JP2014183839A - Diagnostic kit for diagnosis of pancreatic carcinoma in mammals, inspection method using the same and diagnostic marker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biomarker in blood which is useful for diagnosis of pancreatic carcinoma.SOLUTION: A diagnostic kit for diagnosis of pancreatic carcinoma in a mammal is provided which has hTERT detection reagent for quantitative detection of mRNA encoding hTERT in the blood obtained from the mammal, wherein the amount of mRNA encoding hTERT is used as an index for diagnosis of pancreatic carcinoma.

Description

  The present invention relates to a diagnostic kit for diagnosing pancreatic cancer in a mammal, a test method using the kit, and a diagnostic marker.

  According to the pancreatic cancer clinical practice guideline, a definitive diagnosis before the start of pancreatic cancer treatment is desirable. In particular, an ultrasonic endoscopic puncture aspiration biopsy (hereinafter referred to as EUS-FNA) for a pancreatic mass is considered to have a correct diagnosis rate of 85 to 91%, but it is a problem that approximately 10% cannot be correctly diagnosed. In addition, the facilities where EUS-FNA can be performed are limited, and the accuracy rate of biomarkers for current pancreatic cancer is not satisfactory at about 70%. Therefore, there is a need for the development of surrogate markers for pancreatic cancer.

  Some members of the present inventor have established a method for extracting a trace amount of mRNA from human serum and quantifying specific gene expression, and human telomerase reverse transcriptase mRNA (hereinafter, hTERT) as a biomarker for hepatocellular carcinoma. The clinical utility of mRNA) has been reported (Patent Document 1 and Non-Patent Document 1).

  Further, Non-Patent Document 2 reports the results of analyzing the relationship between human nucleotide reverse transcriptase (hereinafter hTERT) gene single nucleotide polymorphisms (SNPs) and pancreatic cancer. Furthermore, Non-Patent Document 3 reports the results of quantifying hTERT mRNA by real-time RT-PCR for samples obtained by microdissection from various cancer tissues obtained by biopsy. Non-Patent Document 4 and Non-Patent Document 5 report the results of quantifying hTERT mRNA in pancreatic juice by RT-PCR (or real-time RT-PCR). Non-Patent Document 6 reports that when hTERT is expressed in an adenovirus vector, it has an antitumor effect due to a synergistic effect with other components.

  On the other hand, Non-Patent Document 7 reports that the amount of carbohydrate antigen 19-9 (hereinafter CA19-9) in serum can be used as a biomarker for pancreatic cancer.

Japanese Patent No. 4761046

BMC gastroenterology, 18; 10:46, 2010 PLoS ONE, November 2011, Volume 6, Issue 11, e27921 Cancer Sci, November 2008, vol. 99, no. 11, 24244-2251 Clinical Cancer Research, Vol. 11, 2855-2292, March 15, 2005 Clinical Cancer Research, Vol. 7, 1976-1981, July 2001 Cancer Sci, March 2010, vol. 101, no. 3,735-742 World J Gastroenterol, 2004; 10 (11): 1675-1677

  However, the prior art described in the above literature has room for improvement in the following points.

  First, although the techniques described in Patent Document 1 and Non-Patent Document 1 have demonstrated that hTERT mRNA has clinical utility as a biomarker for hepatocellular carcinoma, is it useful as a biomarker for pancreatic cancer? It was unknown.

  Second, since the technique of Non-Patent Document 2 merely analyzes the relationship between single nucleotide polymorphisms (SNPs) of hTERT gene and pancreatic cancer, whether hTERT mRNA is useful as a biomarker for pancreatic cancer It was unknown.

  Thirdly, in the technique of Non-Patent Document 3, since hTERT mRNA is quantified by real-time RT-PCR for samples obtained by microdissection from various cancer tissues obtained by biopsy, hTERT in blood or saliva is obtained. It was unclear whether the amount of mRNA was useful as a biomarker for pancreatic cancer.

