Patent Application
20210231669
The text of the computer readable sequence listing filed herewith, titled “38315-251 SEQUENCE LISTING_ST25”, created April 5, 2021, having a file size of 45,012 bytes, is hereby incorporated by reference in its entirety.
The present invention relates to a method for diagnosis of pancreatic cancer using methionyl-tRNA synthetase (MRS) and a kit for diagnosis of pancreatic cancer using the same and, more specifically, to a method for detecting an MRS protein from a pancreatic sample collected from a latent patient to provide information necessary for diagnosis of pancreas cancer, a composition for diagnosis of pancreatic cancer, the composition comprising an agent for measuring the expression level of an MRS protein, and a kit for diagnosis of pancreatic cancer, the kit comprising the composition.
This application claims priority from and the benefit of Korean Patent Application No. 10-2017-0113255 filed on 5 Sep. 2017, which is hereby incorporated in its entirety by reference.
Cancer collectively refers to a group of diseases, which mainly begin with uncontrollable cell proliferation, invade and destroy adjacent normal tissues or organs, and then create new growing sites therein, thereby finally taking the lives of individuals. Although notable progress has been made over the past decade in regulating controlling cell cycles or apoptosis and seeking new targets encompassing carcinogenic genes or cancer-suppressing genes in order to conquer cancer, the incidence of cancer has increased as civilization has advanced.
Out of these, pancreatic cancer is a fatal cancer that has a 5-year survival rate of 1-4% and a median survival period of 5 months, and has an extremely poor prognosis as compared with other human cancers. Pancreatic cancer has a poor prognosis since 80-90% of pancreatic cancer patients are diagnosed in a condition where radical resection of pancreatic cancer for expecting complete recovery is not possible, and moreover, the treatment for pancreatic cancer depends mainly on chemotherapy. Therefore, the development of early diagnosis techniques of pancreatic cancer is urgently requested more than any human cancer. The therapeutic effects of several chemotherapeutic agents, including 5-fluorouracil, gemcitabine, and tarceva, which are known to be effective against pancreatic cancer until today, are extremely disappointing, and the rate of response to chemotherapy is only about 15%. These facts suggest that more accurate and prompt diagnosis techniques are required in order to improve the prognosis of pancreatic cancer patients.
The term “pathological examination” refers to an examination that attempts to elucidate the origin of a disease mainly from a morphological point of view by using extracted cells, tissues, or organs. The pathological examination is an important type of examination applied to the diagnosis of a disease by understanding of macroscopic findings, optical microscopy, electron microscopy, or the like. Such pathological examination includes pathologic histology and pathologic cytology. Meanwhile, biopsy and cytodiagnosis have many differences therebetween, and it has been known that biopsy and cytodiagnosis show many differences in terms of prognostic accuracy, such as diagnostic sensitivity and specificity, in assays using well-known cancer markers. Therefore, it is not sure whether a conventionally known cancer marker can substantially provide effective diagnosis for a specific specimen (tissue or cells).
A difficulty in identifying atypical cells is also one of the obstacles in pathological examination of cells isolated from the body. Since Melamed et al., suggested, as squamous cell atypia, a cell change which is not an inflammatory change but is insufficient to diagnose dysplasia, in 1976, there has been much controversy over the diagnosis, interpretation, and treatment strategy of atypical cells. For the resolution of the controversy, The Bethesda System (TBS) was established. TBS extremely restricts the use of the term “atypical cells” so that the term can be used in only the cases where the cell change cannot be diagnosed to be inflammatory, premalignant, or tumoral, that is, of undetermined significance. Therapeutic strategies for atypical cells are difficult since there may be different views therefor. In particular, there is a problem in that in most cases, a diagnosis result of atypical cells is made in examinations at the tissue or cellular level or a diagnosis result of cancer is made in only tissue specimens although cancer is actually progressing. When it is not clearly discriminated whether the indeterminate tissue structure or cell morphology corresponds to an inflammatory lesion or a neoplasm, it is frequently diagnosed as atypism or cellular atypia. Therefore, several repeated re-examinations by another examination measure or the like are needed, resulting in the consumption of significant time and economic costs.
Since the pancreas is located deep in the body, cancer is difficult to detect when the cancer occurs in the pancreas. In cases of pancreatic cancer that is operable through imaging examinations, a definitive diagnosis of pancreatic cancer is needed prior to surgery through biopsy of mass lesions or cytodiagnosis under endoscopic ultrasound. In addition, in cases of inoperable pancreatic cancer, a biopsy or cytodiagnosis is needed for a histological diagnosis for anticancer therapy or radiotherapy. The representative tumor markers for diagnosing pancreatic cancer are CEA (reference value: 5.0 ng/mL) and CA 19-9 (reference value: 37 U/mL). However, CA19-9 has a problem of low specificity as a pancreatic cancer diagnostic marker. A report says that about 1% of people who had health checkups show an increase in CA19-9 level and only 2% of those showing an increase in CA19-9 level without symptoms were actually found to have cancer. The American Cancer Society guidelines have determined that CA19-9 is less sensitive or specific in screening for pancreatic cancer.
Besides, the development of biomarkers for diagnosing pancreatic cancer is actively ongoing. For example, Korean Patent No. 10-0819122 discloses using matrilin, transthyretin, and stratifin as pancreatic cancer markers, and Korean Patent Publication No. 2012-0082372 discloses using several pancreatic cancer markers. In addition, Korean Patent Publication No. 2009-0003308 discloses a method for diagnosing pancreatic cancer by detecting the expression level of REG4 protein in a blood sample of an individual; Korean Patent Publication No. 2012-0009781 discloses an analysis method for measuring the expression level of XIST RNA in cancer tissues isolated from an individual to provide information necessary for diagnosis of pancreatic cancer of the individual; Korean Patent Publication No. 2007-0119250 discloses a novel gene LBFL313 family that is differently expressed in human pancreatic cancer tissue compared with normal human pancreatic tissue; and US Patent Publication No. 2011/0294136 discloses a method for diagnosis of pancreatic cancer using biomarkers, such as keratin 8 protein.
However, the above-mentioned markers have a limitation in that the markers show a large difference in diagnostic efficiency and accuracy thereof, and are not particularly based on cytological analysis. As for atypical cells that cannot be clearly discriminated whether they are clinically tumor cells or in other disease states, an accurate diagnosis of whether the atypical cells correspond to pancreatic cancer is more important, but there is no marker for accurate diagnosis. Conventionally reported diagnostic markers for pancreatic cancer fail to attain effective diagnosis when applied to cytodiagnosis since such diagnostic markers had poor sensitivity and specificity in cell-level diagnosis unlike when applied to biopsy.
Therefore, in order to diagnose pancreatic cancer, high-priced thorough examinations, such as computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), endoscopic ultrasound (EUS), and angiography, are required in addition to the tumor markers. However, even through these examinations, an accurate diagnosis of pancreatic cancer is very difficult. Moreover, most cases of pancreatic cancer may be advanced cancer cases that have already been unresectable at the time of diagnosis, and only 10-15% of pancreatic cancer cases are operable. A diagnosis of pancreatic cancer is made on inoperable patients through endoscopic ultrasound fine needle aspiration examinations. However, cytodiagnosis through endoscopic ultrasound fine needle aspiration examinations has a limit in definitive diagnosis since the obtained cells cannot be compared with surrounding cells or structures unlike postoperative pancreatic tissues where surrounding structures are intact.
For this reason, the discrimination between pancreatic cancer cells and cells of other diseases (e.g., pancreatitis) still mainly depends on pathological diagnosis based on general staining, such as H&E staining or pap staining. However, the definitive diagnosis of pancreatic cancer by conventional staining may cause different diagnoses according to the experience and interpretation of medical staffs, and especially when the pancreatic cells are identified as atypical cells by pancreatic cell observation through H&E staining or pap staining, it is difficult to discriminate whether the pancreatic cells correspond to pancreatic cancer or a benign disease, and as a result, accurate diagnosis and treatment with respect to diseases of patients are not performed promptly. Since the confirmation on cytodiagnosis is not accurate, appropriate treatment cannot be performed on operable pancreatic cancer patients, and conversely, unnecessary surgery is difficult to prevent. Therefore, to clinically enhance the treatment effect of pancreatic cancer, an accurate diagnosis with respect to cytodiagnosis is needed.
The present inventors performed a careful cytological analysis using pancreatic cell samples obtained from pancreatic cancer clinical patients in order to find markers capable of more accurately diagnosing pancreatic cancer at the cellular level, and as a result, the present inventors first established that malignant tumor cells can be clearly discriminated through the detection of (high) expression of methionyl-tRNA synthetase (MRS) in pancreatic cell samples, and especially, such a discrimination can be made on even cell samples that are identified as atypical cells by conventional pathologic cytology using H&E staining or pap staining and thus cannot be definitively diagnosed for tumor or non-tumor, and completed the present invention.
Therefore, an aspect of the present invention is to provide a method for detecting methionyl-tRNA synthetase (MRS) protein in a pancreatic sample collected from a latent patient, in order to provide information necessary for diagnosis of pancreatic cancer.
An aspect of the present invention is to provide a method for diagnosis of pancreatic cancer, the method comprising:
(a) measuring the expression level of methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) comparing the expression level of the methionyl-tRNA synthetase protein measured in step (a) with that of a negative control, and determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased compared with that of the negative control.
Another aspect of the present invention is to provide: a composition for diagnosis of pancreatic cancer, the composition comprising an agent for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein; and a kit for diagnosis of pancreatic cancer, the kit comprising the composition.
Still another aspect of the present invention is to provide a method for improving sensitivity and specificity in a cytodiagnosis or biopsy of pancreatic cancer, the method comprising:
(a) measuring the expression level of methionyl-tRNA synthetase (MRS) protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
Still another aspect of the present invention is to provide a method for providing information necessary for diagnosis of pancreatic cancer (a method for diagnosis of pancreatic cancer) by performing a method for discrimination of pancreatic cancer cells in combination with a morphological examination in cytodiagnosis or biopsy for pancreatic cancer, wherein the method for discrimination of pancreatic cancer cells comprises:
(a) measuring the expression level of a methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
In accordance with an aspect of the present invention, there is provided a method for detecting a methionyl-tRNA synthetase (MRS) protein in a pancreatic sample collected from a latent patient, in order to provide information necessary for diagnosis of pancreatic cancer.
In accordance with an aspect of the present invention, there is provided a method for diagnosis of pancreatic cancer, the method comprising:
(a) measuring the expression level of methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) comparing the expression level of the methionyl-tRNA synthetase protein measured in step (a) with that of a negative control, and determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased compared with that of the negative control.
In accordance with another aspect of the present invention, there are provided: a composition for diagnosis of pancreatic cancer, the composition comprising an agent for measuring the expression level of methionyl-tRNA synthetase (MRS) protein; and a kit for diagnosis of pancreatic cancer, the kit comprising the composition.
In accordance with still another aspect of the present invention, there is provided a method for improving sensitivity and specificity in a cytodiagnosis or biopsy of pancreatic cancer, the method comprising:
(a) measuring the expression level of methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
In accordance with still another aspect of the present invention, there is provided a method for providing information necessary for diagnosis of pancreatic cancer (a method for diagnosis of pancreatic cancer) by performing a method for discrimination of pancreatic cancer cells in combination with a morphological examination in cytodiagnosis or biopsy for pancreatic cancer, wherein the method for discrimination of pancreatic cancer cells comprises:
(a) measuring the expression level of a methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
Hereinafter, the present invention will be described in detail.
Throughout this disclosure, various aspects or conditions related to the invention can be proposed in a range format. In this specification, the description of a range value encompasses the boundary value unless otherwise stated, in other words, it is meant to encompass all of the values below the lower limit and above the upper limit. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as from 1 to 5 should be considered to have specifically disclosed subranges, such as 1 to 3, 1 to 4, 2 to 5, 2 to 3, 2 to 4, and 3 to 4, as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 3.5, 4.3, and 5. This applies regardless of the breadth of the range.
As used herein, the term “comprising” is used synonymously with “containing” or “being characterized by”, and does not exclude additional elements or steps that are not recited in compositions or methods. The term “consisting of” means excluding additional elements, steps, or ingredients that are not separately described. The term “consisting essentially of” means that in the scope of the compositions or methods, the term includes described ingredients or steps as well as any element or step that does not substantially influence basic characteristics of the compositions or methods.
As used herein, the term “diagnosis” refers to identifying (determining) the presence or characteristics of a pathological condition. Specifically, in the present invention, the diagnosis may be an identification of the presence or absence of pancreatic cancer by determining the expression or non-expression of an MRS protein or the expression level of the MRS protein.
As used herein, the term “MRS” refers to methionyl-tRNA synthetase, and the MRS is an enzyme that mediates aminoacylation of tRNA with amino acid methionine. As long as the MRS protein of the present invention includes an MRS amino acid sequence known in the art, the specific sequence thereof and the biological origin thereof are not particularly limited. For example, MRS is encoded by the MRAS gene in human, and the sequence information of MRS is known by a Genbank accession number, such as NM_004990 (mRNA), NP_004981.2, or P56192.2 (protein). Preferably, the MRS of the present invention may comprise an amino acid sequence of a human MRS protein defined by SEQ ID NO: 1. More preferably, the MRS protein of the present invention may consist of an amino acid sequence defined by SEQ ID NO: 1. The MRS has two isoforms: a cytoplasmic form (cytoplasmic methionyl-tRNA synthetase); and a mitochondrial form (mitochondrial methionyl-tRNA synthetase). The MRS in the present invention may be preferably in a cytoplasmic form.
