JP2016214239A - Pancreatic cancer marker - Google Patents

Abstract

An object of the present invention is to provide a marker exhibiting high sensitivity and specificity for pancreatic cancer. Another object of the present invention is to provide a pancreatic cancer diagnostic kit for detecting the marker and a method for evaluating the progression of pancreatic cancer using the marker. A pancreatic cancer marker according to the present invention is a specific RNA that accumulates in the leaf-like temporary foot of a pancreatic cancer cell. [Selection figure] None

Description

  The present invention relates to a marker exhibiting high sensitivity and specificity for pancreatic cancer, a pancreatic cancer diagnostic kit for detecting the marker, and a method for evaluating the progression of pancreatic cancer using the marker. Is.

  “Tumor” refers to a cell that has proliferated abnormally, and refers to a state in which cell growth continues even if the cause of the abnormal growth disappears or is removed. Among tumors, benign tumors grow slowly and do not metastasize. Therefore, in general, there is no problem if it is excised, and it can be said that there is no difference in life even if it is left without being excised. On the other hand, unlike benign tumors, malignant tumors, that is, cancers, proliferate rapidly and metastasize to lymph nodes and other organs. Therefore, for example, even if removed by surgical operation, cancer cells that remain even slightly, or cancer cells that have already metastasized to lymph nodes or other organs may start to proliferate again. Therefore, cancer has a poor prognosis after treatment is completed, and the survival rate for each cancer has been investigated after 5 years. Generally, 5 years have passed since the cancer disappeared due to treatment. If there is no recurrence until later, it is finally considered a cure.

  Pancreatic cancer is said to have the worst prognosis among cancers. This is because the pancreas is a retroperitoneal organ and is difficult to detect at an early stage, and the pancreatic cancer cells have extremely high motility. Invasion into the digestive tract, nerves, etc., metastasis to nearby lymph nodes, and distant metastasis to the liver. Therefore, it is very important to evaluate the progression of pancreatic cancer.

  A biopsy is accurate in assessing cancer progression, but it can be painful to the patient. Therefore, generally, a test using a cancer marker is performed in advance. A cancer marker is a substance specifically produced in vivo by cancer, and the progression of cancer can be evaluated by measuring the amount in the body fluid. For example, CA125, CA19-9, CEA, elastase 1, SLX, STN are used as pancreatic cancer markers, and CA19-9 is the most common.

  However, since conventional cancer markers are not perfect in terms of sensitivity and specificity, research on better cancer markers is being conducted. Recently, for example, as in the invention described in Patent Document 1, miRNA (microRNA) has been actively developed as a cancer marker.

Japanese Patent Laying-Open No. 2015-51011

  As described above, cancer markers are also used in actual clinical practice, but markers that exhibit higher sensitivity and specificity are always required. For example, the marker described in Patent Document 1 is intended for “cancer except breast cancer”, and it can be said that the specificity is low in this respect.

  Therefore, an object of the present invention is to provide a marker exhibiting high sensitivity and specificity for pancreatic cancer. Another object of the present invention is to provide a pancreatic cancer diagnostic kit for detecting the marker and a method for evaluating the progression of pancreatic cancer using the marker.

This inventor repeated earnest research in order to solve the said subject. As a result, it was found that a specific RNA accumulated in the lamellipodia peculiar to pancreatic cancer cells was extremely useful as a pancreatic cancer marker, and the present invention was completed.
Hereinafter, the present invention will be described.

  [1] CCDC88A mRNA, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA, and a combination of two or more types selected from WASF2 mRNA Pancreatic cancer marker.

[2] The pancreatic cancer marker according to [1], which has any one of the following base sequences 1 to 3.
Base sequence 1: one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25 Base sequence of;
Base sequence 2: One or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: 25 A base sequence in which 1 to 10 bases are deleted, substituted and / or added, and the base sequence of RNA found in the body fluid of pancreatic cancer patients;
Base sequence 3: one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25 A nucleotide sequence having 95% or more sequence identity to the nucleotide sequence of RNA and found in the body fluid of pancreatic cancer patients.

[3] A pancreatic cancer diagnostic kit for detecting specific RNA,
The RNA is one or more selected from the group consisting of CCDC88A mRNA, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA and WASF2 mRNA,
A forward primer and a reverse primer complementary to a part of the base sequence of the cDNA of the RNA, and
A pancreas comprising a fluorescent intercalator that binds to a double-stranded RNA synthesized by PCR and at least one fluorescent compound selected from a fluorescent probe having a partial sequence complementary to the base sequence of the RNA. Cancer diagnostic kit.

