WO2014193159A1 - Method for predicting therapeutic responsiveness of lung cancer with respect to egfr-targeting therapeutic agent - Google Patents

Method for predicting therapeutic responsiveness of lung cancer with respect to egfr-targeting therapeutic agent Download PDF

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WO2014193159A1
WO2014193159A1 PCT/KR2014/004754 KR2014004754W WO2014193159A1 WO 2014193159 A1 WO2014193159 A1 WO 2014193159A1 KR 2014004754 W KR2014004754 W KR 2014004754W WO 2014193159 A1 WO2014193159 A1 WO 2014193159A1
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lung cancer
ant2
cancer
egfr
patients
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김철우
장지영
고현정
김용구
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주식회사 바이오인프라
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Definitions

  • the present invention relates to a method for predicting the reactivity of non-small cell lung cancer patients with drugs targeting Epidermal Growth Factor Receptor (EGFR).
  • EGFR Epidermal Growth Factor Receptor
  • EGFR Epidermal Growth Factor Receptor
  • Cancer treatment can be divided into surgery, radiation therapy, chemotherapy, and biological therapy.
  • patients who are not easy to undergo surgery or radiation therapy (about 50% of all cancer patients) and those who have already metastasized are treated with chemotherapy.
  • drugs used in chemotherapy efforts have been made to separate components having anticancer effects through efficacy evaluation using various types of cancer cell lines and animal tumor models.
  • Anticancer chemotherapy agents are increasingly developed and complicated to treat, but there are no satisfactory effective treatments.
  • One of the obstacles in the treatment of cancer using chemotherapy is chemoresistant.
  • Drug resistance to anticancer drugs means that the cancer cells do not die despite the administration of an amount of anticancer drugs that can reach a blood concentration that can kill the cancer cells.
  • Anticancer drug resistance can vary from patient to patient and can be caused by a variety of factors, including genetic differences between tumors from the same tissue.
  • Extracellular resistance is characterized by severe adverse reactions among patients, and the cancer cells of patients in vitro are abnormally decreased when intestinal absorption is decreased, such as when oral anticancer drugs are administered. It is resistant to anticancer drugs because it has not been exposed to concentrations that can kill cancer cells. Or in a pharmacologic sanctuary where drugs are not penetrated into the tissue due to poor blood flow to cancer tissue or poor blood flow to cancer tissue or physiologically a blood-tissue barrier between blood vessels and cancer tissue. As with cancer cells, cancer cells are ultimately resistant to cancer even when they are not exposed to sufficient levels of cancer.
  • predicting the responsiveness and effectiveness of a specific anticancer agent by determining whether cancer patients acquire anticancer drug resistance is very important in determining a cancer patient treatment policy.
  • lapatinib a breast cancer drug
  • HER2 protein HER2-positive
  • EGRF protein EGRF protein
  • Lung cancer is the leading cause of cancer death worldwide, and can be divided into non-small cell lung cancer and small cell lung cancer according to the type of lung cancer cells. About 80% of lung cancers are classified as non-small cell lung cancers, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Non-small cell lung cancer also generally has slower metastases to other organs than small cell lung cancer.
  • Epidermal growth factor receptor is a transmembrane protein of about 170 kDa that is expressed on the surface of epithelial cells.
  • EGFR belongs to the group of cell cycle regulators tyrosine kinase, and when ligand (EGF or TGF- ⁇ ) is bound to the extracellular domain, it is activated to induce autophosphorylation of intracellular tyrosine kinase domain and eventually signal transduction.
  • the pathway promotes cell proliferation and growth. Genetic modifications that affect the regulation of growth factor receptor function or cause overexpression of receptors and ligands can lead to carcinogenesis.
  • EGFR is a protein product of the oncogene erbB or ErbB1.
  • erbB or ErbB1 is one of the ERBB family of protooncogenes known to be important factors in numerous cancer developments. Increased expression of EGFR has been observed in breast cancer, bladder cancer, gastric cancer and the like, including lung cancer.
  • the ERBB oncogene family encodes four structurally related transmembrane receptors, EGFR, HER-2 / neu (erbB2), HER-3 (erbB3), and HER-4 (erbB4).
  • Gefitinib AstraZeneca UK Ltd., trade name "IRESSA”
  • Erlotinib Genentech, Inc. & OSI Pharmaceuticals, Inc., trade name "TARCEVA”
  • Gefitinib and Erlotinib are quinazoline compounds that inhibit cell growth by inhibiting tyrosine kinase activity of EGFR.
  • the present inventors have completed the present invention as a result of diligently trying to provide a method for predicting treatment responsiveness and effects on specific anticancer drugs by determining whether cancer patients are resistant to cancer.
  • ANT2 as a novel resistance predictive biomarker for gefitinib treatment in lung cancer patients.
  • the present invention provides a method for predicting the reactivity of non-small cell lung cancer patients with a drug targeting the epidermal growth factor receptor (EGFR), comprising the step of identifying the expression level of ANT2 protein or mRNA in lung cancer tissue samples. do.
  • EGFR epidermal growth factor receptor
  • the drug of the method is characterized in that gefitinib (gefitinib) or erlotinib (erlotinib).