As used herein, the term “expression” refers to the production of a protein or a nucleic acid in cells.
As used herein, the term “protein” is used interchangeably with the term “polypeptide” or “peptide”, and refers to, for example, a polymer of amino acid residues, as is usually found in proteins in nature.
As used herein, the term “pancreatic cancer” refers to a malignant neoplasm that has characteristics of a fast proliferation rate, infiltration into surrounding tissues, and metastasis to other organs, including a malignant tumor or cancer occurring in the pancreas. The malignant tumor or cancer is distinguished from a benign tumor having characteristics of a slow growth rate and non-metastasis.
More than 90% of cancer cases occurring in the pancreas are developed in pancreatic duct cells, and pancreatic cancer usually means pancreatic ductal cancer or pancreatic ductal adenocarcinoma. Therefore, the pancreatic cancer in the present invention may preferably mean pancreatic ductal adenocarcinoma.
The pancreatic cancer as a target of diagnosis in the present invention is not particularly limited to the cause thereof regardless of whether the pancreatic cancer is a primary cancer, or a cancer secondarily occurring in the pancreas by metastasis. Preferably, a primary cancer may be a target of diagnosis.
As used herein, the term “normal” refers to a state of not being a malignant tumor or cancer (negative for malignancy), and the term is meant to encompass a completely normal state without any disease, another disease state, such as pancreatitis but not a malignant tumor (cancer), or/and a determination state corresponding to “benign”. As used herein, a benign indication described as “benign” in the determination of the clinical state of a (final) disease is distinguished from a positive indication denoted by “positive” on a corresponding examination, and the positive indication denoted by “positive” means that there is a response in the corresponding examination or there is a result indicating the probability of cancer in the corresponding examination.
Approximately 5-10% of pancreatic cancer patients have genetic predispositions, and the incidence of pancreatic cancer in the family population with pancreatic cancer family history is about 7.8%, which is higher than the incidence of pancreatic cancer in general population, 0.6%. The pancreatic cancer has a 5-year survival rate of 5% or less, showing an extremely poor prognosis. The reasons are that: surgical resection is possible within 20% of cancer cases since most cases of pancreatic cancer are found after cancer progression; the micro-metastasis results in a little improvement in the survival rate although complete resection is made in view of the naked eye; and pancreatic cancer has a low response to chemotherapy and radiotherapy.
At present, pancreatic cancer is diagnosed by a biopsy using computed tomography (CT) or magnetic resonance imaging (MRI), and the imaging examination CT or MRI is used, but corresponds to an indirect diagnosis method. Ultimately, the diagnosis of pancreatic cancer is carried out through a biopsy or cytodiagnosis corresponding to a pathological method. The biopsy enables a definitive diagnosis of cancer in a particular region through a comparison with surrounding structures or cells, but the cytodiagnosis cannot prove a relationship with surrounding tissues since individual cells are collected and smeared. Therefore, there are fundamentally many differences between biopsy and cytodiagnosis.
In recent years, the presence or probability of pancreatic cancer is determined by carrying out protein assay together in a body fluid, such as plasma, as a clinical sample that can be used for diagnosis of pancreatic cancer. However, clinical serology is merely used as a reference material for diagnosing pancreatic cancer, and do not provide conclusive information in the diagnosis of pancreatic cancer. Currently, an example of a tumor indicator that is most commonly used with respect to pancreatic cancer may be carbohydrate antigen 19-9 (CA19-9) or carcinoembryogenic antigen (CEA). However, CA19-9 is known to be unsuitable for the diagnosis of pancreatic cancer since the level of CA19-9 in the blood rises even in noncancerous diseases, such as hepatitis, cirrhosis, and pancreatitis, and CA19-9 is not specific to pancreatic cancer since CA19-9 is a marker that is detectable in the blood but not pancreatic cells per se. Therefore, CA19-9 is used to determine the prognosis of a patient by monitoring the serum CA19-9 level after surgery. CEA also has a limitation in the accurate diagnosis of pancreatic cancer since CEA fails to show sufficient sensitivity, specificity, positive predictive value and/or negative predictive value, and these facts are well described in examples herein.
On the contrary, the present inventors not only first established that MRS is (highly) expressed in pancreatic cancer, but also established that the use of MRS as a pancreatic cancer marker can lead to diagnostic results with high accuracy in cytodiagnosis as well as biopsy. That is, MRS is specifically highly expressed in pancreatic cancer cells rather than normal pancreatic cells (i.e., non-tumoral pancreatic cells), and MRS has been revealed to show remarkable effects that MRS enables the determination of pancreatic cancer on pancreatic cell samples with higher accuracy than that of CEA, which has been most used as a pancreatic cancer marker, and especially, in cytodiagnosis, enables the discrimination of pancreatic cancer cells with high accuracy even on atypical cells that are difficult for definitive diagnosis by conventional pathologic cytology (e.g., H&E staining or pap staining).
Accordingly, the present invention provides a method for detecting methionyl-tRNA synthetase (MRS) protein in a pancreatic sample collected from a latent patient, in order to provide information necessary for the diagnosis of pancreatic cancer. That is, the present invention provides a method for diagnosis of pancreatic cancer by measuring the expression level of a methionyl-tRNA synthetase (MRS) protein in a sample of a subject.
Specifically, the Method Comprises:
(a) measuring the expression level of a methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
The subject of the present invention may be an animal, preferably an animal including a mammal, and especially a human, and includes cells, tissues, organs, and the like derived from the animal. More preferably, the subject may be a human or a patient in need of diagnosis. In the present invention, a step of providing the sample from the subject or the latent patient may be performed before step (a).
As used herein, the term “latent patient” refers to a patient suspected of having pancreatic cancer, and means a patient suspected of having pancreatic cancer through various examinations, such as clinical symptoms, hematological examinations, or imaging examinations.
That is, the latent patient includes a patient who can and cannot be diagnosed with pancreatic cancer through imaging examinations, and also means a patient who is suspected of having pancreatic cancer through clinical symptoms or hematological examinations even though the patient cannot be diagnosed with pancreatic cancer through imaging examinations. Examples of the clinical symptoms that pancreatic cancer patients may include abdominal pain, jaundice, weight loss, indigestion, and diabetes, but these symptoms are not specific only to pancreatic cancer. In addition, the levels of jaundice or diabetes or a tumor marker, such as CEA or CA19-9, may be increased in hematological examinations. Imaging examinations, such as abdominal ultrasound, abdominal CT, abdominal MRI, and PET-CT, may be carried out, and in these imaging examinations, the presence of a pancreatic mass raises a suspicion of pancreatic cancer. These imaging examinations can lead to a suspicion of pancreatic cancer, but not a definitive diagnosis of pancreatic cancer. The final definitive diagnosis of pancreatic cancer is carried out by pathological examinations. An operable patient is definitively diagnosed through tissues obtained after surgery, and an inoperable patient is definitely diagnosed mainly through cytodiagnosis.
Preferably, the latent patient (a patient with suspected pancreatic cancer) of the present invention may mean a patient having general symptoms, such as abdominal pain, jaundice, weight loss, indigestion, and diabetes, and cannot be definitively diagnosed with pancreatic cancer merely through diagnostic equipment, such as CT, ultrasound, and MRI. More preferably, the latent patient may be a patient who cannot or need not have a wide area of invasive biopsy (i.e., the surgical pancreatic biopsy is impossible or unnecessary) and thus is in need of a clear diagnosis of pancreatic cancer on the basis of cytology (cytodiagnosis). In other words, the latent patient may be a patient in need of clear diagnosis of pancreatic cancer through analysis at the cellular level, but is not limited thereto.
The sample is not particularly limited as long as the sample is collected from an individual (patient) or a subject to be diagnosed for the presence or absence of pancreatic cancer; however, for example, the sample may be a pancreatic tissue or pancreatic cells. The pancreatic tissue may be obtained from any site (especially, a suspected lesion site) of the pancreas including pancreatic ducts. The pancreatic tissue may be generally obtained by biopsy or surgery of the pancreas. The isolation method for the pancreatic cells is not particularly limited, and is understood as a concept encompassing current methods used to isolate cells of human tissues in the art as well as new methods to be developed for the same purpose in the future. The isolation method may be preferably brushing cytology or fine needle aspiration (FNA), and most preferably endoscopic ultrasound fine needle aspiration (EUS-FNA).
As used herein, the term “collected by brush cytology” means a manner in which cells are collected by brushing the pancreas, especially, pancreatic duct surface (especially, a suspected lesion site) using an ordinary cytology brush.
As used herein, the term “isolated by fine needle aspiration” means a collection manner in which the cells of a lesion (a suspected pancreas cancer site) are aspired and extracted using ordinary used fine needle in cytodiagnosis.
Preferably, in an embodiment, the pancreatic tissue or pancreatic cell sample essentially comprises pancreatic duct cells. The pancreatic duct is distinguished from the pancreatic parenchyma.
The obtained pancreatic cells or tissue may be pretreated according to an ordinary sample pretreatment manner (e.g., fixation, centrifugation, smearing on slides, etc.) known in the art.
In one preferable embodiment, the pancreatic cell or tissue sample may be pretreated by an ordinary paraffin block or paraffin section manufacturing method before being provided on test slides.
In one preferable embodiment, the pancreatic cell or tissue sample may be prepared after being pretreated by an ordinary liquid-based monolayer slide manufacturing method (a method for manufacturing a slide for liquid-based cytology). For example, the pancreatic cell or tissue sample may be provided on a test slide by a liquid-based monolayer attachment method using ThinPrep, SurePath, CellPrep, or the like.
The method for diagnosis of pancreatic cancer of the present invention may be preferably an analysis of pancreatic cells. Cytological analysis for directly analyzing pancreatic cells has many differences from biopsy, and thus is much different from the pancreatic cancer diagnosis methods reported in the prior art documents described herein. Furthermore, the method of the present invention uses pancreatic cells per se, and thus has no likelihood of confusion with tumors of other organs.
In a conventional biopsy, a target site is endoscopically observed, or a tissue with a predetermined area of about 1 g to 109 cells is collected from a tissue suspected of being transformed into cancer, followed by cancer diagnosis through a biochemical manner, such as staining. It is known that such a biopsy comparatively facilitates a definitive diagnosis of cancer present in a particular region through comparison with surrounding structures or cells. The expression patterns of markers at the tissue level also facilitate a definitive diagnosis through the comparison of overall tendencies with surrounding normal tissues. However, cytodiagnosis cannot prove a relationship with surrounding tissues since separate cells are collected and smeared, and thus cytodiagnosis has considerable difficulty in diagnosis. Therefore, the diagnosis of diseases at the cellular level is significant.
As used herein, the term “detection” is meant to encompass all of a measurement and identification of the presence (expression) or absence of a target substance (a marker protein, MRS or/and CK19 in the present invention), or a measurement and identification of a change in presence level (expression level) of a target substance. In the same context, measuring the expression level of the protein means measuring the expression or non-expression (that is, measuring the presence or absence of expression) or measuring the level of qualitative or quantitative change of the protein. The measurement may be carried out without limitation, including all of qualitative methods (analyses) and quantitative methods. In the measurement at the protein level, the kinds of qualitative and quantitative methods are well known in the art, and the test methods described herein are included therein. Specific comparisons of the protein level for respective methods are well known in the art. Therefore, the detection of the MRS protein is meant to encompass detecting the presence or absence of the MRS protein or identifying the increase in expression level (up-regulation) of the protein.
As used herein, the term “increase in expression (or high expression)” of a protein means that a previously unexpressed one is expressed (that is, a previously undetected one is detected) or an overexpression is relatively shown relative to a normal level (that is, the detected amount is increased). This may imply performing a step comprising a comparison or contrast with a negative control. It can be understood by a person skilled in the art that the meaning of the term opposite thereto has an opposite meaning on the basis of the definition.
In the present invention, the detection of the MRS protein is not particularly limited to a method therefor as long as the detection is carried out by a protein expression level measurement method known in the art, but for example, the detection or measurement may be carried out using an antibody specifically binding to the protein. Specifically, the detection of the protein may be carried out by, for example, any one selected from the group consisting of, but is not limited to, western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining (including immunohistochemical staining, immunocytochemical staining, and immunofluorescence staining), immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS) assay, surface plasmon resonance (SPR) assay, or protein chip assay. Besides, in the measurement method is understood in accordance with the descriptions below of an agent for measuring the expression level of MRS and a kit comprising the same, which are provided in the present invention.
Specifically, the method for detecting a methionyl-tRNA synthetase (MRS) protein in a pancreatic sample collected from a latent patient in order to provide information necessary for the diagnosis of pancreatic cancer may be performed by measuring the expression or non-expression or expression level of a methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient and determining the latent patient to be a pancreatic cancer patient when the methionyl-tRNA synthetase protein is (highly) expressed. The method has an advantage in that pancreatic cancer can be immediately determined with a considerable level of accuracy through the detection of a (high) expression of MRS even without comparison with another control group. This is well described in the examples of the invention.
According to an example of the present invention, MRS was strongly (highly) expressed in malignant tumor cells. That is, it was verified that MRS can be used as a pancreatic cancer diagnostic marker with a considerable predetermined level of accuracy.
