WO2016127239A1 - Method for detecting metastatic cells in lymph nodes of head and neck squamous cell carcinoma patients, and use of micrornas as markers - Google Patents

Method for detecting metastatic cells in lymph nodes of head and neck squamous cell carcinoma patients, and use of micrornas as markers Download PDF

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WO2016127239A1
WO2016127239A1 PCT/BR2016/050027 BR2016050027W WO2016127239A1 WO 2016127239 A1 WO2016127239 A1 WO 2016127239A1 BR 2016050027 W BR2016050027 W BR 2016050027W WO 2016127239 A1 WO2016127239 A1 WO 2016127239A1
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mir
lymph node
metastatic cells
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André Lopes CARVALHO
Ana Carolina DE CARVALHO PETERS
Cristovam SCAPULATEMPO NETO
André Luiz VETTORE
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Hospital De Câncer De Barretos - Fundação Pio Xii
Universidade Federal De São Paulo - Unifesp
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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  • the present invention patent belongs to the field of diagnosis and evaluation of the extent of neoplastic disease by means of markers. More specifically, microRNAs were used as markers for detection of metastatic cells in lymph node and whole lymph node sections obtained from surgical resection, as well as lymph node biopsies obtained through fine needle aspiration (FNA) punctures of patients with head and squamous cell carcinoma. neck (CPB).
  • FNA fine needle aspiration
  • MicroRNAs are promising candidates for diagnostic markers as they: play a role in many pathways involved in tumor metastasis; are reported to have tumor and tissue specific expression; Their expression is evaluated using the sensitive and rapid qRT-PCR technique and is suitable for evaluation on paraffin-embedded tissue, puncture-based biopsies and body fluids.
  • MicroRNAs are small non-coding RNA molecules of approximately 22 nucleotides, playing an important role in virtually all biological pathways in mammals and other multicellular organisms. Similarly, these molecules influence various cancer-relevant processes such as proliferation, apoptosis, cell cycle control, differentiation, migration, and metabolism (Bartel, 2009; Fabian and Sonenberg, 2012; SajandLai, 201 1). Currently, over 1400 human microRNAs have been identified (Griffiths-Jones, 2010), illustrating the enormous potential of these molecules in regulating gene expression, with estimates that about 60% of all mRNAs are under the control of microRNAs (Bartel, 2009).
  • each microRNA can control the expression of several mRNAs and that one mRNA can be targeted by multiple microRNAs, thus aberrant expression of a single microRNA affects multiple transcripts and influences cancer-associated signaling pathways.
  • Tumors usually have reduced levels of mature microRNAs (Lu et al., 2005) as a consequence of genetic loss, epigenetic silencing, defects in their biogenesis or transcriptional repression (Chang et al., 2008).
  • Several studies have shown distinct microRNA expression profiles between normal and tumor samples as well as variations between different tumor types (Lu et al., 2005; Volinia et al., 2006), assigning these molecules a relevant role in tumorigenesis (Squela- Kerscherand Slack, 2006; Hammond, 2007).
  • microRNAs for the diagnosis of various diseases and even in cancer therapy, but as will be seen in none of them there was a deepening in the study of miR-203 and miR-205 microRNAs in the detection of metastatic cells in lymph nodes of patients with head and neck squamous cell carcinoma.
  • WO2010 / 10951 1 describes the miR199b-5p microRNA for anti-cancer therapy and a histopathological tumor diagnosis kit for the detection of metastases in biological samples of medulloblastoma, lymphoma, carcinoma of colon and breast by detecting SEQ ID NO: 1 (disclosed in said document) and at least one of the panel genes in tumor cells.
  • the present invention departs from the above document as the object herein
  • the objective of this study is to use diagnostic markers to identify metastatic lymph node lesions.
  • Both miR-203 and miR-205 markers are capable of detecting the presence of metastases in affected lymph nodes with high sensitivity, accuracy and specificity, especially when evaluated in biopsy specimens obtained from lymph node aspiration punctures.
  • the use of at least 1 of the markers is already sufficient for the diagnosis with high accuracy of metastatic lesions.
  • the evaluation of several markers is necessary, furthermore, said document evaluates primary tumor samples, already in the present invention lymph node samples are evaluated.
  • US 2012/0309638 describes markers useful for determining the risk of developing distant metastases in individuals with lung cancer. For risk to be determined, the expression level of a six-marker panel must be determined. Diagnostic markers are used in the invention described herein, whereas US 2012/0309638 aims at predicting risk. In addition, the patent document evaluates primary tumor samples, while the present invention evaluates lymph node samples. Also in US Patent Document 2012/0309638 the evaluation of six markers is required.
  • Patent document WO 2008/125883 describes markers useful in determining prognosis and the best therapeutic regimen to be used in individuals with cervical cancer from the analysis of DROSHA protein expression level in tumor and biopsy samples.
  • WO20101083464 describes markers based on the expression of microRNAs useful in the diagnosis of dysplastic cervical lesions. Differential expression of these markers is able to identify dysplastic lesions with malignant potential in cholouterine cells.
  • one or many microRNAs must be evaluated.
  • the use of one of the miR-203 or miR-205 markers is already sufficient for the highly accurate diagnosis of metastatic lesions by the rapid and sensitive quantitative PCR technique.
  • marker evaluation by hybridization techniques is often more laborious.
  • Patent Document WO 201 1/014980 describes markers useful for the diagnosis and choice of treatment of uterine cervical dysplastic and malignant lesions and prognostic markers for survival of individuals with cervical cancer and / or dysplasia. according to the level of expression of microRNAs evaluated in cervical, lymph node, blood or serum samples from patients with cervical dysplasia or neoplasia. This document also describes the use of the miR-133b microRNA as a marker for detecting colorectal cancer, tongue squamous cell carcinoma, esophagus and pancreatic adenocarcinoma in tissue, lymph node, blood or serum samples from these tumors. However, in the invention described herein, diagnostic markers of metastatic lymph node lesions were used.
  • WO 201 1/014980 evaluates the use of miR-133b microRNA only in detecting tumor cells in lymph nodes of patients with tongue CE.
  • miR-203 is cited as one of the microRNAs whose differential expression is capable of detecting uterine cervical dysplastic or neoplastic lesions, nothing had been anticipated for CECP.
  • the miR-200 family is composed of the microRNAs that form the miR-200b-200a-429 and miR-200c-141 clusters that are encoded as polycistronic transcripts. (Bracken et al., 2008; Xia et al., 2010).
  • the miR-200 family members target ZEB1 and ZEB2, preventing these factors from inhibiting E-cadherin expression. Maintaining high levels of E-cadherin prevents cell differentiation by preventing the promotion of cell migration and invasion via EMT (Adam et al., 2009; Korpal et al., 2008).
  • miR-203 and miR-205 microRNAs are also important regulators of the epithelial-mesenchymal transition process and are responsible for maintaining the epithelial phenotype.
  • miR-203 has also been associated with this epithelial plasticity (Moes et al., 2012).
  • This microRNA located on chromosome 14, targets SNAI1, another known E-cadherin transcriptional repressor.
  • MiR-203 has been observed to be hypoexpressed in breast and prostate cancer cell lines, thus contributing to tumor cell migration and invasion (Moes et al., 2012).
  • This microRNA also presents differentiated expression between primary and metastatic tumors (Baffa et al., 2009) and its hypoexpression has been associated with the progression of dysplastic lesions to esophageal tumors (Wu et al., 2013).
  • MiR-205 is located on chromosome 1 and targets ZEB2, modulating cell invasion and migration (Gregory et al., 2008a). In esophageal cell lines, the expression of this microRNA is high, but by becoming invasive and migratory via EMT, these cells have decreased miR-205 expression (Matsushima et al., 201 1). This microRNA was described as hypoexpressed in prostate cancer and this hypoexpression was correlated with tumor size, higher Gleason score, metastasis occurrence and shorter overall survival (Hagman et al., 2013; Verdoodt et al., 2013). This microRNA was also hypoexpressed in breast (Baffa et al., 2009) and bladder cancer (Wiklund et al., 201 1a).
  • the present invention aims at a test for the diagnosis of cervical metastasis in patients with CPB by assessing the level of miR-203 and miR-205 microRNA expression.
  • the test can be applied to samples collected using various approaches at different times, such as pre-treatment through diagnosis using FNAB biopsies, intraoperatively through sentinel lymph node biopsy, and after surgery as an auxiliary tool for histopathological evaluation of resected lymph nodes during surgery, helping to choose the treatment of these patients from initial treatment to adjuvant therapy.
  • the present invention then teaches a method of detecting metastases using whole lymph nodes or parts of lymph nodes resected during surgery and specimens collected by lymph node fine needle aspiration (FNA) punctures from patients with CPBP. detection of miR-203 and miR-205 microRNAs. More specifically, the present invention teaches a method for detecting lymph node metastatic cells comprising the steps of: a) processing said lymph nodes in paraffin blocks or fresh lymph nodes or puncture-collected biopsy specimens (FNA) lymph nodes;
  • the present invention describes the analysis of miR-203 and miR-205 microRNA expression as a method of diagnosing metastasis in lymph node samples obtained from surgery and lymph node aspiration biopsy samples from patients with CPB.
  • lymph node specimens obtained from neck dissection ie, surgical resection of lymph nodes
  • all resected lymph nodes are processed, embedded in paraffin, and evaluated in the pathology department of each facility.
  • Routine histopathological evaluation is as follows: after paraffin embedding, the blocks containing the lymph nodes to be evaluated are sectioned on their central axis and the sections containing the sections are stained with hematoxylin & eosin and evaluated by a pathologist to confirm the diagnosis by characterizing the cellular components present in the sections.
  • the present invention provides the possibility to evaluate sections distributed throughout the lymph node or to evaluate the entire lymph node by molecular techniques.
  • paraffin blocks containing the resected lymph nodes are cut at various levels or cut the entire length of the block, reflecting the analysis of the entire lymph node. These cuts are collected in collecting tubes, or placed on slides.
