WO2016205911A1 - Méthode de détection in vitro du cancer de la thyroïde chez un patient, méthode in vitro de différenciation entre le cancer de la thyroïde et le tissu thyroïdien normal ou des lésions thyroïdiennes bénignes, utilisation d'un ensemble de gènes, procédé d'obtention de données pour l'orientation du traitement du cancer de la thyroïde, kit de laboratoire et dispositif pour classer un échantillon biologique de glande thyroïde comme malin ou bénin - Google Patents

Méthode de détection in vitro du cancer de la thyroïde chez un patient, méthode in vitro de différenciation entre le cancer de la thyroïde et le tissu thyroïdien normal ou des lésions thyroïdiennes bénignes, utilisation d'un ensemble de gènes, procédé d'obtention de données pour l'orientation du traitement du cancer de la thyroïde, kit de laboratoire et dispositif pour classer un échantillon biologique de glande thyroïde comme malin ou bénin Download PDF

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Publication number
WO2016205911A1
WO2016205911A1 PCT/BR2016/050142 BR2016050142W WO2016205911A1 WO 2016205911 A1 WO2016205911 A1 WO 2016205911A1 BR 2016050142 W BR2016050142 W BR 2016050142W WO 2016205911 A1 WO2016205911 A1 WO 2016205911A1
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WIPO (PCT)
Prior art keywords
thyroid
seq
lamb3
hmga2
thyroid cancer
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PCT/BR2016/050142
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English (en)
Portuguese (pt)
Inventor
Silvia Regina ROGATTO
Luiz Paulo KOWALSKI
Mateus de Camargo BARROS FILHO
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Fundação Antonio Prudente
Universidade Estadual Paulista Julio De Mesquita Filho
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Publication of WO2016205911A1 publication Critical patent/WO2016205911A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates generally to an in vitro method of detecting thyroid cancer in a patient as well as to an in vitro method of differentiating thyroid cancer from benign or normal tissue lesions. thyroid, as well as some other aspects related to such methods.
  • the invention relates to in vitro methods which are based on the evaluation of expression of the CLDN 10, HMGA2 and LAMB3 gene set contained in a patient's tissue samples, with quantitative molecular detection technique, particularly from RT-qPCR (quantitative reverse polymerase chain reaction transcription) analysis data.
  • quantitative molecular detection technique particularly from RT-qPCR (quantitative reverse polymerase chain reaction transcription) analysis data.
  • PTC papillary thyroid cancer
  • Gene expression The process by which information contained in a gene or DNA sequence is processed into a functional gene product, such as RNA, that can be translated into proteins.
  • - reference gene gene whose expression is maintained at a constant level in both normal and tumor cells and under different experimental conditions
  • messenger RNA molecules formed from the DNA template strand of a given gene
  • primers nucleic acid segments, typically 15 to 30 nucleotides, with sequence complementary to the target DNA or RNA to be amplified by PCR (polymerase chain reaction).
  • Quantitative molecular detection technique means the use of RT-PCR, RT-qPCR, immunohistochemistry or any equivalent purpose-directed technique as known to one skilled in the art.
  • the present invention provides for the reduction of the number of unnecessary surgeries that occur as a result of inaccurate diagnoses, thereby reducing healthcare costs and morbidity for patients.
  • the invention utilizes a set of only 3 molecular markers, sufficient not only to adequately detect but also to distinguish between thyroid cancer and normal or benign thyroid tissue, as well as carcinomas. with a higher risk of lymph node metastases.
  • the invention relates to in vitro methods, namely detecting thyroid cancer in a patient and differentiating between thyroid cancer and benign lesions or normal thyroid tissue.
  • These methods comprise subjecting the patient's suspicious tissue sample to the quantitative molecular detection technique employing, through gene expression analysis, the LAMB3, CLDN 10 and HMGA2 genes, as compared to at least one reference gene.
  • suitable reference genes are those chosen from, for example, EIF2B1, PUM 1, GAPDH, HPRT1, GUSB, ACTB, B2M, HM BS, IP08, PGK1, RPLPO, TBP, TFRC. , UBC, YWHAZ, PPIA, POLR2A, CASC3, CDKN IA, CDKN1B, GADD45A, PSMC4, PES1, ABL1, ELF1, ATP6, M RPL19, POP4, RPL37A, RPL30, RPS17.