  [4] The base sequences of the forward primer and the reverse primer complementary to a part of the base sequence are SEQ ID NO: 2 and SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24, and SEQ ID NO: 26 and SEQ ID NO: 27 bases The diagnostic kit for pancreatic cancer according to the above [3], which is one or more selected from the group consisting of a combination of sequences.

[5] A method for evaluating the progression of pancreatic cancer,
CCDC88A mRNA, SNORA71B snoRNA, SNORA18 snoRNA, SNORA14B snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA, and WASF2 A method comprising the steps of:

  [6] The method according to [5] above, wherein a blood sample is used as the sample.

  [7] The method according to [5] or [6] above, wherein the amount of the RNA is measured by real-time RT-PCR.

  The pancreatic cancer marker according to the present invention is RNA that accumulates on the lamellipodia peculiar to pancreatic cancer cells. Further, the present inventors have found that even if it is RNA, it is released from pancreatic cancer cells in a state of being encapsulated in exosomes. Therefore, since the progression of pancreatic cancer can be evaluated with high sensitivity and specificity if the pancreatic cancer marker according to the present invention is a detection target, the marker of the present invention can replace conventional tumor markers such as CA19-9. You can say that.

FIG. 1 is a graph showing the results of quantitative analysis of CCDC88A mRNA in blood by one-step real-time RT-PCR. The median of each group was 15.19 (2.44 to 175.81, quartile range: 15.02) in the pancreatic cancer patient group, and 6.52 (0.5 to 0.52) in the non-pancreatic cancer patient group. 12.41, interquartile range: 4.51). In addition, when Mann-Whitney U test was conducted to examine whether there was a significant difference in relative mass between the pancreatic cancer patient group and the non-pancreatic cancer patient group obtained by real-time RT-PCR, p = 0.008673 A significant difference was observed. FIG. 2 is a fluorescent photograph for confirming the location of CCDC88A mRNA in pancreatic cancer cells. (1) is a fluorescent photograph showing the presence of CCDC88A mRNA (green), (2) is a fluorescent photograph showing the presence of CD63 (red), an exosome marker, and (3) is the presence of nucleus (blue) It is the photograph which superposed | stacked the fluorescent photograph of (1) and (2) on the fluorescent photograph which shows this. FIG. 3 is a graph showing the results of quantitative analysis of CCDC88A mRNA in blood by one-step real-time RT-PCR. FIG. 4 is a graph showing the results of quantitative analysis of SNORA14B snoRNA in blood by one-step real-time RT-PCR. FIG. 5 is a graph showing the results of quantitative analysis of SNORA25 snoRNA in blood by one-step real-time RT-PCR. FIG. 6 is a graph showing the result of quantitative analysis of SNORA22 snoRNA in blood by one-step real-time RT-PCR. FIG. 7 is a graph showing the results of quantitative analysis of SNORD22 snoRNA in blood by one-step real-time RT-PCR. FIG. 8 is a graph showing the result of quantitative analysis of SNORA74A snoRNA in blood by one-step real-time RT-PCR. FIG. 9 is a graph showing the results of quantitative analysis of ARF6 mRNA in blood by one-step real-time RT-PCR. FIG. 10 is a graph showing the results of quantitative analysis of VAV3 mRNA in blood by one-step real-time RT-PCR. FIG. 11 is a graph showing the results of quantitative analysis of WASF2 mRNA in blood by one-step real-time RT-PCR. FIG. 12 is an ROC curve when CCDC88A mRNA is used as a marker. FIG. 13 is an ROC curve when CA19-9 alone, which is a conventional pancreatic cancer marker, a combination of CCDC88A and SNORA14B, and a combination of CCDC88A, SNORA14B and CA19.9 is used for diagnosis of pancreatic cancer. FIG. 14 is a graph showing the results of quantitative analysis of CCDC88A mRNA in blood for each UICC stage by real-time RT-PCR. FIG. 15 is a graph showing the results of quantitative analysis of ARF6 mRNA in blood for each UICC stage by real-time RT-PCR. FIG. 16 is a graph showing the results of quantitative analysis of VAV3 mRNA in blood for each UICC stage by real-time RT-PCR. FIG. 17 is a graph showing the results of quantitative analysis of WASF2 mRNA in blood for each UICC stage by real-time RT-PCR. FIG. 18 is a graph showing the results of quantitative analysis of CA19-9 in blood for each UICC stage by real-time RT-PCR. FIG. 19 is a graph and ROC curve showing the results of quantitative analysis of CCDC88A mRNA in blood of stage IIA and IIB pancreatic cancer cases and control group by real-time RT-PCR. FIG. 20 is a graph and ROC curve showing the results of quantitative analysis of ARF6 mRNA in the blood of stage IIA and IIB pancreatic cancer cases and control group by real-time RT-PCR. FIG. 21 is a graph and ROC curve showing the results of quantitative analysis of VAV3 mRNA in blood of stage IIA and IIB pancreatic cancer cases and control group by real-time RT-PCR. FIG. 22 is a graph and ROC curve showing the results of quantitative analysis of WASF2 mRNA in blood of stage IIA and IIB pancreatic cancer cases and control group by real-time RT-PCR. FIG. 23 is a graph and ROC curve showing the results of quantitative analysis of CA19-9 in the blood of stage IIA and IIB pancreatic cancer cases and control group by real-time RT-PCR.