  • the region of interest needs to be scraped off with the help of a scalpel or needle and added to a collecting tube.
  • extraction can also be done from fresh material, ie, dried out whole fragments or lymph nodes are collected in cryotubes (low temperature storage resistant tubes) and stored at temperatures below -80 ° C so far. of extraction.
  • RNA assessed by molecular analysis is obtained by processing these collected materials.
  • RNA extraction from fresh frozen whole or paraffin embedded lymph nodes or fragments should be performed using specific protocols and kits for obtaining total RNA following the manufacturer's recommendations. Generally, for fresh samples these protocols / kits begin with processing this sample using a tissue homogenizer. For paraffin-embedded samples a paraffin removal (deparaffinization) step is performed from the sections containing the samples of interest using reagents such as Xylol. The extraction of nucleic acids occurs through digestion with proteinase K (peptidase responsible for degrading cellular proteins) and detergents (which will degrade lipids).
  • proteinase K peptidase responsible for degrading cellular proteins
  • detergents which will degrade lipids.
  • RNA samples are then treated with DNase and purified using chemical reagents and / or columns allowing total RNA free from contamination by DNA, proteins and reagents.
  • chemical Total RNA obtained should be stored in a freezer at a temperature below -80 ° C until use.
  • RNA extraction from FNAB samples specific RNA extraction reagents that contain phenol and guanidine isothiocyanate should be used.
  • RNA extracted from the samples to be analyzed After obtaining the total RNA extracted from the samples to be analyzed, it should be quantified and about 10 nanograms should be used in the reverse transcription (RT) reaction.
  • This reaction is based on the synthesis of a complementary DNA strand (cDNA) to the RNA of interest.
  • cDNA complementary DNA strand
  • cDNA synthesis of endogenous U6 and U47 RNAs and microRNAs of interest miR-203 and miR-205 is required. That is, for each sample to be evaluated 4 RT reactions are required.
  • RT reactions For RT reactions, specific primers are used for each endogenous microRNA or RNA and the reverse transcriptase enzyme, as well as other reagents required for the reaction.
  • the present invention describes the use of detection of miR-203 and miR-205 microRNA expression level as a diagnostic tool for lymph node metastasis of patients with CPB.
  • the evaluation of microRNA expression level in total RNA samples obtained from surgically resected lymph node samples and from FNA of suspicious lymph nodes should be performed using real-time PCR analysis. Various equipment is available and can be used to perform these analyzes.
  • RNA or endogenous RNA PCR reaction should be performed, ie there are 4 real-time PCR reactions for each sample.
  • reactions For PCR reactions, the addition of specific reagents and primers and probes is required for the amplification of cDNA obtained by said reverse transcription reaction. Reactions should be performed in triplicate in a real-time PCR equipment that uses temperature cycling for specific periods for the amplification reaction (as recommended for the reagents, primers and probes used) and is capable of detecting presence of the cDNA of interest and provide data that allow the quantification of their expression in the evaluated samples. For each sample evaluated, when there is the presence of the microRNA of interest, the primers and probe are able to bind to the microRNA and initiate amplification.
  • an amplification curve occurs which allows the expression to be quantified by determining for each sample a Ct (Threshold Cycle) value, ie the PCR cycle in which the generated fluorescence reaches a pre-set point. threshold of the log phase of the reaction.
  • Ct Gate Cycle
  • the primers and probe do not bind and the amplification reaction does not occur.
  • the 2 'àACt method or the Ct comparative method should be used.
  • This method is based on the normalization of expression of the microRNAs of interest (in this case miR-203 and miR-205) by the expression of normalizing RNAs (U6 and U47) in the same biological sample, and changes in microRNA expression are calculated based on the differences between reference samples and experimental samples.
  • the reference group used was non-metastatic lymph node samples.
  • the value inferred to ACt is equivalent to the difference between the mean value of the Cts of the microRNAs of interest, miR-203 and miR-205 (Ct (miR-203) and Ct (miR-205), respectively).
  • Ct (U6> and Ct (U47>, respectively) the mean Cts value of the U6 and U47 normalizing RNAs obtained by evaluating the expression of these markers in the suspect samples (in this case, lymph nodes and FNA of suspected lymph nodes) and reference sample group (non-metastatic lymph nodes).
  • ACt (miR-203 sample) Ct (miR-203 sample) - [Ct (U6 sample) + Ct (U47 sample)] / 2
  • ACt (miR-205 sample) Ct (miR-205 sample) - [Ct ( U6 sample) + Ct (U47 sample)] / 2 [036]
  • AACt (miR-203) ACt (miR-203 sample) - ACt (miR-203 reference)
  • AACt (miR-203) ACt (miR-203 sample) —11,1656545
  • AACt (miR-205) ACt (miR-205 sample) - ACt (miR-205 reference)
  • AACt (miR-205) ACt (miR-205 sample) - 10.688645
  • the already normalized Ct values of the miR-203 and miR-205 microRNAs obtained after real-time PCR reaction in the suspect samples will always be compared to the normalized Ct value of a previously evaluated non-metastatic lymph node group. used as a reference.
  • the result is expressed in fold-change or the number of times the expression of the microRNA of interest is increased in the suspect samples relative to the reference sample group.
  • a cut-off value of 2-fold should be used to consider a positive sample for the presence of metastatic cells ie At least 2-fold expression means that the suspected lymph node evaluated contains metastatic cells.
  • a 10-fold cutoff should be used to consider a metastatic sample.
  • lymph node or lymph node FNA in metastatic and non-metastatic.
  • the fold-change value of at least one of the microRNAs is above the 2-fold cut for lymph node samples or 10 times for the lymph node samples. puncture.
  • FIG. 1 Schematic representation of lymph node processing in sections for molecular analysis:
  • Example Material Processing for Molecular Analysis For the processing of paraffin-embedded lymph node samples it is possible to evaluate sections or the entire lymph node. For slice analysis, one option is to subject the block (1) to 3 to 5 serial histological sections (2) of 5 to 10 pm in thickness, distributed at approximately 6 levels at 50 pm intervals (3). For the analysis of the entire lymph node, the whole block (4) can be submitted to 5 to 10 pm cuts (5). In both cases, the obtained cuts are collected in 1.5 ml tubes for further extraction.
  • the collecting needle attached to the syringe can be washed in 100 to 400 ⁇ of solution containing EDTA and sodium chloride.
  • the collection solution can be prepared as follows: Fill a blood collection tube containing EDTA with 0.9% sodium chloride (saline), mix using a vortex tube shaker and prepare 100 to 400 ⁇ aliquots of the collection solution. in cryotubes (tubes resistant to storage at low temperatures). The collected material is frozen in liquid nitrogen and stored in an ultrafreezer at temperatures below -80 ° C until RNA extraction.
  • RNA extraction from paraffin material is suggested.
  • the only modification should be made in the deparaffinization phase for material obtained from the entire lymph node. Because of the large amount of paraffin In these samples, 3 washes with Xylol (instead of just one, as recommended in the kit).
  • any other kit capable of extracting total RNA from paraffin-embedded samples may be used, always following the manufacturer's recommendations.
  • Trizol LS reagent may be used with some modifications.
  • This reagent was chosen because it was specifically formulated to obtain RNA from liquid samples. However, other reagents of the same composition may also be used. Extraction began with the addition of Trizol LS to the tube containing the puncture material, homogenization in vortex shaker and incubation at room temperature. Then chloroform was added, the material was homogenized and incubated at room temperature. The tubes were then centrifuged and the supernatant aqueous phase containing the RNA was transferred to a new tube to which sodium acetate at 3M concentration and pH 5.2, glycogen at 20mg / ml concentration and isopropanol were added. The material was precipitated at -20 ° C and centrifuged for pellet recovery.
  • the supernatant was discarded by inversion and the pellet was washed with 70% ice cold ethanol. After further centrifugation, the supernatant was discarded and the pellet was dried at room temperature for 15 to 20 minutes. The pellet was resuspended in RNase free water, and the total RNA obtained was stored at -80 ° C.
  • reagents, primers and probes for detection of miR-203 and miR-205 microRNAs and endogenous U6 and U47 RNAs by Real Time PCR are commercially available.
  • these reactions were performed using TaqMan Universal PCR Master Mix 2X, a solution containing all reagents necessary for the amplification reaction (such as DNA polymerase, buffer, magnesium, deoxyribonucleotides) and TaqMan MicroRNA Assay 20X assays. contain primers and probe specific for each of the evaluated microRNAs and RNAs.

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Abstract

The present invention is directed to a test for diagnosing cervical metastasis in head and neck squamous cell carcinoma patients by evaluating the level of expression of the micro-RNAs miR-203 and miR-205. The test can be applied to samples collected using various methods at different points in time, for example during pre-treatment, for diagnosing using fine-needle aspiration biopsy (FNAB), in the intra-operative period through sentinel lymph node biopsy and after surgery, as an auxiliary tool of histopathological evaluation of lymph nodes resected during surgery, assisting in the choice of treatment of such patients from the initial treatment to adjuvant therapy.

Description

Relatório Descritivo de Patente de Privilégio de Invenção para "Método de detecção de células metastáticas em linfonodos de pacientes com carcinoma epidermóide de cabeça e pescoço e uso de microRNAs como marcadores" Campo da Invenção  Field Report of the Invention for "Method of Detecting Lymph Node Metastatic Cells in Patients with Head and Neck Squamous Cell Carcinoma and Using MicroRNAs as Markers" Field of the Invention
[001 ] A presente patente de privilégio de invenção pertence ao campo do diagnóstico e avaliação da extensão da doença neoplásica, por meio de marcadores. Mais especificamente foram utilizados microRNAs como marcadores para detecção de células metastáticas em cortes de linfonodos e linfonodos inteiros obtidos a partir de ressecção cirúrgica, além de biópsias colhidas de linfonodos através de punções aspirativas por agulha fina (PAAF) de pacientes com carcinoma epidermóide de cabeça e pescoço (CECP). The present invention patent belongs to the field of diagnosis and evaluation of the extent of neoplastic disease by means of markers. More specifically, microRNAs were used as markers for detection of metastatic cells in lymph node and whole lymph node sections obtained from surgical resection, as well as lymph node biopsies obtained through fine needle aspiration (FNA) punctures of patients with head and squamous cell carcinoma. neck (CPB).
