer (BC) CTCs, selected from biological samples, such as peripheral blood, from patients with BC or DBM through a simplified and error-free diagnostic method. enrichment step using flow cytometry.
  • the analysis of the expression of the selected markers allows the identification and separation of populations of CTCs with mesenchymal (CD45-, EpCAM-, CD44+ and vimentin+), intermediate (CD45-, EpCAM+, CD44+ and vimentin+) and epithelial profile (CD45-, EpCAM+, CD44- and vimentin-).
  • mesenchymal CD45-, EpCAM-, CD44+ and vimentin+
  • intermediate CD45-, EpCAM+, CD44+ and vimentin+
  • epithelial profile CD45-, EpCAM+, CD44- and vimentin-
  • the present invention proposes the use of a simplified method for detecting populations of CTCs, exempt from the need for a previous enrichment step of the CTCs, based on: (i) the selection of CTCs absent from CD45 (CD45-) and (ii) in the identification of the profile of CTCs through the presence or absence of vimentin, CD44 and EpCAM, the identification of these populations composes a simplified panel for diagnosis and treatment monitoring.
  • the present patent application proposes more particularly the use of a diagnostic method and monitoring of breast cancer treatment, based on the detection, identification and analysis of populations of CTCs, preferably by flow cytometry, which comprises the steps of: obtaining a biological sample, preferably blood, more preferably peripheral blood;
  • CD45- selection of CTCs absent from CD45
  • the step of identifying the profiles of the CTCs includes identifying the profiles:
  • mesenchymal CD45-, EpCAM-, CD44+ and vimentin4.
  • the CTC profile analysis stage comprises the use of defined cut offs in relation to the percentage of CTC populations in each of the identified profiles, for the diagnosis of benign disease (BMD) or breast cancer (CM), or monitoring of breast cancer with chemotherapy (CM with CT) and breast cancer without chemotherapy (CM without CT).
  • BMD benign disease
  • CM breast cancer
  • CM with CT breast cancer without chemotherapy
  • the present patent application further describes a panel of markers for the diagnosis and monitoring of the treatment of patients with breast cancer comprising fluorochrome-labeled anti-CD45 antibody, fluorochrome-labeled anti-CD44 antibody, fluorochrome-labelled antibody anti-vimentin and anti-EpCAM fluorochrome-labelled antibody for detection of CTC populations, where the panel allows the identification of CTC profiles:
  • mesenchymal CD45-, EpCAM-, CD44+ and vimentin+
  • epithelial CD45-, EpCAM+, CD44- and vimentin-).
  • the Panel further comprises a diagnostic panel and a monitoring panel based on the analysis of the CTC profiles, in which the diagnostic panel comprises:
  • the monitoring panel comprises:
  • This patent application further describes a kit for the diagnosis and monitoring of breast cancer treatment comprising, combined or separated in one or more containers: antibody labeled with an anti-CD45 fluorochrome, antibody labeled with a fluorochrome anti-CD44, anti-vimentin fluorochrome-labeled antibody, and anti-EpCAM fluorochrome-labeled antibody; in which it still understands instructions for use.
  • Said kit may further comprise, in another embodiment, said panel of markers described in the present patent application, as well as may comprise instructions for use in said method described in the present patent application.
  • This example refers to the characteristics of the study subjects, who are grouped into patients with breast cancer who have or have not undergone chemotherapy (CM with CT or CM without CT group), patients with benign breast disease (CM group DBM).
  • Table 1 presents the clinical, hormonal, diagnostic and therapeutic characteristics of patients with breast cancer.
  • Table 1 presents the clinical, hormonal, diagnostic and therapeutic characteristics of patients with breast cancer.
  • This example refers to the RT-qPCR analysis of the transcriptional levels of the breast cancer and benign breast disease groups.
  • Table 2 shows the comparison of transcriptional levels between breast cancer (CM) and benign breast disease (BMD).
  • Table 3 shows the comparison of levels between breast cancer with chemotherapy (CM with CT), breast cancer without chemotherapy (CM without CT) and benign breast disease (BMD).
  • FIG. 1 shows the percentages of CTCs with mesenchymal, intermediate and epithelial profile in the blood of patients with benign breast disease (BMD), breast cancer with chemotherapy (CM with CT) and breast cancer without chemotherapy (CM without CT).
  • Trizol reagent Invitrogen, Life Technologies, Carlsbad, CA, USA
  • the solution was incubated in a PTC-100 thermocycler (MJ Research) at 37°C for 1 hour and heated to 95°C for 5 minutes. Control reactions (without RNA) were performed to verify possible exogenous contaminants.
  • the cDNA was stored at -20°C for further amplification.
  • Ep-CAM, MMP-2, MMP-9, N-cadherin and vimentin) in relation to the endogenous gene (B2M) were estimated using real-time PCR from the cDNA obtained.
  • the samples were amplified in duplicate and the detection was based on the fluorescence emission of the SYBRGreen dye according to the use of the Master Mix SYBR®Green PCR Core Reagents kit (Applied Biosystems).
  • the monolayer was incubated with AB serum to block the FC portions, preventing unspecific binding, and then labeled with fluorochrome-labeled monoclonal antibodies anti-CD45 (304008, PE) (Biolegend, San Diego, CA, USA), anti-CD44 (338816 , PE/Cy7) (Biolegend, San Diego, CA, USA) and anti-EpCAM (324208, APC) (Biolegend, San Diego, CA, USA). Then, erythrocytes were lysed with BD lysis solution FACS (Becton, Dickinson, and Company (BD), Franklin Lakes
  • washing solution (1x Phosphate Buffer Saline, 1% Bolvin Serum Albumin and 0.1% Sodium Azide).
  • Cells were resuspended in 150pL of washing solution.
  • BD FACS permeabilization solution Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA).
  • the Kolmogorov-Smirnov normality test was performed. Depending on the behavior of the variables, parametric tests were performed for variables with normal distribution or non-parametric tests for variables without normal distribution. To compare the expression (mRNA and protein) of the target genes between the established groups the Mann-Whitney test or t test for independent samples was used. The 95% confidence interval was considered and p values ⁇ 0.05 were considered significant. Statistical analyzes were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) and SPSS version 21.0 (SPSS, IBM, Chicago, IL, USA).
  • KRUSPE S.
  • DICKEY D.D.
  • URAK K. T. et al. Rapid and sensitive detection of breast cancer cells in patient blood with nuclease-activated probe technology. Mol Ther Nucleic Acids., v.8, p.542-557, 2017.
  • KLINGBEIL P.; NATRAJAN. R.; EVERITT, G.; VATCHEVA, R.; MARCHIO, C.; PALACIOS, J.; BUERGER, H.; REIS-FILHO, JS;
  • ISACKE, C.M. CD44 is overexpressed in basal-like breast cancers but is not a driver of llplS amplification.

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Abstract

The invention comprises a method for detecting subpopulations of CTCs based on (i) the absence of CD45 and (ii) the presence of vimentin, CD44 and EpCAM analysed by flow cytometry in biological samples, for example peripheral blood, to be used as biomarkers for breast cancer (BC). These markers were analysed without a prior step of CTC enrichment, optimising the process. Furthermore, this invention relates to a panel of markers and a kit for rapid diagnostic testing of breast cancer and rapid treatment monitoring of breast cancer patients, having the potential to be used as a complementary tool to clinical diagnosis and treatment monitoring.

Description

Relatório descritivo para a patente de invenção: "MÉTODO,Descriptive report for the invention patent: "METHOD,
PAINEL E KIT PARA O DIAGNÓSTICO E MONITORAMENTO DO TRATAMENTO DE PACIENTES COM CÂNCER DE MAMA". PANEL AND KIT FOR THE DIAGNOSIS AND MONITORING OF THE TREATMENT OF PATIENTS WITH BREAST CANCER".
Campo da Invenção Field of Invention
[0001] O presente pedido de patente refere-se à um método de diagnóstico e monitoramento do tratamento de pacientes com câncer de mama, mais particularmente em que o método compreende a detecção de subpopulações de células tumorais circulantes (CTCs), baseado na presença ou ausência de proteínas analisadas por meio de citometria de fluxo no sangue periférico para serem utilizadas como biomarcadores para o câncer de mama (CM). Além disso, o presente pedido de patente refere-se à um painel de marcadores e um kit para teste diagnóstico rápido de câncer de mama e monitoramento rápido do tratamento de pacientes com câncer de mama. Especificamente, o presente pedido de patente descreve um método para detecção de subpopulações de CTCs baseado (i) na ausência de CD45 e (ii) na presença de vimentina, CD44 e EpCAM. A análise desses marcadores foi realizada sem enriquecimento prévio das CTCs, otimizando o processo. Sendo assim, o método, painel de marcadores e kit para detecção baseado no resultado da análise de CTCs por citometria de fluxo, tem o potencial de ser utilizado como ferramenta complementar ao diagnóstico clinico, podendo também ser utilizado para monitoramento do tratamento. Ressalta-se ainda que a metodologia aqui proposta compreende uma técnica rápida e não invasiva. [0001] This patent application refers to a method of diagnosing and monitoring the treatment of patients with breast cancer, more particularly in which the method comprises the detection of subpopulations of circulating tumor cells (CTCs), based on the presence or absence of proteins analyzed by flow cytometry in peripheral blood to be used as biomarkers for breast cancer (BC). In addition, the present patent application refers to a panel of markers and a kit for rapid diagnostic testing of breast cancer and rapid monitoring of the treatment of patients with breast cancer. Specifically, the present patent application describes a method for detecting subpopulations of CTCs based on (i) the absence of CD45 and (ii) the presence of vimentin, CD44 and EpCAM. The analysis of these markers was performed without prior enrichment of the CTCs, optimizing the process. Therefore, the method, marker panel and detection kit based on the result of CTC analysis by flow cytometry, has the potential to be used as a tool complementary to the clinical diagnosis, and can also be used to monitor the treatment. It should also be noted that the methodology proposed here comprises a fast and non-invasive technique.