  • EIF2B1 and PUM 1 genes are used for reference.
  • the aforementioned methods of the invention are particularly directed to follicular epithelium-derived thyroid carcinomas, in particular papillary thyroid carcinomas (PTC).
  • PTC papillary thyroid carcinomas
  • the patient tissue sample to be evaluated may be fresh, fixed in paraffin blocks or frozen.
  • tissue sample is obtained via fine needle aspiration biopsy (FNAB).
  • FNAB fine needle aspiration biopsy
  • the basis of this invention was the profiling of gene expression using microarrays in 61 tissues with PTC and 13 adjacent normal (NT) tissues.
  • a reliable list of transcripts was used for inter-study validation (138 PTC / NT cases in external public databases). The results were then collectively interpreted by in silico analysis.
  • a panel of 28 transcripts were selected and evaluated using the RT-qPCR (quantitative reverse transcription polymerase chain reaction) technique, including benign thyroid lesions (BTL) and other follicular cell-derived thyroid carcinomas (OFDTC).
  • the diagnostic algorithm of the invention was developed (training set: 23 NT, 8 BTL and 86 PTC), validated (independent set: 10 NT, 140 BTL, 120 PTC and 12 OFDTC) and associated with clinical features.
  • the quantitative molecular detection technique employed used, in particular, RT-qPCR analysis to evaluate the target markers detected in the analysis of large scale gene expression data.
  • RT-qPCR or equivalent quantitative molecular detection technique
  • PFAFFL Pfaffl MW.
  • PFAFFL Pfaffl MW.
  • e45 quantification in real-time RT-PCR Nucleic Acids Res. 2001 May 1.29 (9): e45). This method is based on dividing the ACq of the transcript of interest by the geometric mean ACq of the selected reference transcripts.
  • the numerical value (score) obtained with this algorithm relates to the probability of malignancy as indicated below, and illustrated in fig. 1;
  • CLDN10 which encodes a membrane integral protein, shows increased expression in PTC as compared to tissue normal, but low expression in benign thyroid lesion (BTL) tissue, or in other follicular cell-derived thyroid carcinomas (OFDTC) such as those poorly differentiated, anaplastic, follicular, and Hurthle cell carcinoma.
  • BTL benign thyroid lesion
  • OFDTC follicular cell-derived thyroid carcinomas
  • HMGA2 product which showed increased PTC expression, alters the regulatory structure of chromatin by affecting the transcription of various genes.
  • RT-qPCR a marker evaluated by RT-qPCR.
  • the LAMB3 gene product belongs to a family of basement membrane proteins (laminins), with an important association between its increased expression and lymph node metastasis. This fact is confirmed in the higher risk of lymph node metastasis in patients with PTC who had higher scores.
  • Necessary and sufficient diagnostic markers of the invention are the CLDN 10, HMGA2 and LAMB3 transcripts used in the thyroid cancer detection method and in vitro method of differentiating thyroid cancer from normal or thyroid tissue. benign lesions.
  • the quantitative molecular detection technique particularly RT-qPCR, is used, where tissue samples suspected of the patient's tumor are used.
  • the method of the invention is performed in a particular embodiment by combining the results obtained in molecular detection. with a risk scale, for example with the methods of Pfaffl (2001) or Livak & Schmittgen (2001) mentioned above.
  • the patient sample to be evaluated according to the methods of the invention may be freshly woven, paraffin blocks or frozen.
  • the tissue sample is obtained via fine needle aspiration biopsy (FNAB).
  • the invention is used in thyroid nodule biopsies to guide surgery and clinical follow-up, while alerting which malignant cases are most at risk for lymph node involvement, particularly when there is increased expression of LAMB3.
  • the invention also relates to the use of a set of transcripts characterized in that it is in an in vitro diagnostic method of thyroid cancer, said set comprising the transcripts LAMB3, CLDN10 and HMGA2 compared to at least one reference transcript.
  • the invention further relates to the use of a set of transcripts characterized in that it is in a method of analysis to differentiate between thyroid cancer and normal thyroid tissue. or of benign lesions, said set comprising the transcripts LAMB3, CLDNIO and HMGA2 compared to at least one reference transcript.