  • the expression level of the ANT2 protein is immunohistochemical staining, characterized in that the expression level of ANT2 mRNA is confirmed using qRT-PCR.
  • the immunohistochemical staining is characterized by using an anti-ANT2 antibody.
  • the present invention it is possible to predict in advance whether a lung cancer patient will respond to a drug treatment, thereby solving the problem of low treatment success rate due to resistance.
  • IHC immunohistochemical staining
  • the expression level of ANT2 which is highly expressed in most cancer cells, and particularly in cancer cells that have acquired anticancer drug resistance, determines whether the anticancer drug resistance is obtained and the therapeutic responsiveness and effects on specific anticancer drugs.
  • ANT2 immunohistochemical staining (ANT2 protein), qRT-PCR (ANT2 mRNA) were performed on lung cancer tissues of lung cancer patients, and anticancer drug reactivity, ANT2 expression, and anticancer drugs The degree of ANT2 expression before and after obtaining resistance was compared.
  • the cancer patients selected for the study were lung cancer patients treated with gefitinib, a lung cancer drug.
  • Lung cancer is a very fatal cancer with the highest cancer mortality rate worldwide.
  • Non-small cell lung cancer which accounts for most of the lung cancer, is almost impossible to diagnose early, and has a high mortality rate due to early distant metastasis.
  • the main therapeutic agent is a conventional anticancer agent, and epidermal growth factor receptor (EGFR) -targeted therapeutic agents such as gefitinib and erlotinib have recently been used as target therapeutics.
  • EGFR epidermal growth factor receptor
  • gefitinib is a very important drug for treating lung cancer and is very effective when used in lung cancer patients with EGFR mutations, but in all patients, acquired resistance and death eventually occur on average 6-8 months.
  • the acquisition-resistant mechanisms have been identified, including the T790M gatekeeper mutation and amplification of MET oncogene. These mechanisms have been identified and suggested to overcome resistance by using inhibitors in patients with acquired resistance. Therefore, the mechanism of acquisition resistance is essential for the development of follow-up treatment after failure of gefitinib treatment. .
  • the present invention uses ANT2 as a novel resistance predictive biomarker.
  • the adenine nucleotide translocator (ANT) is an enzyme present in the inner membrane (IM) of the mitochondrial membrane (IM) through the voltage dependent anion channel (VDAC) of the outer membrane (OM) from the cytoplasm to the mitochondria inside the mitochondria. It is known as an enzyme that performs the function of importing and exporting ATP generated through an electron transfer chain system to the cytoplasm.
  • ANT which plays an essential role in cellular energy metabolism, is known as three kinds of isoforms, ANT1, ANT2 and ANT3.
  • ANT2 has a low expression in normal cells but very high in cancer cells or high proliferative cells. It is characterized by being expressed, which is closely related to glycolysis in anaerobic conditions and has recently been suggested as a target for new cancer treatments.
  • the present invention can predict drug reactivity of lung cancer patients by confirming the increased expression of ANT2 protein using immunohistochemical staining.
  • immunohistochemistry is a method of analyzing protein expression of cells using an antigen-antibody reaction.
  • a tumor cell expresses a specific antigenic protein, it reacts with a complementary antibody to the antibody, and the antibody binds specifically to the complementary antigen, and the antigen-antibody binding product is treated with a secondary antibody to amplify the reaction and develop color.
  • a secondary antibody When observed under a microscope, it can be objectively determined whether tumor cells express (positive) or do not express (negative) antigens.
  • Immunohistochemical staining was performed on the ANT2 protein in the tissues of patients with lung cancer who obtained data on the therapeutic effect and resistance to gefitinib.
  • the specific method is as follows.
  • Lung cancer tissues were surgically removed from 10 lung cancer patients at Seoul National University Hospital and stored in liquid nitrogen until analysis.
  • the lung cancer tissue was made into a paraffin embedding block, it was cut into 4 ⁇ m thickness using a microtome and attached onto a glass slide to prepare a tissue slide. At this time, the slide was placed in a dry oven at 65 ° C. for 1 hour to prevent tissue from falling off. Then, 40 minutes of deparaffinization with xylene and alcohol.
  • microwaves were sectioned three times for 5 minutes to expose the ANT2 protein antigen.
  • 3% H 2 O 2 was treated for 15 minutes to remove blood, an endogenous factor present in the tissue, and then blocked with normal serum for 1 hour to prevent nonspecific binding of the antibody.
  • the primary antibody used for immunization was an anti-ANT2 antibody (Creative BioMart, Cat No. CAB4666MH, 1: 100 dilution), and the primary antibody was treated at room temperature for 1 hour without washing with water. Thereafter, after washing with water, the secondary antibody (biotinylated antibody) and streptavidin enzyme complex were treated with water for 20 minutes, washed with water, and then colored with DAB. After Mayor Hemtoxylin® staining, the cells were washed with water, dried and sealed in permount.
  • ANT2 expression was relatively low in the tissues of the patient group with high anti-cancer drug treatment, and ANT2 expression was high in the tissues of the patient group with low anti-cancer drug treatment. Confirmed. In addition, in the case of patients who initially had good therapeutic responsiveness but acquired anticancer drug resistance after treatment, and the treatment responsiveness became low, the expression of ANT2 may be markedly increased after treatment. Pathology S05-25201 and S07-2403 were analyzed in tissues before and after the same patient tolerated gefitinib.