Estado da Técnica State of the art
[0002] O câncer de mama é o tipo de câncer mais incidente entre as mulheres. Na última estimativa mundial, publicada em 2018, houve 18 milhões de novos casos de câncer e destes 11,6% foram de mama (BRAY et al.,2018). No Brasil, a estimativa para cada ano do triénio 2020-2022 é de 625 mil novos casos de câncer, sendo que o câncer de mama representará 29,7% dos cânceres em mulheres (INCA, 2019). [0002] Breast cancer is the most incident type of cancer among women. In the last world estimate, published in 2018, there were 18 million new cases of cancer and of these 11.6% were breast cancer (BRAY et al., 2018). In Brazil, the estimate for each year of the 2020-2022 period is 625,000 new cases of cancer, with breast cancer accounting for 29.7% of cancers in women (INCA, 2019).
[0003] A carcinogênese caracteriza-se como um processo de múltiplas etapas orientadas por alterações tanto em nivel de genótipo quanto de fenótipo e o crescimento descontrolado das células que sofreram tais alterações é definido como câncer (BARTOSCH et al., 2017; WEIGELT et al., 2014). A progressão metastática é uma sequência de eventos não determinados, que envolve a migração das células por meio da membrana basal e tecido circundante, deslocamento pela corrente sanguínea para locais distantes, readaptação ao novo ambiente tecidual e por fim a formação de metástases clinicamente detectáveis (VANHARANTA & MASSAGUE, 2013). Para que as primeiras etapas ocorram, evidências sugerem que as células tumorais sofrem a transição de um fenótipo epitelial para mesenquimal, o que desencadeará a liberação e disseminação destas células na corrente sanguinea (WERNER et al., 2017). [0003] Carcinogenesis is characterized as a multi-step process driven by changes both at the genotype and phenotype levels, and the uncontrolled growth of cells that have undergone such changes is defined as cancer (BARTOSCH et al., 2017; WEIGELT et al. ., 2014). Metastatic progression is an undetermined sequence of events that involves migration of cells across the basement membrane and surrounding tissue, displacement through the bloodstream to distant sites, readaptation to the new tissue environment, and ultimately the formation of clinically detectable metastases. & MASSAGUE, 2013). For Before the first steps occur, evidence suggests that tumor cells undergo the transition from an epithelial to mesenchymal phenotype, which will trigger the release and dissemination of these cells into the bloodstream (WERNER et al., 2017).
[0004] As mortes em decorrência do câncer são consequência, em sua maioria, das metástases em órgãos e tecidos distantes (HANAHAN & WEINBERG, 2000). O padrão ouro para avaliar a recidiva do câncer tem sido os mesmos testes utilizados para o diagnóstico do tumor, quando possivel a coleta do material. As técnicas de histopatologia, mamografia, ultrassonografia e imageamento por ressonância magnética são as análises mais utilizadas na rotina clinica, porém são demoradas e exigem protocolos rigidos (TRIA et al., 2013). [0004] Deaths due to cancer are a consequence, for the most part, of metastases in distant organs and tissues (HANAHAN & WEINBERG, 2000). The gold standard for evaluating cancer recurrence has been the same tests used for tumor diagnosis, when possible, the collection of material. Histopathology, mammography, ultrasonography and magnetic resonance imaging techniques are the most used analyzes in the clinical routine, but they are time-consuming and require strict protocols (TRIA et al., 2013).
[0005] A biópsia do tecido é utilizada para o diagnóstico e fornece informações histológicas e permite estabelecer o perfil imuno-histoquimico dos receptores hormonais e do status do receptor HER-2. Porém, possui limitações e pode fornecer informações de apenas um momento e apenas do tecido tumoral coletado (TAY & TAN, 2019). Mas, os tumores são heterogêneos e ainda podem sofrer alterações de acordo com o tipo de tratamento realizado (GERLINGER et al., 2012). A possibilidade de utilizar CTCs para complementar ou substituir biópsias de tecidos tumorais para o diagnóstico e terapêutica torna tal metodologia relevante e aplicável como "biópsias liquidas em tempo real" (PANTEL & ALIX-PANABIERES, 2010; BARDELLI & PANTEL, 2017). A biópsia liquida pode suprir tanto a carência de dados em relação a diferentes tempos e ainda detectar a inerente heterogeneidade do tecido tumoral. [0005] Tissue biopsy is used for diagnosis and provides histological information and allows establishing the immunohistochemical profile of hormone receptors and HER-2 receptor status. However, it has limitations and can provide information from just one moment and only from the collected tumor tissue (TAY & TAN, 2019). However, tumors are heterogeneous and can still undergo changes according to the type of treatment performed (GERLINGER et al. al., 2012). The possibility of using CTCs to complement or replace tumor tissue biopsies for diagnosis and therapy makes such a methodology relevant and applicable as "real-time liquid biopsies" (PANTEL & ALIX-PANABIERES, 2010; BARDELLI & PANTEL, 2017). Liquid biopsy can fill both the lack of data in relation to different times and still detect the inherent heterogeneity of the tumor tissue.
[0006] O único método para realizar a biópsia liquida aprovado pelo Food and Drug Administration (EDA) é o CellSearch. Este método se baseia na expressão de moléculas de adesão celular epitelial (EpCAM), para seleção das CTCs, seguido por caracterização das células selecionadas em: i) negativo para CD45 e ii) positivo para citoqueratinas. Porém, estudos demonstraram que esta técnica não captura CTCs com baixa expressão de EpCAM adequadamente. Assim, CTCs que sofrem o processo de transição epitélio-mesenquimal (EMT), perdem seus antigenos epiteliais e não são detectadas pelo CellSearch. Além disso, essas CTCs negativas para EpCAM parecem ser um subtipo altamente agressivo e invasivo (PUNNOOSE et al., 2010; GEORGES et al., 2012). [0006] The only method to perform liquid biopsy approved by the Food and Drug Administration (EDA) is CellSearch. This method is based on the expression of epithelial cell adhesion molecules (EpCAM) for selection of CTCs, followed by characterization of selected cells as: i) negative for CD45 and ii) positive for cytokeratins. However, studies have shown that this technique does not adequately capture CTCs with low EpCAM expression. Thus, CTCs that undergo the process of epithelial-mesenchymal transition (EMT), lose their epithelial antigens and are not detected by CellSearch. Furthermore, these EpCAM-negative CTCs appear to be a highly aggressive and invasive subtype (PUNNOOSE et al., 2010; GEORGES et al., 2012).
[0007] Diante desse quadro, fica evidente a necessidade de novos métodos para a detecção de CTCs, que possibilitem não somente a identificação de CTCs com diferentes fenótipos, mas também que seja exequivel e que não requeira muitos processos até a obtenção do resultado final. A presente invenção busca atender essas exigências. [0007] Given this situation, the need for new methods for detecting CTCs is evident, which allow not only the identification of CTCs with different phenotypes, but also that it is feasible and does not require many processes until obtaining the final result. The present invention seeks to meet these requirements.
[0008] Apesar das limitações do CellSearch, vários estudos que utilizaram essa técnica, comprovaram a relevância da análise de CTCs para estabelecimento do prognóstico. Pacientes com câncer de mama e com valores de ≥ 5 CTCs por 7.5 mL de sangue total em comparação com aqueles com valores de < 5 CTCs apresentaram significativamente menor sobrevida livre de progressão e sobrevida global (CRISTOFANILLI et al.,2004; BUDD et al., 2006; PIERGA et al.,2012; ROSSI et al., 2018). [0008] Despite the limitations of CellSearch, several studies that used this technique, proved the relevance of the analysis of CTCs for establishing the prognosis. Patients with breast cancer with values of ≥ 5 CTCs per 7.5 mL of whole blood compared to those with values of < 5 CTCs had significantly lower progression-free survival and overall survival (CRISTOFANILLI et al.,2004; BUDD et al. , 2006; PIERGA et al., 2012; ROSSI et al., 2018).
[0009] Para o diagnóstico do câncer de mama, a utilização de CTCs ainda precisa ser melhor investigada pois evidencia-se a existência de resultados muito pouco significativos. Porém, sondas ativadas por nucleases derivadas de CTCs foram capazes de discriminar pacientes com câncer de mama metastático de controles saudáveis (KRUSPE et al., 2017). [0009] For the diagnosis of breast cancer, the use of CTCs still needs to be better investigated as it shows the existence of very insignificant results. However, probes activated by CTC-derived nucleases were able to discriminate patients with metastatic breast cancer from healthy controls (KRUSPE et al., 2017).
[00010] Os documentos de patente US9671407B2, EP283121, CN105518464B, WO201600431IA, CN104781007B,[00010] Patent documents US9671407B2, EP283121, CN105518464B, WO201600431IA, CN104781007B,
JP2018079327A, TW201920928A e US20200055048A1 utilizam um dispositivo (chip microfluido ou microfiltros) para capturar e/ou detectar CTCs. Apesar de também utilizarem anticorpos, porém imobilizados no microchip ou filtro, verifica-se a necessidade da utilização de técnicas após a captura para caracterização das células alvo, tais como imunohistoquimica, citometria de fluxo, hibridização in situ, análise de mutações no DNA e imunofluorescência. JP2018079327A, TW201920928A and US20200055048A1 use a device (microfluid chip or microfilters) to capture and/or detect CTCs. Although they also use antibodies, but immobilized on the microchip or filter, there is a need to use post-capture techniques to characterize the target cells, such as immunohistochemistry, flow cytometry, in situ hybridization, analysis of DNA mutations and immunofluorescence .