1. Pancreatic Cancer Marker The pancreatic cancer marker according to the present invention is a specific RNA that accumulates on the leaf-like temporary foot of pancreatic cancer cells. By using the RNA as a detection target, the progression of pancreatic cancer can be evaluated with high sensitivity and specificity.

  The pancreatic cancer marker according to the present invention is selected from CCDC88A mRNA, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA and WASF2 mRNA. Only one type of pancreatic cancer marker according to the present invention may be selected, or two or more types may be used in combination. By combining two or more of the above RNAs, there is a possibility that progression of pancreatic cancer can be evaluated with higher sensitivity and specificity.

  Pancreatic cancer cells are characterized by high motility and easy metastasis, and have a protrusion site involved in cell motility called lamellipodia. The specific RNA that is a marker according to the present invention is accumulated in the leaf-like temporary foot of pancreatic cancer cells. Moreover, the present inventors have found that the above RNA is released out of pancreatic cancer cells in a state of being encapsulated in exosomes. Therefore, it is possible to evaluate the progression of pancreatic cancer with high sensitivity and specificity by measuring the amount of RNA in the body fluid sample of the subject.

  Furthermore, CA19-9, which is currently used mainly as a pancreatic cancer marker, is an antibody against sugar chains derived from cell membrane glycolipids that increase with the progression of pancreatic cancer and the like. Whereas the sensitivity to pancreatic cancer is low, the pancreatic cancer marker according to the present invention is an RNA characteristic of pancreatic cancer cells as described above. May show high sensitivity.

  In addition to CCDC88A mRNA, which is one of the markers according to the present invention, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA, and WASF2 mRNA are similar to CCDC88A mRNA. Accumulated in lamellipodia and secreted from pancreatic cancer cells encapsulated in exosomes. Preliminary experiments have confirmed the same effect as CCDC88A mRNA, and are useful as pancreatic cancer markers. it is conceivable that.

  CCDC88A mRNA, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA and WASF2 mRNA, which are markers of the present invention, are generally SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 4, respectively. It has the base sequences of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: 25. Depending on the pancreatic cancer patient, the RNA found in the body fluid may have another sequence in addition to the base sequence, or a part of the base sequence may be mutated. That is, the scope of the present invention includes a case where RNA has the following base sequence.

Base sequence 1: one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25 Base sequence of;
Base sequence 2: One or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: 25 A base sequence in which 1 to 10 bases are deleted, substituted and / or added, and the base sequence of RNA found in the body fluid of pancreatic cancer patients;
Base sequence 3: one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25 A nucleotide sequence having 95% or more sequence identity to the nucleotide sequence of RNA and found in the body fluid of pancreatic cancer patients.

  In the base sequence 2, the number of deletions, substitutions and / or additions is preferably 8 or less, 6 or less, more preferably 5 or less or 4 or less, and even more preferably 2 or less. The sequence identity in the base sequence 3 is preferably 96% or more or 98% or more, more preferably 99% or more, and still more preferably 99.5% or more or 99.8% or more. Since mRNA has a larger number of bases than snoRNA, the number of mutations and sequence identity may differ between snoRNA and mRNA. For example, compared to snoRNA, the number of mutations in mRNA may be large and the sequence identity may be low.

  In the base sequence 2 and the base sequence 3, the presence / absence and position of deletion, substitution and / or addition, and sequence identity can be analyzed by direct comparison of the sequences. Analysis can be performed using sequence analysis software or the like.

  However, there are only a few studies on RNA accumulated in the foliate foot of pancreatic cancer cells, and there has been no report that the mRNA and snoRNA according to the present invention have base sequences other than the above base sequences so far. It is preferable that the base sequence of RNA consists of said sequence number 1, sequence number 4, sequence number 7, sequence number 10, sequence number 13, sequence number 16, sequence number 19, sequence number 22, and sequence number 25.