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Abstract

The present invention relates to a method for predicting whether non-small-cell lung-cancer patients will be responsive to a drug targeting epidermal growth factor receptor (EGFR), thereby increasing therapeutic effectiveness in acquired-resistance patients. The present invention makes it possible to predict in advance whether a lung-cancer patient will react to a medical therapeutic agent, and overcomes the problem of a low rate of therapeutic success due to resistance, and at the same time is useful in estimating anticancer agent prognosis and determining future treatment strategy.

Description

EGFR 표적 치료제에 대한 폐암의 치료 반응성을 예측하는 방법How to predict the therapeutic responsiveness of lung cancer to EGFR-targeted therapeutics
본 발명은, 표피성장인자 수용체(Epidermal Growth Factor Receptor; EGFR)를 표적으로 하는 약물에 대하여 비소세포 폐암 환자의 반응성 유무를 예측하는 방법에 관한 것이다. The present invention relates to a method for predicting the reactivity of non-small cell lung cancer patients with drugs targeting Epidermal Growth Factor Receptor (EGFR).
보다 상세하게는, ANT2(Adenine Nucleotide Translocator 2) 단백질 또는 mRNA의 발현 증가를 확인함으로써 표피성장인자 수용체(Epidermal Growth Factor Receptor; EGFR) 표적 약물에 내성을 보이는 비소세포 폐암 환자를 식별 또는 예측하는 방법에 관한 것이다.More specifically, the method for identifying or predicting non-small cell lung cancer patients showing resistance to Epidermal Growth Factor Receptor (EGFR) target drugs by confirming increased expression of Adenine Nucleotide Translocator 2 (ANT2) protein or mRNA It is about.
암을 치료하는 방법은 크게 수술, 방사선 치료, 항암화학요법, 그리고 생물학적 치료법 등으로 나눌 수 있다. 이 중 수술이나 방사선치료가 용이하지 않는 환자(전체 암 환자의 약 50%)와 이미 암이 전이된 환자들은 주로 화학요법으로 치료한다. 항암화학요법에 사용되는 약제의 경우 다양한 종류의 암 세포주 및 동물 종양 모델을 이용한 효능평가를 통해 항암효과를 갖는 성분을 분리해내기 위한 노력이 계속되고 있다. Cancer treatment can be divided into surgery, radiation therapy, chemotherapy, and biological therapy. Among these, patients who are not easy to undergo surgery or radiation therapy (about 50% of all cancer patients) and those who have already metastasized are treated with chemotherapy. In the case of drugs used in chemotherapy, efforts have been made to separate components having anticancer effects through efficacy evaluation using various types of cancer cell lines and animal tumor models.
이와 같이, 항암화학요법제는 점점 더 많이 개발되고 아울러 처치하는 요법도 복잡해지고 있지만 아직까지 만족할만한 효과적인 치료법은 나타나고 있지 않다. 항암화학요법을 이용한 암 치료에 있어서 장애가 되는 요소 중의 하나는 항암제내성이다. 항암제에 대한 약제내성은 암세포를 죽일 수 있는 혈중농도에 도달할 수 있는 양의 항암제를 투여했음에도 불구하고 암세포가 죽지 않는 경우를 말한다. 항암제내성은 환자 개개에 따라 다를 수 있으며 심지어 같은 조직으로부터 유래된 종양들 사이의 유전적 차이 등을 포함한 다양한 인자들에 의해 유발될 수 있다. As such, anticancer chemotherapy agents are increasingly developed and complicated to treat, but there are no satisfactory effective treatments. One of the obstacles in the treatment of cancer using chemotherapy is chemoresistant. Drug resistance to anticancer drugs means that the cancer cells do not die despite the administration of an amount of anticancer drugs that can reach a blood concentration that can kill the cancer cells. Anticancer drug resistance can vary from patient to patient and can be caused by a variety of factors, including genetic differences between tumors from the same tissue.
항암제 내성 기작은 일반적으로 세포외적 내성과 세포내적 내성으로 크게 분류될 수 있다. 세포외적 내성은 환자에 따라 특이적으로 심한 부작용을 나타내 충분한 농도의 항암제를 투여하지 못한 경우나, 경구용 항암제를 투여했을 때처럼 장내 흡수가 비정상적으로 저하된 경우에, 환자의 암세포가 in vitro에서 암세포를 죽일 수 있는 농도에 노출되지 못했기 때문에 항암제에 대해 내성을 보일 수 있다. 또는 충분한 혈중농도에는 도달했으나 암 조직으로의 혈류분포가 좋지 않거나 생리학적으로 혈관과 암 조직 사이에 장벽(blood-tissue barrier)이 있음으로 해서 약물이 조직 내로 침투가 되지 못하는 부위(pharmacologic sanctuary)에 암세포가 존재하는 경우처럼, 궁극적으로 암세포가 충분한 농도의 항암제에 노출되지 못할 때에도 항암제 내성 현상을 보인다.Mechanisms of cancer resistance can be broadly classified into extracellular resistance and intracellular resistance. Extracellular resistance is characterized by severe adverse reactions among patients, and the cancer cells of patients in vitro are abnormally decreased when intestinal absorption is decreased, such as when oral anticancer drugs are administered. It is resistant to anticancer drugs because it has not been exposed to concentrations that can kill cancer cells. Or in a pharmacologic sanctuary where drugs are not penetrated into the tissue due to poor blood flow to cancer tissue or poor blood flow to cancer tissue or physiologically a blood-tissue barrier between blood vessels and cancer tissue. As with cancer cells, cancer cells are ultimately resistant to cancer even when they are not exposed to sufficient levels of cancer.