[00011] O documento de patente US20190107542A1 fornece métodos para detectar e isolar CTCs por anticorpos ligados a esferas magnéticas e posterior caracterização da expressão gênica destas células. Os documentos de patentes EP2948776B, CA2798250A1 e US20140154799A1 selecionam CTCs com base em um conjunto de anticorpos, tais como EpCAM, CD45, CD44, CD14, CD24, CD34, Citoqueratinas como 8, 18 e 19, EGFR (receptor do fator de crescimento epidérmico) , ALDH1 (aldeidesidrogenase 1), vimentina, dentro outros, e em seguida as coloca em cultura para enriquecimento, com o objetivo principal de selecionar fármacos para a terapia dos pacientes, além de poder caracterizar as CTCs em cultura por imagem. [00011] The patent document US20190107542A1 provides methods to detect and isolate CTCs by antibodies linked to magnetic beads and subsequent characterization of the gene expression of these cells. Patent documents EP2948776B, CA2798250A1 and US20140154799A1 select CTCs based on a set of antibodies such as EpCAM, CD45, CD44, CD14, CD24, CD34, Cytokeratins like 8, 18 and 19, EGFR (epidermal growth factor receptor) , ALDH1 (aldehyde dehydrogenase 1), vimentin, among others, and then put them in culture for enrichment, with the main objective of selecting drugs for patient therapy, in addition to being able to characterize the CTCs in culture by imaging.
[00012] O documento de patente AU2016228165A1 fornece métodos para obter CTCs e a possibilidade de analisar uma única célula. Essa identificação se dá por imunohistoquimica com a aspiração das células por pipetagem e posterior análise morfológica, genômica, epigenética ou proteômica. Utiliza marcadores epiteliais e mesenquimais. Da mesma forma, o documento de patente JP2006509185A fornece um método para detectar e descriminar mudanças nos niveis de expressão de marcadores moleculares, utiliza, entre outros marcadores, o CD44. [00012] The patent document AU2016228165A1 provides methods to obtain CTCs and the possibility of analyzing a single cell. This identification is made by immunohistochemistry. with aspiration of cells by pipetting and subsequent morphological, genomic, epigenetic or proteomic analysis. Uses epithelial and mesenchymal markers. Likewise, the patent document JP2006509185A provides a method to detect and discriminate changes in the expression levels of molecular markers, using, among other markers, CD44.
[00013] O documento de patente WO2017180499A2 utiliza um método para capturar CTCs de fluidos biológicos que compreende o contato do material biológico com a lectina presa na superfície magnética. A separação ocorre em um aparato magnético. A análise das CTCs ocorre a posteriori por técnicas como imunhistoquimica, genômica e metabolômica, por exemplo. De uma maneira semelhante, o documento de patente CN107428837A utiliza um método para enriquecimento e detecção de CTCs baseado em anticorpo anti-TROP2, porém tal análise é valida apenas para tumores positivos para esse marcador. Análises posteriores incluem imunofluorescência, FISH e RT-PCR. [00013] The patent document WO2017180499A2 uses a method to capture CTCs from biological fluids that comprises the contact of the biological material with the lectin trapped on the magnetic surface. The separation takes place in a magnetic apparatus. The analysis of CTCs occurs a posteriori by techniques such as immunohistochemistry, genomics and metabolomics, for example. Similarly, patent document CN107428837A uses a method for enrichment and detection of CTCs based on anti-TROP2 antibody, however such analysis is valid only for tumors positive for this marker. Further analyzes include immunofluorescence, FISH and RT-PCR.
[00014] O documento de patente US20170038389A1 utiliza um método de detecção de CTCs visando sua diferenciação de células endoteliais circulantes e confirma que a vimentina é expressa nestas últimas células, mas quando analisada em conjunto com outros marcadores (CD45 e CD144) é possível separar as populações. O documento de patente CN108588018A utiliza uma membrana de eritrócitos para capturar CTCs, sendo uma captura mais efetiva que os métodos disponíveis . [00014] The patent document US20170038389A1 uses a method of detection of CTCs aiming at their differentiation from circulating endothelial cells and confirms that vimentin is expressed in these last cells, but when analyzed together with other markers (CD45 and CD144) it is possible to separate the populations. The patent document CN108588018A uses an erythrocyte membrane to capture CTCs, being a more effective capture than the available methods .
[00015] O documento de patente EP3160654A2 identifica ácidos nucleicos de CTCs utilizando a combinação de PCR e FACS (seleção ativada por PCR), os primers podem ser de EpCAM, CD44 e vimentina, entre outros. Em outra abordagem, o documento de patente CN106868101A utiliza marcadores semelhantes para detecção de CTCs por sonda e hibridização in situ, mas não houveram análises deste método para o monitoramento do tratamento ou diagnóstico. [00015] The patent document EP3160654A2 identifies nucleic acids of CTCs using the combination of PCR and FACS (PCR-activated selection), the primers can be EpCAM, CD44 and vimentin, among others. In another approach, the patent document CN106868101A uses similar markers for detection of CTCs by probe and in situ hybridization, but there were no analyzes of this method for monitoring treatment or diagnosis.
[00016] Os documentos de patente JP2017207516A, US20130129681A1 e EP2529223B1 utilizam marcadores epiteliais e mesenquimais e detecção por citometria de fluxo, mas utilizam métodos de enriquecimento como passos iniciais, esferas magnéticas, RosetteSep e CellSearch, respectivamente . [00016] Patent documents JP2017207516A, US20130129681A1 and EP2529223B1 use epithelial and mesenchymal markers and detection by flow cytometry, but use enrichment methods such as initial steps, magnetic beads, RosetteSep and CellSearch, respectively.
[00017] Nenhum dos ensinamentos do estado da técnica demonstram ou sugerem um método que utilize da combinação dos marcadores: vimentina, CD44, CD45 e EpCAM, nem da detecção destes 4 marcadores utilizando a técnica de citometria de fluxo, e nem muito menos, que tal método permitisse a análise de diferentes populações de CTCs sem a necessidade de uma etapa de inicial de enriquecimento. O método proposto pelo presente pedido de patente se mostrou eficaz na detecção direta de diferentes populações de CTCs em amostras biológicas, por exemplo, amostras de sangue periférico. Proporcionando então o desenvolvimento de um método, painel de marcadores e kit para o diagnóstico do câncer de mama e monitoramento do tratamento, sendo que a técnica utilizada e descrita no presente pedido de patente fornece uma ferramenta simples e poderosa para analisar amostras biológicas com pouca preparação, permitindo que a amostra seja analisada diretamente, sem a necessidade de etapas adicionais de enriquecimento das CTCs. Além disso, o método , painel e kit aqui descritos facilitam ainda mais a utilização na rotina clinica, produzindo um ponto de inflexão (cut off) e informando o diagnóstico positivo ou negativo para o câncer de mama e a possibilidade de acompanhamento do perfil destas células durante o tratamento. [00017] None of the teachings of the state of the art demonstrate or suggest a method that uses the combination of markers: vimentin, CD44, CD45 and EpCAM, nor the detection of these 4 markers using the technique of flow cytometry, and not least that such a method would allow the analysis of different populations of CTCs without the need for an initial enrichment step. The method proposed by the present patent application proved to be effective in the direct detection of different populations of CTCs in biological samples, for example, peripheral blood samples. Providing then the development of a method, panel of markers and kit for the diagnosis of breast cancer and monitoring of the treatment, and the technique used and described in the present patent application provides a simple and powerful tool to analyze biological samples with little preparation , allowing the sample to be analyzed directly, without the need for additional CTC enrichment steps. In addition, the method, panel and kit described here facilitate even more use in the clinical routine, producing a point of inflection (cut off) and informing the positive or negative diagnosis for breast cancer and the possibility of monitoring the profile of these cells during treatment.
[00018] Dessa forma, apesar dos documentos do estado da técnica revelarem à busca pela captura e detecção de CTCs no diagnóstico de câncer, há ainda a necessidade de um método de diagnóstico sensível, especifico e simples que possa ser implementado na rotina clinica, para o câncer de mama. [00018] Thus, despite the state of the art documents revealing the search for the capture and detection of CTCs in the diagnosis of cancer, there is still a need for a sensitive, specific and simple diagnostic method that can be implemented in the clinical routine for breast cancer.
Objetivo da invenção Purpose of the invention
[00019] O presente pedido de patente teve como objetivo de resolver um problema no estado da técnica desenvolvendo um método, painel de marcadores e kit para o diagnóstico e monitoramento do tratamento de pacientes com câncer de mama, sensível, especifico e simples que possa ser implementado na rotina clinica de forma otimizada. [00019] This patent application aimed to solve a problem in the state of the art by developing a method, panel of markers and kit for the diagnosis and monitoring of the treatment of patients with breast cancer, sensitive, specific and simple that can be implemented in the clinical routine in an optimized way.