Histórico da Invenção Invention History
[002] A presença de doença metastática em linfonodos cervicais de pacientes com CECP é um dos determinantes mais importantes na escolha da terapia e na determinação do prognóstico. [002] The presence of metastatic disease in cervical lymph nodes of patients with CPBP is one of the most important determinants in choosing therapy and determining prognosis.
[003] O adequado estadiamento de metástases em linfonodos regionais é crítico para a resposta ao tratamento e o sucesso do controle loco-regional da doença. O estadiamento clínico realizado na rotina nem sempre é eficiente, pois pode não se detectar metástases ocultas e a avaliação patológica pós- cirúrgica dos linfonodos ressecados por hematoxilina & eosina (H&E) não é sensível na detecção de pequenos depósitos metastáticos. O correto estadiamento influencia diretamente na sobrevida global dos pacientes, dessa forma, é imperativo o uso de marcadores de diagnóstico e técnicas que possibilitem uma detecção rápida e precisa de células metastáticas em nódulos linfáticos cervicais de forma a guiar os médicos na escolha da terapia mais adequada para os pacientes. [004] Os microRNAs são candidatos promissores para marcadores de diagnóstico uma vez que: desempenham papel em muitas vias envolvidas em metástase tumoral; são relatados como tendo expressão tumor e tecido específica; sua expressão é avaliada usando a técnica sensível e rápida de qRT-PCR e são adequados para serem avaliadas em tecido incluído em parafina, biópsias baseadas em punção e fluidos corporais. Proper staging of regional lymph node metastases is critical for treatment response and the success of locoregional control of the disease. Routine clinical staging is not always efficient because hidden metastases may not be detected and postoperative pathological evaluation of hematoxylin & eosin (H&E) resected lymph nodes is not sensitive in detecting small metastatic deposits. The correct staging directly influences the overall survival of the patients, thus Therefore, it is imperative to use diagnostic markers and techniques that enable rapid and accurate detection of metastatic cells in cervical lymph nodes in order to guide physicians in choosing the most appropriate therapy for patients. MicroRNAs are promising candidates for diagnostic markers as they: play a role in many pathways involved in tumor metastasis; are reported to have tumor and tissue specific expression; Their expression is evaluated using the sensitive and rapid qRT-PCR technique and is suitable for evaluation on paraffin-embedded tissue, puncture-based biopsies and body fluids.
[005] Os microRNAs são pequenas moléculas de RNA não-codificantes de aproximadamente 22 nucleotídeos, com papel importante em virtualmente todas as vias biológicas em mamíferos e outros organismos multicelulares. Da mesma forma, estas moléculas influenciam diversos processos relevantes ao câncer como proliferação, apoptose, controle do ciclo celular, diferenciação, migração e metabolismo (Bartel, 2009; Fabian e Sonenberg, 2012; SajandLai, 201 1 ). Atualmente, mais de 1400 microRNAs humanos foram identificados (Griffiths-Jones, 2010), ilustrando o enorme potencial destas moléculas na regulação da expressão gênica, com estimativas de que cerca de 60% de todos os mRNAs estejam sob o controle de microRNAs (Bartel, 2009). MicroRNAs are small non-coding RNA molecules of approximately 22 nucleotides, playing an important role in virtually all biological pathways in mammals and other multicellular organisms. Similarly, these molecules influence various cancer-relevant processes such as proliferation, apoptosis, cell cycle control, differentiation, migration, and metabolism (Bartel, 2009; Fabian and Sonenberg, 2012; SajandLai, 201 1). Currently, over 1400 human microRNAs have been identified (Griffiths-Jones, 2010), illustrating the enormous potential of these molecules in regulating gene expression, with estimates that about 60% of all mRNAs are under the control of microRNAs (Bartel, 2009).
[006] Estima-se que cada microRNA possa controlar a expressão de diversos mRNAs e que um mRNA possa ser alvo de múltiplos microRNAs, desta forma, a expressão aberrante de um único microRNA afeta diversos transcritos e influencia vias de sinalização associadas ao câncer. It is estimated that each microRNA can control the expression of several mRNAs and that one mRNA can be targeted by multiple microRNAs, thus aberrant expression of a single microRNA affects multiple transcripts and influences cancer-associated signaling pathways.
[007] Tumores geralmente apresentam níveis reduzidos de microRNAs maduros (Lu et al., 2005) como consequência de perdas genéticas, silenciamento epigenético, defeitos em sua biogênese ou repressão transcricional (Chang et al., 2008). Diversos estudos têm mostrado perfis distintos de expressão de microRNA entre amostras normais e tumorais e também variações entre tipos tumorais diferentes (Lu et al., 2005; Volinia et al., 2006), atribuindo a estas moléculas um papel relevante na tumorigênese (Esquela-Kerscherand Slack, 2006; Hammond, 2007). [007] Tumors usually have reduced levels of mature microRNAs (Lu et al., 2005) as a consequence of genetic loss, epigenetic silencing, defects in their biogenesis or transcriptional repression (Chang et al., 2008). Several studies have shown distinct microRNA expression profiles between normal and tumor samples as well as variations between different tumor types (Lu et al., 2005; Volinia et al., 2006), assigning these molecules a relevant role in tumorigenesis (Squela- Kerscherand Slack, 2006; Hammond, 2007).
[008] Dessa forma, diversas publicação mencionam o uso de microRNAs para o diagnóstico de diversas doenças e até mesmo na terapia do câncer, mas como será visto em nenhuma delas houve o aprofundamento no estudo dos microRNAs miR-203 e miR-205 na detecção de células metastáticas em linfonodos de pacientes com carcinoma epidermóide de cabeça e pescoço. Thus, several publications mention the use of microRNAs for the diagnosis of various diseases and even in cancer therapy, but as will be seen in none of them there was a deepening in the study of miR-203 and miR-205 microRNAs in the detection of metastatic cells in lymph nodes of patients with head and neck squamous cell carcinoma.
Estado da Técnica State of the Art
[009] O documento de patente WO2010/10951 1 descreve o microRNA miR199b-5p com a finalidade de terapia anti-cancer e um kit de diagnóstico do estágio histopatológico de tumores para a detecção de metástases em amostras biológicas de meduloblastoma, linfoma, carcinoma de cólon e de mama através da detecção de SEQ ID NO:1 (revelada no referido documento) e de pelo menos um dos genes de um painel em células tumorais. A presente invenção se distancia do documento acima citado, uma vez que o objeto aqui ensinado tem como objetivo a utilização de marcadores de diagnóstico para identificar lesões metastáticas em linfonodos. Ambos os marcadores miR-203 e miR-205 são capazes de detectar a presença de metástases em linfonodos afetados com alta sensibilidade, acurácia e especificidade, principalmente ao serem avaliados em amostras de biópsias obtidas a partir de punções aspirativas dos linfonodos. Ou seja, a utilização de pelo menos 1 dos marcadores (miR-203 e miR-205) já é suficiente para o diagnóstico com alta acurácia de lesões metastáticas. No documento WO 2010/10951 1 A1 é necessário a avaliação de diversos marcadores, além disso, o referido documento avalia amostras de tumores primários, já na presente invenção são avaliadas amostras de linfonodos. WO2010 / 10951 1 describes the miR199b-5p microRNA for anti-cancer therapy and a histopathological tumor diagnosis kit for the detection of metastases in biological samples of medulloblastoma, lymphoma, carcinoma of colon and breast by detecting SEQ ID NO: 1 (disclosed in said document) and at least one of the panel genes in tumor cells. The present invention departs from the above document as the object herein The objective of this study is to use diagnostic markers to identify metastatic lymph node lesions. Both miR-203 and miR-205 markers are capable of detecting the presence of metastases in affected lymph nodes with high sensitivity, accuracy and specificity, especially when evaluated in biopsy specimens obtained from lymph node aspiration punctures. That is, the use of at least 1 of the markers (miR-203 and miR-205) is already sufficient for the diagnosis with high accuracy of metastatic lesions. In WO 2010/10951 1 A1 the evaluation of several markers is necessary, furthermore, said document evaluates primary tumor samples, already in the present invention lymph node samples are evaluated.
[010] O documento US 2012/0309638 descreve marcadores úteis para determinar o risco de desenvolvimento de metástases à distância em indivíduos portadores de câncer de pulmão. Para que o risco seja determinado, o nível de expressão de um painel de seis marcadores precisa ser determinado. Na invenção aqui descrita são utilizados marcadores de diagnóstico, já no documento US 2012/0309638 objetiva-se a predição de risco. Além disso, o documento de patente avalia amostras de tumores primários, já a presente invenção avalia amostras de linfonodo. Ainda no documento de patente US 2012/0309638 é necessária a avaliação de seis marcadores. [010] US 2012/0309638 describes markers useful for determining the risk of developing distant metastases in individuals with lung cancer. For risk to be determined, the expression level of a six-marker panel must be determined. Diagnostic markers are used in the invention described herein, whereas US 2012/0309638 aims at predicting risk. In addition, the patent document evaluates primary tumor samples, while the present invention evaluates lymph node samples. Also in US Patent Document 2012/0309638 the evaluation of six markers is required.
[01 1 ] O documento de patente WO 2008/125883 descreve marcadores úteis em determinar o prognóstico e o melhor regime terapêutico a ser utilizado em indivíduos portadores de câncer de colo uterino a partir da análise do nível de expressão da proteína DROSHA em amostras tumorais e de biópsias. [01 1] Patent document WO 2008/125883 describes markers useful in determining prognosis and the best therapeutic regimen to be used in individuals with cervical cancer from the analysis of DROSHA protein expression level in tumor and biopsy samples.