  • the invention relates to a method of obtaining data for directing the treatment of Thyroid cancer characterized by the fact that it comprises subjecting a patient's suspicious tissue sample to the quantitative molecular detection technique that uses, for gene expression analysis, the set of LAMB3, CLDNIO and HMGA2 transcripts, compared to the expression of at least a reference gene, optionally associated with a risk scale.
  • the invention in another aspect, relates to a laboratory kit comprising reagents for detecting the expression of the LAMB3, CLDNIO, and HMGA2 genes related to the diagnosis of PTC, as well as for evaluating or assisting in the evaluation. of its expression with respect to at least one reference gene, optionally containing an instruction manual for use.
  • RNA extraction from a patient's tissue sample a- suitable component for RNA extraction from a patient's tissue sample
  • dNTPs triphosphate c-deoxyribonucleotides
  • LAMB3 and for one or more reference transcripts are LAMB3 and for one or more reference transcripts
  • i- means to calculate the scores according to a risk table that indicates the results by applying the diagnostic algorithm:
  • the invention relates to a device for classifying a biological sample of the thyroid gland as malignant or benign, comprising:
  • means for measuring expression level are, for example, RT-PCR, RT-qPCR or any other equivalent;
  • Means for correlating expression level with thyroid disease status are, for example, the methodologies of Pfaffl (2001) or Livak & Schmittgen (2001), already mentioned above;
  • Disease status are any, for example a display where digits, codes, words, colors or images appear without excluding any other suitable means or devices.
  • transcript set of the invention including one or more reference transcript, commercial primers or specific primers may be used as described below.
  • Example 1 Methods of the invention using commercial primers in the quantitative molecular detection technique.
  • RNA is converted to complementary DNA or cDNA (day 1).
  • the protocol for converting RNA to cDNA began with the addition of oligo dT 12 18 primers (from US Healthcare Life Sciences) and / or random primers (from Invitrogen), triphosphate deoxyribonucleotides (dNTPs) ) (from US company Invitrogen), Super Script III buffer First Strand Buffer (from US company Invitrogen), 5 mM diethyltreitol (DTT) (from US company Invitrogen) and 200 units of the enzyme Super Script III Reverse Transcriptase (from the US company Invitrogen), following a series of incubations at varying temperatures.
  • RNA to cDNA can be used.
  • PCR amplification was carried out by using 20 ng of cDNA, IX TaqMan Gene Expression assays (probes and primers, the North American company Applied Biosystems), IX TaqMan Universal Master Mix ® II (North American company Applied Biosystems) supplementing with ultrapure water (Ambion®, from the US company Life Technologies) to 15 final volume (day 2).
  • Example 3 Methods of the invention applied to real case.
  • MCS patient had a suspected thyroid nodule and, according to clinical, laboratory and imaging tests, fine-needle aspiration biopsy was indicated;
  • RNA extraction was subjected to RNA extraction followed by conversion to cDNA (estimated time 5 hours); PCR was performed with the primers according to example 1 (40 amplification cycles: 1 minute at 95 ° C, 30 seconds at 60 ° C) (estimated time 2 hours);
  • Example 4 Method of the invention applied to real case.
  • Patient TBB had a suspected thyroid nodule and, according to clinical, laboratory and imaging tests, fine-needle aspiration biopsy was indicated;
  • the remaining biopsy material was subjected to RNA extraction followed by conversion to cDNA (estimated time 5 hours).
  • PCR was performed as per example 2 (40 amplification cycles: 1 minute at 95 ° C, 30 seconds at 60 ° C) (estimated time 2 hours);
  • the diagnostic algorithm When applied the diagnostic algorithm obtained a score of -1.5, presenting high risk of malignancy.
  • Example 5 Method of the invention applied to real case.
  • Patient WS had a suspicious thyroid nodule and fine-needle aspiration biopsy was indicated on imaging studies;
  • RNA extraction followed by conversion to cDNA as per example 2 (estimated time 5 hours);
  • PCR was performed according to example 2 (40 amplification cycles: 1 minute at 95 ° C, 30 seconds at 60 ° C) (estimated time 2 hours);