In particular, it was verified that MRS was highly expressed and detected even in pancreatic cells of a patient wherein the pancreatic cells were identified as atypical cells by conventional cytopathological diagnosis (based on H&E staining, pap staining, or the like) and thus undiagnosable but afterward the patient was finally diagnosed with pancreatic cancer as a result of follow-up observation of the patient. Considering that it is very difficult to discriminate whether atypical cells correspond to a tumor or other benign disease, such as pancreatitis, by conventional cytopathological diagnosis (based on H&E staining, pap staining, or the like) generally used for cancer diagnosis, the determination of whether such atypical cells correspond to a tumor is clinically very important, and it is very meaningful that atypical cells can be determined to be tumor cells when a (high) expression of MRS is observed in the atypical cells.
Previously reported pancreatic cancer diagnostic markers fail to attain effective diagnosis when applied to cytodiagnosis, unlike when applied to biopsy, since such diagnostic markers had poor sensitivity and specificity in the diagnosis at the cellular level. According to another example of the present invention, in cases of CEA as one of the diagnostic markers most generally used in the diagnosis of pancreatic cancer, the expression of CEA was not observed in pancreatic cells, or the extent of expression of CEA, if any, was very weak. In addition, CEA was not expressed in cells identified as atypical cells (afterward, finally diagnosed with pancreatic cancer) as a result of H&E staining. In other words, previously reported pancreatic cancer diagnostic markers do not show consistent expression in atypical cells that are not clearly identified as pancreatic cancer cells or normal cells by conventional cytopathological diagnosis using H&E staining or the like, failing to attain a clear diagnosis, but the MRS of the present invention can attain a clear diagnosis of whether even pancreatic cells identified to be atypical are tumor cells, leading to a more accurate diagnosis of pancreatic cancer.
Conventionally, for diagnosis of pancreatic cancer, high-priced thorough examinations, such as computed tomography (CT), endoscopic retrograde cholangiopancreatography (ERCP), endoscopic ultrasound (EUS), and angiography, are required, but even through these examinations, an accurate diagnosis of pancreatic cancer is very difficult. Ultimately, a pathological method, such as biopsy or cytodiagnosis, needs to be performed for definitive diagnosis of pancreatic cancer. Meanwhile, most cases of pancreatic cancer may be advanced cancer cases that have already become unresectable at the time of diagnosis, and only 10-15% of pancreatic cancer cases are operable. A diagnosis of pancreatic cancer is carried out for inoperable patients through endoscopic ultrasound fine needle aspiration examinations. However, cytodiagnosis through endoscopic ultrasound fine needle aspiration examinations has a limit in definitive diagnosis since the obtained cells cannot be compared with surrounding cells or structures unlike postoperative pancreatic tissues where surrounding structures are intact.
Conventional cytopathological diagnosis mainly depends on general staining, such as H&E staining or pap staining, to discriminate pancreatic cancer cells from cells of other diseases (e.g., pancreatitis). However, when a definitive diagnosis of pancreatic cancer is made by a conventional cytopathological diagnostic method based on such conventional general staining, different diagnoses are made depending on experiences and analysis techniques of medical staffs, and these conventional examinations often show pathological findings as atypical cells that are not clearly identified as pancreatic cancer cells or normal cells (having a comprehensive meaning of non-pancreatic cancer cells, for example, including pancreatitis cells or the like), and thus requiring a plurality of additional and repetitive re-examinations.
Especially, when pancreatic cells are identified as atypical cells in the pancreatic cell observation through H&E staining or pap staining, it is very difficult to discriminate whether the pancreatic cells correspond to pancreatic cancer or other benign disease, failing to make a fast accurate diagnosis and treatment for a diseased patient. Since the accurate diagnosis in cytodiagnosis is delayed, an appropriate treatment cannot be performed on operable pancreatic cancer patients, and conversely, unnecessary surgery is difficult to prevent. Therefore, to clinically enhance the treatment effect of pancreatic cancer, an accurate diagnosis with respect to cytodiagnosis is needed.
Considering that it is very difficult to discriminate whether atypical cells correspond to a tumor or other benign disease by H&E staining or pap staining that is generally used for cancer diagnosis, the determination of whether such atypical cells correspond to a tumor is clinically very important, and it is very meaningful that atypical cells can be determined to be tumor cells when a (high) expression of MRS is observed in the atypical cells. Furthermore, considering that compared with cytodiagnosis, a biopsy requiring relatively large amounts of biopsy material increases the physical burden on patients in terms of sample acquisition, and in some cases, surgery is impossible and tissue collection is impossible depending on the progression of cancer, the MRS marker of the present invention, which provides an accurate diagnosis even at the cellular level, has even greater advantages.
In the present invention, a pathological examination based on morphological diagnosis that has been used may be carried out before, simultaneously with, or after the detection of the MRS protein (or the step of measuring the expression level of the MRS protein. That is, steps (i) and (ii) below may be further performed before, simultaneously with, or after the detection of the MRS protein (or the step of measuring the expression level of the MRS protein):
(i) staining the pancreatic cells collected from the latent patient with: at least one staining solution selected from the group consisting of 4′,6-diamidino-2-phenylindole (DAPI), methylene blue, acetocarmine, toluidine blue, hematoxylin, and Hoechst, which stain nuclei; and at least one solution selected from the group consisting of eosin, crystal violet, and Orange G, which stain cytoplasm; and
(ii) identifying the pancreatic cells as malignant tumor cells, atypical cells, or normal cells, through the staining of the pancreatic cells.
The atypical cells identified in step (ii) are understood as undiagnosed and un-diagnosable cells, and specifically, the atypical cells may be meant to also encompass, but are not limited to, a determination of being suspicious of malignancy in the pathological examination based on morphological diagnosis.
The steps (i) and (ii) above are involved in the cytodiagnosis method according to a pathological examination based on morphological diagnosis, and follow H&E staining and pap staining used in an example of the present invention. As used herein, the term “pathological examination based on morphological diagnosis” or “morphological examination” refers to an examination of an abnormal morphological change when normal cells are transformed into cancer.
Specific examination items or their criteria with respect to the abnormal morphological change are not particularly limited as long as the abnormal morphological change is a kind of morphological change of cancer cells in the art, but at least one selected from the group consisting of: cell clustering; the nuclear to cytoplasmic ratio (N/C ratio); the shape of the nuclear membrane (irregularity of the nuclear membrane shape); chromatin clumping; an appearance of nucleoli in the nucleus; and an appearance of mitosis may be examined. The morphological examination may be performed simultaneously with, separately from, or sequentially with the foregoing method for providing information necessary for the diagnosis of pancreatic cancer.
In step (ii), the identifying of the pancreatic cell sample as malignant tumor cells, atypical cells, or normal cells from the cell-staining results in step (i) may be made on the basis of abnormal morphological changes when normal cells are transformed into cancer, and the specific criteria for identification are well known in the art. The “atypical cells” means cells that cannot be clearly identified as malignant tumor cells (cancer cells) or normal cells by a morphological change.
In one preferable embodiment of the present invention, in step (ii), the identifying of the pancreatic cell sample to be malignant tumor cells, atypical cells, or normal cells from the cell-staining results in step (i) may be performed according to the following criteria:
the pancreatic cells are identified as malignant tumor cells when the pancreatic cells show two or more types of morphological abnormality selected from the group consisting of: a three-dimensional smear of cells; a high nuclear to cytoplasmic ratio (N/C ratio); an appearance of chromatin clumping; a rough-shaped nuclear membrane; an appearance of nucleoli, and an appearance of mitosis;
the pancreatic cells are identified as normal cells when the pancreatic cells are smeared in one layer; the nuclear to cytoplasmic ratio (N/C ratio) is small; and the nuclear membrane has a smooth shape; and
the pancreatic cells are identified as atypical cells when the pancreatic cells have neither a cell change leading to malignant tumor cells, nor can be determined to be normal.
When steps (i) and (ii) are further performed in parallel before, simultaneously with, or after the MRS detection (expression level measurement) step, diagnostic results with very high accuracy can be obtained merely through an examination at the cellular level (that is, cytodiagnosis). For example, pancreatic cells, which are identified as malignant tumor cells or normal cells through the cell staining by performing steps (i) and (ii) before the detection of MRS, can be determined more accurately to correspond to pancreatic cancer or be normal by further re-analyzing the expression level (or expression or non-expression) of the MRS protein in the MRS detection step that is subsequently performed, leading to a significant reduction in diagnostic error. Alternatively, pancreatic cells, which are identified as atypical cells through the cell staining, can be clearly determined to correspond to a tumor or not by analyzing the expression level (or expression or non-expression) of MRS in the MRS detection step. As such, a definitive diagnosis with high accuracy can be made at the cellular level, thereby dramatically resolving problems of conventional pathological examinations, for example, the troubles of again performing a biopsy to carry out a re-diagnosis upon the receipt of examination results indicating atypical cells, and the physical burden on a patient attributable to the need for multiple tissue specimens for accurate definitive determination of pancreatic cancer from a biopsy tissue sample, so that a very excellent diagnostic effect can be produced.
The level of MRS detection (level of MRS expression, especially, a level of MRS increase), which is a standard for diagnosis of pancreatic cancer, may be determined by the presence or absence of detection (expression) or by grading the level of detection (expression) according to a measurement method selected by those skilled in the art. For example, a normal range, a pancreatic cancer occurrence range, and the like are classified according to the level of MRS detection (expression) by measuring the expression levels of MRS in samples of multiple normal persons and patients, followed by data storage and analysis, so that an appropriate criterion for diagnosis can be provided.
In addition, the method may be performed in comparison with a negative control sample (especially, a normal control). Therefore, the method may further comprise a step of, after the detection of MRS (the measurement of the expression level of MRS), comparing the level of the MRS protein detected in the pancreatic sample collected from the latent patient with a negative control sample. As used herein, the term “normal control” is meant to encompass all of a pancreatic sample collected from a normal site in the pancreas of a latent patient to be examined (the same individual as the patient to be examined in step (a)) or a pancreatic sample collected from another normal individual (an individual without pancreatic cancer). If the level of MRS protein detected in the pancreatic sample collected from the latent patient is higher than the level in the negative control (especially, a normal control), the latent patient can be determined to be a pancreatic cancer patient.
The information about such a control (e.g., the intensity of detection or expression) may be provided in the form specified in an agent for measuring the expression level of MRS and a kit comprising the same, which are provided in the present invention described later, or may be provided subordinately in another form. If the expression level of MRS in a sample to be examined is higher compared with such a control, the sample can be determined to correspond to a pancreatic cancer patient.
Therefore, according to an aspect, the present invention provides a method for diagnosis of pancreatic cancer, the method comprising:
(a) measuring the expression level of a methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) comparing the expression level of the methionyl-tRNA synthetase protein measured in step (a) with that of a negative control, and determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased compared with the negative control.
As used herein, the term “normal pancreatic cells” or “normal control” refers to non-tumoral (cancerous) pancreatic cells, and the term is meant to encompass all of pancreatic cells in a state of not a tumor (cancer) but a difference disease (e.g., pancreatitis), benign cells, and fully healthy pancreatic cells (disease-free cells).
In addition, the present invention can provide a method for discriminating atypical cells into cancer cells (malignant tumor cells) and normal cells (non-cancer cells) by comprising steps (a) and (b).
As used herein, the term “sensitivity” refers to a proportion of the determination of pancreatic cancer made through a target examination (e.g., an examination of the present invention) in a sample or patient having a final clinicopathological diagnosis of pancreatic cancer.
As used herein, the term “specificity” refers to a proportion of the determination of being normal made through a target examination (e.g., an examination of the present invention) in a sample or patient having a final clinicopathological diagnosis of being normal.
When the diagnosis of pancreatic cancer employs a manner in which an increase in MRS alone as a marker is detected, the sensitivity of diagnosis shows a level of 80% or higher (80-100%, preferably 80-99%, more specifically, 80-98%) in examinations at the tissue and cellular levels. A specific value for the level includes all of range values each having, as boundary values, two numbers selected from the group consisting of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. In one embodiment of the present invention, out of the numerical ranges, specifically, 80% and 95% may be selected as boundary values, and thus it would be obvious to a person skilled in the art that all of the values in the range of 80% to 95% are intended in the present invention. In another embodiment, the sensitivity may be preferably 80-93%, more preferably 90-92%, and most preferably 85-92%, but is not limited thereto.
In addition, the use of MRS alone as a marker may show a specificity of 60-70% in examinations at the tissue and cellular levels. More preferably, a specificity of 62-68% may be shown. A specific value for the level includes all of range values each having, as boundary values, two numbers selected from the group consisting of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, and 70%. In one embodiment of the present invention, out of the numerical ranges, specifically, 60% and 65% may be selected as boundary values, and thus it would be obvious to a person skilled in the art that all of the values in the range of 60% to 65% are intended in the present invention. In still another embodiment, the specificity may be preferably 60-64%, but is not limited thereto.
Therefore, the present invention provides a method for improving sensitivity and specificity in a cytodiagnosis or biopsy of pancreatic cancer, the method comprising:
(a) measuring the expression level of methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
It would be obvious to a person skilled in the art that the improvements in sensitivity and specificity lead to an improvement in accuracy. Therefore, the method of the present invention can be understood as a method for improving accuracy, and the use of MRS as a marker may lead to an accuracy of 80-100%, preferably 82-98%, and more preferably 85-95%. A specific value for the level includes all of range values each having, as boundary values, two numbers selected from the group consisting of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. In one embodiment of the present invention, out of the numerical ranges, specifically, 80% and 90% may be selected as boundary values, and thus it would be obvious to a person skilled in the art that all of the values in the range of 80% to 90% are intended in the present invention. In still another embodiment, the accuracy may be preferably 80-88%, but is not limited thereto.