[012] O documento de patente WO20101083464 descreve marcadores baseados na expressão de microRNAs úteis no diagnóstico de lesões displásicas de colo uterino. A expressão diferencial destes marcadores é capaz de identificar lesões displásicas com potencial maligno em células de colouterino. No referido documento, para que o diagnóstico seja realizado, um ou inúmeros microRNAs devem ser avaliados. Na presente invenção, a utilização de um dos marcadores miR-203 ou miR-205 já é suficiente para o diagnóstico com alta acurácia de lesões metastáticas através da técnica rápida e sensível de PCR quantitativa. Além disso, avaliação de marcadores por técnicas de hibridização costuma ser mais laboriosa. WO20101083464 describes markers based on the expression of microRNAs useful in the diagnosis of dysplastic cervical lesions. Differential expression of these markers is able to identify dysplastic lesions with malignant potential in cholouterine cells. In that document, for the diagnosis to be made, one or many microRNAs must be evaluated. In the present invention, the use of one of the miR-203 or miR-205 markers is already sufficient for the highly accurate diagnosis of metastatic lesions by the rapid and sensitive quantitative PCR technique. In addition, marker evaluation by hybridization techniques is often more laborious.
[013] O Documento de patente WO 201 1/014980 descreve marcadores úteis para o diagnóstico e escolha do tratamento de lesões displásicas e malignas de colo uterino e marcadores de prognóstico para a sobrevida de indivíduos com câncer e/ou displasia no colo uterino, de acordo com o nível de expressão de microRNAs avaliados em amostras de colo uterino, linfonodo, sangue ou soro de pacientes com displasia ou neoplasia de colo de útero. Esse documento também descreve a utilização do microRNA miR-133b como marcador de detecção de câncer colorretal, carcinoma epidermóide de língua, esôfago e adenocarcinoma de pâncreas em amostras de tecido, linfonodo, sangue ou soro destes tumores. Entretanto, na invenção aqui descrita foram utilizados marcadores de diagnóstico de lesões metastáticas em linfonodos de pacientes com CECP, já no documento WO 201 1/014980 são avaliados tecido, sangue, linfonodo ou soro de pacientes com displasia ou neoplasia de colo uterino. Além disso, na presente invenção são avaliadas a expressão dos microRNAs miR-203 e miR-205 em amostras de linfonodo, possíveis sítios de metástases regionais de pacientes com carcinomas epidermóides de cabeça e pescoço. O documento de patente WO 201 1/014980 A1 avalia o uso somente do microRNA miR-133b em detectar células tumorais em linfonodos de pacientes com CE de língua. Embora o miR-203 seja citado como um dos microRNAs cuja expressão diferencial é capaz de detectar lesões displásicas ou neoplásicas de colo uterino, nada fora antecipado para CECP. Patent Document WO 201 1/014980 describes markers useful for the diagnosis and choice of treatment of uterine cervical dysplastic and malignant lesions and prognostic markers for survival of individuals with cervical cancer and / or dysplasia. according to the level of expression of microRNAs evaluated in cervical, lymph node, blood or serum samples from patients with cervical dysplasia or neoplasia. This document also describes the use of the miR-133b microRNA as a marker for detecting colorectal cancer, tongue squamous cell carcinoma, esophagus and pancreatic adenocarcinoma in tissue, lymph node, blood or serum samples from these tumors. However, in the invention described herein, diagnostic markers of metastatic lymph node lesions were used. of patients with CPB, already in WO 201 1/014980 are evaluated tissue, blood, lymph node or serum of patients with cervical dysplasia or neoplasia. In addition, in the present invention the expression of miR-203 and miR-205 microRNAs in lymph node samples, possible sites of regional metastases of patients with head and neck squamous cell carcinoma, are evaluated. Patent document WO 201 1/014980 A1 evaluates the use of miR-133b microRNA only in detecting tumor cells in lymph nodes of patients with tongue CE. Although miR-203 is cited as one of the microRNAs whose differential expression is capable of detecting uterine cervical dysplastic or neoplastic lesions, nothing had been anticipated for CECP.
[014] Além dos documentos de patentes, a literatura científica nos ensina que a família do miR-200 é composta pelos microRNAs que formam os clusters miR-200b-200a-429 e miR-200c-141 que são codificados na forma de transcritos policistronicos (Bracken et al., 2008; Xia et al., 2010). Os membros da família miR-200 tem como alvo ZEB1 e ZEB2, impedindo que estes fatores inibam a expressão de E-caderina. A manutenção de níveis altos de E- caderina impede a diferenciação celular, evitando a promoção da migração e invasão da célula via EMT (Adam et al., 2009; Korpal et al., 2008). In addition to patent documents, the scientific literature teaches that the miR-200 family is composed of the microRNAs that form the miR-200b-200a-429 and miR-200c-141 clusters that are encoded as polycistronic transcripts. (Bracken et al., 2008; Xia et al., 2010). The miR-200 family members target ZEB1 and ZEB2, preventing these factors from inhibiting E-cadherin expression. Maintaining high levels of E-cadherin prevents cell differentiation by preventing the promotion of cell migration and invasion via EMT (Adam et al., 2009; Korpal et al., 2008).
[015] Assim como os microRNAs da família miR-200, os microRNAs miR-203 e miR-205 também são reguladores importantes do processo de transição epitélio-mesênquima, sendo responsáveis pela manutenção do fenótipo epitelial. Recentemente, o miR-203 também foi associado a esta plasticidade epitelial (Moes et al., 2012). Este microRNA, localizado no cromossomo 14, tem como alvo SNAI1 , outro conhecido repressor transcricional da E-caderina. O miR-203 já foi observado hipoexpresso em linhagens celulares de câncer de mama e de próstata, contribuindo, assim, para a migração e invasão de células tumorais (Moes et al., 2012). Este microRNA também apresenta expressão diferenciada entre tumores primários e metastáticos (Baffa et al., 2009) e sua hipoexpressão mostrou-se associada à progressão de lesões displásicas para tumores de esôfago (Wu et al., 2013). [015] Like miR-200 family microRNAs, miR-203 and miR-205 microRNAs are also important regulators of the epithelial-mesenchymal transition process and are responsible for maintaining the epithelial phenotype. Recently, miR-203 has also been associated with this epithelial plasticity (Moes et al., 2012). This microRNA, located on chromosome 14, targets SNAI1, another known E-cadherin transcriptional repressor. MiR-203 has been observed to be hypoexpressed in breast and prostate cancer cell lines, thus contributing to tumor cell migration and invasion (Moes et al., 2012). This microRNA also presents differentiated expression between primary and metastatic tumors (Baffa et al., 2009) and its hypoexpression has been associated with the progression of dysplastic lesions to esophageal tumors (Wu et al., 2013).
[016] O miR-205 encontra-se no cromossomo 1 e tem como alvo ZEB2, modulando a invasão e migração celular (Gregory et al., 2008a). Em linhagens celulares de esôfago, a expressão deste microRNA é alta, mas, ao se tornar invasiva e migratória, via EMT, estas células tem a expressão do miR-205 diminuída (Matsushima et al., 201 1 ). Este microRNA foi descrito como hipoexpresso em câncer de próstata e esta hipoexpressão mostrou correlação com o tamanho dos tumores, maior escore de Gleason, ocorrência de metástases e menor sobrevida global (Hagman et al., 2013; Verdoodt et al., 2013). Este microRNA também mostrou-se hipoexpresso em câncer de mama (Baffa et al., 2009) e de bexiga (Wiklund et al., 201 1 a). MiR-205 is located on chromosome 1 and targets ZEB2, modulating cell invasion and migration (Gregory et al., 2008a). In esophageal cell lines, the expression of this microRNA is high, but by becoming invasive and migratory via EMT, these cells have decreased miR-205 expression (Matsushima et al., 201 1). This microRNA was described as hypoexpressed in prostate cancer and this hypoexpression was correlated with tumor size, higher Gleason score, metastasis occurrence and shorter overall survival (Hagman et al., 2013; Verdoodt et al., 2013). This microRNA was also hypoexpressed in breast (Baffa et al., 2009) and bladder cancer (Wiklund et al., 201 1a).
[017] Apesar do importante papel destes microRNAs na carcinogênese de diversos tumores, especificamente com relação ao processo de formação de metástases, poucos trabalhos avaliaram sua expressão em CECP. [017] Despite the important role of these microRNAs in the carcinogenesis of various tumors, specifically in relation to the process of metastasis formation, few studies have evaluated their expression in CPB.