이처럼 암 환자의 항암제 내성의 획득 여부를 판단함으로써 특정 항암제에 대한 치료 반응성과 효과를 예측하는 것은 암 환자 치료방침을 정하는데 매우 중요하다. 대표적인 예로, 유방암 치료제인 라파티닙(lapatinib)은 HER2 단백질의 수치가 높고 (HER2 양성) EGRF 단백질의 수치가 낮은 경우에 치료효과가 있는 것으로 밝혀진 바 있다. 그러나 전이성의 HER2 음성 유방암은 라파티닙에 반응을 하지 않아 라파티닙이 효과가 없는 것으로 드러났다. 이러한 연구결과를 참고하면, 일단 유방암 환자들은 치료를 받기 전에 정확한 검사를 통하여 HER2 음성인지 혹은 양성인지를 확실히 밝혀야 적절한 치료를 선택할 수 있음을 알 수 있다.As such, predicting the responsiveness and effectiveness of a specific anticancer agent by determining whether cancer patients acquire anticancer drug resistance is very important in determining a cancer patient treatment policy. As a representative example, lapatinib, a breast cancer drug, has been shown to be effective in the case of high levels of HER2 protein (HER2-positive) and low levels of EGRF protein. However, metastatic HER2-negative breast cancers do not respond to lapatinib, indicating that lapatinib is not effective. These findings suggest that breast cancer patients can select appropriate treatment by confirming whether they are HER2-negative or positive before they are treated.
폐암은 전세계적으로 암 사망의 주원인이며, 폐암세포의 종류에 따라 비소세포(non-small cell) 폐암과 소세포(small cell) 폐암으로 나눌 수 있다. 폐암의 약 80%가 비소세포 폐암으로 분류되며, 선암종(adenocarcinoma), 편평세포암종(squamous cell carcinoma), 및 대세포암종(large cell carcinoma)이 이에 속한다. 또한, 비소세포 폐암은 일반적으로 소세포 폐암 보다 다른 기관으로의 전이가 느리다. Lung cancer is the leading cause of cancer death worldwide, and can be divided into non-small cell lung cancer and small cell lung cancer according to the type of lung cancer cells. About 80% of lung cancers are classified as non-small cell lung cancers, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Non-small cell lung cancer also generally has slower metastases to other organs than small cell lung cancer.
표피성장인자 수용체(EGFR)는 약 170kDa의 막단백질(transmembrane protein)로서 상피세포의 표면에서 발현된다. EGFR은 세포주기 조절인자인 티로신 키나제(tyrosine kinase) 군에 속하며, 세포외 도메인에 리간드(EGF 또는 TGF-α)가 결합하면 활성화되어 세포내 티로신 키나제 도메인의 자가 인산화 반응을 유도하고, 결국 신호전달경로를 통하여 세포증식 및 성장을 촉진하게 된다. 성장인자수용체 기능의 조절에 영향을 주거나, 수용체, 리간드의 과다발현을 유발하는 유전자 변형이 일어나면 발암으로 이어질 수 있다.Epidermal growth factor receptor (EGFR) is a transmembrane protein of about 170 kDa that is expressed on the surface of epithelial cells. EGFR belongs to the group of cell cycle regulators tyrosine kinase, and when ligand (EGF or TGF-α) is bound to the extracellular domain, it is activated to induce autophosphorylation of intracellular tyrosine kinase domain and eventually signal transduction. The pathway promotes cell proliferation and growth. Genetic modifications that affect the regulation of growth factor receptor function or cause overexpression of receptors and ligands can lead to carcinogenesis.
EGFR은, 종양유전자(oncogene)인 erbB 또는 ErbB1의 단백질 산물이다. erbB 또는 ErbB1은 수많은 암 발생에 있어 중요 인자로 알려진 원발암유전자(protooncogenes)인 ERBB 군의 하나이다. 폐암을 포함하여, 유방암, 방광암, 위암 등에서 EGFR의 발현이 증가되는 것이 관찰되었다. ERBB 종양유전자 군은 구조적으로 연관된 4개의 transmembrane 수용체들, 즉, EGFR, HER-2/neu(erbB2), HER-3(erbB3), 및 HER-4(erbB4)를 암호화하고 있다. EGFR is a protein product of the oncogene erbB or ErbB1. erbB or ErbB1 is one of the ERBB family of protooncogenes known to be important factors in numerous cancer developments. Increased expression of EGFR has been observed in breast cancer, bladder cancer, gastric cancer and the like, including lung cancer. The ERBB oncogene family encodes four structurally related transmembrane receptors, EGFR, HER-2 / neu (erbB2), HER-3 (erbB3), and HER-4 (erbB4).