Furthermore, the present invention provides a method for providing information necessary for diagnosis of pancreatic cancer (a method for diagnosis of pancreatic cancer) by performing a method for discrimination of pancreatic cancer cells in combination with a morphological examination in cytodiagnosis or biopsy for pancreatic cancer, wherein the method for discrimination of pancreatic cancer cells comprises:
(a) measuring the expression level of methionyl-tRNA synthetase protein in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
The morphological examination is meant to encompass, as a preferable example, an examination conducted by comprising the foregoing steps (i) and (ii), and encompass all of other morphological examinations following such a manner. A description thereof will be made with reference to the above details, and a person skilled in the art could use the above manner through appropriate selection.
The method comprising steps (a) and (b), when performed adjunctively (i.e., as an adjuvant therapy) in addition to a morphological examination, may be performed simultaneously with, separately from, or sequentially with the morphological examination. In addition, the method comprising steps (a) and (b) may be performed before, simultaneously with, or after the morphological examination.
As described above, MRS has an excellent pancreatic tumor (cancer) diagnostic effect as a marker for cytodiagnosis. Therefore, the present invention provides a composition for diagnosis of pancreatic cancer, the composition comprising an agent for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein, or a kit for diagnosis of pancreatic cancer.
In addition, the present invention provides a composition for diagnosis of pancreatic cancer, the composition consisting of an agent for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein, or a kit for diagnosis of pancreatic cancer.
In addition, the present invention provides a composition for diagnosis of pancreatic cancer, the composition consisting essentially of an agent for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein, or a kit for diagnosis of pancreatic cancer.
In addition, the present invention provides use of an agent for measuring the expression level of a methionyl-tRNA synthetase (MRS) protein in the manufacture of an agent for diagnosis of pancreatic cancer.
The agent for measuring the expression level of an MRS protein is not particularly limited to the kind thereof as long as the agent is known to be usable in the measurement of protein expression levels in the art. Preferably, the agent may be an antibody or an aptamer specifically binding to an MRS protein.
As used herein, the term “antibody” refers to an immunoglobulin specifically binding to an antigenic site. More specifically, the term indicates a glycoprotein comprising at least two heavy (H) chains and at least two light (L) chains, which are held together by disulfide bonds. Each of the heavy chains is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2 and CH3. Each of the light chains is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (termed complementarity determining regions (CDRs)), with further conserved regions called framework regions (FRs) distributed therebetween. VH and VL each are composed of three CDRs and four FRs, arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant regions of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
An anti-MRS antibody in the present invention is an antibody that specifically binds only to an MRS protein but does not respond to the other proteins including different types of aminoacyl-tRNA synthetases. The antibody specifically binding to the MRS protein in the present invention may be preferably an antibody specifically binding to a protein (MRS) comprising an amino acid sequence defined by SEQ ID NO: 1. The anti-MRS antibody may be produced by an ordinary method in the art, for example, the MRS gene is cloned into an expression vector to obtain a protein encoded by the gene and the protein thus obtained is injected into an animal to produce the antibody. The MRS antibody may be produced through the MRS full-length sequence protein. Alternatively, an MRS protein-specific antibody can be produced using an MRS protein fragment comprising an MRS antigenic site. The specific sequence and form of the antibody of the present invention are not particularly limited, and include a polyclonal antibody or a monoclonal antibody. In addition, the antibody is not particularly limited to the type of immunoglobulin as provided, and for example, may be selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and may be preferably an IgG antibody. Furthermore, the antibody of the present invention includes special antibodies and recombinant antibodies, such as a humanized antibody and a chimeric antibody, as long as the antibody can specifically bind to an MRS protein. In addition, a part of the whole antibody is also included in the antibody of the present invention as long as the part has antigen-antibody binding properties (response), and all types of immunoglobulin antibodies that specifically bind to MRS are included in the antibody of the present invention. For example, the antibody of the present invention may include not only the full-form antibody having two full-length light chains and two full-length heavy chains but also functional fragments of the antibody molecule, that is, Fab, F(ab′), F(ab′)2, Fv, diabody, scFv, and the like, which have an antigen-binding function.
Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, and is composed of a heavy chain and a light chain each consisting of one variable domain and one constant domain. F(ab′)2 is a fragment produced by pepsin hydrolysis of an antibody, and F(ab′)2 has a form in which two Fab molecules are linked via disulfide bonds at the heavy-chain hinge region. F(ab′) is a monomeric antibody fragment in which a heavy-chain hinge is added to a Fab separated from F(ab′)2 fragment by the reduction of disulfide bonds thereof. Fv (variable fragment) is an antibody fragment composed of only respective variable regions of the heavy and light chains. scFv (single chain variable fragment) is a recombinant antibody fragment in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked to each other via a flexible peptide linker. The term “diabody” refers to a fragment in which VH and VL of scFv, which cannot bind to each other due to the linkage thereof via a very short linker, bind to VL and VH of another scFv in the same form, respectively, to form a dimer. For the purpose of the present invention, the fragment of the antibody is not limited to the structure or form thereof as long as the fragment of the antibody retains binding specificity to the human-derived MRS protein.
In the present invention, the anti-MRS antibody (including functional fragments thereof) is not particularly limited to the site of MRS, which the antibody interacts with (that is, binds to) as long as the antibody can specifically bind to the MRS protein, but preferably, the antibody may be an antibody or a functional fragment thereof, which specifically binds to an epitope region comprising an amino acid sequence defined by SEQ ID NO: 2 in MRS. More preferably, the antibody of the present invention may be an antibody or a fragment thereof, which specifically binds to an epitope containing the 861st to 900th amino acid region in the methionyl-tRNA synthetase (MRS) protein defined by SEQ ID NO: 1.
In an example of the present invention, the present inventors verified that for high-sensitivity detection (staining) for MRS in pancreatic cancer cells, an antibody having, as an epitope, a region of an amino acid sequence defined by SEQ ID NO: 2 in MRS was obtained, and that such an antibody could offer high-sensitivity detection ability for MRS.
The antibody specifically binding to an epitope region comprising an amino acid sequence defined by SEQ ID NO: 2 is not particularly limited to the specific sequence thereof as long as the antibody has desired specific binding ability, but preferably, the antibody may comprise:
a light chain variable region (VL) comprising: light chain complementarity-determining region 1 (CDR1) comprising an amino acid sequence defined by SEQ ID NO: 4 or SEQ ID NO:16; a light chain complementarity-determining region 2 (CDR2) comprising an amino acid sequence defined by SEQ ID NO: or SEQ ID NO: 18; and a light chain complementarity-determining region 3 (CDR3) comprising an amino acid sequence defined by SEQ ID NO: 8 or SEQ ID NO: 20, and
a light chain variable region (VH) comprising: heavy chain complementarity-determining region 1 (CDR1) comprising an amino acid sequence defined by SEQ ID NO: 10 or SEQ ID NO: 22; a heavy chain complementarity-determining region 2 (CDR2) comprising an amino acid sequence defined by SEQ ID NO: 12 or SEQ ID NO: 24; and a heavy chain complementarity-determining region 3 (CDR3) comprising an amino acid sequence defined by SEQ ID NO: 14 or SEQ ID NO: 26.
In the antibody (including functional fragments thereof) of the present invention, as a preferable example having the CDR configuration, the light chain variable region may comprise an amino acid sequence defined by SEQ ID NO: 28 and the heavy chain variable region may include the amino acid sequence defined by SEQ ID NO: 30.
In the antibody (including functional fragments thereof) of the present invention, as another preferable example having the CDR configuration, the light chain variable region may comprise an amino acid sequence defined by SEQ ID NO: 32 and the heavy chain variable region may comprise an amino acid sequence defined by SEQ ID NO: 34.
As the most preferable example, the present invention provides an antibody composed of a light chain consisting of the amino acid sequence of SEQ ID NO: 36 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 37.
As another most preferable example, the present invention provides an antibody composed of a light chain consisting of the amino acid sequence of SEQ ID NO: 38 and a heavy chain consisting of the amino acid sequence of SEQ ID NO: 39.
In the present invention, detecting agents (typically, antibodies and functional fragments thereof) may be labeled with general detectable moieties for “detection” thereof. For example, the detection agents may be labeled with a radioisotope or a fluorescent label by using the technique described in literature [Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N. Y., Pubs]. In addition, various enzyme-substrate labels are usable, and examples of the enzymatic label include: luciferase, such as drosophila luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazine dionese, malate dehydrogenase, urase, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in, for example, literature [O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzyme. (J. Langone & H. Van Vunakis, eds.), Academic press, N. Y., 73: 147-166]. The labels may be directly or indirectly conjugated to antibodies by using various known techniques. For example, an antibody may be conjugated to biotin, and any labels pertaining to three classes of widespread categories cited above may be conjugated to avidin or vice versa. Biotin may selectively bind to avidin, and therefore, this label may be conjugated to an antibody in such an indirect manner. Alternatively, in order to attain the indirect conjugation of a label to an antibody, the antibody may be conjugated to a small hapten (e.g., digoxin), and one of the different types of labels set forth above may be conjugated to an anti-hapten antibody (e.g., an anti-digoxin antibody). Therefore, the indirect conjugation of labels to antibodies can be attained.
As used herein, the term “aptamer” refers to a substance capable of specifically binding to an analyte to be detected in a sample, wherein the aptamer means a single-stranded nucleic acid having a stable three-dimensional structure per se (DNA, RNA, or a modified nucleic acid), and the presence of a target protein in a sample can be specifically identified by an aptamer. An aptamer may be prepared according to a general aptamer preparation method by determining and synthesizing a sequence of an oligonucleotide having selectivity and high binding ability to a target protein to be identified and then modifying the 5′-terminus or 3′-terminus of the oligonucleotide to have —SH, —COOH, —OH, or —NH2 so as to bind the 5′-terminus or 3′-terminus to a functional group of an aptamer chip, but is not limited thereto.
In order to measure the expression level of the MRS protein, the kit for diagnosis of pancreatic cancer of the present invention may comprise not only an antibody or aptamer selectively recognizing an MRS protein but also one or more kinds of other constituent compositions, solutions, or devices suitable for analysis.
In a specific aspect, the kit may be a diagnostic kit comprising known essential elements and subsidiary elements needed for performing western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining assay, immunoprecipitation assay, complement fixation assay, FACS, SPR, or a protein chip assay, but is not limited thereto.
For example, the kit may comprise an antibody specific to an MRS protein. The antibody may be a monoclonal, polyclonal, or recombinant antibody, which has high specificity and affinity to a target marker protein and has little cross-reactivity to other proteins. In addition, the kit may comprise an antibody specific to any control protein. The antibody provided in the kit may itself be labeled with a detectable moiety, which is as described above. Additionally, the kit may further comprise a separate reagent capable of detecting a bound antibody, for example, a labeled secondary antibody, a chromophore, an enzyme (e.g., in the form of being conjugated with the antibody) and a substrate thereof, or other materials capable of binding to the antibody. In addition, the kit of the present invention may comprise a wash or an eluent, which can remove excess chromogenic substrates, unbound proteins, and the like but retain only a protein marker bound to an antibody.
The agent for measuring the expression level of an MRS protein may also be meant to encompass an agent for detecting the expression level of the MRS gene (MARS). An increase in expression level of a protein is accompanied by an increase in transcripts (e.g., mRNA) from a gene encoding the protein, and therefore a person skilled in the art could obviously understand that not only a means of detecting the MRS protein itself but also a means of indirectly detecting transcripts directly related to the expression of the MRS protein can be used.
The agent for detecting MRS mRNA is not particularly limited to the type thereof as long as the agent is a ligand specifically binding to or hybridizing with MRS mRNA, but may be, for example, a primer (pair) or a probe.
The term “primer” refers to a short nucleic acid sequence having a short free 3′ hydroxyl group, wherein the nucleic acid sequence can form base pairs with a complementary template and act as a starting point for template strand replication. Primers may initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in appropriate buffer and temperature conditions. PCR conditions and the length of sense and antisense primers can be appropriately selected according to a technique known in the art.
The sequences of the primers do not necessarily need to be perfectly complementary to a part of the nucleotide sequences of a template, and the primers are favorable as long as the primers hybridize with the template to have sufficient complementarity within a range in which the primers can perform inherent actions thereof. Therefore, the primers for measuring the expression level of MRS mRNA in the present invention do not necessarily need to be perfectly complementary to the MRS coding gene sequence, and the primers are sufficient as long as the primers have a length and complementarity that are fit for the purpose of measuring the amount of MRS mRNA by amplifying a specific section of MRS mRNA or MRS cDNA through DNA synthesis. Primers for the amplification consist of one set (or pair) of primers that bind complementarily to a template (or sense) and an opposite side (antisense), respectively, at both ends of a specific region of the MRS mRNA to be amplified. A person skilled in the art could easily design primers with reference to the nucleotide sequence of MRS mRNA or cDNA.
The term “probe” refers to a fragment of a polynucleotide, such as RNA or DNA, capable of specifically binding to mRNA or complementary DNA (cDNA) of a specific gene and having a length from several to several hundreds of base pairs. Since the probe is labeled, the probe can be used to check for the presence or absence of the target mRNA or cDNA to be bound or the expression level thereof. For the purpose of the present invention, a probe complementary to MRS mRNA can be used for diagnosis by measuring the expression level of MRS mRNA through hybridization with a sample from a subject. A selection of probes and hybridization conditions may be appropriately made according to a technique known in the art.