[018] Um estudo identificou que a expressão deste microRNA é restrita a epitélio escamoso e analisou a expressão deste microRNA em 8 amostras de linfonodos de pacientes com CECP portando macrometástases, encontrando elevada expressão em relação a linfonodos não-metastáticos (Fletcher et al., 2008). Kozaki e colaboradores (Kozaki et al., 2008) observaram uma associação entre a hipoexpressão de miR-203 e a hipermetilação deste microRNA em amostras de CECP. [019] Como pode ser verificado diversos documentos de patentes e publicações científicas têm explorado o papel dos microRNAs como marcadores, mas em nenhum deles foi antecipado o método de detecção de células metastáticas em linfonodos de pacientes com carcinoma epidermóide de cabeça e pescoço que será a seguir revelado, [018] One study identified that the expression of this microRNA is restricted to squamous epithelium and analyzed the expression of this microRNA in 8 lymph node samples from CPEC patients with macrometastases, finding high expression compared to non-metastatic lymph nodes (Fletcher et al., 2008). Kozaki et al. (Kozaki et al., 2008) observed an association between miR-203 hypoexpression and hypermethylation of this microRNA in CECP samples. As can be seen several patent documents and scientific publications have explored the role of microRNAs as markers, but none of them anticipated the method of detecting metastatic cells in lymph nodes of patients with head and neck squamous cell carcinoma. follow revealed,
Sumário da Invenção Summary of the Invention
[020] A presente invenção tem como objetivo um teste para o diagnóstico de metástase cervical em pacientes com CECP através da avaliação do nível de expressão dos microRNAs miR-203 e miR-205. O teste pode ser aplicado em amostras coletadas utilizando-se várias abordagens em diferentes momentos, como no pré-tratamento através do diagnóstico utilizando biópsias de PAAF, no intra-operatório através de biópsia do linfonodo sentinela e após a cirurgia como uma ferramenta auxiliar para a avaliação histopatológica de linfonodos ressecados durante a cirurgia, ajudando na escolha do tratamento destes pacientes desde o tratamento inicial até a terapia adjuvante. [021 ] A presente invenção ensina então um método de detecção de metástases utilizando linfonodos inteiros ou partes de linfonodos ressecados durante a cirurgia e amostras coletadas por punções aspirativas por agulha fina (PAAF) de linfonodos de pacientes com CECP, utilizando para essa detecção os microRNAs miR-203 e miR-205. De forma mais específica, a presente invenção ensina um método de detecção de células metastáticas em linfonodos caracterizado por compreender as etapas de: a) Processamento dos ditos linfonodos em blocos de parafina ou linfonodos frescos ou ainda amostras de biópsia coletadas por punção (PAAF) de linfonodos; [020] The present invention aims at a test for the diagnosis of cervical metastasis in patients with CPB by assessing the level of miR-203 and miR-205 microRNA expression. The test can be applied to samples collected using various approaches at different times, such as pre-treatment through diagnosis using FNAB biopsies, intraoperatively through sentinel lymph node biopsy, and after surgery as an auxiliary tool for histopathological evaluation of resected lymph nodes during surgery, helping to choose the treatment of these patients from initial treatment to adjuvant therapy. [021] The present invention then teaches a method of detecting metastases using whole lymph nodes or parts of lymph nodes resected during surgery and specimens collected by lymph node fine needle aspiration (FNA) punctures from patients with CPBP. detection of miR-203 and miR-205 microRNAs. More specifically, the present invention teaches a method for detecting lymph node metastatic cells comprising the steps of: a) processing said lymph nodes in paraffin blocks or fresh lymph nodes or puncture-collected biopsy specimens (FNA) lymph nodes;
b) Extração de RNA;  b) RNA extraction;
c) Quantificação do RNA total extraído;  c) Quantification of total extracted RNA;
d) Síntese de cDNA dos RNAs endógenos U6 e U47 e dos microRNAs miR-203 e miR-205;  d) cDNA synthesis of endogenous U6 and U47 RNAs and miR-203 and miR-205 microRNAs;
e) Avaliação do nível de expressão dos microRNAs miR-203 e miR-205 nas amostras de RNA total por meio de análises de PCR em tempo real;  e) Evaluation of miR-203 and miR-205 microRNA expression level in total RNA samples by real-time PCR analysis;
f) Análise dos dados de expressão e diagnóstico.  f) Analysis of expression and diagnosis data.
Descrição Detalhada da Invenção Obtenção do material para análise molecular Detailed Description of the Invention Obtaining Molecular Analysis Material
[022] A presente invenção descreve a análise da expressão dos microRNAs miR-203 e miR-205 como método de diagnóstico de metástases em amostras de linfonodos obtidas a partir de cirurgia e amostras de biópsia por punção aspirativa de linfonodos de pacientes com CECP. [022] The present invention describes the analysis of miR-203 and miR-205 microRNA expression as a method of diagnosing metastasis in lymph node samples obtained from surgery and lymph node aspiration biopsy samples from patients with CPB.
[023] Na análise de rotina de amostras de linfonodo obtidas a partir do esvaziamento cervical, ou seja, da ressecção cirúrgica dos linfonodos cervicais deste paciente, todos os linfonodos ressecados são processados, incluídos em parafina e avaliados no setor de patologia de cada serviço. A avaliação histopatológica de rotina ocorre da seguinte forma: após a inclusão em parafina, os blocos contendo os linfonodos a serem avaliados são submetidos a cortes no seu eixo central e as lâminas contendo os cortes são submetidas a coloração por hematoxilina & eosina e avaliadas por um patologista para confirmação do diagnóstico através da caracterização dos componentes celulares presentes nos cortes. A presente invenção traz a possibilidade de avaliar cortes distribuídos ao longo do linfonodo ou de avaliar o linfonodo inteiro a partir de técnicas moleculares. Assim, blocos de parafina contendo os linfonodos ressecados são submetidos a cortes em diversos níveis ou a cortes em toda a extensão do bloco, refletindo a análise do linfonodo inteiro. Estes cortes são coletados em tubos coletores, ou colocados em lâminas. Para a extração do material a partir de lâminas, a região de interesse precisa ser raspada com a ajuda de um bisturi ou agulha e adicionada a um tubo coletor. De forma alternativa, a extração também pode ser feita a partir de material fresco, ou seja, fragmentos ou linfonodos inteiros ressecados são coletados em criotubos (tubos resistentes ao armazenamento a baixas temperaturas) e armazenados a temperaturas inferiores a -80 °C até o momento da extração. O RNA avaliado pela análise molecular é obtido através do processamento destes materiais coletados. [023] In routine analysis of lymph node specimens obtained from neck dissection, ie, surgical resection of lymph nodes In this patient's cervical arteries, all resected lymph nodes are processed, embedded in paraffin, and evaluated in the pathology department of each facility. Routine histopathological evaluation is as follows: after paraffin embedding, the blocks containing the lymph nodes to be evaluated are sectioned on their central axis and the sections containing the sections are stained with hematoxylin & eosin and evaluated by a pathologist to confirm the diagnosis by characterizing the cellular components present in the sections. The present invention provides the possibility to evaluate sections distributed throughout the lymph node or to evaluate the entire lymph node by molecular techniques. Thus, paraffin blocks containing the resected lymph nodes are cut at various levels or cut the entire length of the block, reflecting the analysis of the entire lymph node. These cuts are collected in collecting tubes, or placed on slides. For extraction of material from blades, the region of interest needs to be scraped off with the help of a scalpel or needle and added to a collecting tube. Alternatively, extraction can also be done from fresh material, ie, dried out whole fragments or lymph nodes are collected in cryotubes (low temperature storage resistant tubes) and stored at temperatures below -80 ° C so far. of extraction. RNA assessed by molecular analysis is obtained by processing these collected materials.
[024] Para a avaliação das amostras de biópsias a partir de punção, linfonodos cervicais suspeitos de pacientes com CECP são puncionados utilizando-se um citoaspirador de Valeri, de acordo com a rotina dos ambulatórios de punção. O material coletado é utilizado na confecção de lâminas de esfregaço que são coradas com H&E e avaliadas por um citopatologista para determinação do diagnóstico através da caracterização dos componentes celulares presentes na lâmina. A presente invenção traz a possibilidade de avaliar o restante do material coletado que permanece na agulha após a preparação das lâminas de esfregaço, por técnicas moleculares. Assim, a agulha coletora conectada à seringa deve ser lavada em solução contendo EDTA e cloreto de sódio e o material coletado deve ser congelado em nitrogénio líquido e armazenado em ultrafreezer a temperaturas inferiores a - 80°C até o momento da extração do RNA. [024] For the evaluation of biopsy specimens from puncture, suspected cervical lymph nodes from patients with CPB are punctured using a Valeri cytoaspirator according to routine outpatient clinics. puncture. The collected material is used to make smear slides that are stained with H&E and evaluated by a cytopathologist to determine the diagnosis by characterizing the cellular components present in the slide. The present invention provides the possibility to evaluate the remaining collected material that remains in the needle after preparation of the smear slides by molecular techniques. Thus, the collecting needle connected to the syringe should be washed in a solution containing EDTA and sodium chloride and the collected material should be frozen in liquid nitrogen and stored in an ultrafreezer at temperatures below -80 ° C until RNA extraction.
Extração de RNA e Síntese de cDNA RNA Extraction and cDNA Synthesis
[025] A extração do RNA a partir de fragmentos ou linfonodos inteiros congelados a fresco ou ainda linfonodos incluídos em parafina deve ser realizada utilizando protocolos e kits específicos para a obtenção de RNA total seguindo as recomendações do fabricante. De forma geral, para as amostras frescas estes protocolos/kits iniciam com o processamento desta amostra utilizando um homogeneizador de tecido. Para as amostras incluídas em parafina é realizado um passo de remoção da parafina (desparafinização) dos cortes contendo as amostras de interesse, utilizando reagentes como o Xilol. A extração de ácidos nucléicos ocorre através de digestão com proteinase K (peptidase responsável por degradar proteínas celulares) e detergentes (que irão degradar lipídeos). Em seguida, amostras são tratadas com DNase e purificadas utilizando-se reagentes químicos e/ou colunas que permitem a obtenção de RNA total livre de contaminações por DNA, proteínas e reagentes químicos. O RNA total obtido deve ser armazenado em freezer a temperatura inferior a -80°C até o momento de utilização. [025] RNA extraction from fresh frozen whole or paraffin embedded lymph nodes or fragments should be performed using specific protocols and kits for obtaining total RNA following the manufacturer's recommendations. Generally, for fresh samples these protocols / kits begin with processing this sample using a tissue homogenizer. For paraffin-embedded samples a paraffin removal (deparaffinization) step is performed from the sections containing the samples of interest using reagents such as Xylol. The extraction of nucleic acids occurs through digestion with proteinase K (peptidase responsible for degrading cellular proteins) and detergents (which will degrade lipids). Samples are then treated with DNase and purified using chemical reagents and / or columns allowing total RNA free from contamination by DNA, proteins and reagents. chemical Total RNA obtained should be stored in a freezer at a temperature below -80 ° C until use.
[026] Para a extração do RNA das amostras de PAAF, devem ser utilizados reagentes específicos para a extração de RNA que contenham fenol e isotiocianato de guanidina. [026] For RNA extraction from FNAB samples, specific RNA extraction reagents that contain phenol and guanidine isothiocyanate should be used.