Breve descrição das figuras Brief description of figures
[00020] A presente invenção será melhor compreendida com base na descrição, a seguir, tomada em conjunto com as figuras anexas, nas quais: [00020] The present invention will be better understood based on the description, below, taken together with the attached figures, in which:
[00021] A FIGURA 1 a) mostra a porcentagem de CTCs mesenquimal. Teste de Mann-Whitney com *p<0.05; [00021] FIGURE 1 a) shows the percentage of mesenchymal CTCs. Mann-Whitney test with *p<0.05;
[00022] A FIGURA 1 b) mostra a porcentagem de CTCs intermediário. Teste de Mann-Whitney com *p<0.05; [00022] FIGURE 1 b) shows the percentage of intermediate CTCs. Mann-Whitney test with *p<0.05;
[00023] A FIGURA 1 c) mostra a porcentagem de CTCs epitelial. Teste de Mann-Whitney com *p<0.05. [00023] FIGURE 1 c) shows the percentage of epithelial CTCs. Mann-Whitney test with *p<0.05.
Descrição detalhada da invenção Detailed description of the invention
[00024] Os inventores do presente pedido de patente, por meio de experimentos utilizando amostras biológicas de sangue periférico e tecidos de pacientes com doença benigna da mama ou com câncer de mama submetidos ou não à quimioterapia, foram capazes de identificar que a expressão de somente quatro marcadores especificos seria útil no desenvolvimento de um método, painel de marcadores e kit de diagnóstico e monitoramento do tratamento de câncer de mama simplificado. Mais particularmente, a análise dos referidos marcadores dentro do método desenvolvido é capaz de indicar, através de um cut off definido especificamente para separação dos grupos, se o paciente apresenta: doença benigna da mama (DBM), câncer de mama sem tratamento por quimioterapia (CM sem QT) ou câncer de mama com tratamento por quimioterapia (CM com QT). [00024] The inventors of this patent application, through experiments using biological samples of peripheral blood and tissues from patients with benign breast disease or breast cancer submitted or not to chemotherapy, were able to identify that the expression of only four specific markers would be useful in the development of a method, panel of markers and diagnostic and monitoring kit for simplified breast cancer treatment. More particularly, the analysis of the referred markers within the developed method is capable of indicating, through a cut-off defined specifically for the separation of groups, whether the patient has: benign breast disease (BMD), breast cancer not treated by chemotherapy ( BC without CT) or breast cancer with chemotherapy treatment (CM with CT).
[00025] O painel de marcadores propostos pela presente invenção engloba a detecção de: CD45, um marcador para seleção negativa, definido como um marcador pan- leucocitário; Vimentina, marcador tipico do fenótipo mesenquimal e com estudos demonstrando que maiores niveis dessa proteína estão associados ao maior poder de invasão de células tumorais in vitro e consequentemente, o seu silenciamento reduz a formação de metástases (ZELENKO et al., 2017); CD44, marcador estabelecido para células tronco, sendo uma molécula de adesão, relacionada às interações célula a célula e célula-matriz (KLINGREIL et al., 2010).[00025] The panel of markers proposed by the present invention encompasses the detection of: CD45, a marker for negative selection, defined as a pan-leukocyte marker; Vimentin, a typical marker of the mesenchymal phenotype and with studies demonstrating that higher levels of this protein are associated with greater invasive power of tumor cells in vitro and, consequently, its silencing reduces the formation of metastases (ZELENKO et al., 2017); CD44, established marker for stem cells, being an adhesion molecule, related to cell-to-cell and cell-matrix interactions (KLINGREIL et al., 2010).
Por meio das interações anteriormente citadas, o CD44 promove a invasão e migração das CTCs (SENBANJO et al., 2017); EpCAM, uma glicoproteina transmembrana, que possui função de adesão e está relacionada com a sinalização, proliferação, diferenciação e migração celular (OSTA et al.,2004). Through the aforementioned interactions, CD44 promotes the invasion and migration of CTCs (SENBANJO et al., 2017); EpCAM, a transmembrane glycoprotein, which has an adhesion function and is related to cell signaling, proliferation, differentiation and migration (OSTA et al., 2004).
[00026] Frente a estes fatos, o presente pedido de patente propõe a utilização de um painel de marcadores, que compreende anticorpos marcados anti-CD45 (para seleção negativa), anti-CD44, anti-vimentina e anti-EpCAM. Esse painel de marcadores é capaz de, identificar subpopulações de CTCs do câncer de mama (CM), selecionadas a partir de amostras biológicas, como por exemplo sangue periférico, de pacientes com CM ou DBM por meio de um método de diagnóstico simplificado e isento de etapa de enriquecimento, usando a citometria de fluxo. [00026] In view of these facts, the present patent application proposes the use of a panel of markers, comprising labeled antibodies anti-CD45 (for negative selection), anti-CD44, anti-vimentin and anti-EpCAM. This panel of markers is capable of identifying subpopulations of breast cancer (BC) CTCs, selected from biological samples, such as peripheral blood, from patients with BC or DBM through a simplified and error-free diagnostic method. enrichment step using flow cytometry.
[00027] A utilização de um método de detecção baseado na expressão de vimentina, CD44 e EpCAM em CTCs que são CD45 negativas, de amostras biológicas de pacientes com doença benigna da mama ou com câncer de mama submetidos ou não à quimioterapia, demonstrou possibilitar não somente o diagnóstico de CM ou DBM, mas também a possibilidade de monitoramento do tratamento com a identificação dos grupos: câncer de mama sem tratamento por quimioterapia (CM sem QT) e câncer de mama com tratamento por quimioterapia (CM com QT). [00027] The use of a detection method based on the expression of vimentin, CD44 and EpCAM in CTCs that are CD45 negative, of biological samples from patients with benign breast disease or with breast cancer submitted or not to chemotherapy, demonstrated to make possible not only the diagnosis of CM or DBM, but also the possibility of monitoring the treatment with the identification of the groups: breast cancer without chemotherapy treatment (CM without chemotherapy) and breast cancer with chemotherapy treatment (CM with QT).
[00028] Mais particularmente, a análise da expressão dos marcadores selecionados possibilita a identificação e separação das populações de CTCs com perfil mesenquimal (CD45-, EpCAM-, CD44+ e vimentina+), intermediário (CD45-, EpCAM+, CD44+ e vimentina+) e epitelial (CD45-, EpCAM+, CD44- e vimentina-). Tal identificação dos perfis de CTCs a partir da análise da expressão dos referidos marcadores, com a determinação de cut offs específicos em relação a porcentagem de populações de CTCs em cada perfil, mostrou um alto potencial no diagnóstico e monitoramento do tratamento de câncer de mama. [00028] More particularly, the analysis of the expression of the selected markers allows the identification and separation of populations of CTCs with mesenchymal (CD45-, EpCAM-, CD44+ and vimentin+), intermediate (CD45-, EpCAM+, CD44+ and vimentin+) and epithelial profile (CD45-, EpCAM+, CD44- and vimentin-). Such identification of the CTCs profiles from the analysis of the expression of the mentioned markers, with the determination of specific cut offs in relation to the percentage of populations of CTCs in each profile, showed a high potential in the diagnosis and monitoring of the treatment of breast cancer.
[00029] Assim, a presente invenção propõe a utilização de um método simplificado para detecção de populações de CTCs, isento da necessidade de uma etapa de enriquecimento prévio das CTCs, baseado: (i) na seleção de CTCs ausentes de CD45 (CD45-) e (ii) na identificação do perfil das CTCs através da presença ou ausência de vimentina, CD44 e EpCAM, a identificação destas populações compõem um painel simplificado para diagnóstico e monitoramento do tratamento . [00029] Thus, the present invention proposes the use of a simplified method for detecting populations of CTCs, exempt from the need for a previous enrichment step of the CTCs, based on: (i) the selection of CTCs absent from CD45 (CD45-) and (ii) in the identification of the profile of CTCs through the presence or absence of vimentin, CD44 and EpCAM, the identification of these populations composes a simplified panel for diagnosis and treatment monitoring.
[00030] Para o diagnóstico, a análise do perfil mesenquimal para os três grupos (DBM, CM com QT e CM sem QT) apresentou uma sensibilidade de 73% e especificidade de 83% para CM vs DBM e odds ratio de 13.33 (p=0.0443). A análise do perfil intermediário para os três grupos (DBM, CM com QT e CM sem QT) apresentou uma sensibilidade de 100% e especificidade de 83% para CM vs DBM e odds ratio de 84.33 (p=0.0096). A análise do perfil mesenquimal para DBM vs CM sem tratamento, apresentou uma sensibilidade de 83% e especificidade de 100% e odds ratio de 47.6667 (p=0.0257). A análise do perfil epitelial para DBM vs CM sem QT apresentou uma sensibilidade de 100% e especificidade de 83% e odds ratio de 47.6667 (p=0.0257). [00030] For diagnosis, analysis of the mesenchymal profile for the three groups (DBM, CM with CT and CM without CT) showed a sensitivity of 73% and specificity of 83% for CM vs DBM and an odds ratio of 13.33 (p=0.0443). The intermediate profile analysis for the three groups (DBM, CM with CT and CM without CT) showed a sensitivity of 100% and specificity of 83% for CM vs DBM and an odds ratio of 84.33 (p=0.0096). Analysis of the mesenchymal profile for DBM vs CM without treatment showed a sensitivity of 83% and specificity of 100% and an odds ratio of 47.6667 (p=0.0257). The analysis of the epithelial profile for DBM vs CM without CT showed a sensitivity of 100% and specificity of 83% and an odds ratio of 47.6667 (p=0.0257).