  In the following, the description of CCDC88A mRNA having the base sequence of SEQ ID NO: 1 according to the present invention also applies to the other RNAs described above.

  In the present invention, the above “sensitivity” refers to sensitivity to pancreatic cancer, and means that, for example, when the RNA is a detection target, the probability that a pancreatic cancer patient is determined to be negative is low. In the present invention, the “specificity” is specificity for pancreatic cancer, and there is a strong correlation between the amount of RNA and the presence or absence of pancreatic cancer, while the amount of RNA is It means that there is no or low correlation with the presence or absence of benign pancreatic tumors such as pancreatic cystic tumors (IPMN) as well as cancer types other than pancreatic cancer.

2. Next, a method for evaluating the progression of pancreatic cancer using the above-mentioned pancreatic cancer marker as a detection target will be described step by step. The “evaluation of the progression of pancreatic cancer” in the present invention includes the determination of the presence or absence of pancreatic cancer in addition to the evaluation of the degree of progression of pancreatic cancer.

(1) Sample acquisition process In this process, a sample is acquired from a subject. The sample herein refers to a sample obtained by treating collected body fluid such as serum or plasma in addition to the body fluid of the subject such as blood, lymph and urine.

  As a sample used in this step, a blood sample is preferable. A blood sample refers to the blood itself, serum or plasma.

(2) RNA amount measuring step In this step, the amount of the mRNA and / or snoRNA in the collected sample is measured. The amount to be measured may be not only the absolute amount of the RNA in a predetermined amount of sample, but also the concentration of the RNA in the sample.

  The measuring means is not particularly limited as long as it can measure the amount of RNA in the sample. For example, real-time RT-PCR, ELISA, microarray analysis, Northern blot, etc. can be mentioned. These can be carried out by a conventional method. Among the measurement means, real-time RT-PCR is particularly preferable because it can be easily carried out at a low cost. Hereinafter, the real-time RT-PCR will be described representatively.

(2-1) Reverse Transcription Reaction In the present invention, since the amount of the RNA in the sample is measured, in order to perform PCR, it is first necessary to synthesize cDNA from the RNA in the sample by a reverse transcription reaction.

  As a primer used in the reverse transcription reaction, an oligo dT primer or a random primer for reverse transcription of the RNA according to the present invention over the entire length, or a reverse primer among gene-specific primers used in subsequent PCR can be used. In the case of one-step RT-PCR described below, it is necessary to use a specific reverse primer for the RNA according to the present invention.

  In this step, the RNA is reverse-transcribed using a primer specific for the RNA according to the present invention. The conditions for the reverse transcription reaction may be in accordance with conventional methods.

(2-2) Real-time RT-PCR
Next, real-time PCR is performed using cDNA reverse transcribed from the RNA according to the present invention as a template. Real-time RT-PCR includes one-step real-time RT-PCR that performs reverse transcription reaction and PCR continuously in one pot, and two-step real-time RT that performs PCR by dispensing the reaction solution of reverse transcription reaction into another reactor. -There is PCR.

  Two-step real-time RT-PCR is considered to be more reactive than one-step real-time RT-PCR. On the other hand, since one-step real-time RT-PCR performs reverse transcription reaction and PCR continuously in one pot, it is possible to efficiently measure many samples.

  Primers used in the real-time PCR are complementary to a part of the base sequence of the cDNA of the RNA, and are designed to amplify cDNA corresponding to all or part of the RNA. For example, the nucleotide sequence characteristic of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 and / or SEQ ID NO: 25 is amplified. Design what you can. In general, it is preferable to use a primer set in which the size of DNA to be amplified is about 80 bp to 150 bp, and the length of each primer is about 17 bases to 25 bases. If the size of the DNA to be amplified is about 80 bp to 150 bp, high amplification efficiency can be obtained. If the length of each primer is about 17 bases to 25 bases, sufficient specificity can be secured and synthesis is easy. It is. Furthermore, in order to prevent the formation of secondary structure in the primer and the formation of complementary strands between primers, do not complement more than 3 bases inside or between primers, align Tm values of forward primer and reverse primer, and BLAST search. It is also important to confirm the characteristics of each primer by, for example.