폐암, 유방암, 방광암, 위암 등의 상피세포암을 치료하기 위하여 다양한 EGFR 표적 약물이 개발되었으며, 특히 게피티닙(Gefitinib)(AstraZeneca UK Ltd., 상표명 "IRESSA")와 얼로티닙(Erlotinib)(Genentech, Inc. & OSI Pharmaceuticals, Inc., 상표명 "TARCEVA")이 대표적인 약물이다. 게피티닙(Gefitinib)과 얼로티닙(Erlotinib)은 퀴나졸린계 화합물로서, EGFR의 티로신 키나제 활성을 저해하여 인산화를 억제함으로써 세포성장을 막는다.Various EGFR target drugs have been developed for the treatment of epithelial cell carcinoma such as lung cancer, breast cancer, bladder cancer and gastric cancer, in particular Gefitinib (AstraZeneca UK Ltd., trade name "IRESSA") and Erlotinib ( Genentech, Inc. & OSI Pharmaceuticals, Inc., trade name "TARCEVA") are representative drugs. Gefitinib and Erlotinib are quinazoline compounds that inhibit cell growth by inhibiting tyrosine kinase activity of EGFR.
이들 표적 치료제들이 매우 유효한 약물임에도, 계속 사용시 치료효과들에 대하여 내성을 나타내거나, 측정가능한 정도의 반응을 보이지 않는 문제가 있다. 실제, 비소세포 폐암 환자들의 약 10% 정도만이 이들 약물에 반응성을 보이고 있다.Although these targeted therapies are very effective drugs, there is a problem in that they continue to be resistant to therapeutic effects or do not show measurable response when used continuously. In fact, only about 10% of non-small cell lung cancer patients are responsive to these drugs.
이에, 본 발명자들은 암 환자의 항암제 내성 여부를 판단하여 특정 항암제에 대한 치료 반응성과 효과의 예측방법을 제공하고자 예의 노력한 결과, 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention as a result of diligently trying to provide a method for predicting treatment responsiveness and effects on specific anticancer drugs by determining whether cancer patients are resistant to cancer.
구체적으로, 본 발명의 목적은 폐암 환자의 게피티닙 치료에 대하여 ANT2를 신규의 내성 예측 바이오마커로 제공하는 것이다.In particular, it is an object of the present invention to provide ANT2 as a novel resistance predictive biomarker for gefitinib treatment in lung cancer patients.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명은, 폐암조직 샘플에서 ANT2 단백질 또는 mRNA의 발현량을 확인하는 단계를 포함하는, 표피성장인자 수용체(EGFR)를 표적으로 하는 약물에 대하여 비소세포 폐암 환자의 반응성 유무를 예측하는 방법을 제공한다.The present invention provides a method for predicting the reactivity of non-small cell lung cancer patients with a drug targeting the epidermal growth factor receptor (EGFR), comprising the step of identifying the expression level of ANT2 protein or mRNA in lung cancer tissue samples. do.
본 발명의 일 구체예로, 상기 방법의 약물은 게피티닙(gefitinib) 또는 얼로티닙(erlotinib)인 것을 특징으로 한다.In one embodiment of the invention, the drug of the method is characterized in that gefitinib (gefitinib) or erlotinib (erlotinib).
본 발명의 다른 구체예로, 상기 ANT2 단백질의 발현량은 면역조직화학염색, ANT2 mRNA의 발현량은 qRT-PCR을 이용하여 확인하는 것을 특징으로 한다.In another embodiment of the present invention, the expression level of the ANT2 protein is immunohistochemical staining, characterized in that the expression level of ANT2 mRNA is confirmed using qRT-PCR.
본 발명의 또 다른 구체예로, 상기 면역조직화학염색은 항-ANT2 항체를 이용하는 것을 특징으로 한다.In another embodiment of the present invention, the immunohistochemical staining is characterized by using an anti-ANT2 antibody.
본 발명에 의하면, 폐암 환자가 약물 치료제에 반응할 것인지의 여부를 미리 예측할 수 있어, 내성으로 인한 낮은 치료 성공율의 문제를 해결할 수 있다.According to the present invention, it is possible to predict in advance whether a lung cancer patient will respond to a drug treatment, thereby solving the problem of low treatment success rate due to resistance.
또한, 본 발명에 의하면, 폐암을 치료하는데 사용된 약물에 환자가 반응적일지의 여부를 결정하여, 항암제 예후를 추정하고 향후의 치료방침을 정할 수 있다.In addition, according to the present invention, it is possible to determine whether a patient is responsive to a drug used to treat lung cancer, to estimate the anticancer drug prognosis and to set a future treatment policy.
도 1은, 폐암 조직 샘플에서 ANT2 단백질의 발현정도를 확인하기 위해 면역조직화학염색(IHC)을 실시한 결과이며, 폐암 환자의 게피티닙 치료에 대하여 ANT2를 신규의 내성 예측 바이오마커로 이용할 수 있음을 확인하였다.1 is a result of immunohistochemical staining (IHC) to confirm the expression level of ANT2 protein in lung cancer tissue samples, and can use ANT2 as a novel resistance predictive biomarker for gefitinib treatment in lung cancer patients. It was confirmed.