[027] Após a obtenção do RNA total extraído a partir das amostras a serem analisadas, esse deve ser quantificado e cerca de 10 nanogramas deve ser utilizado na reação de transcrição reversa (RT). Esta reação se baseia na síntese de uma fita de DNA complementar (cDNA) ao RNA de interesse. Na presente invenção, para que possa ser diagnosticada a presença ou não de metástases, é necessária a síntese de cDNA dos RNAs endógenos U6 e U47 e dos microRNAs de interesse miR-203 e miR-205. Ou seja, para cada amostra a ser avaliada são necessárias 4 reações de RT. [027] After obtaining the total RNA extracted from the samples to be analyzed, it should be quantified and about 10 nanograms should be used in the reverse transcription (RT) reaction. This reaction is based on the synthesis of a complementary DNA strand (cDNA) to the RNA of interest. In the present invention, in order to diagnose the presence or absence of metastases, cDNA synthesis of endogenous U6 and U47 RNAs and microRNAs of interest miR-203 and miR-205 is required. That is, for each sample to be evaluated 4 RT reactions are required.
[028] Para as reações de RT, são utilizados primers específicos para cada microRNA ou RNA endógeno e a enzima transcriptase reversa, além de outros reagentes necessários para a reação. [028] For RT reactions, specific primers are used for each endogenous microRNA or RNA and the reverse transcriptase enzyme, as well as other reagents required for the reaction.
Expressão dos microRNAs MicroRNA Expression
[029] A presente invenção descreve a utilização da detecção do nível de expressão dos microRNAs miR-203 e miR-205 como ferramenta de diagnóstico de metástases em linfonodos de pacientes com CECP. A avaliação do nível de expressão dos microRNAs nas amostras de RNA total obtidas a partir de amostras de linfonodos ressecados cirurgicamente e de PAAF de linfonodos suspeitos deve ser feita utilizando análises de PCR em tempo Real. Diversos equipamentos estão disponíveis e podem ser utilizados para a realização destas análises. [029] The present invention describes the use of detection of miR-203 and miR-205 microRNA expression level as a diagnostic tool for lymph node metastasis of patients with CPB. The evaluation of microRNA expression level in total RNA samples obtained from surgically resected lymph node samples and from FNA of suspicious lymph nodes should be performed using real-time PCR analysis. Various equipment is available and can be used to perform these analyzes.
[030] Para cada amostra a ser avaliada, deve ser feita uma reação de PCR por microRNA ou RNA endógeno, ou seja, são 4 reações de PCR em tempo real para cada amostra. [030] For each sample to be evaluated, a microRNA or endogenous RNA PCR reaction should be performed, ie there are 4 real-time PCR reactions for each sample.
[031 ] Para as reações de PCR, é necessária a adição de reagentes e de primers e sondas específicos para a amplificação do cDNA obtido através da referida reação de transcrição reversa. As reações devem ser realizadas em triplicata em um equipamento de PCR em tempo real, que utiliza ciclagens de temperaturas por períodos específicos para a reação de amplificação (de acordo com o recomendado para os reagentes, primers e sondas utilizados) e é capaz de detectar a presença do cDNA de interesse e fornecer dados que permitem a quantificação da expressão destes nas amostras avaliadas. [032] Para cada amostra avaliada, quando há a presença do microRNA de interesse, os primers e sonda são capazes de se ligar ao microRNA e iniciar a amplificação. Neste caso, ocorre a formação de uma curva de amplificação que permite a quantificação da expressão através da determinação para cada amostra de um valor de Ct (Threshold Cycle), ou seja, do ciclo da PCR no qual a fluorescência gerada atinge um ponto pré-estabelecido (threshold) da fase logarítmica da reação. Quando a amostra não contém o microRNA de interesse, os primers e sonda não se ligam e a reação de amplificação não ocorre. [033] Para a análise dos dados de expressão obtidos de cada microRNA nas amostras de linfonodos e de PAAF de linfonodos suspeitas, deve ser utilizado o método 2'àACt ou método comparativo de Ct. Este método se baseia na normalização da expressão dos microRNAs de interesse (neste caso, miR- 203 e miR-205) pela expressão de RNAs normalizadores (U6 e U47) na mesma amostra biológica, e as mudanças na expressão dos microRNAs são calculadas baseadas nas diferenças entre amostras referência e amostras experimentais. Para a presente invenção, o grupo referência utilizado foi composto por amostras de linfonodos não metastáticos. [034] Desta forma, o valor inferido à ACt equivale à diferença entre o valor das médias dos Cts dos microRNAs de interesse, miR-203 e miR-205 (Ct(miR-203) e Ct(miR-205), respectivamente), e o valor da média dos Cts dos RNAs normalizadores U6 e U47 (Ct(U6> e Ct(U47>, respectivamente) obtidos através da avaliação da expressão destes marcadores nas amostras suspeitas (no caso, linfonodos e PAAF de linfonodos suspeitos) e do grupo de amostras referência (linfonodos não metastáticos). [031] For PCR reactions, the addition of specific reagents and primers and probes is required for the amplification of cDNA obtained by said reverse transcription reaction. Reactions should be performed in triplicate in a real-time PCR equipment that uses temperature cycling for specific periods for the amplification reaction (as recommended for the reagents, primers and probes used) and is capable of detecting presence of the cDNA of interest and provide data that allow the quantification of their expression in the evaluated samples. For each sample evaluated, when there is the presence of the microRNA of interest, the primers and probe are able to bind to the microRNA and initiate amplification. In this case, an amplification curve occurs which allows the expression to be quantified by determining for each sample a Ct (Threshold Cycle) value, ie the PCR cycle in which the generated fluorescence reaches a pre-set point. threshold of the log phase of the reaction. When the sample does not contain the microRNA of interest, the primers and probe do not bind and the amplification reaction does not occur. For the analysis of expression data obtained from each microRNA in lymph node and FNA samples from suspected lymph nodes, the 2 'àACt method or the Ct comparative method should be used. This method is based on the normalization of expression of the microRNAs of interest (in this case miR-203 and miR-205) by the expression of normalizing RNAs (U6 and U47) in the same biological sample, and changes in microRNA expression are calculated based on the differences between reference samples and experimental samples. For the present invention, the reference group used was non-metastatic lymph node samples. Thus, the value inferred to ACt is equivalent to the difference between the mean value of the Cts of the microRNAs of interest, miR-203 and miR-205 (Ct (miR-203) and Ct (miR-205), respectively). , and the mean Cts value of the U6 and U47 normalizing RNAs (Ct (U6> and Ct (U47>, respectively)) obtained by evaluating the expression of these markers in the suspect samples (in this case, lymph nodes and FNA of suspected lymph nodes) and reference sample group (non-metastatic lymph nodes).
[035] Para as amostras suspeitas: [035] For suspect samples:
ACt(miR-203 amostra) = Ct(miR-203 amostra) - [Ct(U6 amostra) + Ct(U47 amostra)]/2 ACt(miR-205 amostra) = Ct(miR-205 amostra) - [Ct(U6 amostra) + Ct(U47 amostra)]/2 [036] Os valores de ACt para as amostras referência foram obtidos a partir da análise de linfonodos não metastáticos e padronizou-se que os valores adequados a serem utilizados nos cálculos são os seguintes: ACt(miR-203 referência) =1 1 ,156545; ACt(miR-205 referência) = 10,688645. [037] Já o cálculo ΔΔθί envolve a subtração entre o valor de ACt(amostra) de cada amostra experimental (no caso, linfonodos e PAAF de linfonodos suspeitos) e o valor médio de ACt(referência) do grupo de amostras referência (linfonodos não metastáticos), ou seja, ACt(miR -203 referência) =1 1 , 156545; ACt(miR- 205 referência) = 10,688645.. ACt (miR-203 sample) = Ct (miR-203 sample) - [Ct (U6 sample) + Ct (U47 sample)] / 2 ACt (miR-205 sample) = Ct (miR-205 sample) - [Ct ( U6 sample) + Ct (U47 sample)] / 2 [036] The ACt values for the reference samples were obtained from non-metastatic lymph node analysis and it was standardized that the appropriate values to be used in the calculations are as follows: ACt (miR-203 reference) = 11, 156545; ACt (miR-205 reference) = 10.688645. [037] The ΔΔθί calculation involves the subtraction between the ACt value (sample) of each experimental sample (in this case lymph nodes and FNA of suspected lymph nodes) and the mean ACt value (reference) of the reference sample group (non-lymph nodes). metastatic), ie ACt (miR-203 reference) = 11, 156545; ACt (miR-205 reference) = 10.688645 ..
[038] Para miR-203: [038] For miR-203:
AACt(miR-203) = ACt(miR-203 amostra) - ACt(miR-203 referência) AACt(miR-203) = ACt(miR-203 amostra)—1 1 ,156545 AACt (miR-203) = ACt (miR-203 sample) - ACt (miR-203 reference) AACt (miR-203) = ACt (miR-203 sample) —11,1656545
[039] Para miR-205: AACt(miR-205) = ACt(miR-205 amostra) - ACt(miR-205 referência) AACt(miR-205) = ACt(miR-205 amostra) - 10,688645 For miR-205: AACt (miR-205) = ACt (miR-205 sample) - ACt (miR-205 reference) AACt (miR-205) = ACt (miR-205 sample) - 10.688645
[040] Assim, os valores de Ct já normalizados dos microRNAs miR-203 e miR- 205 obtidos após reação de PCR em tempo real nas amostras suspeitas será sempre comparado ao valor de Ct normalizado de um grupo de linfonodos não-metastáticos já avaliados previamente utilizado como referência. O resultado é expresso em fold-change ou o número de vezes o qual a expressão do microRNA de interesse está aumentada nas amostras suspeitas em relação ao grupo de amostras referências. Thus, the already normalized Ct values of the miR-203 and miR-205 microRNAs obtained after real-time PCR reaction in the suspect samples will always be compared to the normalized Ct value of a previously evaluated non-metastatic lymph node group. used as a reference. The result is expressed in fold-change or the number of times the expression of the microRNA of interest is increased in the suspect samples relative to the reference sample group.