  As a suitable primer used for detecting CCDC88A mRNA which is one of the pancreatic cancer markers according to the present invention, primers having the nucleotide sequences of SEQ ID NO: 2 (forward primer) and SEQ ID NO: 3 (reverse primer) are used. Can be mentioned. When other RNAs according to the present invention are quantified, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 15, Primers having the nucleotide sequences of SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24, and SEQ ID NO: 26 and SEQ ID NO: 27 can be preferably used. As a set of a forward primer and a reverse primer, only 1 type may be selected from the said combination, and 2 or more types may be selected.

  In the present invention, in addition to the amount of RNA described above, the amount of CA19-9, which is a conventional pancreatic cancer marker, is also measured, so that the progression of pancreatic cancer can be evaluated with higher sensitivity and specificity. There is. In the present invention, it is possible to evaluate the progression of pancreatic cancer with higher sensitivity and specificity by measuring two or more specific amounts of the above RNA, such as a combination of CCDC88A mRNA and SNORA14B snoRNA. There is sex. Therefore, it is preferable to measure the amount of two or more RNAs simultaneously in real-time RT-PCR. In that case, in the reverse transcription reaction, when using a reverse primer on the 3 ′ end side among reverse primers for measuring the amount of two or more RNAs, or when performing two-step real-time RT-PCR Requires reverse transcription of the entire length of the RNA of interest using an oligo dT primer or a random primer in a reverse transcription reaction.

  In real-time PCR, the amount of amplification product is measured by fluorescence intensity. Therefore, the reaction is carried out in the presence of a fluorescent intercalator that binds to double-stranded RNA synthesized by PCR or a fluorescent probe having a partial sequence complementary to the base sequence of the DNA to be amplified.

  A fluorescent intercalator is a compound that has a fluorescent labeling group and binds to a double strand (including a double strand of a template DNA and a primer and a double strand in the middle of extension) generated by PCR. Examples of the fluorescent intercalator include SYBR (registered trademark) Green series manufactured by Takara Bio Inc.

  The fluorescent probe used in real-time PCR is a compound having a fluorescent labeling group at one end of a DNA having a base sequence complementary to a part of the template DNA and a quencher group at the other end. There is. One is not fluorescent due to the quencher group when present alone and forms a duplex with the template DNA with primer annealing, but is extended by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase. When it is decomposed during the reaction, the fluorescent labeling moiety is liberated and fluorescence is emitted. The other has a stem-loop structure, and when present alone, it does not show fluorescence because the fluorescent coloring group and the quencher group are close to each other. When formed, this is a type that exhibits fluorescence because the fluorescent coloring group and the quencher group are separated. The other, when present alone and when forming a duplex with the template DNA together with primer annealing, does not exhibit fluorescence due to the quencher group, but is cleaved by the action of RNase H after primer annealing, This is a type in which a portion having a fluorescent coloring group is released to exhibit fluorescence.

  Whether to use a fluorescent intercalator or a fluorescent probe, or what type of fluorescent probe to use, may be appropriately determined according to the detection sensitivity of DNA corresponding to the amount of RNA to be measured.

  In real-time PCR, a calibration curve is created in advance between the amount to be detected and the Ct value (Threshold Cycle), and from the Ct value corresponding to the threshold (Threshold) on the amplification curve of the measurement sample, The amount of the detection target in is obtained.

  In the present invention, aqueous solutions of various concentrations are prepared for the RNA that is the pancreatic cancer marker according to the present invention, and real-time RT-PCR is performed for each aqueous solution to obtain an amplification curve from the fluorescence intensity with respect to the number of cycles. Next, an appropriate threshold value is set, and a Ct value at which each amplification curve and the threshold value intersect is obtained. Since there is a linear relationship between the obtained Ct value and the target RNA concentration of each solution, a calibration curve can be created. Next, real-time RT-PCR is performed on the sample under the same conditions, the Ct value is similarly determined, and the amount of target RNA in the measurement sample is determined from the calibration curve.

(3) Judgment process Progression of pancreatic cancer is evaluated from the target RNA amount obtained in the above process. In practice, samples are obtained from pancreatic cancer patients and non-pancreatic cancer patients, as well as pancreatic cancer patients of various degrees of progression, and real-time RT-PCR is performed under the same conditions to measure the amount of target RNA in the sample. Data is accumulated. The accumulated data is compared with the measurement result to determine the presence or absence of pancreatic cancer and the degree of progression of pancreatic cancer in the subject.

3. Pancreatic cancer diagnostic kit The pancreatic cancer diagnostic kit according to the present invention can be used in the above-described method for evaluating the progression of pancreatic cancer. More specifically, it is for detecting the RNA, and has the forward primer and reverse primer for amplifying the base sequence characteristic of the RNA to be detected, and the fluorescent intercalator or fluorescent probe.