본 발명은 대부분의 암 세포에서 높은 발현을 나타내며, 특히 항암제 내성을 획득한 암 세포에서는 더욱 높은 발현을 나타내는 ANT2의 발현 정도가, 항암제 내성의 획득 여부를 판단하고 특정 항암제에 대한 치료 반응성과 효과를 예측하는 좋은 기준 인자가 될 것으로 기대하고, 폐암 환자의 폐암 조직을 대상으로 ANT2 면역조직화학염색(ANT2 단백질), qRT-PCR(ANT2 mRNA)을 실시하여, 항암제 치료 반응성과 ANT2 발현 정도, 그리고 항암제 내성을 획득하기 전과 후의 ANT2 발현 정도를 비교하였다. In the present invention, the expression level of ANT2, which is highly expressed in most cancer cells, and particularly in cancer cells that have acquired anticancer drug resistance, determines whether the anticancer drug resistance is obtained and the therapeutic responsiveness and effects on specific anticancer drugs. ANT2 immunohistochemical staining (ANT2 protein), qRT-PCR (ANT2 mRNA) were performed on lung cancer tissues of lung cancer patients, and anticancer drug reactivity, ANT2 expression, and anticancer drugs The degree of ANT2 expression before and after obtaining resistance was compared.
이 때, 연구 대상으로 정한 암 환자는 폐암치료제인 게피티닙(gefitinib)으로 치료를 진행한 폐암 환자군이다. 폐암은 전세계적으로 암 사망율 1위인 매우 치명적인 암이다. 폐암의 대부분을 차지하는 비소세포폐암은 조기 진단이 거의 불가능하며, 조기에 원격전이를 잘하여 사망률이 높다. 폐암 1기에 발견되어 수술적인 치료를 받았어도 약 40%-50% 정도의 환자가 재발하여 사망에 이른다. 주된 치료제는 고식적인 항암제를 사용하며, 최근 표적 치료제로 게피티닙(gefitinib), 엘로티닙(erlotinib) 등의 상피세포성장인자수용체(Epidermal Growth Factor Receptor;EGFR)-표적 치료제들이 많이 사용되고 있다. 이러한 표적 치료제는 종양에 특정 바이오마커(biomarker)가 발현하고 있을 때에만 좋은 효과를 거둘 수 있기때문에, 예측 바이오마커(predictive biomarker)를 발굴하는 것은 당업계의 초미의 관심사이다.At this time, the cancer patients selected for the study were lung cancer patients treated with gefitinib, a lung cancer drug. Lung cancer is a very fatal cancer with the highest cancer mortality rate worldwide. Non-small cell lung cancer, which accounts for most of the lung cancer, is almost impossible to diagnose early, and has a high mortality rate due to early distant metastasis. Even in the first stage of lung cancer and surgical treatment, about 40% -50% of patients relapse and die. The main therapeutic agent is a conventional anticancer agent, and epidermal growth factor receptor (EGFR) -targeted therapeutic agents such as gefitinib and erlotinib have recently been used as target therapeutics. The discovery of predictive biomarkers is of paramount concern because such targeted therapies can only have a good effect when certain biomarkers are expressed in tumors.
특히 게피티닙은 폐암 치료제로서 매우 중요한 약제로 EGFR 돌연변이가 있는 폐암 환자에 사용할 경우 매우 효과적이지만, 모든 환자에서 평균 6-8개월 후에 획득 내성이 생기고 결국 사망하게 된다. 현재까지 획득 내성기전으로 밝혀진 것으로 T790M gatekeeper mutation과 MET oncogene의 amplification이 있다. 이러한 기전이 밝혀지고 획득 내성이 생긴 환자에게 이에 대한 억제제를 사용함으로써 내성을 극복할 수 있는 방안을 제시해주고 있으며, 따라서 획득 내성 기전을 밝히는 것은 게피티닙 치료 실패 후 후속 치료제 개발을 위해 필수적인 연구이다.In particular, gefitinib is a very important drug for treating lung cancer and is very effective when used in lung cancer patients with EGFR mutations, but in all patients, acquired resistance and death eventually occur on average 6-8 months. To date, the acquisition-resistant mechanisms have been identified, including the T790M gatekeeper mutation and amplification of MET oncogene. These mechanisms have been identified and suggested to overcome resistance by using inhibitors in patients with acquired resistance. Therefore, the mechanism of acquisition resistance is essential for the development of follow-up treatment after failure of gefitinib treatment. .
본 발명은 ANT2를 신규의 내성 예측 바이오마커로 이용한다. 이때, 아데닌 뉴클레오티드 트랜스로케이터(adenine nucleotide translocator, ANT)는 미토콘드리아 내막(inner membrane, IM)에 존재하는 효소로서 외막(outer membrane, OM)의 VDAC(voltage dependent anion channel)를 통하여 세포질로부터 미토콘드리아 내부로 ADP를 전달(import)하고 전자전달계(electron transfer chain system)를 거쳐 생성되는 ATP를 세포질로 방출(export)하는 기능을 수행하는 효소로 알려져 있다.The present invention uses ANT2 as a novel resistance predictive biomarker. In this case, the adenine nucleotide translocator (ANT) is an enzyme present in the inner membrane (IM) of the mitochondrial membrane (IM) through the voltage dependent anion channel (VDAC) of the outer membrane (OM) from the cytoplasm to the mitochondria inside the mitochondria. It is known as an enzyme that performs the function of importing and exporting ATP generated through an electron transfer chain system to the cytoplasm.