The primers or probes of the present invention may be chemically synthesized using phosphoramidite solid-phase synthesis or other well-known methods. In addition, the primers or probes may be variously modified by a method known in the art within the scope within which hybridization with MRS mRNA is not impeded. Examples of the modification are methylation, capping, substitution of at least one natural nucleotide with an analogue thereof, modification between nucleotides, for example, modification with an uncharged linker (e. g., methyl phosphonate, phosphotriester, phosphoramidate, carbamate, etc.) or a charged linker (e. g., phosphorothioate, phosphorodithioate, etc.), and binding with a labeling material using a fluorescence or an enzyme.
The kit for diagnosis of the present invention may comprise not only primers or probes recognizing MRS mRNA as a marker to measure the expression level of MRS but also one or more kinds of other constituent compositions, solutions, or devices suitable for analysis. The kit is not particularly limited to the kind thereof as long as the kit is a known diagnostic kit providing primers (primer pairs) or probes as constituents, and examples thereof may include a kit for polymerase chain reaction (PCR), RNase protection assay, northern blotting, southern blotting, or a DNA microarray chip.
As an example, the kit for diagnosis may comprise essential elements required to perform polymerase chain reaction. The kit for polymerase chain reaction comprises respective primer pairs specific to a marker gene (mRNA). The primers are nucleotides having sequences specific to nucleic acid sequences of each marker gene (mRNA), and have a length of about 7-50 bp, and more preferably, about 10-30 bp. In addition, the kit for polymerase chain reaction may comprise primers specific to the nucleic acid sequences of a control gene. Besides, the kit for polymerase chain reaction may comprise test tubes or appropriate containers, buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), DNA polymerase (e.g., Taq-polymerase) and reverse transcriptase, DNAse and RNAse inhibitors, DEPC-water, sterilized water, and the like.
The present inventors found that in the diagnosis of pancreatic cancer, MRS was not expressed in most of normal cells, but a small number of normal cells often showed a false positive by MRS expression, and such a false-positive determination resulted from a high expression of MRS in normal acinar cells. As a result of testing various marker combinations in order to exclude such false-positive reactions and find a method with higher accuracy (including sensitivity and specificity), a dual staining method for MRS and CK19 of the present invention was developed. The present inventors first established that when CK19, besides MRS, is specifically used as an additional double marker in the diagnosis of pancreatic cancer, the diagnostic accuracy of pancreatic cancer in cytodiagnosis is substantially (almost) 100%. Therefore, a sample classified as atypical cells in conventional cytodiagnosis can be discriminated into pancreatic cancer and non-pancreatic cancer (normal).
Accordingly, the present invention provides a method for detecting methionyl-tRNA synthetase (MRS) and cytokeratin 19 (CK19) proteins in a pancreatic sample collected from a latent patient, in order to provide information necessary for the diagnosis of pancreatic cancer. That is, the present invention provides a method for diagnosis of pancreatic cancer by measuring the expression levels of methionyl-tRNA synthetase (MRS) and cytokeratin 19 (CK19) proteins in a sample of a subject.
Specifically, the method may comprise:
(a) measuring the expression levels of MRS and CK19 proteins in a pancreatic sample collected from a latent patient; and
(b) determining the latent patent to be a pancreatic cancer patient when the CK19 protein is expressed and the expression level of the MRS protein is increased in step (a).
As used herein, “CK19” is meant to indicate cytokeratin 19, and CK19 is an intermediate filament protein belonging to type I keratin and is mainly expressed in epithelial cells and is responsible for structural integrity of the epithelial cells. The CK19 protein of the present invention is particularly limited to the specific sequence and a biological origin thereof as long as the CK protein includes the CK19 amino acid sequence known in the art. For example, CK19 is encoded by the KRT19 gene in humans, and the sequence information of CK19 is known by a Genbank accession number, such as NM_002276.4 (mRNA) or NP_002267.2 (protein). Preferably, the CK19 of the present invention may comprise an amino acid sequence of the human CK19 protein defined by SEQ ID NO: 3. More preferably, the CK19 protein of the present invention may consist of the amino acid sequence defined by SEQ ID NO: 3.
In the present invention, the detection of the MRS and CK19 proteins is not particularly limited to a method therefor as long as the detection is carried out by a protein expression level measurement method known in the art. For example, the MRS and CK19 proteins can be detected or measured using antibodies specifically binding to the proteins, respectively. Specifically, the detection of the proteins in the present invention may be carried out by, for example, any one selected from the group consisting of, but is not limited to, western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining (including immunohistochemical staining, immunocytochemical staining, and immunofluorescence staining), immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS) assay, surface plasmon resonance (SPR), or protein chip assay.
Specifically, the method for detecting MRS and CK19 proteins in a pancreatic sample collected from a latent patient in order to provide information necessary for the diagnosis of pancreatic cancer may be performed by measuring the presence or absence of the expression of MRS and CK19 proteins or the expression levels of MRS and CK19 proteins in a pancreatic sample collected from a latent patient and determining the latent patient to be a pancreatic cancer patient when the CK19 protein is expressed and the MRS protein is (highly) expressed in the pancreatic sample. The method has an advantage in that a direct determination of pancreatic cancer (especially, pancreatic ductal cancer or pancreatic ductal adenocarcinoma) with very excellent accuracy can be attained through the detection of (high) expression of the MRS protein together with the CK19 protein even without a particular comparison procedure. This is well described in the examples of the invention.
In addition, the determination can be performed through the comparison with a negative control sample (especially, a normal control sample), and such a comparison of the intensity of detection or expression is understood with reference to the above description. Specifically, cells showing an expression of CK19 protein and an increase in expression of MRS protein compared with a negative control can be determined to be pancreatic cancer cells.
Therefore, according to an aspect, the present invention provides
A method for diagnosis of pancreatic cancer, the method comprising:
(a) measuring the expression levels of MRS and CK19 proteins in a pancreatic sample collected from a latent patient; and
(b) comparing the expression level of the methionyl-tRNA synthetase protein measured in step (a) with that of a negative control, and determining the latent patient to be a pancreatic cancer patient when the expression level of the methionyl-tRNA synthetase protein is increased compared with that of the negative control and the CK19 protein is expressed.
In particular, it was verified that MRS and CK19 were highly expressed and detected even in pancreatic cells of a patient wherein the pancreatic cells were identified as atypical cells by conventional cytopathological diagnosis (based on H&E staining, pap staining, or the like) and thus undiagnosable but the patient was finally diagnosed with pancreatic cancer as a result of follow-up observation of the patient afterward. Considering that it is very difficult to discriminate whether atypical cells correspond to a tumor or other benign disease by conventional pathological cytology that is generally used for cancer diagnosis, the determination of whether such atypical cells correspond to a tumor is clinically very important, and it is very meaningful that atypical cells can be determined to be tumor cells when a (high) expression of MRS is observed in the atypical cells. In particular, the combinative use of MRS with CK19 notably reduces the error of the false-positive determination due to acinar cells, thereby significantly increasing the accuracy in diagnosis of pancreatic cancer.
Furthermore, considering that compared with cytodiagnosis, a biopsy requiring relatively large amounts of biopsy material increases the physical burden on patients in terms of sample acquisition, and in some cases, surgery is impossible and the collection of large amounts of tissue is impossible depending on the progression of cancer, the method of the present invention, which provides an accurate diagnosis even at the cellular level, has even greater advantages.
When the diagnosis of pancreatic cancer employs a manner in which both MRS and CK19 are used as markers and increases thereof are detected, the sensitivity, specificity, positive predictive value and/or negative predictive value is substantially (almost) 100% in the examinations at the tissue and cellular level.
Specifically, at least one selected from the group consisting of the sensitivity, specificity, positive predictive value, and negative predictive value shows a level of 80% or higher (80-100%, preferably 85-99%, and more preferably 90-98%). A specific value for the level includes all of range values each having, as boundary values, two numbers selected from the group consisting of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. In one embodiment of the present invention, out of the numerical ranges, specifically, 80% and 95% may be selected as boundary values, and thus it would be obvious to a person skilled in the art that all of the values in the range of 80% to 95% are intended in the present invention. In another embodiment, the sensitivity, specificity, positive predictive value and/or negative predictive value shows a level of 85% or higher (85-100%, preferably 87-99%, and more preferably 90-98%), but is not limited thereto.
Therefore, the present invention provides a method for improving sensitivity and specificity in a cytodiagnosis or biopsy of pancreatic cancer, the method comprising:
(a) measuring the expression levels of methionyl-tRNA synthetase and cytokeratin 19 (CK19) proteins in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the expression level of the cytokeratin 19 (CK19) protein is detected (expressed) and the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
It would be obvious to a person skilled in the art that the improvements in sensitivity, specificity, positive predictive value and/or negative predictive value lead to an improvement in accuracy. Therefore, the method of the present invention can be understood as a method for improving accuracy, and the accuracy may show a level of preferably 90-100%, and more preferably 90-99%. A specific value for the level includes all of range values each having, as boundary values, two numbers selected from the group consisting of 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, and 100%. In one embodiment of the present invention, out of the numerical ranges, specifically, 92.5% and 99.5% may be selected as boundary values, and thus it would be obvious to a person skilled in the art that all of the values in the range of 93.5% to 99.5% are intended in the present invention. In still another embodiment, the accuracy may show a level of more preferably 93-98%, and most preferably 95-98%, but is not limited thereto.
Furthermore, the present invention provides a method for providing information necessary for diagnosis of pancreatic cancer (a method for diagnosis of pancreatic cancer) by performing a method for discrimination of pancreatic cancer cells in combination with a morphological examination in cytodiagnosis or biopsy for pancreatic cancer, wherein the method for discrimination of pancreatic cancer cells comprises:
(a) measuring the expression levels of methionyl-tRNA synthetase and cytokerain 19 (CK19) proteins in a pancreatic sample collected from a latent patient; and
(b) determining the latent patient to be a pancreatic cancer patient when the cytokerain 19 (CK19) protein is detected (expressed) and the expression level of the methionyl-tRNA synthetase protein is increased in step (a).
The morphological examination is meant to encompass, as a preferable example, an examination conducted by comprising the foregoing steps (i) and (ii), and to encompass all of other morphological examinations following such a manner. A description thereof will be made with reference to the above details, and a person skilled in the art could use the above manner through appropriate selection.
A specific description of steps (a) and (b) is as described above, and the steps may be performed simultaneously with, separately from, or sequentially with the morphological examination when such steps are performed adjunctively (i.e., as an adjuvant therapy). In addition, the determination method comprising steps (a) and (b) may be performed before, simultaneously with, or after the morphological examination.
Furthermore, the present invention provides a composition for diagnosis of pancreatic cancer or a kit for diagnosis of pancreatic cancer, the composition and the kit each comprising agents for measuring the expression levels of methionyl-tRNA synthetase (MRS) and cytokeratin 19 (CK19) proteins (especialy, each agent for MRS or CK19).
In addition, the present invention provides a composition for diagnosis of pancreatic cancer or a kit for diagnosis of pancreatic cancer, the composition and the kit each consisting of agents for measuring the expression levels of MRS and CK19 proteins.
In addition, the present invention provides a composition for diagnosis of pancreatic cancer or a kit for diagnosis of pancreatic cancer, the composition and the kit each consisting essentially of agents for measuring the expression levels of MRS and CK19 proteins.
Furthermore, the present invention provides use of use of an agent for measuring the expression level of an MRS) protein in the manufacture of an agent for diagnosis of pancreatic cancer.
In the present invention, the agent for measuring the expression level of the CK19 protein is not particularly limited to the kind thereof as long as the agent is known to be usable in the measurement of the expression level of a protein in the art. Preferably, the agent may be an antibody or aptamer specifically biding to the CK19 protein, and a specific description thereof follows the descriptions of the above anti-MRS antibody and aptamer.
The agent for measuring the expression level of the CK19 protein is meant to encompass an agent for detecting the expression level of the CK19 gene (KRT19). An increase in expression level of a protein is accompanied by an increase in transcripts (e.g., mRNA) from a gene encoding the protein, and therefore a person skilled in the art could obviously understand that not only a means of detecting the CK19 protein itself but also a means of indirectly detecting transcripts directly related to the expression of the CK19 protein can be used. The agent for detecting CK19 mRNA is not particularly limited to the kind thereof as long as the agent is a ligand specifically attached or hybridized with CK19 mRNA, and may be for example a primer (pair) or probe, a description of which follows the description of the above primer (pair) or probe for MRS mRNA.
In order to measure the expression levels of the MRS protein or/and CK19 protein, the kit for diagnosis of pancreatic cancer of the present invention may comprise not only antibodies or aptamers selectively recognizing the proteins, ligands specifically attaching to (hybridizing with) mRNA encoding the proteins, or primer (pairs), probes, but also one or more kinds of other constituent compositions, solutions, or devices suitable for analysis. The kind of kit according to a specific detection method and the specific components resulting therefrom are understood with reference to the above description.
The methionyl-tRNA synthetase (MRS) as a pancreatic cancer marker shows a much higher diagnostic accuracy than a conventional pancreatic cancer marker, such as CEA, so that a clear determination of pancreatic cancer or non-pancreatic cancer can be made by analyzing the expression or non-expression of methionyl-tRNA synthetase (MRS) in pancreatic cells, and an equivalent effect is achieved in even the cells identified as atypical cells by ordinary staining, and thus MRS can be very helpfully used in the diagnosis of pancreatic cancer.