[041 ] Para as amostras de linfonodo (incluídas em parafina ou frescas) deve ser utilizado um valor de corte de 2 vezes, para considerar uma amostra positiva para a presença de células metastáticas, ou seja, um valor de expressão de pelo menos 2 vezes, significa que o linfonodo suspeito avaliado contém células metastáticas. Para as amostras de PAAF, deve-se utilizar um corte de 10 vezes para considerar uma amostra metastática. [041] For lymph node samples (paraffin-embedded or fresh) a cut-off value of 2-fold should be used to consider a positive sample for the presence of metastatic cells ie At least 2-fold expression means that the suspected lymph node evaluated contains metastatic cells. For FNAB samples, a 10-fold cutoff should be used to consider a metastatic sample.
[042] Estes valores de corte foram validados após diversos cálculos estatísticos de sensibilidade, especificidade, acurácia e construção de curvas ROC e determinação do valor de AUC (área abaixo da curva), que comprovaram que sua utilização era adequada para a correta classificação de amostras de linfonodo ou de PAAF de linfonodos em metastáticas e não metastáticas. Para que uma amostra seja considerada metastática, basta que o valor de fold-change de pelo menos um dos microRNAs (miR-203 ou miR- 205) esteja acima do corte de 2 vezes para as amostras de linfonodo ou 10 vezes para as amostras de punção. [042] These cut-off values were validated following various statistical calculations of sensitivity, specificity, accuracy and construction of ROC curves and determination of AUC value (area below the curve), which proved that their use was adequate for the correct classification of samples. lymph node or lymph node FNA in metastatic and non-metastatic. For a sample to be considered metastatic, it is sufficient that the fold-change value of at least one of the microRNAs (miR-203 or miR-205) is above the 2-fold cut for lymph node samples or 10 times for the lymph node samples. puncture.
Breve descrição das figuras Brief Description of the Figures
[043] Figura 1 - Representação esquemática do processamento dos linfonodos em cortes para análise molecular: [043] Figure 1 - Schematic representation of lymph node processing in sections for molecular analysis:
A) 3 a 5 cortes de 5 a 10 m são realizados e coletados a cada 50 pm; A) 3 to 5 cuts of 5 to 10 m are made and collected every 50 pm;
B) Cortes de 5 a 10 m são realizados por toda a extensão do linfonodo e coletados. B) Cuts of 5 to 10 m are performed over the entire lymph node and collected.
Exemplo Processamento do material para análise molecular [044] Para o processamento das amostras de linfonodo incluídas em parafina é possível a avaliação de cortes ou do linfonodo inteiro. Para a análise de cortes, uma opção é submeter o bloco (1 ) a 3 a 5 cortes histológicos (2) seriados de 5 a 10 pm de espessura, distribuídos em aproximadamente 6 níveis com intervalos de 50 pm de distância (3). Já para a análise do linfonodo inteiro, todo o bloco (4) pode ser submetido a cortes de 5 a 10 pm (5). Em ambos os casos, os cortes obtidos são coletados em tubos de 1 ,5 ml para posterior extração. Example Material Processing for Molecular Analysis [044] For the processing of paraffin-embedded lymph node samples it is possible to evaluate sections or the entire lymph node. For slice analysis, one option is to subject the block (1) to 3 to 5 serial histological sections (2) of 5 to 10 pm in thickness, distributed at approximately 6 levels at 50 pm intervals (3). For the analysis of the entire lymph node, the whole block (4) can be submitted to 5 to 10 pm cuts (5). In both cases, the obtained cuts are collected in 1.5 ml tubes for further extraction.
[045] Para a coleta das amostras de biópsia por punção, a agulha coletora conectada à seringa pode ser lavada em 100 a 400μΙ de solução contendo EDTA e cloreto de sódio. A solução de coleta pode ser preparada da seguinte forma: preencher um tubo de coleta de sangue contendo EDTA com cloreto de sódio (soro fisiológico) 0.9% , misturar utilizando um agitador de tubos tipo vortex e preparar alíquotas de 100 a 400μΙ da solução de coleta em criotubos (tubos resistentes ao armazenamento a baixas temperaturas). O material coletado é congelado em nitrogénio líquido e armazenado em ultrafreezer a temperaturas inferiores a -80°C até o momento da extração do RNA. [045] For puncture biopsy specimen collection, the collecting needle attached to the syringe can be washed in 100 to 400μΙ of solution containing EDTA and sodium chloride. The collection solution can be prepared as follows: Fill a blood collection tube containing EDTA with 0.9% sodium chloride (saline), mix using a vortex tube shaker and prepare 100 to 400μΙ aliquots of the collection solution. in cryotubes (tubes resistant to storage at low temperatures). The collected material is frozen in liquid nitrogen and stored in an ultrafreezer at temperatures below -80 ° C until RNA extraction.
Extração de RNA e Síntese de cDNA RNA Extraction and cDNA Synthesis
[046] Para a realização da presente invenção sugere-se a utilização do kit Recoverall Total Nucleic Acid Isolation de acordo com as recomendações do fabricante para a extração de RNA a partir de material parafinado. A única modificação deve ser feita na fase de desparafinização para o material obtido a partir do linfonodo inteiro. Por conta da grande quantidade de parafina nestas amostras, 3 lavagens com Xilol (ao invés de apenas uma, conforme recomendando no kit). Para a extração e purificação do RNA, qualquer outro kit capaz de extrair RNA total de amostras incluídas em parafina pode ser utilizado, sempre seguindo as recomendações do fabricante. [047] Para a extração de RNA a partir das amostras de PAAF, o reagente Trizol LS pode ser utilizado com algumas modificações. Este reagente foi escolhido por ter sido formulado especificamente para a obtenção de RNA a partir de amostras líquidas. No entanto, outros reagentes com mesma composição também podem ser utilizados. A extração iniciou-se com a adição de Trizol LS ao tubo contendo o material da punção, homogeneização em agitador vortex e incubação a temperatura ambiente. Em seguida foi adicionado clorofórmio, o material foi homogeneizado e incubado à temperatura ambiente. Os tubos foram então centrifugados e a fase aquosa sobrenadante contendo o RNA foi transferida para um novo tubo ao qual foi adicionado acetato de sódio à concentração 3M e pH 5.2, glicogênio à concentração de 20mg/ml e isopropanol. O material foi precipitado a -20°C e centrifugado para recuperação do pellet. O sobrenadante foi descartado por inversão e o pellet foi lavado com etanol 70% gelado. Após nova centrifugação, o sobrenadante foi descartado e o pellet foi seco à temperatura ambiente por 15 a 20 minutos. O pellet foi ressuspendido em água livre de RNase, e o RNA total obtido foi armazenado a -80°C. [046] For the purpose of the present invention, the use of the Recoverall Total Nucleic Acid Isolation Kit according to the manufacturer's recommendations for the extraction of RNA from paraffin material is suggested. The only modification should be made in the deparaffinization phase for material obtained from the entire lymph node. Because of the large amount of paraffin In these samples, 3 washes with Xylol (instead of just one, as recommended in the kit). For RNA extraction and purification, any other kit capable of extracting total RNA from paraffin-embedded samples may be used, always following the manufacturer's recommendations. [047] For RNA extraction from FNAB samples, Trizol LS reagent may be used with some modifications. This reagent was chosen because it was specifically formulated to obtain RNA from liquid samples. However, other reagents of the same composition may also be used. Extraction began with the addition of Trizol LS to the tube containing the puncture material, homogenization in vortex shaker and incubation at room temperature. Then chloroform was added, the material was homogenized and incubated at room temperature. The tubes were then centrifuged and the supernatant aqueous phase containing the RNA was transferred to a new tube to which sodium acetate at 3M concentration and pH 5.2, glycogen at 20mg / ml concentration and isopropanol were added. The material was precipitated at -20 ° C and centrifuged for pellet recovery. The supernatant was discarded by inversion and the pellet was washed with 70% ice cold ethanol. After further centrifugation, the supernatant was discarded and the pellet was dried at room temperature for 15 to 20 minutes. The pellet was resuspended in RNase free water, and the total RNA obtained was stored at -80 ° C.
[048] Diversos kits estão comercialmente disponíveis para a fase de síntese do cDNA. Na presente invenção, esta reação foi realizada utilizando o kit Taqman microRNA Kit e ensaios TaqMan MicroRNA Assays que contêm sequências de primers específicas para a reação de transcrição reversa dos microRNAs miR-203 e miR-205 e para os RNA endógenos U6 e U47. [048] Several kits are commercially available for the cDNA synthesis phase. In the present invention, this reaction was performed using the Taqman microRNA Kit and TaqMan MicroRNA Assays containing specific primer sequences for the miR-203 and miR-205 reverse transcriptional reaction and endogenous RNAs U6 and U47.
Expressão dos microRNAs MicroRNA Expression
[049] Diversas opções de reagentes, primers e sondas para a detecção dos microRNAs miR-203 e miR-205 e dos RNAs endógenos U6 e U47 por PCR em tempo Real estão comercialmente disponíveis. Na presente invenção, estas reações foram feitas utilizando-se o TaqMan Universal PCR Master Mix 2X, solução que contém todos os reagentes necessários para a reação de amplificação (como DNA polimerase, tampão, magnésio, desoxirribonucleotídeos) e os ensaios TaqMan MicroRNA Assay 20X que contêm primers e sonda específicos para cada um dos microRNAs e RNAs avaliados. Several options for reagents, primers and probes for detection of miR-203 and miR-205 microRNAs and endogenous U6 and U47 RNAs by Real Time PCR are commercially available. In the present invention, these reactions were performed using TaqMan Universal PCR Master Mix 2X, a solution containing all reagents necessary for the amplification reaction (such as DNA polymerase, buffer, magnesium, deoxyribonucleotides) and TaqMan MicroRNA Assay 20X assays. contain primers and probe specific for each of the evaluated microRNAs and RNAs.