  The pancreatic cancer diagnostic kit according to the present invention is preferably one that measures the amount of two or more of the above RNAs, and also preferably one that can also measure the amount of CA19-9. By measuring the amount of two or more of the above RNAs, or by combining the amount of one or more of the above RNAs with the amount of CA19-9, the progression of pancreatic cancer can be evaluated with higher sensitivity and specificity. obtain. For example, in addition to the amount of CCDC88A mRNA, in order to measure the amount of SNORA14B snoRNA and the amount of CA19-9, which is a conventional pancreatic cancer marker, the sequence number 2 and the sequence for quantifying CCDC88A mRNA Qualitatively or quantitatively determine the primer set having the base sequence of No. 3, the primer set having the base sequences of SEQ ID NO: 5 and SEQ ID NO: 6 for quantifying the SNORA14B snoRNA, and the amount of these amplified DNAs The thing which has the fluorescent compound set for this, and also what has the kit for quantitative determination of CA19-9 are also preferable.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1
5 pancreatic cancer patients with stage I-III, 13 pancreatic cancer patients with stage IV, and 13 patients with chronic hepatitis, colon polyps or gastric polyps The purpose of this experiment was explained to 12 non-pancreatic cancer patients without cancer, blood was collected after obtaining consent. Next, serum was rapidly obtained by a three-stage centrifugation method. The obtained serum was stored in a dedicated freezer until the start of the following experiment.

  Using BLAST bioinformatics, a primer set highly specific to CCDC88A mRNA was designed. Furthermore, one-step real-time RT-PCR using CCDC88A mRNA as a template was performed, and it was confirmed in advance that the CCDC88A mRNA was specifically amplified by the primer set.

  MRNA contained in exosomes was purified from a part (100 μL) of the serum sample using a Norgen kit. Using purified mRNA, a primer set specific to CCDC88A mRNA (SEQ ID NO: 2 and 3), and a one-step RT-PCR kit (Takara Bio Inc. “One Step SYBR (registered trademark) PrimeScript RT-PCR Kit”) One-step real-time RT-PCR was performed to amplify a base sequence specific to CCDC88A mRNA. FIG. 1 shows the relative mass of CCDC88A mRNA in serum samples obtained from a pancreatic cancer patient group and a non-pancreatic cancer patient group.

  When the Mann-Whitney U test was performed on the results shown in FIG. 1, a significant difference was observed between the pancreatic cancer patient group and the non-pancreatic cancer patient group at p = 0.008673. In addition, the fluorescence photograph which shows presence (green part) of CCDC88A mRNA in a pancreatic cancer cell is shown in FIG. 2 (1), and the fluorescence photograph which shows presence (red part) of CD63 which is an exosome marker is shown in FIG. 2 (2). FIG. 2 (3) shows a superposition of these fluorescent photographs and a fluorescent photograph showing the presence of DAPI as a nuclear staining reagent. When green and red are mixed, it becomes yellow, and it can be confirmed from these fluorescent photographs that CCDC88A mRNA is localized in exosomes.

Comparative Example 1
Since non-pancreatic cancer patients were not allowed to measure the cancer marker CA19-9 in insurance practice, CA19-9 measurement was performed only on pancreatic cancer patients (18 patients). The results are shown in Table 1. In addition, the case where CA19-9 value is over 37 is an abnormal value.

Example 2
In the ROC (Receiver Operating Characteristic) curve obtained by the real-time RT-PCR in Example 1 above, the AUC value, which is one of the indexes for evaluating the diagnostic ability, was 0.79. According to a paper report, the AUC value of CA19-9 is 0.7 to 0.8, and the AUC value according to the method of the present invention is considered to be equal to or higher than that of CA19-9. In the measurement result, the threshold value was set to 7.98 from the obtained ROC curve. Table 2 shows the result of the χ ² test when the threshold is 7.98.

  In the experimental result of the comparative example 1, the sensitivity of CA19-9 was 55.56. On the other hand, as described above, the sensitivity when CCDC88A mRNA was used as a pancreatic cancer marker was 72.22, which exceeded CA19-9. In addition, since CA19-9 measurement is not performed with respect to a non-pancreatic cancer patient group, the comparison of specificity is not performed.

Example 3
The correlation between the CCDC88A mRNA relative mass in the serum of the pancreatic cancer patient group obtained in Example 1 and the CA19-9 value in Comparative Example 1 was analyzed by the Spearman rank correlation number test. The results are shown in Table 3.