[00031] Para o monitoramento do tratamento, a análise do perfil epitelial para os três grupos (DBM, CM com QT e CM sem QT) apresentou uma sensibilidade de 100% e especificidade de 91% para câncer de mama sem QT vs doença benigna da mama e CM com QT, e odds ratio de 91 (p=0.0082). A análise do perfil intermediário para CM sem QT vs CM com QT apresentou uma sensibilidade de 100% e especificidade de 100% e odds ratio de 143 (p=0.00172). A análise do perfil epitelial para CM sem QT vs CM com QT apresentou uma sensibilidade de 100% e especificidade de 100% e odds ratio de 143 (p=0.00172). [00031] For treatment monitoring, the analysis of the epithelial profile for the three groups (DBM, CM with CT and CM without CT) showed a sensitivity of 100% and specificity of 91% for breast cancer without CT vs. breast and BC with CT, and odds ratio of 91 (p=0.0082). The intermediate profile analysis for CM without CT vs CM with CT showed a sensitivity of 100% and specificity of 100% and an odds ratio of 143 (p=0.00172). The analysis of the epithelial profile for CM without CT vs CM with CT showed a sensitivity of 100% and specificity of 100% and an odds ratio of 143 (p=0.00172).
[00032] Assim, o presente pedido de patente propõe mais particularmente a utilização de um método de diagnóstico e monitoramento do tratamento de câncer de mama, baseado na detecção, identificação e análise de populações de CTCs, preferencialmente por citometria de fluxo, em que compreende as etapas de: obtenção da amostra biológica, preferencialmente sangue, mais preferencialmente sangue periférico; [00032] Thus, the present patent application proposes more particularly the use of a diagnostic method and monitoring of breast cancer treatment, based on the detection, identification and analysis of populations of CTCs, preferably by flow cytometry, which comprises the steps of: obtaining a biological sample, preferably blood, more preferably peripheral blood;
- preparação da amostra para receber os anticorpos marcadores, em que a preparação é isenta da necessidade de uma etapa de enriquecimento prévio; - preparation of the sample to receive the marker antibodies, in which the preparation is exempt from the need for a previous enrichment step;
- por em contado a amostra preparada com os anticorpos marcados com fluorocromos anti-CD45, anti-CD44, anti-Epcam e anti-vimentina, - to count the sample prepared with the antibodies labeled with fluorochromes anti-CD45, anti-CD44, anti-Epcam and anti-vimentin,
- seleção de CTCs ausentes de CD45 (CD45-); e - selection of CTCs absent from CD45 (CD45-); It is
- identificação e análise do perfil das CTCs através da presença ou ausência de vimentina, CD44 e EpCAM. - identification and analysis of the profile of CTCs through the presence or absence of vimentin, CD44 and EpCAM.
[00033] A etapa de identificação dos perfis das CTCs compreende a identificação dos perfis: [00033] The step of identifying the profiles of the CTCs includes identifying the profiles:
(i) mesenquimal (CD45-, EpCAM-, CD44+ e vimentina4);(i) mesenchymal (CD45-, EpCAM-, CD44+ and vimentin4);
(ii) intermediária (CD45-, EpCAM4, CD444 e vimentina4); e (ii) intermediate (CD45-, EpCAM4, CD444 and vimentin4); It is
(iii) epitelial (CD45-, EpCAM4, CD44- e vimentina-), por citometria de fluxo. (iii) epithelial (CD45-, EpCAM4, CD44- and vimentin-), by flow cytometry.
[00034] A identificação e análise do perfil das CTCs compõem então um painel de marcadores simples para diagnóstico e monitoramento do tratamento, a etapa de análise dos perfis das CTCs compreende o uso de cut offs definidos em relação a porcentagem de populações de CTCs em cada um dos perfis identificados, para o diagnóstico de doença benigna da mama (DBM) ou câncer de mama (CM), ou monitoramento de câncer de mama com quimioterapia (CM com QT) e câncer de mama sem quimioterapia (CM sem QT). [00034] Identification and analysis of the profile of CTCs then compose a panel of simple markers for diagnosis and treatment monitoring, the CTC profile analysis stage comprises the use of defined cut offs in relation to the percentage of CTC populations in each of the identified profiles, for the diagnosis of benign disease (BMD) or breast cancer (CM), or monitoring of breast cancer with chemotherapy (CM with CT) and breast cancer without chemotherapy (CM without CT).
[00035] O presente pedido de patente descreve ainda um painel de marcadores para o diagnóstico e monitoramento do tratamento de pacientes com câncer de mama em que compreende anticorpo marcado com fluorocromos anti-CD45, anticorpo marcado com fluorocromos anti-CD44, anticorpo marcado com fluorocromos anti-vimentina e anticorpo marcado com fluorocromos anti-EpCAM para detecção de populações de CTCs, em que o painel permite a identificação dos perfis das CTCs: [00035] The present patent application further describes a panel of markers for the diagnosis and monitoring of the treatment of patients with breast cancer comprising fluorochrome-labeled anti-CD45 antibody, fluorochrome-labeled anti-CD44 antibody, fluorochrome-labelled antibody anti-vimentin and anti-EpCAM fluorochrome-labelled antibody for detection of CTC populations, where the panel allows the identification of CTC profiles:
(i) mesenquimal (CD45-, EpCAM-, CD44+ e vimentina+);(i) mesenchymal (CD45-, EpCAM-, CD44+ and vimentin+);
(ii) intermediária (CD45-, EpCAM+, CD44+ e vimentina+); e (ii) intermediate (CD45-, EpCAM+, CD44+ and vimentin+); It is
(iii) epitelial (CD45-, EpCAM+, CD44- e vimentina-). (iii) epithelial (CD45-, EpCAM+, CD44- and vimentin-).
[00036] O Painel ainda compreende um painel de diagnóstico e um painel de monitoramento com base na análise dos perfis das CTCs, em que o painel de diagnóstico compreende : [00036] The Panel further comprises a diagnostic panel and a monitoring panel based on the analysis of the CTC profiles, in which the diagnostic panel comprises:
- a análise do perfil mesenquimal para os três grupos (DBM, CM com QT e CM sem QT), com um cut off acima de 4,5% de células tumorais circulantes mesenquimais indicando diagnóstico de câncer de mama; - analysis of the mesenchymal profile for the three groups (DBM, CM with CT and CM without CT), with a cut off above 4.5% of circulating mesenchymal tumor cells indicating a diagnosis of breast cancer;
- a análise do perfil intermediário para os três grupos (DBM, CM com QT e CM sem QT), com um cut off abaixo de 0,04% de células tumorais circulantes intermediárias indicando diagnóstico de câncer de mama; - analysis of the intermediate profile for the three groups (DBM, CM with CT and CM without CT), with a cut off below 0.04% of intermediate circulating tumor cells indicating a diagnosis of breast cancer;
- a análise do perfil mesenquimal para DBM vs CM sem QT, com um cut off acima de 5,5% de células tumorais circulantes mesenquimais indicando diagnóstico de câncer de mama; e - analysis of the mesenchymal profile for DBM vs CM without CT, with a cut off above 5.5% of circulating mesenchymal tumor cells indicating a diagnosis of breast cancer; It is
- a análise do perfil epitelial para DBM vs CM sem QT, com um cut off abaixo de 0,28% de células tumorais circulantes epiteliais indicando diagnóstico de câncer de mama; e o painel de monitoramento compreende: - analysis of the epithelial profile for DBM vs CM without CT, with a cut off below 0.28% of circulating epithelial tumor cells indicating a diagnosis of breast cancer; and the monitoring panel comprises:
- a análise do perfil epitelial para os três grupos (DBM, CM com QT e CM sem QT), com um cut off abaixo de 0,27% de células tumorais circulantes epiteliais indicando tumor sem tratamento; a análise do perfil intermediário para CM sem QT vs- the analysis of the epithelial profile for the three groups (DBM, CM with CT and CM without CT), with a cut off below 0.27% of epithelial circulating tumor cells indicating tumor without treatment; the analysis of the intermediate profile for CM without QT vs.
CM com QT, com um cut off acima de 0,0004% de células tumorais circulantes intermediárias indicando tumor sem tratamento; e CM with CT, with a cut off above 0.0004% of intermediate circulating tumor cells indicating untreated tumor; It is
- a análise do perfil epitelial para CM sem QT vs CM com QT, com um cut off abaixo de 0,6% de células tumorais circulantes epiteliais indicando tumor sem tratamento. - the analysis of the epithelial profile for CM without CT vs CM with CT, with a cut off below 0.6% of circulating epithelial tumor cells indicating untreated tumor.
[00037] O presente pedido de patente descreve ainda um kit para o diagnóstico e monitoramento do tratamento de câncer de mama em que compreende, combinados ou separados em um ou mais recipientes: anticorpo marcado com um fluorocromo anti-CD45, anticorpo marcado com um fluorocromo anti-CD44, anticorpo marcado com fluorocromo anti-vimentina e anticorpo marcado com fluorocromo anti-EpCAM; em que ainda compreende instruções de uso. [00037] This patent application further describes a kit for the diagnosis and monitoring of breast cancer treatment comprising, combined or separated in one or more containers: antibody labeled with an anti-CD45 fluorochrome, antibody labeled with a fluorochrome anti-CD44, anti-vimentin fluorochrome-labeled antibody, and anti-EpCAM fluorochrome-labeled antibody; in which it still understands instructions for use.
[00038] O referido kit ainda pode compreender em outra modalidade o referido painel de marcadores descrito no presente pedido de patente, bem como pode compreender instruções para uso no referido método descrito no presente pedido de patente. [00038] Said kit may further comprise, in another embodiment, said panel of markers described in the present patent application, as well as may comprise instructions for use in said method described in the present patent application.