세포의 에너지 대사에 필수적인 역할을 수행하는 ANT는 3종의 유사체(isoform)인 ANT1, ANT2 및 ANT3가 알려져 있으며 이 중에서 특히 ANT2는 정상세포에서는 그 발현이 낮지만 암세포 또는 증식능이 높은 세포에서는 매우 높게 발현되는 특징을 가지고 있는데 이는 혐기성 상태(anaerobic condition)에서의 해당(glycolysis)반응과 밀접한 관련이 있으며, 또한 최근에는 새로운 암 치료의 표적으로 제시된 바 있다. ANT, which plays an essential role in cellular energy metabolism, is known as three kinds of isoforms, ANT1, ANT2 and ANT3. Among them, ANT2 has a low expression in normal cells but very high in cancer cells or high proliferative cells. It is characterized by being expressed, which is closely related to glycolysis in anaerobic conditions and has recently been suggested as a target for new cancer treatments.
본 발명은 면역조직화학염색을 이용하여 ANT2 단백질의 발현 증가를 확인함으로써 폐암환자의 약물 반응성을 예측할 수 있다. 이때, "면역조직화학염색(immunohistochemistry)"이란, 항원-항체 반응을 이용하여 세포의 단백질 발현 양상을 분석하는 방법이다. 종양세포가 특정 항원 단백질을 발현하는 경우 이에 대한 상보적인 항체로 반응시키면 이 항체는 상보적인 항원에만 특이적으로 결합하게 되고, 이 항원-항체 결합 산물을 2차 항체로 처리하여 반응을 증폭시키고 발색시켜 현미경으로 관찰하면, 종양세포가 항원을 발현하는지(양성) 또는 발현하지 않는지(음성)를 객관적으로 판단할 수 있다.The present invention can predict drug reactivity of lung cancer patients by confirming the increased expression of ANT2 protein using immunohistochemical staining. In this case, "immunohistochemistry" is a method of analyzing protein expression of cells using an antigen-antibody reaction. When a tumor cell expresses a specific antigenic protein, it reacts with a complementary antibody to the antibody, and the antibody binds specifically to the complementary antigen, and the antigen-antibody binding product is treated with a secondary antibody to amplify the reaction and develop color. When observed under a microscope, it can be objectively determined whether tumor cells express (positive) or do not express (negative) antigens.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 일례일 뿐, 본 발명이 하기 실시예에 한정된 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only examples of the present invention, and the present invention is not limited to the following examples.
[실시예]EXAMPLE
실시예 1 : 폐암 조직에서 ANT2 단백질에 대한 면역조직화학염색Example 1 Immunohistochemical Staining for ANT2 Protein in Lung Cancer Tissues
폐암 환자군에서 게피티닙에 대한 치료 효과와 내성획득 유무에 대한 자료가 확보된 환자 조직을 대상으로, ANT2 단백질에 대한 면역조직화학염색(IHC)을 실시하였다. 구체적인 방법은 다음과 같다.Immunohistochemical staining (IHC) was performed on the ANT2 protein in the tissues of patients with lung cancer who obtained data on the therapeutic effect and resistance to gefitinib. The specific method is as follows.
1-1. 폐암 조직의 준비1-1. Preparation of Lung Cancer Tissue
서울대학교병원에서 10명의 폐암 환자로부터 폐암 조직을 외과적으로 제거한 후 분석때까지 액체질소에 보관하였다.Lung cancer tissues were surgically removed from 10 lung cancer patients at Seoul National University Hospital and stored in liquid nitrogen until analysis.
1-2. 조직 슬라이드의 제조1-2. Preparation of Tissue Slides
상기 폐암 조직을 파라핀 포매 블록으로 만든 후, 마이크로톰을 이용하여 4μm 두께로 잘라 유리슬라이드상에 붙여 조직 슬라이드를 제조하였다. 이때, 슬라이드를 드라이오븐(dry oven)에서 65℃, 1시간동안 두어 조직이 떨어지지 않게 하였다. 그 다음, 크실렌과 알코올로 40분간 탈파라핀 과정을 거쳤다.After the lung cancer tissue was made into a paraffin embedding block, it was cut into 4 μm thickness using a microtome and attached onto a glass slide to prepare a tissue slide. At this time, the slide was placed in a dry oven at 65 ° C. for 1 hour to prevent tissue from falling off. Then, 40 minutes of deparaffinization with xylene and alcohol.