  As the result shown in Table 3, it was p = 0.39 and there was no correlation. Therefore, it can be said that there is a possibility that the correct diagnosis rate can be improved by using CA19-9 together in the method of the present invention. In addition, although the test was conducted regarding the age and sex of the subject in the results of the method of the present invention, there was no significant difference regarding the age and sex.

Example 4
20 patients with pancreatic cancer (stage IIA: 2, IIB: 4, III: 6, IV: 8) according to UICC (International Association for Cancer) classification, chronic hepatitis, colon polyps or gastric polyps but pancreas Targeting 30 non-pancreatic disease patients without cancer and 13 IPMN patients, the relative mass of specific mRNA or snoRNA in the serum samples between the patient groups was determined in the same manner as in Example 1 above. . The detection of CCDC88A mRNA (SEQ ID NO: 1) uses the primer set of SEQ ID NO: 2 and 3, and the detection of SNORA14B snoRNA (SEQ ID NO: 4) uses the primer set of SEQ ID NO: 5 and 6, and SNORA25 snoRNA (SEQ ID NO: 7 ) Is used for detection of SNOR22 snoRNA (SEQ ID NO: 10), and for detection of SNORD22 snoRNA (SEQ ID NO: 13). The primer set of Nos. 14 and 15 was used, the primer set of SEQ ID Nos. 17 and 18 was used for detection of SNORA74A snoRNA (SEQ ID No. 16), and the primer of SEQ ID Nos. 20 and 21 was used for detection of ARF6 mRNA (SEQ ID No. 19). Using set, VAV3 mRNA Detection using a primer set of SEQ ID NO: 23 and 24 in SEQ ID NO: 22), the detection of WASF2 mRNA (SEQ ID NO: 25) using primer set of SEQ ID NO: 26, 27.

  Each result is shown in FIGS. In FIGS. 3 to 11, “*” indicates that a significant difference is recognized between the pancreatic cancer patient group and the non-pancreatic cancer patient group at p <0.05 by Mann-Whitney U test. In addition, Table 4 shows specific p values of the respective markers.

  As shown in FIGS. 3 to 11 and Table 4, pancreatic cancer can be significantly detected by using the mRNA and snoRNA as markers.

Comparative Example 2
The CA19-9 measurement was performed on the serum samples of each patient group targeted for Example 4 above. Table 5 shows the results of the pancreatic cancer patient group (20 patients), Table 6 shows the results of the control non-pancreatic disease patient group (30 persons), and Table 7 shows the results of the IPMN patient group (13 persons). In addition, the case where CA19-9 value is over 37 is an abnormal value.

  According to the results shown in Tables 5 to 7, there are cases where CA19-9 values are small even in patients with advanced pancreatic cancer, and cases where CA19-9 values are large even in patients with non-pancreatic diseases and IPMN patients, and the sensitivity of CA19-9 Was 60 and the specificity was 81.

Example 5
A ROC (Receiver Operating Characteristic) curve was created from the results of experiments on real-time RT-PCR of the target mRNA and snoRNA in Example 4 above, and an AUC value, which is one of indices for evaluating diagnostic ability, was obtained. FIG. 12 representatively shows an ROC curve when CCDC88A mRNA is used as a marker. Table 8 shows the AUC values comparing pancreatic cancer with controls using each mRNA and snoRNA as markers.

  According to a paper report, the AUC value of CA19-9 was 0.7 to 0.8, and 0.810 in this experiment. Regarding AUC values, ARF6 and WASF2 are significantly superior to CA19-9 at p <0.05, and other RNAs are considered equivalent to CA19-9. Therefore, it was shown that mRNA and snoRNA according to the present invention can be serum markers that can diagnose pancreatic cancer with sensitivity and specificity equivalent to or higher than those of CA19-9.

Moreover, from the ROC curve obtained using CCDC88A mRNA, ARF6 mRNA, VAV3 mRNA and WASF2 mRNA as markers, a threshold value for pancreatic cancer diagnosis was determined and χ ² test was performed. Table 9 shows the results of sensitivity, specificity, positive predictive value, and negative predictive value.

  In the experimental results of Comparative Example 2, the sensitivity of CA19-9 was 60 and the specificity was 81. On the other hand, as shown above, when CCDC88A mRNA, ARF6 mRNA, VAV3 mRNA, and WASF2 mRNA were used as pancreatic cancer markers, the specificity was 75 or more, and the sensitivity of all mRNAs was CA19-9. It was higher. In particular, the positive predictive value of ARF6 mRNA, VAV3 mRNA, and WASF2 mRNA was 90% or higher, and it was shown that the positive predictive value can be a marker capable of diagnosing pancreatic cancer with higher sensitivity and specificity than CA19-9.