[00039] A presente invenção será ainda melhor compreendida por meio da descrição dos resultados obtidos. Os procedimentos experimentais envolvidos estão detalhados no final desse relatório descritivo. [00039] The present invention will be even better understood through the description of the results obtained. The experimental procedures involved are detailed at the end of this descriptive report.
Exemplo 1 Example 1
[00040] Esse exemplo refere-se às características dos sujeitos do estudo, sendo estes agrupados em pacientes com câncer de mama que foram ou não submetidos a quimioterapia (grupo CM com QT ou CM sem QT), pacientes com doença benigna da mama (grupo DBM). [00040] This example refers to the characteristics of the study subjects, who are grouped into patients with breast cancer who have or have not undergone chemotherapy (CM with CT or CM without CT group), patients with benign breast disease (CM group DBM).
[00041] A Tabela 1 apresenta as características clinicas, hormonais, diagnósticas e terapêuticas de pacientes com câncer de mama.
Figure imgf000021_0001
Figure imgf000022_0001
[00041] Table 1 presents the clinical, hormonal, diagnostic and therapeutic characteristics of patients with breast cancer.
Figure imgf000021_0001
Figure imgf000022_0001
[00042] A Tabela 1 apresenta as características clinicas, hormonais, diagnósticas e terapêuticas de pacientes com câncer de mama. [00042] Table 1 presents the clinical, hormonal, diagnostic and therapeutic characteristics of patients with breast cancer.
Exemplo 2 Example 2
[00043] Esse exemplo refere-se a análise por RT-qPCR dos níveis transcricionais dos grupos câncer de mama e doença benigna da mama. [00044] A Tabela 2 apresenta a comparação dos níveis transcricionais entre câncer de mama (CM) e doença benigna da mama (DBM).
Figure imgf000023_0001
[00043] This example refers to the RT-qPCR analysis of the transcriptional levels of the breast cancer and benign breast disease groups. [00044] Table 2 shows the comparison of transcriptional levels between breast cancer (CM) and benign breast disease (BMD).
Figure imgf000023_0001
1Teste de Mann-Whitney 2Teste t para amostras independentes1Mann-Whitney test 2T test for independent samples
[00045] A Tabela 3 apresenta a comparação dos níveis transcricionais entre câncer de mama com quimioterapia (CM com QT), câncer de mama sem quimioterapia (CM sem QT) e doença benigna da mama (DBM).
Figure imgf000024_0001
[00045] Table 3 shows the comparison of levels between breast cancer with chemotherapy (CM with CT), breast cancer without chemotherapy (CM without CT) and benign breast disease (BMD).
Figure imgf000024_0001
1Teste de Mann-Whitney 2Teste t para amostras independentes.1Mann-Whitney test 2T test for independent samples.
As medianas horizontais seguidas de letras diferentes diferiram estatisticamente de acordo com o teste de post-hoc de Tukey a 5% de probabilidade; *p <0,005. Horizontal medians followed by different letters differed statistically according to Tukey's post-hoc test at 5% probability; *p<0.005.
Exemplo 3 Example 3
[00046] Para análise da expressão dos marcadores em nivel proteico, com os resultados obtidos na RT-qPCR e dados da literatura, os marcadores EpCAM, CD44 e vimetina foram selecionados . [00046] For analysis of the expression of the markers at protein level, with the results obtained in RT-qPCR and data from the literature, the markers EpCAM, CD44 and vimetina were selected.
[00047] As diferentes populações de CTCs (mesenquimal, intermediário e epitelial) apresentam diferentes porcentagens para cada grupo.A figura 1 apresenta as porcentagens de CTCs com perfil mesenquimal, intermediário e epitelial do sangue de pacientes com doença benigna da mama (DBM), câncer de mama com quimioterapia (CM com QT) e câncer de mama sem quimioterapia (CM sem QT). [00047] The different populations of CTCs (mesenchymal, intermediate and epithelial) show different percentages for each group. Figure 1 shows the percentages of CTCs with mesenchymal, intermediate and epithelial profile in the blood of patients with benign breast disease (BMD), breast cancer with chemotherapy (CM with CT) and breast cancer without chemotherapy (CM without CT).
[00048] Para o diagnóstico, a análise do perfil mesenquimal para os três grupos (DBM, CM com QT e CM sem QT), com um cut off acima de 4,5% de CTCs mesenquimais indicando diagnóstico de câncer de mama, apresentou uma sensibilidade de 73% e especificidade de 83% para câncer de mama vs doença benigna da mama (DBM) e odds ratio de 13.33 (p=0.0443). A análise do perfil intermediário para os três grupos (DBM, CM com QT e CM sem QT), com um cut off abaixo de 0,04% de CTCs intermediárias indicando diagnóstico de câncer de mama, apresentou uma sensibilidade de 100% e especificidade de 83% para câncer de mama vs doença benigna da mama (DBM) e odds ratio de 84.33 (p=0.0096). A análise do perfil mesenquimal para DBM vs CM sem QT, com um cut off acima de 5,5% de CTCs mesenquimais indicando diagnóstico de câncer de mama, apresentou uma sensibilidade de 83% e especificidade de 100% e odds ratio de 47.6667 (p=0.0257). A análise do perfil epitelial para DBM vs CM sem QT, com um cut off abaixo de 0,28% de CTCs epiteliais indicando diagnóstico de câncer de mama, apresentou uma sensibilidade de 100% e especificidade de 83% e odds ratio de 47.6667 (p=0.0257). Para o monitoramento do tratamento, a análise do perfil epitelial para os três grupos (DBM, CM com QT e CM sem QT), com um cut off abaixo de 0,27% de CTCs epiteliais , apresentou uma sensibilidade de 100% e especificidade de 91% para CM sem QT vs DBM e CM com QT, e odds ratio de 91 (p=0.0082). A análise do perfil intermediário para CM sem QT vs CM com QT, com um cut off acima de 0,0004% de CTCs intermediárias indica tumores sem tratamento prévio, apresentou uma sensibilidade de 100% e especificidade de 100% e odds ratio de 143 (p=0.00172). A análise do perfil epitelial para CM sem QT vs CM com QT, com um cut off abaixo de 0,6% de CTCs epiteliais indicando tumor sem tratamento prévio, apresentou uma sensibilidade de 100% e especificidade de 100% e odds ratio de 143 (p=0.00172). [00048] For the diagnosis, the analysis of the mesenchymal profile for the three groups (DBM, CM with CT and CM without CT), with a cut off above 4.5% of mesenchymal CTCs indicating a diagnosis of breast cancer, showed a sensitivity of 73% and specificity of 83% for breast cancer vs benign breast disease (BMD) and odds ratio of 13.33 (p=0.0443). The analysis of the intermediate profile for the three groups (DBM, CM with CT and CM without CT), with a cut off below 0.04% of intermediate CTCs indicating a diagnosis of breast cancer, had a sensitivity of 100% and specificity of 83% for breast cancer vs benign breast disease (BMD) and an odds ratio of 84.33 (p=0.0096). Mesenchymal profile analysis for DBM vs CM without CT, with a cut off above 5.5% of mesenchymal CTCs indicating breast cancer diagnosis, showed a sensitivity of 83% and specificity of 100% and odds ratio of 47.6667 (p =0.0257). Analysis of the epithelial profile for DBM vs CM without CT, with a cut off below 0.28% of epithelial CTCs indicating breast cancer diagnosis, showed a sensitivity of 100% and specificity of 83% and odds ratio of 47.6667 (p =0.0257). For treatment monitoring, the analysis of the epithelial profile for the three groups (DBM, CM with CT and CM without CT), with a cut off below 0.27% of epithelial CTCs, showed a sensitivity of 100% and a specificity of 91% for CM without CT vs DBM and CM with CT, and odds ratio of 91 (p=0.0082). The analysis of the intermediate profile for CM without CT vs CM with CT, with a cut off above 0.0004% of intermediate CTCs indicates tumors without previous treatment, presented a sensitivity of 100% and specificity of 100% and odds ratio of 143 ( p=0.00172). Epithelial profile analysis for CM without CT vs CM with CT, with a cut off below 0.6% of epithelial CTCs indicating untreated tumor previous study, had a sensitivity of 100% and specificity of 100% and an odds ratio of 143 (p=0.00172).
Metodologia Empregada Methodology Employed
[00049] A seguir, é descrita a metodologia para o desenvolvimento do método de detecção a partir da análise de populações de CTCs por citometria de fluxo em amostras de sangue de pacientes com CM sem QT, CM com QT e DBM. [00049] Next, we describe the methodology for developing the detection method based on the analysis of populations of CTCs by flow cytometry in blood samples from patients with CM without CT, CM with CT and DBM.
Aspectos éticos e participantes do estudo Ethical aspects and study participants
[00050] A presente invenção foi conduzida no Hospital de Clinicas da Universidade Federal de Uberlândia e aprovado pelo Comitê de Ética em Pesquisa com Seres Humanos (número do protocolo: 174.009/2013) e baseado na Declaração de Helsinque. Termo de Consentimento Livre e Esclarecido foi obtido de todas as participantes. Os critérios de exclusão foram idade inferior a 18 anos, sitio do tumor primário diferente da mama e incapacidade fisica ou mental para responder o questionário. [00050] The present invention was conducted at the Clinical Hospital of the Federal University of Uberlândia and approved by the Ethics Committee for Research with Human Beings (protocol number: 174.009/2013) and based on the Declaration of Helsinki. Free and Informed Consent Term was obtained from all participants. Exclusion criteria were age below 18 years, primary tumor site other than the breast and physical or mental inability to answer the questionnaire.