[050] Os exemplos acima não devem ser interpretados como um limitante para invenção, servindo apenas para ilustrar a forma preferível de realização da invenção. O alcance da proteção da invenção será interpretado com base nas reivindicações descritas a seguir. [050] The above examples are not to be construed as limiting the invention, but merely to illustrate the preferred embodiment of the invention. The scope of the protection of the invention will be interpreted based on the claims described below.

Claims

Reivindicações Claims
1 . Um método de detecção de células metastáticas em linfonodos caracterizado por compreender as etapas de: 1 . A method for detecting lymph node metastatic cells, comprising the steps of:
a) Processamento dos ditos linfonodos em blocos de parafina ou linfonodos frescos ou ainda amostras de biópsia coletadas por punção (PAAF) de linfonodos;  (a) processing said lymph nodes into paraffin blocks or fresh lymph nodes or lymph node puncture-collected biopsy specimens (FNA);
b) Extração de RNA;  b) RNA extraction;
c) Quantificação do RNA total extraído;  c) Quantification of total extracted RNA;
d) Síntese de cDNA dos RNAs endógenos U6 e U47 e dos microRNAs miR-203 e miR-205;  d) cDNA synthesis of endogenous U6 and U47 RNAs and miR-203 and miR-205 microRNAs;
e) Avaliação do nível de expressão dos microRNAs miR-203 e miR-205 nas amostras de RNA total por meio de análises de PCR em tempo real;  e) Evaluation of miR-203 and miR-205 microRNA expression level in total RNA samples by real-time PCR analysis;
f) Análise dos dados de expressão e diagnóstico.  f) Analysis of expression and diagnosis data.
2. O método de detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato dos blocos de parafina contendo os ditos linfonodos ressecados serem submetidos a 3 a 5 cortes histológicos seriados de 5 a 10 pm de espessura, distribuídos em preferivelmente 6 níveis com intervalos preferíveis de 50 pm de distância. The method of detecting lymph node metastatic cells according to claim 1, wherein the paraffin blocks containing said resected lymph nodes are subjected to 3 to 5 serial histological sections of 5 to 10 pm thickness, distributed in preferably 6 levels with preferable intervals of 50 pm apart.
3. O método de detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato de que na análise de linfonodos inteiros todo o bloco é submetido a cortes de 5 a 10 pm. The method of detecting lymph node metastatic cells according to claim 1, characterized in that in whole lymph node analysis the whole block is subjected to 5 to 10 pm sections.
4. O método de detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato do processamento ser feito a partir linfonodos fresco, preferivelmente, fragmentos ou linfonodos inteiros ressecados que são coletados em criotubos e armazenados a temperaturas inferiores a -80°C até o momento da extração do RNA. The method of detecting lymph node metastatic cells according to claim 1, characterized in that the processing is done from fresh lymph nodes, preferably resected fragments or whole lymph nodes that are collected in cryotubes and stored at temperatures below - 80 ° C until RNA extraction.
5. O método de detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato que a coleta das amostras de biópsia por punção é realizada por meio de uma agulha coletora conectada à seringa que é lavada em 100 a 400μΙ de solução contendo EDTA e cloreto de sódio, o material coletado é congelado em nitrogénio líquido e armazenado a temperaturas inferiores a -80°C até o momento da extração do RNA. The method of detecting lymph node metastatic cells according to claim 1, characterized in that the collection of biopsy specimens by puncture is performed by means of a collecting needle connected to the syringe which is flushed at 100 to 400μΙ. solution containing EDTA and sodium chloride, the collected material is frozen in liquid nitrogen and stored at temperatures below -80 ° C until the time of RNA extraction.
6. O método de detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato de que na extração de RNA para os linfonodos em blocos de parafina é realizado de um a três passos de desparafinização dos cortes utilizando-se Xilol. The method of detecting lymph node metastatic cells according to claim 1, characterized in that in the extraction of RNA for paraffin block lymph nodes it is One to three steps of deparaffinization of the cuts were performed using Xylol.
7. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato de que na extração do RNA das amostras deve-se utilizar, metodologias, reagentes e kits apropriados para a dita extração. The method of detecting lymph node metastatic cells according to claim 1, characterized in that the RNA extraction from the samples must use appropriate methodologies, reagents and kits for said extraction.
8. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato de que o RNA total obtido deve ser armazenado em temperatura inferior a - 80°C até o momento de utilização. The method of detecting lymph node metastatic cells according to claim 1, characterized in that the total RNA obtained must be stored below -80 ° C until the time of use.
9. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato de 1 a 10 nanogramas do RNA total extraído ser utilizado na reação de transcrição reversa. The method of detecting lymph node metastatic cells according to claim 1, wherein 1 to 10 nanograms of total extracted RNA is used in the reverse transcription reaction.
10. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato de serem utilizados kits, na etapa de síntese, que contenham reagentes e primers com sequência específicas para a reação de transcrição reversa dos microRNAs miR-203 e miR-205 e para os RNA endógenos U6 e U47. The method of detecting lymph node metastatic cells according to claim 1, characterized in that kits are used in the synthesis step containing reagents and primers with sequence specific for the miR-203 reverse transcription reaction. and miR-205 and for endogenous RNAs U6 and U47.
1 1 . O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 10, caracterizado pelo fato de que a dita síntese do cDNA ser realizada preferivelmente utilizando-se o kit Taqman microRNA e ensaios TaqMan MicroRNA Assays. 1 1. The method of detecting lymph node metastatic cells according to claim 10, wherein said cDNA synthesis is preferably performed using the Taqman microRNA kit and TaqMan MicroRNA Assays assays.
12. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 1 , caracterizado pelo fato das reações de PCR serem realizadas em triplicata em um equipamento de PCR em tempo real, que utilize ciclagens de temperaturas por períodos específicos para a reação de amplificação, de acordo com o recomendado para os reagentes, primers e sondas utilizados e seja capaz de detectar a presença do cDNA de interesse. The method of lymph node metastatic cell detection according to claim 1, characterized in that the PCR reactions are performed in triplicate in a real-time PCR equipment using temperature cycling for specific periods for the reaction of amplification as recommended for the reagents, primers and probes used and be able to detect the presence of the cDNA of interest.
13. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 12, caracterizado pelo fato de que na presença dos microRNAs miR-203 e miR-205 inicia-se o processo de amplificação, extração da curva de amplificação e quantificação da expressão por meio da determinação de um valor de Ct (Threshold Cycle) para cada amostra.  The method of detecting lymph node metastatic cells according to claim 12, characterized in that in the presence of the miR-203 and miR-205 microRNAs the process of amplification, extraction of the amplification curve and quantification of the expression by determining a Ct (Threshold Cycle) value for each sample.
14. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 13, caracterizado pelo fato de que a análise dos dados de expressão obtidos de cada microRNA nas amostras de linfonodos e de PAAF de linfonodos suspeitas é realizada por meio do método 2_ΔΔα ou método comparativo de Ct. The method of lymph node metastatic cell detection according to claim 13, characterized in that the analysis of expression data obtained from each microRNA in lymph node and FNA samples of suspected lymph nodes is performed by method 2. _ΔΔα or Ct comparative method.
15. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 14, caracterizado pelo fato de que os valores de ACt para as amostras referência foram obtidos a partir da análise de linfonodos não metastáticos, sendo seus valores preferíveis ACt(miR-203 referência)=1 1 , 156545 e ACt(miR-205 referência) = 10,688645. The method of detecting lymph node metastatic cells according to claim 14, characterized in that the ACt values for the reference samples were obtained from the analysis of non-metastatic lymph nodes, and their preferred ACt values (miR- 203 reference) = 11, 156545 and ACt (miR-205 reference) = 10.688645.
16. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 14, caracterizado pelo fato de que nas amostras de linfonodo incluídas em parafina ou frescas é utilizado um valor de expressão de pelo menos 2 vezes, sendo esse o valor de corte, para considerar uma amostra positiva para a presença de células metastáticas. The method of detecting lymph node metastatic cells according to claim 14, characterized in that at least 2-fold expression value is used for paraffin-embedded or fresh lymph node samples, which is the cut-off value. , to consider a positive sample for the presence of metastatic cells.
17. O método detecção de células metastáticas em linfonodos, de acordo com a reivindicação 14, caracterizado pelo fato de que nas amostras de PAAF é utilizado um valor de expressão de pelo menos 10 vezes, sendo esse o valor de corte, para considerar uma amostra positiva para a presença de células metastáticas. The method of detecting lymph node metastatic cells according to claim 14, wherein at least 10-fold expression value is used for FNA samples, which is the cut-off value to consider a sample. positive for the presence of metastatic cells.
18. O método detecção de células metastáticas em linfonodos, de acordo com as reivindicações 16 ou 17, caracterizado pelo fato de que para uma amostra ser considerada metastática o valor de fold- change de pelo menos um dos microRNAs (miR-203 ou miR-205) deve estar acima do referido valor de corte. The method of detecting lymph node metastatic cells according to claim 16 or 17, characterized in that for a sample the fold-change value of at least one of the microRNAs (miR-203 or miR- 205) must be above said cut-off value.
19. Uso dos microRNA miR-203 em método de diagnóstico in vitro caracterizado pelo fato de serem capazes de detectar células metastáticas em linfonodos de pacientes com carcinoma epidermóide de cabeça e pescoço. 19. Use of miR-203 microRNA in in vitro diagnostic method characterized by being able to detect metastatic cells in lymph nodes of patients with head and neck squamous cell carcinoma.
20. Uso dos microRNA miR-205 em método de diagnóstico in vitro caracterizado pelo fato de serem capazes de detectar células metastáticas em linfonodos de pacientes com carcinoma epidermóide de cabeça e pescoço. 20. Use of miR-205 microRNA in an in vitro diagnostic method characterized by being able to detect metastatic cells in lymph nodes of patients with head and neck squamous cell carcinoma.
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