Hereinafter, the present invention will be described in detail.
However, the following examples are merely for illustrating the present invention, and are not intended to limit the scope of the present invention.
It has been known that in vivo, methionyl-tRNA synthetase (MRS) is present in a state of binding with aminoacyl-tRNA synthetase complex-interacting multifunctional protein 3 (AIMP3) and such binding is broken by UV irradiation or the like. Therefore, for substantially accurate detection of MRS, only MRS needs to be specifically detected even in situations where MRS binds with AIMP3. However, current AIMP types and ARS types have many similarities in terms of protein structures, and thus commercial antibodies have a problem of showing cross-reactivity with other AIMP and ARS types. For diagnostic accuracy in the pancreatic cancer examination method of the present invention, the present inventors produced high-sensitivity MRS antibody having no cross-activity with the other proteins as below.
1-1. MRS-AIMP3 Protein Production
MRS-AIMP3 co-purified protein was expressed and purified from E. coli, and specific experiment methods are as follows. BL21DE3 strain was transformed so as to express MRS (SEQ ID: 1) and AIMP3 (SEQ ID NO: 40, NCBI ref.NM_004280.4), and cultured in LB medium, and then single colony was cultured to reach an OD600 value of 0.6-0.8 in 5 ml of LB liquid medium containing ampicillin. After 1 mM IPTG was added, the cells were incubated at 37° C. for 3 h, and then only the cells were obtained by centrifugation for 10 minutes. SDS-PAGE was performed with the cell solution to check for the expression of the proteins using Coomassie staining. Thereafter, the cell solution having IPTG-induced overexpression was collected and centrifuged to obtain cells. The cells were loosened with 1 ml of DPBS, followed by cell lysis using an ultrasonicator, and then the lysed cells were centrifuged to separate MRS-AIMP3 co-purified protein.
1-2. Mouse Immunization through Injection of MRS-AIMP3 Protein
To obtain immunized mice necessary for the preparation of hybridoma cells, the MRS-AIMP3 co-purified protein obtained in Example 1-1 was primarily injected into the abdominal cavity of four 8- to 10-week old mice. The 10-week old BALB/c mice weighing 25-30 g were purchased from Orient Bio Co. (Sungnam, KyungKi-Do, Republic of Korea). The animals were sufficiently acclimated under predetermined conditions (temperature: 20±2° C., humidity: 40-60%, light cycle: 12-h light/dark cycle), and then used in the present study. Animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee of Seoul National University. Two weeks later, the same dose of MRS-AIMP3 co-purified protein was secondarily injected into the abdominal cavity of the mice to enhance the immunity of the mice after the primary immunization. One week later, the MRS-AIMP3 co-purified protein was booster-injected into the tail vein of the mice three days before cell fusion. After the immunized mice were anesthetized with ether, blood was drawn from the heart using a heparinized syringe. Thereafter, the blood was allowed to stand overnight at 4° C., and then centrifuged to separate serum. The separated serum was appropriately divided and stored at −80° C.
1-3. Preparation of Hybridoma Cells
First, myeloma cells were prepared for cell fusion. The myeloma cells were cultured to a cell density of 2.5 to 5×104 cell/ml. The myeloma cells were prepared by a ⅓ dilution 24 hours before cell fusion. The mice immunized in Example 1-2 were anesthetized with ether, and then spleens were harvested, followed by isolation of B cells. The spleens were washed with SF-DMEM2 (DMEM+2×AA), followed by cell elution. The cell suspension was collected, placed in a tube, and allowed to stand to settle heavy masses. The supernatant was transferred to a new tube, and then centrifuged at 1500 rpm for 5 m. The centrifuged splenocytes were collected by removing the supernatant, and the tube was tapped and then filled with SF-DMEM2. The B cells and the myeloma cells were separately centrifuged and washed, and then washing was repeated once more. The supernatant of the washed myeloma cells was removed, and the tube was tapped and then filled with SF-DMEM2. In addition, the supernatant of the washed B cells was removed, and the tube was tapped, and treated with 1 ml of lysis buffer (LB) to lyse red blood cells (RBCs), and then filled with SF-DMEM2. Then, the B cells and the myeloma cells were separately centrifuged, and the supernatants of the centrifuged splenocytes and myeloma cells were removed, and then the tubes were tapped and filled with 10 ml of SF-DMEM2. The B cells and myeloma cells were diluted 100-fold in e-tubes, respectively, and counted to determine concentrations thereof [B cell concentrations (1×108, 8×107, 5×107), myeloma cell concentrations (1×107, 8×106, 5×106)]. The B cells and the myeloma cells were determined at a ratio of 10:1. The B cells and the myeloma cells with the determined concentrations were placed together in a tube and centrifuged. The supernatant of the centrifuged cells was removed, and then the tube was put upside down on an alcohol pad and semi-dried for 30 s to 1 min, and tapped. PEG (2 ml) was slowly added for 1 min to the tube while pipetting, and the tube was shaken with the addition of SF-DMEM2, followed by centrifugation. After the centrifugation, the supernatant was removed and, without tapping, HT medium [1 ml of HT50×(HT(sigma) 1 vial+SF-DMEM1 10 ml), 10 ml of FBS, 30 ml of SF-DMEM1(DMEM+1×AA)] was added dropwise with gradually increasing speed, to reach 50 ml. This suspension was again incubated in a 5% CO2 incubator at 37° C. for 3 h.
1-4. Selection of Hybridoma Cells Producing MRS-Specific Monoclonal Antibodies
To select cells specifically recognizing MRS but not AIMP3 in the fusion cell groups prepared in Example 1-3 above and investigate the production or non-production of an antibody, the following test was conducted.
First, the medium was exchanged 8-9 days after cell fusion, and incubated in cDMEM2 until the cells were well grown in 96 wells and 24 wells. After medium exchange, the supernatants in wells in which the color has changed were collected and filled with cDMEM2 on day 5-7, and then an ELISA test was performed for the binding of an antibody produced from each fusion cell with MRS and AIMP3. After the ELISA test, wells were selected, and the cells in the selected well were transferred in 24 wells, followed by incubation. After the incubation in 24 wells, an ELISA test was again performed. Specifically, the concentration of fusion cells in 24 wells was checked, and the fusion cells were diluted in 15 ml of the culture so as to reach 0.5 cells/well in the 96-well plate. The fusion cell dilution was dispensed at 150 μl per well. The wells containing one cell were checked by microscopy. The supernatant in the wells containing cells that grew to some extent was collected, and primary screening was conducted by investigating the binding of the antibody, produced from respective fusion cells, with MRS and AIMP3 through ELISA and western blotting. The fusion cells selected on the basis of the primary screening were transferred and incubated in 24 wells, followed by centrifugation, and thereafter, the supernatant was collected, and secondary screening was conducted by ELISA and western blotting. The absorbance (OD value) of the fusion cells grown in the 24 wells was checked by ELISA to select only fusion cells having an absorbance exceeding 1.0. The selected cells were transferred and incubated in 25T/C culture flask and centrifuged, and then the supernatant was collected, and tertiary screening was conducted by ELISA and western blotting. The fusion cells selected on the basis of the tertiary screening were again transferred and incubated in 75T/C culture flask, and then the absorbance was checked by ELISA to select cells well recognizing MRS but not AIMP3, thereby finally securing “1E8” and “8A12” clones.
1-5. Culture of Hybridoma Cells Producing MRS-Specific Monoclonal Antibodies and Purification of Antibodies
Monoclonal antibodies to MRS can be obtained from the final fusion cells (hybridoma cells “1E8” and “8A12”) selected in Example 1-4 by the following two methods.
1) Female mice aged 7-8 weeks were injected with 500 μl of pristane through the abdominal cavity. The fusion cells cultured in the 75T/C culture flask were collected and centrifuged, followed by supernatant removal, and then the cells were placed in a phosphate buffer and pipetted. After 7-10 days of pristane administration, the fusion cells selected in Example 1-4 were injected in an amount of 8×105 to 4×107 cells into the abdominal cavity of the mice. When the abdominal cavity of the mice was full of ascites 12 weeks later, the ascites were extracted using an 18G-syringe needle. The ascites were kept at 4° C. overnight and then centrifuged the next day, thereby removing a mass material containing a yellow layer of fat and separating only the supernatant. The separated supernatant was dispensed and stored at −20° C.
For the purification of antibodies from the ascites, a column was packed with an appropriate amount of protein A, which has been stored in a stock solution (20% ethanol), and 20% ethanol was allowed to flow through the column, and then the column was washed with a 5-bed volume of a binding buffer (20 mM sodium phosphate, pH 7.0). The ascites were diluted with an appropriate amount of a phosphate buffer, and was then loaded on the protein A column. After binding was conducted using a 3-bed volume of a binding buffer (20 mM sodium phosphate, pH 7.0), 5 ml of fractions were eluted with a 3-bed volume of an elution buffer (0.1 M glycine buffer, pH 3.0-2.5). Each fraction was neutralized with 35 μl of a neutralization buffer (1M Tris-HCl, pH 9.0). The fractions were checked for purity through SDS-PAGE, and desalted with Ammersharm GE column.
2) The hybridoma cells obtained in Example 1-4 were acclimated in serum-free medium (Thermo) supplemented with GlutaMAX (Gibco) (final 5 mM) and lx cholesterol lipid concentrate (Gibco) (8A12 antibody-producing hybridoma cells are also named 34-8F2). Thereafter, the cells were cultured in a maximum culture volume of 860 ml using Cellstack-5 (Corning, Corning, NY). GlutaMAX (Gibco) (final 5 mM) and 1× cholesterol lipid concentrate (Gibco) were added to the serum-less medium (Thermo), and the cells were inoculated at an initial cell concentration of 1.4-2.0×105 cells/ml. After 4-5 days of the inoculation, the cells were removed by centrifugation at 2000 rpm for 10 m and the culture supernatant was recovered. The pH of the supernatant was checked, and then the pH was adjusted to 7.6 using the prepared 20X binding solution (1M potassium phosphate dibasic, pH 9.0). Thereafter, the supernatant was filtered using a 0.22-μm filter to obtain a neutralized antibody culture solution.
The obtained antibody culture solution was purified through a protein A column. After 10 column volumes of distilled water were allowed to flow through the protein A column, an equal amount of 1X binding solution (50 mM potassium phosphate dibasic, pH 9.0) was allowed to flow therethrough. Thereafter, the obtained antibody culture solution was allowed to flow therethrough to bind antibodies to protein A, followed by washing with 1X binding solution (50 mM potassium phosphate dibasic, pH 9.0). To elute the antibodies bound to protein A, 2 column volumes of an elution solution (0.2 M citric acid, pH 3.0) were allowed to flow therethrough, thereby obtaining an eluate. After the eluate was neutralized with 1 M Tris, the concentration of antibodies was determined by measurement of the absorbance at 280 nm.
Thereafter, the GE PD-10 column was equilibrated with 25 ml of physiological saline and then centrifuged (1,000 g, 2 min). Thereafter, 2.5 ml of the antibody eluate obtained from the protein A column was added to the GE PD-10 column, followed by centrifugation (1000 g, 2 min), thereby collecting an antibody solution exchanged with physiological saline. The antibody concentration was determined by measurement of the absorbance at 280 nm, and then the antibody solution was dispensed and stored at −80° C.
1-6. Antibody Sequencing and Cloning
The 1E8 and 8A12 clone-expression antibodies were cloned and sequenced by YBIO Inc. and Abclon Inc. (Korea), respectively. Briefly, RNA was first extracted from 1E8 or 8A12 hybridoma cells to synthesize cDNA. Then, PCR was performed using primers specific to VL, CL, VH, and CH1. PCR products with expected sizes were purified on agarose gel, the sequences thereof were identified through sequencing, and CDR regions were identified through Kabat numbering. The sequencing results of the antigen-binding region of 1E8 antibody are shown in Table 1, and the sequencing results of the antigen-binding region of 8A12 antibody are shown in Table 2. The Fab molecules were synthesized from the identified sequences, and were verified to show high binding ability to MRS through ELISA.
It was also verified that the above identified sequences are consistent with the protein sequencing results (mass spectrometry results) of the antibodies, which were obtained through ascites purification after the hybridoma cells were injected into the abdominal cavity of the mice in Example 1-5.
The obtained 1E8 Fab or 8A12 Fab sequences were cloned into the mouse IgG heavy chain sequence vector (pFUSE-mIgG2a-Fc, InvivoGen) and mouse light chain (pFUSE2-CLIg-mK, InvivoGen) sequence vector. Then, the vectors were co-transformed into freestyle 293F cells using PEI (Polysciences, 23966-2), so that antibody heavy and light chains were co-expressed together in the cells. The transformed 293F cells were cultured under conditions of 37° C. and 8% CO2 for 7 days. Then, the cells were obtained and centrifuged, and the supernatant was obtained. The pH of the supernatant was checked, and then the pH of the supernatant was adjusted to 7.6 using the prepared 20X binding solution (1M potassium phosphate dibasic, pH 9.0). Thereafter, the supernatant was filtered using a 0.22-um filter to obtain a neutralized antibody culture solution. An antibody was obtained from the antibody culture solution by the method described in 2) of Example 1-5. It was confirmed that the whole 1E8 IgG antibody thus obtained is composed of a light chain comprising an amino acid sequence of SEQ ID NO: 36 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 37. It was also confirmed that the whole 8A12 IgG antibody thus obtained is composed of a light chain comprising an amino acid sequence of SEQ ID NO: 38 and a heavy chain comprising an amino acid sequence of SEQ ID NO: 39.