[00051] Os dados foram coletados em duas etapas de diferentes pacientes. Na primeira etapa, o perfil transcricional foi avaliado de 2013 a 2016, com 62 pacientes com câncer de mama (49 não submetidas a quimioterapia e 13 submetidas a quimioterapia) e 25 pacientes com doença benigna da mama. Na segunda etapa de 2019 a 2020, o perfil proteico foi analisado com 6 pacientes com câncer de mama e não submetidas a quimioterapia, 5 pacientes com câncer de mama e submetidas a quimioterapia e 6 pacientes com câncer de mama. [00051] Data were collected in two steps from different patients. In the first stage, the transcriptional profile was evaluated from 2013 to 2016, with 62 patients with breast cancer (49 not undergoing chemotherapy and 13 undergoing chemotherapy) and 25 patients with benign breast disease. In the second stage from 2019 to 2020, the protein profile was analyzed with 6 patients with breast cancer and not undergoing chemotherapy, 5 patients with breast cancer and undergoing chemotherapy and 6 patients with breast cancer.
Coleta da amostra e processamento Sample collection and processing
[00052] Sangue periférico foi coletado em tubos Vacutainer™ contendo 7.2 mg de K2EDTA. O tecido obtido foi armazenado a -80°C submergido em RNAlater (Invitrogen™) para extração de RNA. [00052] Peripheral blood was collected into Vacutainer™ tubes containing 7.2 mg of K2EDTA. The tissue obtained was stored at -80°C submerged in RNAlater (Invitrogen™) for RNA extraction.
[00053] O RNA foi extraído usando o reagente Trizol (Invitrogen, Life Technologies, Carlsbad, CA, USA) e seguindo as recomendações do fabricante. O produto da extração foi analisado por eletroforese em gel de agarose (1,5% de agarose). Para a transcrição reversa foi utilizado Ipg de RNA total, 10 U de inibidor de RNase (Invitrogen), 40 U de MMLV-RT (Amersham Biosciences), IX de Tampão da MMLV- RT (Amersham Biosciences), 200 pM de dNTPs (dGTP, dATP, dTTP e dCTP) e 126 pmoles. A solução foi incubada em termociclador PTC-100 (MJ Research) a 37°C por 1 hora e aquecida a 95°C por 5 minutos. Reações controle (sem RNA) foram realizadas para a verificação de possíveis contaminantes exógenos. O cDNA foi estocado a -20°C para posterior amplificação. [00053] The RNA was extracted using the Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) and following the manufacturer's recommendations. The extraction product was analyzed by agarose gel electrophoresis (1.5% agarose). For reverse transcription, Ipg of total RNA, 10 U of RNase inhibitor (Invitrogen), 40 U of MMLV-RT (Amersham Biosciences), IX of MMLV-RT Buffer (Amersham Biosciences), 200 pM of dNTPs (dGTP) were used , dATP, dTTP and dCTP) and 126 pmoles. The solution was incubated in a PTC-100 thermocycler (MJ Research) at 37°C for 1 hour and heated to 95°C for 5 minutes. Control reactions (without RNA) were performed to verify possible exogenous contaminants. The cDNA was stored at -20°C for further amplification.
[00054] As quantificações transcricionais relativas dos genes alvos (citoqueratinas, ALDH1, CD44, E-caderina,[00054] Relative transcriptional quantifications of target genes (cytokeratins, ALDH1, CD44, E-cadherin,
Ep-CAM, MMP-2, MMP-9, N-caderina e vimentina) em relação ao gene endógeno (B2M) foram estimadas por meio de PCR em tempo real a partir do cDNA obtido. As amostras foram amplificadas em duplicata e a detecção ocorreu a partir da emissão de fluorescência do corante dye SYBRGreen de acordo com o uso do kit Master Mix SYBR®Green PCR Core Reagents (Applied Biosystems) . Ep-CAM, MMP-2, MMP-9, N-cadherin and vimentin) in relation to the endogenous gene (B2M) were estimated using real-time PCR from the cDNA obtained. The samples were amplified in duplicate and the detection was based on the fluorescence emission of the SYBRGreen dye according to the use of the Master Mix SYBR®Green PCR Core Reagents kit (Applied Biosystems).
[00055] A análise das proteínas envolvidas na EMT em CTCs foi realizada por citometria de fluxo usando BD ACCURITM C6 (Becton, Dickinson and Company (BD), Franklin Lakes, FL, USA). Para este experimento dois tubos (4mL) contendo sangue periférico estabilizado com EDTA foi utilizado. O primeiro tubo foi descartado para evitar contaminação com células epiteliais. A amostra foi primeiramente submetida a centrifugação e então a monocamada de leucócitos coletada. A monocamada foi incubada com soro AB para bloquear as porções FC, evitando ligações inespecificas e então marcadas com os anticorpos monoclonais marcados com fluorocromos anti-CD45 (304008, PE) (Biolegend, San Diego, CA, USA), anti- CD44 (338816, PE/Cy7) (Biolegend, San Diego, CA, USA) e anti- EpCAM (324208, APC) (Biolegend, San Diego, CA, USA). Em seguida, os eritrócitos foram lisados com solução de lise BD FACS (Becton, Dickinson, and Company (BD), Franklin Lakes[00055] The analysis of proteins involved in EMT in CTCs was performed by flow cytometry using BD ACCURITM C6 (Becton, Dickinson and Company (BD), Franklin Lakes, FL, USA). For this experiment two tubes (4mL) containing peripheral blood stabilized with EDTA were used. The first tube was discarded to avoid contamination with epithelial cells. The sample was first subjected to centrifugation and then the leukocyte monolayer collected. The monolayer was incubated with AB serum to block the FC portions, preventing unspecific binding, and then labeled with fluorochrome-labeled monoclonal antibodies anti-CD45 (304008, PE) (Biolegend, San Diego, CA, USA), anti-CD44 (338816 , PE/Cy7) (Biolegend, San Diego, CA, USA) and anti-EpCAM (324208, APC) (Biolegend, San Diego, CA, USA). Then, erythrocytes were lysed with BD lysis solution FACS (Becton, Dickinson, and Company (BD), Franklin Lakes
FL, USA) e lavagem da amostra duas vezes com solução de lavagem (Tampão fosfato salino lx, Albumina do soro bolvino 1% e Azida sódica 0,1%). As células foram ressuspendidas em 150pL de solução de lavagem. Para marcação intracelular, as células foram incubadas com solução de permeabilização BD FACS (Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA). Em seguida, as amostras foram incubadas com soro AB, lavadas duas vezes com solução de lavagem e então incubadas com o anticorpo monoclonal marcado com fluorocromo anti-vimentina (562338, Alexa fluor 488) (Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA) e lavadas duas vezes com a solução de lavagem e ressuspendidas em 150 pL de solução de lavagem. BD™ CompBeads (Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA) foram utilizadas para compensação da fluorescência, assim como a marcação com iodeto de propidio realizada para avaliação da viabilidade celular. Células não marcadas e anticorpos isotipos controles foram utilizados para controle. Para cada tubo, 300.000 eventos foram coletados. FL, USA) and washing the sample twice with washing solution (1x Phosphate Buffer Saline, 1% Bolvin Serum Albumin and 0.1% Sodium Azide). Cells were resuspended in 150pL of washing solution. For intracellular labeling, cells were incubated with BD FACS permeabilization solution (Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA). Samples were then incubated with AB serum, washed twice with washing solution, and then incubated with the fluorochrome-labeled anti-vimentin monoclonal antibody (562338, Alexa fluor 488) (Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA) and washed twice with the washing solution and resuspended in 150 µl of washing solution. BD™ CompBeads (Becton, Dickinson, and Company (BD), Franklin Lakes, FL, USA) were used for fluorescence compensation, as well as labeling with propidium iodide performed to assess cell viability. Unlabeled cells and isotype control antibodies were used for control. For each tube, 300,000 events were collected.
[00056] A estratégia de gate seguiu uma ordem especifica para analisar os dados: (i) eliminação dos debris celulares, (ii) eliminação dos doublets, (iii) eliminação dos leucócitos totais (CD45+), e (iv) seleção positiva para CTCs mesenquimais (CD45-EpCAM-CD44+Vimentina+), intermediárias (CD45-EpCAM+CD44+Vimentin+) e epiteliais (CD45-EpCAM+CD44-Vimentin-). Análise Estatistica [00056] The gate strategy followed a specific order to analyze the data: (i) elimination of cellular debris, (ii) elimination of doublets, (iii) elimination of total leukocytes (CD45+), and (iv) positive selection for mesenchymal (CD45-EpCAM-CD44+Vimentin+), intermediate (CD45-EpCAM+CD44+Vimentin+) and epithelial (CD45-EpCAM+CD44-Vimentin-) CTCs. Statistical analysis
[00057] Inicialmente, o teste de normalidade de Kolmogorov-Smirnov foi realizado. Dependendo do comportamento das variáveis, testes paramétricos foram realizados para variáveis com distribuição normal ou teste não paramétricos para variáveis sem distribuição normal. Para comparar a expressão (mRNA e proteína) dos genes alvo entre os grupos estabelecidos o teste Mann-Whitney ou teste t para amostras independentes foi utilizado. O intervalo de confiança de 95% foi considerado e valores de p<0.05 foi considerado significante. Analises estatísticas foram realizadas no GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) e SPSS versão 21.0 (SPSS, IBM, Chicago, IL, USA). [00057] Initially, the Kolmogorov-Smirnov normality test was performed. Depending on the behavior of the variables, parametric tests were performed for variables with normal distribution or non-parametric tests for variables without normal distribution. To compare the expression (mRNA and protein) of the target genes between the established groups the Mann-Whitney test or t test for independent samples was used. The 95% confidence interval was considered and p values <0.05 were considered significant. Statistical analyzes were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) and SPSS version 21.0 (SPSS, IBM, Chicago, IL, USA).