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Abstract

La présente invention concerne, de manière générale, une méthode in vitro de détection du cancer de la thyroïde chez un patient ainsi qu'une méthode in vitro de différenciation entre le cancer de la thyroïde et des lésions bénignes ou du tissu normal de la thyroïde, basées sur l'évaluation de l'expression de l'ensemble de gènes CLDN10, HMGA2 e LAMB3, une technique de détection moléculaire étant utilisée. L'invention concerne également d'autres aspects associés à ces méthodes, soit l'utilisation d'un ensemble de gènes, un procédé d'obtention de données pour l'orientation du traitement du cancer de la thyroïde, un kit de laboratoire et un dispositif pour classer un échantillon biologique de glande thyroïde comme malin ou bénin.
PCT/BR2016/050142 2015-06-23 2016-06-22 Méthode de détection in vitro du cancer de la thyroïde chez un patient, méthode in vitro de différenciation entre le cancer de la thyroïde et le tissu thyroïdien normal ou des lésions thyroïdiennes bénignes, utilisation d'un ensemble de gènes, procédé d'obtention de données pour l'orientation du traitement du cancer de la thyroïde, kit de laboratoire et dispositif pour classer un échantillon biologique de glande thyroïde comme malin ou bénin WO2016205911A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BR102015015096A BR102015015096A2 (pt) 2015-06-23 2015-06-23 método de detecção in vitro de câncer de tireoide em um paciente, método in vitro de diferenciação entre câncer de tireoide e tecido de tireoide normal ou de lesões benignas de tireoide, uso de um conjunto de genes, método de obtenção de dados para direcionamento do tratamento do cancer de tireoide, kit laboratorial e dispositivo para classificar uma amostra biológica de glândula tireoide como maligna ou benigna
BRBR102015015096-2 2015-06-23

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107858438A (zh) * 2017-10-12 2018-03-30 华南农业大学 异色瓢虫在不同因子下稳定表达的内参基因组合及其应用
CN109609661A (zh) * 2018-12-28 2019-04-12 山东中医药大学 一种肾阳虚外感小鼠模型肺组织qPCR内参基因组合及其筛选方法

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US20080044824A1 (en) * 2005-10-11 2008-02-21 Regents Of The University Of Michigan Expressed profile of thyroid cancer
EP2366800A1 (fr) * 2010-03-01 2011-09-21 Centrum Onkologii-Instytut im M. Sklodowskiej-Curie Oddzial w Gliwicach Kit, méthode et l'utilisation pour le diagnostic de cancer papillaire de la thyroïde en utilisant un profil d'expression génique

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US20080044824A1 (en) * 2005-10-11 2008-02-21 Regents Of The University Of Michigan Expressed profile of thyroid cancer
EP2366800A1 (fr) * 2010-03-01 2011-09-21 Centrum Onkologii-Instytut im M. Sklodowskiej-Curie Oddzial w Gliwicach Kit, méthode et l'utilisation pour le diagnostic de cancer papillaire de la thyroïde en utilisant un profil d'expression génique

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ARORA, N. ET AL.: "Identification of borderline thyroid tumors by gene expression array analysis.", CÂNCER., vol. 115, no. 23, 2009, pages 5421 - 5231, XP055340511 *
BARROS-FILHO, M. C. ET AL.: "High Diagnostic Accuracy Based on CLDN10, HMGA2, and LAMB3 Transcripts in Papillary Thyroid Carcinoma.", J CLIN ENDOCRINOL METAB., vol. 100, no. 6, 2015, pages E890 - 9., XP055340493 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107858438A (zh) * 2017-10-12 2018-03-30 华南农业大学 异色瓢虫在不同因子下稳定表达的内参基因组合及其应用
CN109609661A (zh) * 2018-12-28 2019-04-12 山东中医药大学 一种肾阳虚外感小鼠模型肺组织qPCR内参基因组合及其筛选方法

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