1-7. Verification on Comparison of Binding Specificity of Antibody to MRS—Western Blotting
To investigate the MRS binding ability of the 1E8 and 8A12 antibodies obtained in the above example, the following western blot test was conducted. H460 cells were incubated in DMEM (HyClone, GE Life Sciences) containing 10% fetal bovine serum (FBS, HyClone, GE Life Sciences) and 1% penicillin (HyClone, GE Life Sciences). All the cells were incubated under conditions of 5% CO2 and 37° C. The incubated H460 cells were treated with si-MRS for 72 h. Then, the H460 cells were obtained and lysed, and then the H460 cell lysate was subjected to western blotting. The test was repeated twice. The 1E8 or 8A12 antibody as a primary antibody was diluted to 1:5000 (0.2 μg/ml) before use and, for comparison of binding ability, a currently commercially available MRS antibody (Abcam, Ab50793) was used by the same method, and tubulin was used as a control.
As a result of the test, as shown in
In addition, the MRS-binding ability of the 8A12 and 1E8 antibodies was further investigated using the PANC-1 pancreatic cancer cell line and SCK cell line (non-pancreatic cancer cell line). A commercially available MRS antibody (Abcam, Ab137105) was used as a control (antibodies were diluted to 1:1000 before use, 0.137flg/m1), and western blotting was carried out in the same manner as described above except for the si-MRS treatment procedure.
As a result of the test, as shown in
1-8. Verification on Cross-Reactivity with other Proteins—ELISA
To investigate whether the 8A12 antibody obtained in the above example had cross-reactivity with other aminoacyl-tRNA synthetase (ARS) proteins, the following test was conducted.
On 96-well plates (Corning 3690 flat bottom, 96-well half-area plates), MRS proteins (His-MRS and MRS full) and other ARS proteins (DX2 tag free, 34S-DX2, 34S-AIMP2, His-CRS, His-AIMP1, His-GRS, His-WRS, and His-KRS) were each coated at a concentration of 1 μg/ml. The 1E8 or 8A12 antibody was added at a concentration of 500 ng/ml to the 96-well plates coated with the respective ARS proteins, followed by incubation for 1 hour. Thereafter, HRP-conjugated anti-mouse IgG secondary antibody was added, followed by incubation for 1 h, and then the absorbance at 450 nm was measured by ELISA. As a substrate, 3,3′,5,5′-tetramethylbenzidine (TMB) was used.
As a result, as shown in
1-9. Verification on Antibody Affinity using Surface Plasmon Resonance
To investigate MRS-specific affinity of the 1E8 and 8A12 antibodies, a surface plasmon resonance (SPR) test was conducted using MRS-AIMP3 co-purified protein (hereinafter, MRS+AIMP3 protein) and AIMP3 protein. MRS+AIMP3 or AIMP3 protein was coated on a CM5 chip, and different concentrations of the 1E8 or 8A12 antibody were allowed to flow down, thereby measuring the degree of binding response with the protein. An analyte sample or buffer was injected at a flow rate of 30 μl/min for 8 minutes, and washed for 20 minutes.
As a result, as shown in
As shown in
1-10. Verification on Binding Site of MRS Antibodies
To investigate the binding site (epitope, main binding site) of the 1E8 or 8A12 antibody, the following test was conducted.
First, several MRS fragments with different lengths and loci in the MRS whole protein were constructed in consideration of GST, catalytic domain, and tRNA binding domain sites, and the MRS whole protein or each MRS fragment was cloned into the pcDNA3 vector (EV). The loci of the respective MRS fragments were selected at loci containing several small-unit domains, including the fragment of aa 1-266, the fragment of aa 267-597, the fragment of aa 1-598, the fragment of aa 598-900, the fragment of aa 660-860, the fragment of aa 660-900, the fragment of aa 730-900, and the like, in the whole amino acid sequence of MRS of SEQ ID NO: 1. In the fragments, the Myc protein was conjugated to the N-terminus of each peptide, and the Myc protein was used as a control. Then, 2 μg of the cloned vector DNA was transfected into H460 cells by using Turbofect (Thermo) according to the instruction of the manufacturer. After 24 h, the cells were harvested and western blotting was carried out. For the primary antibody, 1E8 and 8A12 antibodies were diluted at 1:5000 (0.2 μg/mL) before use.
Through the test, the epitope of the 1E8 and 8A12 antibodies was shown to be present in at least a region of aa 598-900 in the MRS protein of SEQ ID NO: 1.
Therefore, several small-unit domains, including the fragment of aa 811-840, the fragment of aa 821-850, the fragment of aa 831-860, the fragment of aa 841-870, the fragment of aa 846-875, the fragment of aa 851-880, the fragment of aa 856-885, the fragment of aa 861-890, the fragment of aa 866-895, and the fragment of aa 871-900, were constructed, and each peptide was coated at 300 ng/well on 96 well ELISA plates and ELISA was carried out according to the common protocol. The 1E8 or 8A12 antibody as a primary antibody was diluted to 10 nM (lx PBST-Tween 0.05%), and HRP-conjugated Goat anti-mouse IgG (Thermo) as a secondary antibody was diluted at 1:10000 (1xPBST-Tween0.05%). The absorbance was measured at 450 nm.
The test results verified that the 1E8 and 8A12 antibodies specifically recognized, as an epitope, the region of aa 861-900 (AQKADKNEVA AEVAKLLDLK KQLAVAEGKP PEAPKGKKKK, SEQ ID NO: 2) in the MRS protein. These test results indicate that other binding molecules (other antibodies and functional fragments thereof) recognizing the region of aa 861-900 as an epitope would also have excellent MRS-specific binding ability and MRS-identifying ability.
Hereinafter, an example employing antibodies with excellent MRS detection ability constructed above will be described for the novel pancreatic cancer examination method invented by the present inventors.
1-11. Pancreatic Cancer Cell Staining using Anti-MRS Antibody of Present Invention and Comparison of Effect with Commercially Available Antibody
As for the PANC-1 pancreatic cancer cell line and the SCK cell line (non-pancreatic cancer cell line), MRS fluorescence staining was carried out using 1E8 or 8A12 antibody. A commercially available MRS antibody (Abcam, Ab137105) was used as a control. Specifically, staining was carried out as follows. Each type of target cells prepared on slides was treated with PBS containing 0.2% Tween 20 to improve cell permeability thereof, and then blocked with 2% goat serum for 1 h. The slides were incubated with 1 μg/ml of the antibody (1E8 or 8A12 antibody, produced by Oncotag) of the present invention as a primary antibody or Ab137105 antibody (Abcam) at 37° C. for 1 h, and washed three times with 0.05% TBST (500 μl). Thereafter, the Alexa-488-conjugated secondary antibody (purchased from Molecular Probes, Cat. No. A11001) as the fluorescent substance-conjugated secondary antibody was diluted to 1:100, and the slides were incubated with the secondary antibody at room temperature in the dark place, and washed three times with 0.05% TBST (500 pl). The tissue on the slides was treated with 20 μl of DAPI-added mounting solution (ProLong Gold antifade regent with DAPI/ Molecular Probes, Cat. No. P36931), and then the slides were covered with coverslips, followed by observation using a confocal laser microscope and a fluorescence microscope.
As shown in
In addition, the staining effects of the 1E8 and 8A12 antibodies of the present invention were investigated by utilizing the Thinprep device (Hologic. Inc) used in patient sample processing in clinical sites to manufacture Thinprep slides similar to clinical conditions of the PANC-1 cell line (see Example 2 below).
As a result, as shown in
Methods
1) Pancreatic cells as a specimen were obtained by endoscopic ultrasound fine needle aspiration (EUS-FNA). First, a linear array echoendoscope EUS (product name: GF-UCT140 or GF-UCT180, Olympus, Japan) was guided to the stomach or duodenum, and an ultrasound device mounted on the front end of the EUS was used to identify pancreatic masses through ultrasound imaging. The pancreatic cells were collected by allowing a needle for fine needle aspiration (product name: 22G Echo-ultra™, Cook Medical, Cork, Ireland) to enter the ultrasound-guided masses.
Thereafter, the collected pancreatic cells were provided, as Cellient paraffin sections, on slides by a common method using the Cellient Automated Cell Block System (Hologic) (Antonio Ieni et al., Cell-block procedure in endoscopic ultrasound-guided-fine-needle-aspiration of gastrointestinal solid neoplastic lesions, World J Gastrointest Endosc 2015 August 25; 7(11): 1014-1022). Alternatively, the pancreatic cancer cells may be smeared or provided through direct smearing on ThinPrep slides by a common method using ThinPrep (Hologic.Inc) (de Luna R et al., Comparison of ThinPrep and conventional preparations in pancreatic fine-needle aspiration biopsy. Diagnostic Cytopathology, 2004 Feb;30(2):71-6). The cell samples each were subjected to the following examination method, and the results thereof were compared.
2) Pathological findings by a conventional cytological examination may be made by the staining results using H&E staining that has frequently been used up to now. The H&E staining was carried out using hematoxylin and eosin according to a common protocol (see protocol details in Example 3 below). Alternatively, the pathological findings may be made by the staining results through Pap staining. The Pap staining was carried out using hematoxylin, OG-6(Orange G-6), and eosin azure according to a common protocol (see protocol details in Example 3 below). The paraffin section samples were treated with staining substances after paraffin removal and hydration were carried out through common methods.
The cells were determined to be benign (normal) cells when: the cells were smeared in one layer on the slides; the nuclear to cytoplasmic ratio (N/C ratio) is small; and the nuclear membrane has a smooth shape. The cells were determined to be malignant cells when: the cells were three-dimensionally smeared; the nucleus/cytoplasm ratio was high; chromatin clumping appeared; the nuclear membrane had a rough shape; and nucleoli and mitosis appeared. The cells were identified as atypical cells when the cell change did not reach malignant cells but could not be diagnosed with benign.
3) The final clinical diagnosis results were made by doctors through a comprehensive final determination on the basis of the measurement results by imaging examinations (abdominal ultrasound, abdominal computed tomography, abdominal magnetic resonance imaging, endoscopic retrograde cholangiogram, and positron emission tomography) and pathological examinations (cytodiagnosis and biopsy).
4) The present inventors developed the immunohistochemistry (especially, immunofluorescence staining) for measuring the expression level of MRS in pancreatic cells and normal pancreatic cells (including benign pancreatitis cells but not cancer cells) as follows. Specifically, the paraffin sections were treated as follows.
{circle around (1)} Paraffin removal: dissolve paraffin in oven at 60° C.
{circle around (2)} Hydration: wash with xylene for 5 min three times, wash with 100% ethanol two times, wash with 95% ethanol for 2 min, wash with 90% ethanol for 2 min, wash with 70% ethanol for 2 min, wash with D.W for 2 min, and wash with PBS for 5 min
{circle around (3)} Treatment for permeability increase: treat with 0.2% Triton X-100 for 30 min, and wash with PBS for 5min
{circle around (4)} Pretreatment: block with 2% goat serum for 1 h
{circle around (5)} Primary antibody treatment: dilute anti-MRS antibody (the 8A12 antibody of the present invention being representatively used, Oncotag) to 1 : 300, incubated with the diluted antibody at 4° C., and wash with PBS for 5 min three times
{circle around (6)} Color development: The Alexa-488-conjugated secondary antibody (purchased from Molecular Probes, Cat. No. A11001) as a secondary antibody to which a fluorescent substance is conjugated to 1:200 - 1:300, incubate with the secondary antibody at room temperature in the dark for 1 h, and wash with PBS for 5 min three times
{circle around (7)} Treat the tissue on the slides with 20 pl of DAPI-added mounting solution (ProLong Gold antifade regent with DAPI/Molecular Probes, Cat. No. P36931), and cover the slides with coverslips.
The Thinprep slides were treated by a method comprising steps {circle around (3)} to {circle around (7)}, without paraffin removal, and reference samples prepared by the method were observed by a confocal laser microscope and a fluorescence microscope.
The MRS staining intensity was determined in the reference samples on the basis of the positive cell sample (PANC-1, see
Results
Specific test results are shown in Tables 3, 4, and 5 below. As a result of applying the pancreatic cancer discrimination method through MRS staining of the present invention to 14 benign pancreatic cell samples and 94 pancreatic cancer (malignancy) cell samples, the conventional pathologic cytology results using H&E staining showed a sensitivity of about 75.5%, whereas the MRS staining of the present invention showed a sensitivity of 92.6%. In particular, even cell samples that have been classified as atypia through a conventional cytopathological examination could be discriminated for pancreatic cancer or non-pancreatic cancer, and thus the negative predictive value (NPV) through the conventional pathologic cytology is 36%, whereas the negative predictive value through MRS staining was significantly high. These results indicate that the staining method using MRS as a marker in pancreatic cancer in the present invention shows high discrimination ability (diagnostic ability) even at the cellular level, and as shown in Example 3 to be described later, the MRS used as a marker of the present invention was clearly distinguished from conventional commercially available pancreatic cancer markers (known pancreatic cancer markers, such as CEA) of which effectiveness had been difficult to exhibit in the diagnosis at the cellular level (that is, cytodiagnosis).