1-3. 면역조직화학염색의 전처리1-3. Pretreatment of immunohistochemical staining
상기 준비된 조직 슬라이드에 대하여, ANT2 단백질 항원을 노출시키기 위해 microwave를 5분간 3번 쪼였다. 그 다음, 조직 내에 존재하는 내인성 인자인 혈액을 제거하기 위해 3% H2O2 를 15분간 처리한 후, 항체의 비특이적 결합을 막기 위해 정상 혈청으로 1시간 동안 블록킹(blocking)을 실시하였다.For the prepared tissue slides, microwaves were sectioned three times for 5 minutes to expose the ANT2 protein antigen. Next, 3% H 2 O 2 was treated for 15 minutes to remove blood, an endogenous factor present in the tissue, and then blocked with normal serum for 1 hour to prevent nonspecific binding of the antibody.
1-4. 면역조직화학염색1-4. Immunohistochemical Staining
면역에 사용된 1차 항체는 항-ANT2 항체(Creative BioMart, Cat No. CAB4666MH, 1:100 dilution) 이며, 수세 과정 없이 상기　1차 항체를 실온에서 1시간 동안 처리하였다. 이후, 수세를 한 다음 2차 항체를(biotinylated antibody) 및 streptavidin enzyme complex를 20분간 처리하여 수세를 한 다음 DAB로 발색을 하고, 발색여부를 현미경으로 확인하였다. Mayer Hemtoxylin으로　대조 염색후에 다시 수세 및 건조시켜 permount로 봉입하였다. The primary antibody used for immunization was an anti-ANT2 antibody (Creative BioMart, Cat No. CAB4666MH, 1: 100 dilution), and the primary antibody was treated at room temperature for 1 hour without washing with water. Thereafter, after washing with water, the secondary antibody (biotinylated antibody) and streptavidin enzyme complex were treated with water for 20 minutes, washed with water, and then colored with DAB. After Mayor Hemtoxylin® staining, the cells were washed with water, dried and sealed in permount.
그 결과, 하기 표 1 및 도 1에 나타낸 바와 같이, 항암제의 치료 반응성이 높은 환자 그룹의 조직에서는 비교적 ANT2 발현이 낮음을 확인하였고, 항암제의 치료 반응성이 낮은 환자 그룹의 조직에서는 ANT2 발현이 높음을 확인하였다. 뿐만 아니라, 초기에는 치료 반응성이 좋았으나 치료 후 항암제 내성을 획득하여 치료 반응성이 낮아진 환자의 경우에, 치료 후 ANT2 발현이 현저히 증가되어 있음을 할 수 있다. 병리번호 S05-25201와 S07-2403는 동일 환자로 게피티닙에 대한 내성을 갖기 전 후의 조직으로 분석하였다. As a result, as shown in Table 1 and FIG. 1, it was confirmed that ANT2 expression was relatively low in the tissues of the patient group with high anti-cancer drug treatment, and ANT2 expression was high in the tissues of the patient group with low anti-cancer drug treatment. Confirmed. In addition, in the case of patients who initially had good therapeutic responsiveness but acquired anticancer drug resistance after treatment, and the treatment responsiveness became low, the expression of ANT2 may be markedly increased after treatment. Pathology S05-25201 and S07-2403 were analyzed in tissues before and after the same patient tolerated gefitinib.
표 1
Figure PCTKR2014004754-appb-T000001
 Table 1
Figure PCTKR2014004754-appb-T000001
무radish

Claims (5)

  1. 하기의 단계를 포함하는, 표피성장인자 수용체(Epidermal Growth Factor Receptor; EGFR)를 표적으로 하는 약물에 대하여 비소세포 폐암 환자의 반응성을 예측하는 방법:A method of predicting responsiveness of a non-small cell lung cancer patient to a drug targeting an Epidermal Growth Factor Receptor (EGFR), comprising the following steps:
    폐암조직 샘플에서 ANT2(Adenine Nucleotide Translocator 2) 단백질 또는 mRNA의 발현량을 확인하는 단계, 및Confirming the expression level of ANT2 (Adenine Nucleotide Translocator 2) protein or mRNA in lung cancer tissue samples, and
    대조군에 비하여 ANT2의 발현이 높을 경우, EGFR을 표적으로 하는 약물에 대하여 비소세포 폐암 환자의 반응성이 낮을 것으로 판단하는 단계.When the expression of ANT2 is higher than that of the control group, it is determined that the reactivity of the non-small cell lung cancer patients to the drug targeting EGFR is low.
  2. 제 1 항에 있어서, 상기 약물은 게피티닙(gefitinib) 또는 얼로티닙(erlotinib)인 것을 특징으로 하는 방법.The method of claim 1, wherein the drug is gefitinib or erlotinib.
  3. 제 1 항에 있어서, 상기 ANT2 단백질의 발현량은 면역조직화학염색을 이용하여 확인하는 것을 특징으로 하는 방법.The method of claim 1, wherein the expression level of the ANT2 protein is confirmed using immunohistochemical staining.
  4. 제 3 항에 있어서, 상기 면역조직화학염색은 항-ANT2 항체를 이용하는 것을 특징으로 하는 방법.4. The method according to claim 3, wherein said immunohistochemical staining uses an anti-ANT2 antibody.
  5. 제 1 항에 있어서, 상기 ANT2 mRNA의 발현량은 qRT-PCR을 이용하여 확인하는 것을 특징으로 하는 방법.The method of claim 1, wherein the expression level of the ANT2 mRNA is confirmed using qRT-PCR.
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