  Similarly to Example 3 above, the results of markers other than CCDC88A mRNA were also analyzed by the Spearman rank correlation number test. As a result, all the results were the same as those of CCDC88A mRNA and were not related to CA19-9.

Example 6
There is a possibility that pancreatic cancer can be diagnosed with high sensitivity and specificity by combining a plurality of RNA markers and CA19-9. FIG. 13 shows ROC curves when CA19-9 alone, which is a conventional pancreatic cancer marker, a combination of CCDC88A and SNORA14B, and a combination of CCDC88A, SNORA14B and CA19.9 are used for diagnosis of pancreatic cancer. As shown in the results of FIG. 13, it was revealed that by combining CCDC88A and SNORA14B as markers, the AUC value is higher than that of CA19-9 alone, and pancreatic cancer can be diagnosed with high sensitivity and specificity. Furthermore, it has been clarified that by combining CCDC88A and SNORA14B with CA19-9, diagnosis can be made with significantly higher sensitivity and specificity at p = 0.0108 compared to CA19-9 alone.

Example 7
CCDC88A, ARF6, VAV3 and WASF2, which are mRNAs used as pancreatic cancer markers in the present invention, have a diagnostic ability compared with CA19-9 in pancreatic cancer cases in which tumors remain in the pancreas of UICC stages I and II. There was a tendency to be excellent. Serum CCDC88A, ARF6, VAV3 and WASF2 were measured by real-time RT-PCR. The graph which shows the result of having quantitatively analyzed each said mRNA in the blood for every UICC stage by real-time RT-PCR is shown to FIGS. 14-17, and CA19-9 value is shown in FIG. Moreover, the graph and ROC curve which show the result of having quantitatively analyzed each said mRNA in the blood of the stage IIA and IIB pancreatic cancer case and control group by real-time RT-PCR are shown in FIGS. 19-22, and CA19-9 value And the ROC curve is shown in FIG.

  Stage I-II pancreatic cancer cases have a better life prognosis after surgery than stage III-IV cases, and it is clinically significant that pancreatic cancer can be diagnosed at stages I-II. large. As shown in FIGS. 14 to 23, the diagnostic ability of UICC stage I to II pancreatic cancer by CCDC88A, ARF6, VAV3 and WASF2 may be higher than that of CA19-9, and can be used in future screening. The result was suggested.

Claims (7)

  CCDC88A mRNA, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA, and a combination of two or more types characterized by WASF2 mRNA Marker. The pancreatic cancer marker of Claim 1 which has either the following base sequences 1-3.
Base sequence 1: one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25 Base sequence of;
Base sequence 2: One or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: 25 A base sequence in which 1 to 10 bases are deleted, substituted and / or added, and the base sequence of RNA found in the body fluid of pancreatic cancer patients;
Base sequence 3: one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25 A nucleotide sequence having 95% or more sequence identity to the nucleotide sequence of RNA and found in the body fluid of pancreatic cancer patients. A pancreatic cancer diagnostic kit for detecting specific RNA,
The RNA is one or more selected from the group consisting of CCDC88A mRNA, SNORA14B snoRNA, SNORA25 snoRNA, SNORA22 snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA and WASF2 mRNA,
A forward primer and a reverse primer complementary to a part of the base sequence of the cDNA of the RNA, and
A pancreas comprising a fluorescent intercalator that binds to a double-stranded RNA synthesized by PCR and at least one fluorescent compound selected from a fluorescent probe having a partial sequence complementary to the base sequence of the RNA. Cancer diagnostic kit.   The base sequences of forward primer and reverse primer complementary to a part of the base sequence are SEQ ID NO: 2 and SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, and SEQ ID NO: 11, respectively. SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 24, and SEQ ID NO: 26 and SEQ ID NO: 27, The pancreatic cancer diagnostic kit according to claim 3, wherein the kit is one or more selected from the group consisting of: A method for assessing the progression of pancreatic cancer,
CCDC88A mRNA, SNORA71B snoRNA, SNORA18 snoRNA, SNORA14B snoRNA, SNORD22 snoRNA, SNORA74A snoRNA, ARF6 mRNA, VAV3 mRNA, and WASF2 A method comprising the steps of:   The method according to claim 5, wherein a blood sample is used as the sample.   The method according to claim 5 or 6, wherein the amount of RNA is measured by real-time RT-PCR.