Methods
1) Patient Groups and Obtaining Pancreatic Cell Samples for Cytodiagnosis
The present study was performed using 26 cases of pancreatic cell samples collected and obtained from 26 patients with suspected pancreatic cancer through cell aspiration (fine needle aspiration) under endoscopic ultrasound, and approved by the Research Ethics Committee of the Gangnam Severance Hospital. The pancreatic cell samples were obtained from the patients by the same method as in Example 2 through endoscopic ultrasound fine needle aspiration (EUS-FNA). Thereafter, the collected pancreatic cells were prepared in a state of Cellient paraffin sections or Thinprep by a common method using the Cellient Automated Cell Block System (Hologic) (see Example 2).
The 26 patients with suspected pancreatic cancer were finally diagnosed with pancreatic cancer (13 cases) and normal pancreas (13 cases) through follow-up observation. Among 13 patients finally diagnosed with pancreatic cancer, seven cases were classified as having pancreatic cancer cells through histological observation by pathologists, and the other six cases were classified as having atypical cells by histological observation and finally diagnosed with pancreatic cancer through follow-up observation.
The patients provided written informed consent at the time of collection of pancreatic cells, and pancreatic cells, atypical cells, and normal cells were histologically confirmed by pathologists (see the standard for determination in Example 2). Respective types of representative diagnostic cases for normal cells, tumor cells and atypical cells among the 26 obtained samples are shown in the drawing for each test.
2) Conventional Cytodiagnosis Staining
H&E staining: The paraffin section samples were subjected to paraffin removal and hydration by treatment with xylene for 5 min three times, treatment with 100% ethanol for 2 min, treatment with 95% ethanol for 2 min, treatment with 90% ethanol for 2 min, treatment with 70% ethanol for 2 min, and treatment with tap water for 10 min. The samples were incubated with hematoxyline at room temperature for 30 s, and washed with tap water for 10 min. The samples were incubated with eosin at room temperature for 1 min, and washed with tap water for 10 min. The samples were subjected to dehydration and clearing by treatment with 70% ethanol for 1 min, 90% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min, and xylene for 5 min three times. After the mounting solution was dropped on the tissue on the slides, the slides were covered with cover slides, and then the samples were observed under an optical microscope.
Pap staining: Papanicolaou staining was carried out using Varistain 24-4 stainer of Thermo Scientific according to the protocol embedded in the device. The protocol is shown in Table 6 below.
3) Criteria of Determination in Cytodiagnosis According to Morphological Pathology by Conventional Staining
The most critical evidence in the morphological diagnosis of malignancies is an invasive growth into surrounding normal tissues, but unlike the biopsy facilitating to prove the relationship between cancer and surrounding tissues, the cytodiagnosis cannot prove the relationship with the surrounding tissues since individual cells were extracted and smeared. Therefore, as an alternative, the atypia of individual cells is evaluated. The atypia refers to a high nuclear to cytoplasmic ratio (N/C ratio) resulting from cell nucleus enlargement and cytoplasm reduction, chromatin partially clumping without uniform distribution in the nucleus, appearance of nucleoli in the nucleus, appearance of mitosis, or the like. The diagnosis of malignancy can be made when all of these findings of atypia are shown and the degrees thereof are severe, but cells should be classified as atypical cells when the findings of the cells are partially shown or the degrees thereof are weak. The reason is that such findings may be partially shown even in benign lesions, such as severe inflammation. On the contrary, cells not showing any of the findings can be determined to be normal cells. It should be of course assumed that the cells can be observed at the site where the cells are collected.
4) Comparative Staining using CEA, known Commercially Available Pancreatic Cancer Marker, and Present Inventive MRS
Immunostaining for MRS was carried out in the same manner as described in Example 2. The immunostaining for MRS was carried out by the same procedures as in Example 2 except that Carcinoembryonic Antigen (CEA) antibody (purchased from Dako, Cat. No. M7072) was used for an optimal treatment condition.
5) Statistical Analysis
The test results are expressed as mean±standard deviation on the basis of three or more independent test results. For statistical significance, the Student's t test was used to analyze data to compare the difference among multiple groups. The test results were determined statistically significant when P-value was less than 0.05.
Results
3-1. Immunostaining of Normal Cells
The immunofluorescence staining using MRS or CEA antibody was carried out on the cells that have been determined to be normal as a result of analysis of H&E staining of the cells isolated from the pancreas of normal patients or pancreatic cancer patients by EUS-FNA. The results are shown in
As shown in
3-2. Immunostaining of Tumor Cells
The immunofluorescence staining using MRS or CEA antibody was carried out on the cells that have been determined to be tumor cells as a result of analysis of H&E staining of the cells isolated from the pancreas of pancreatic cancer patients. The results are shown in
As shown in
3-3. Immunostaining of Atypical Cells
MRS or CEA staining was carried out on the pancreatic cells of patients, which are atypical cells that could not be clearly determined to be tumor cells by only H&E staining and have been finally determined to be tumor cells as a result of follow-up observation of the prognosis of the patients afterward. The results are shown in
As shown in
It can be seen from the results of Example 3 that performing both H&E staining and MRS staining on pancreatic cells isolated from patients with suspected pancreatic cancer can diagnose pancreatic cancer with much higher accuracy than other tumor markers. In addition, conventional tumor markers (e.g., CEA) cannot clearly discriminate whether the pancreatic cells, which cannot be determined to be tumor cells or normal cells even through H&E staining, are tumor cells or normal cells, but MRS is a very meaningful pancreatic cancer diagnostic marker in that MRS can clearly discriminate, with significantly high accuracy, whether the atypical cells are tumor cells.
4-1. Finding cause of False-Positive Results
As shown in Example 3, it has been proved that MRS enables a diagnosis of pancreatic cancer with higher accuracy than conventional existing pancreatic cancer markers, such as CEA. However, the tests in the above-described examples were conducted on all the samples that have already been definitively determined, and therefore, it is necessary to investigate how accurately MRS can diagnose in a blind state after tissue was collected from patients suspected of developing pancreatic cancer. In addition, as shown in Example 2, MRS showed very excellent sensitivity in the diagnosis of pancreatic cancer, but MRS alone showed a somewhat low tendency of specificity as compared with sensitivity in the determination of pancreatic cancer. The present inventors, while testing the ability of MRS to discriminate pancreatic cancer in pancreatic samples obtained from new patients, observed that out of ten samples determined to be negative (non-pancreatic cancer) in pathologic cytology, three samples seemed to show MRS expression. That is, it was confirmed that the expression of MRS is high in some normal pancreatic cell samples, causing an error of false-positive results (a determination may be made diagnosing non-pancreatic cancer samples as a pancreatic cancer).
As a result of checking a cause of the false-positive determination by discriminate the overall specimens into cancer tissues and normal tissues through H&E staining and then carrying out MRS staining, it was verified as shown in
4-2. Establishment of Strategies for Improving MRS Diagnostic Accuracy
As shown in the results in Example 4-1, a high level of MRS was expressed in even normal acinar cells, and thus MRS alone contributes to a false-positive rate in the determination of pancreatic cancer (i.e., the specificity of diagnosis is relatively low in the determination using MRS alone). Therefore, a strategy for reducing the false-positive rate was sought by excluding such acinar cells from the interpretation of the results.
After various trials, the present inventors first established that pancreatic cancer (especially pancreatic ductal cancer) can be discriminated with very high accuracy at the cellular level by using, as a double marker, MRS and cytokeratin 19 (CK19) among several subsidiary marker candidates. Therefore, double staining using MRS and CK19 was invented as follows.
4-3. Establishment of Double Staining
As for paraffin section samples, paraffin was melted in an oven at 60° C., and subjected to paraffin removal and hydration by washing with xylene for 5 min three times, washing with 100% ethanol two times, washing with 95% ethanol for 2 min, washing with 90% ethanol for 2 min, washing with 70% ethanol for 2 min, and washing with D.W for 2 min.
For double staining of MSR and CK19, a specific test protocol was established such that MRS was detected in green (Alexa Fluor 488) and CK19 was detected in red (Texas Red). First, cells were treated with 0.2% Triton X-100 to increase cell permeability. An incubation was conducted with 2% goat serum, a blocking solution, for 1 h. Thereafter, the pancreatic cells (specimen) was incubated with the primary antibody, methionyl-tRNA synthetase antibody (1:200) and anti-CK19 antibody(1:100) at 4° C. overnight. Thereafter, the cells were washed three times or more by performing at least 30 times of dipping in 1X TBST (assumed as one time), and then incubated with Alexa Fluor 488- and Texas Red-conjugated secondary antibodies (1:200 to 1:300, ThermoFisher Scientific, catalog no. A-11001/SANTA CRUZ BIOTECHNOLOGY, INC., catalog no. sc-278) at room temperature in a dark place for 1 h. The cells were washed three times or more by performing at least times of dipping in 1X TBST (assumed as one time). Thereafter, the DAPI-added mounting solution (ProLong Gold antifade regent with DAPI, Molecular probes, Cat. No. P36931) was dropped onto the tissue on slides, and then the cells were covered with cover slides, and the samples were observed by a fluorescent microscope.
The standard for pancreatic cancer discrimination is as shown in Table 7 below. The cells were determined to be pancreatic cancer cells when high levels of MRS and CK19 were detected (increasingly expressed). That is, the cells were classified as Positive for MRS (+) and CK19 (+) and classified as Negative for the others.
When the level of MRS in a specimen sample was increased by 2-fold or more as compared with the negative cell sample (CT-26), together with expression of CK19, the cells were determined to be pancreatic cancer cells. The intensity of staining was based on those of positive cell sample (PANC-1) and the negative cell sample (CT-26).
The discrimination of pancreatic cancer was carried out by applying double staining of the present invention to 29 new pancreatic cancer cell specimens obtained from patients different from those used in the above-described examples. These pancreatic cancer cells were collected by EUS-FNA in the same manner as described above. The double staining of the present invention was carried out while pathological diagnosis results or final clinical diagnosis results were blinded.
5-1. Staining Pattern in Patient Classified as Atypical Cells by Conventional Pathologic Cytology and thus Undiagnosable but Finally Diagnosed with Pancreatic Cancer
In the cell sample that has been classified as atypical cells by conventional pathologic cytology through pap staining and diagnosed with malignancy in the final clinical diagnosis, the double staining of the present invention was applied to discriminate whether it is pancreatic cancer or not.
As shown in
5-2. Staining Pattern in Patient Undiagnosed Due to being Suspicious of Pancreatic cancer by Conventional Pathologic cytology but finally diagnosed with pancreatic cancer
In the cell sample that has not been diagnosed with pancreatic cancer by conventional pathologic cytology through pap staining and thus temporarily diagnosed in a state of suspicious of pancreatic cancer (malignancy) but diagnosed with malignant tumor cells in the final clinical diagnosis, the double staining of the present invention was applied to discriminate between pancreatic cancer cells and benign cells.
As shown in
5-3. Staining Pattern in the Specimen from Patient Diagnosed with Pancreatic Cancer by Conventional Pathologic Cytology and Finally Diagnosed with Pancreatic Cancer
In the cell sample that has been identified as atypical cells as a result of analysis by conventional pathological examinations through pap staining and diagnosed with malignant tumor cells in the final clinical diagnosis, the double staining of the present invention was applied to discriminate between pancreatic cancer cells and benign cells.
As shown in
5-4. Overall Result
A comparison was made among the determination results obtained by carrying out the double staining of the present invention, the final clinicopathological diagnosis results, and the pathologic cytology diagnosis results, for each cell specimen, and the comparison results are shown in Table 8. As a result of double staining of the present invention, the cell specimen was classified as Positive of pancreatic cancer for MRS (+) and CK19 (+) and Negative of pancreatic cancer for the other case (i.e., either MRS or CK19 being stained, or MRS (−) and CK19 (−))
As shown in Table 8 above, the determination of pancreatic cancer through MRS and CK19 double staining of the present invention showed a sensitivity of 100%, a specificity of 100%, a positive predictive value (PPV) of 100%, and a negative predictive value (NPV) of 100%, indicating an improvement in diagnostic accuracy.
As shown in Example 5, the double staining of the present invention can clearly discriminate between malignant tumor cells and non-tumor cells from the cells that have been undiagnosable and undiagnosed by a conventional method, including atypical cells, thereby significantly improving the diagnostic efficiency in cytodiagnosis.
The double staining of the present invention was applied to clinical tissues containing both pancreatic cancer and surrounding normal pancreas area. The tissues obtained by surgery were paraffin-embedded by a common method, and then sliced. The double staining and determination results according to the present invention are shown in Table 9 below. As shown in Table 9 below, the tissue samples, unlike cytodiagnosis samples, were discriminated into acinar and pancreatic duct in the pancreatic tissues and thus can be separately determined according to the region.
As shown in Table 9 above, the test results verified that the accuracy in discrimination between pancreatic cancer and normal pancreas through the double staining of the present invention reached 100% (sensitivity 100%, specificity 100%). It was especially verified that the double staining of the present invention can be used to provide information about normal acinar cells, thereby significantly increasing the diagnostic accuracy.
As set forth above, the present invention relates to a method for diagnosis of pancreatic cancer by using methionyl-tRNA synthetase (MRS). Methionyl-tRNA synthetase (MRS) as a pancreatic cancer marker shows a much higher diagnostic accuracy than a conventional pancreatic cancer marker, such as CEA, so that a clear determination of pancreatic cancer can be made by analyzing the expression or non-expression of methionyl-tRNA synthetase (MRS) in pancreatic cells, and an equivalent effect is achieved in even the cells identified as atypical cells by ordinary staining, and thus MRS can be very helpfully used in the diagnosis of pancreatic cancer and thus has high industrial applicability in the field of in-vitro diagnostic industry.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0113255 | Sep 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/010369 | 9/5/2018 | WO | 00 |