[00058] Para análise da sensibilidade, especificidade e odds ratio foi definido um cut off para separação dos grupos em relação a porcentagem de população de CTCs especificas, e calculados por meio do site https://www.medcalc .org. [00058] For analysis of sensitivity, specificity and odds ratio, a cut off was defined for separating the groups in relation to the percentage of the population of specific CTCs, and calculated through the website https://www.medcalc .org.
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Claims

Reivindicações Claims
1- Método de diagnóstico e monitoramento do tratamento de pacientes com câncer de mama caracterizado pelo fato de que compreende as etapas de: 1- Method for diagnosing and monitoring the treatment of patients with breast cancer characterized by the fact that it comprises the steps of:
- obtenção da amostra biológica, - obtaining the biological sample,
- preparação da amostra para receber os anticorpos marcadores, em que a preparação é isenta da necessidade de uma etapa de enriquecimento prévio; - preparation of the sample to receive the marker antibodies, in which the preparation is exempt from the need for a previous enrichment step;
- por em contado a amostra preparada com os anticorpos marcados com fluorocromos anti-CD45, anti-CD44, anti-Epcam e anti-vimentina, - to count the sample prepared with the antibodies labeled with fluorochromes anti-CD45, anti-CD44, anti-Epcam and anti-vimentin,
- seleção de CTCs ausentes de CD45 (CD45-); e - selection of CTCs absent from CD45 (CD45-); It is
- identificação e análise dos perfis das CTCs através da presença ou ausência de vimentina, CD44 e EpCAM. - identification and analysis of CTCs profiles through the presence or absence of vimentin, CD44 and EpCAM.
2- Método de diagnóstico e monitoramento caracterizado pelo fato de que a etapa de identificação dos perfis das CTCs compreende a identificação dos perfis: 2- Diagnosis and monitoring method characterized by the fact that the step of identifying the profiles of the CTCs comprises the identification of the profiles:
(i) mesenquimal (CD45-, EpCAM-, CD44+ e vimentina4);(i) mesenchymal (CD45-, EpCAM-, CD44+ and vimentin4);
(ii) intermediária (CD45-, EpCAM4, CD444 e vimentina4); e (ii) intermediate (CD45-, EpCAM4, CD444 and vimentin4); It is
(iii) epitelial (CD45-, EpCAM4, CD44- e vimentina-), por citometria de fluxo. (iii) epithelial (CD45-, EpCAM4, CD44- and vimentin-), by flow cytometry.
3- Método de diagnóstico e monitoramento caracterizado pelo fato de que a etapa de análise dos perfis das CTCs compreende o uso cut offs definidos em relação a porcentagem de populações de CTCs especificas em cada um dos perfis identificados, para o diagnóstico de doença benigna da mama (DBM) ou câncer de mama (CM), ou monitoramento de câncer de mama com quimioterapia (CM com QT) e câncer de mama sem quimioterapia (CM sem QT). 3- Diagnosis and monitoring method characterized by the fact that the analysis stage of CTC profiles comprises the use of defined cut offs in relation to the percentage of specific CTC populations in each of the identified profiles, for the diagnosis of benign breast disease (BMD) or breast cancer (CM), or monitoring of breast cancer with chemotherapy (CM with CT) and breast cancer without chemotherapy (CM without CT).
4- Método de diagnóstico e monitoramento caracterizado pelo fato de que a amostra biológica é uma amostra de sangue, mais preferencialmente sangue periférico. 4- Diagnosis and monitoring method characterized by the fact that the biological sample is a blood sample, more preferably peripheral blood.
5- Painel de marcadores para o diagnóstico e monitoramento do tratamento de pacientes com câncer de mama caracterizado pelo fato de que compreende anticorpos marcados com fluorocromos anti-CD45, anticorpos marcados com fluorocromos anti-CD44, anticorpos marcados com fluorocromos anti-vimentina e anticorpos marcados com fluorocromos anti- EpCAM para detecção de populações de CTCs, em que o painel permite a identificação dos perfis das CTCs: 5- Panel of markers for the diagnosis and monitoring of the treatment of patients with breast cancer characterized by the fact that it comprises antibodies labeled with fluorochromes anti-CD45, antibodies labeled with fluorochromes anti-CD44, antibodies labeled with fluorochromes anti-vimentin and labeled antibodies with anti-EpCAM fluorochromes for detection of CTC populations, where the panel allows the identification of CTC profiles:
(i) mesenquimal (CD45-, EpCAM-, CD44+ e vimentina+);(i) mesenchymal (CD45-, EpCAM-, CD44+ and vimentin+);
(ii) intermediária (CD45-, EpCAM+, CD44+ e vimentina+); e (ii) intermediate (CD45-, EpCAM+, CD44+ and vimentin+); It is
(iii) epitelial (CD45-, EpCAM+, CD44- e vimentina-).(iii) epithelial (CD45-, EpCAM+, CD44- and vimentin-).
7- Painel caracterizado pelo fato de que o painel ainda compreende um painel de diagnóstico e um painel de monitoramento com base na análise dos perfis das CTCs, em que o painel de diagnóstico compreende: a análise do perfil mesenquimal para os três grupos (DBM, CM com QT e CM sem QT), com um cut off acima de 4,5% de células tumorais circulantes mesenquimais indicando diagnóstico de câncer de mama; a análise do perfil intermediário para os três grupos (DBM, CM com QT e CM sem QT), com um cut off abaixo de 0,04% de células tumorais circulantes intermediárias indicando diagnóstico de câncer de mama a análise do perfil mesenquimal para DBM vs CM sem QT, com um cut off acima de 5,5% de células tumorais circulantes mesenquimais indicando diagnóstico de câncer de mama; e a análise do perfil epitelial para DBM vs CM sem QT, com um cut off abaixo de 0,28% de células tumorais circulantes epiteliais indicando diagnóstico de câncer de mama; e o painel de monitoramento compreende: a análise do perfil epitelial para os três grupos (DBM, CM com QT e CM sem QT), com um cut off abaixo de 0,27% de células tumorais circulantes epiteliais indicando tumor sem tratamento; a análise do perfil intermediário para CM sem QT vs CM com QT, com um cut off acima de 0,0004% de células tumorais circulantes intermediárias indicando tumor sem tratamento; e a análise do perfil epitelial para CM sem QT vs CM com QT, com um cut off abaixo de 0,6% de células tumorais circulantes epiteliais indicando tumor sem tratamento. 7- Panel characterized by the fact that the panel also comprises a diagnostic panel and a monitoring panel based on the analysis of CTC profiles, in which the diagnostic panel comprises: the analysis of the mesenchymal profile for the three groups (DBM, CM with CT and CM without CT), with a cut off above 4.5% of circulating mesenchymal tumor cells indicating diagnosis of breast cancer; the analysis of the intermediate profile for the three groups (DBM, CM with CT and CM without CT), with a cut off below 0.04% of intermediate circulating tumor cells indicating a diagnosis of breast cancer the analysis of the mesenchymal profile for DBM vs. CM without CT, with a cut off above 5.5% of circulating mesenchymal tumor cells indicating a diagnosis of breast cancer; and the analysis of the epithelial profile for DBM vs CM without CT, with a cut off below 0.28% of circulating epithelial tumor cells indicating a diagnosis of breast cancer; and the monitoring panel comprises: the analysis of the epithelial profile for the three groups (DBM, CM with CT and CM without CT), with a cut off below 0.27% of epithelial circulating tumor cells indicating tumor without treatment; analysis of the intermediate profile for CM without CT vs CM with CT, with a cut off above 0.0004% of intermediate circulating tumor cells indicating untreated tumor; and analysis of the epithelial profile for CM without CT vs CM with CT, with a cut off below 0.6% of epithelial circulating tumor cells indicating untreated tumor.
8- Kit para o diagnóstico e monitoramento do tratamento de câncer de mama caracterizado pelo fato de que compreende, combinados ou separados em um ou mais recipientes: anticorpos marcados com um fluorocromo anti- CD45; anticorpos marcados com um fluorocromo anti-CD44; anticorpos marcados com fluorocromo anti-vimentina; anticorpos marcados com fluorocromo anti-EpCAM; e instruções de uso. 8- Kit for the diagnosis and monitoring of the treatment of breast cancer characterized by the fact that comprises, combined or separated in one or more containers: antibodies labeled with an anti-CD45 fluorochrome; antibodies labeled with an anti-CD44 fluorochrome; fluorochrome-labeled anti-vimentin antibodies; fluorochrome-labeled anti-EpCAM antibodies; and instructions for use.
9- Kit caracterizado pelo fato de que compreende o painel de marcadores tal como definido no método apresentado neste pedido de patente. 9- Kit characterized by the fact that it comprises the panel of markers as defined in the method presented in this patent application.
10- Kit caracterizado pelo fato de que as instruções de uso são para utilização tal como definido no método apresentado neste pedido de patente. 10- Kit characterized by the fact that the instructions for use are for use as defined in the method presented in this patent application.
PCT/BR2021/050561 2021-12-20 2021-12-20 Method, panel and kit for the diagnosis and treatment monitoring of breast cancer patients WO2023115171A1 (en)

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