  Fourth, in the technique of Non-Patent Document 4 or Non-Patent Document 5, since the hTERT mRNA in pancreatic juice is quantified by real-time RT-PCR, the amount of hTERT mRNA in blood or saliva is useful as a biomarker for pancreatic cancer Whether it was unknown.

  Fifth, the technique of Non-Patent Document 6 only shows that when hTERT is expressed in an adenovirus vector, it has an antitumor effect due to a synergistic effect with other components. It was unclear whether it was useful as a biomarker.

  Sixth, in the technique of Non-Patent Document 7, the sensitivity as a biomarker of pancreatic cancer is only 77% even when combined with other diagnostic markers, and in particular, the sensitivity in the first stage and second stage of pancreatic cancer However, there was room for improvement in practical use because of the low values of 40.0% and 58.3%.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a biomarker in blood or saliva useful for diagnosis of pancreatic cancer.

  According to the present invention, there is provided a diagnostic kit for diagnosing pancreatic cancer in a mammal, the hTERT detection reagent for quantitatively detecting mRNA encoding TERT in blood or saliva obtained from the mammal. A diagnostic kit is provided in which the amount of mRNA encoding the TERT serves as an index for diagnosis of pancreatic cancer.

  Further, according to the present invention, there is provided a diagnostic marker for diagnosing pancreatic cancer in a mammal, the diagnostic marker including the amount of mRNA encoding TERT in blood or saliva obtained from the mammal. The

  According to these diagnostic kits or diagnostic markers, since the amount of mRNA encoding TERT in blood or saliva is a suitable index for diagnosis of pancreatic cancer, as shown in the examples described later, medical personnel such as doctors However, when diagnosing pancreatic cancer in mammals including humans, accurate diagnosis can be easily performed using blood or saliva without performing a biopsy or the like.

  According to the present invention, the diagnostic kit further includes a CA19-9 detection reagent for quantitatively detecting CA19-9 in blood or saliva obtained from the mammal, and the TERT It is preferable to show a comprehensive index including the amount of mRNA encoding and the amount of CA19-9 as an index for diagnosis of pancreatic cancer.

  According to the present invention, the diagnostic marker further includes the amount of CA19-9 in blood or saliva obtained from the mammal, the amount of mRNA encoding the TERT, and the amount of CA19-9. It is preferable to show a comprehensive index including the amount as an index for diagnosis of pancreatic cancer.

  According to these diagnostic kits or diagnostic markers, as shown in the examples described later, the amount of mRNA encoding TERT in blood or saliva and the amount of CA19-9 are further used for further diagnosis of pancreatic cancer. Therefore, even if a medical staff such as a doctor diagnoses pancreatic cancer in mammals including humans, it can be easily and more accurately diagnosed using blood or saliva without performing a biopsy or the like.

  Further, according to the present invention, there is provided a test method for testing pancreatic cancer in a mammal, comprising the step of obtaining an index for diagnosis of pancreatic cancer in blood obtained from the mammal using the diagnostic kit. An inspection method is provided.

  According to this method, as shown in the examples described later, medical indicators such as doctors and the like are used by medical personnel such as doctors in order to perform an examination of a diagnostic index for pancreatic cancer in blood or saliva using the above-described diagnostic kit. When diagnosing pancreatic cancer in a mammal, accurate diagnosis can be easily performed using blood or saliva without performing a biopsy or the like.

  According to the present invention, when a medical staff such as a doctor diagnoses pancreatic cancer in mammals including humans, accurate diagnosis can be easily performed using blood or saliva without performing a biopsy or the like.

It is a graph which shows the result of real-time RT-PCR of hTERTmRNA performed in the Example. It is a graph which shows the calibration line of real-time RT-PCR of hTERTmRNA used in the Example. 10 is a graph showing ROC curve used in Example 2.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

<Explanation of terms>
A “diagnostic marker” includes a concept commonly referred to as a “biomarker” and is a definition of an NIH biomarker “normal biological process, pathological process, or pharmacological response to a therapeutic intervention. It is a concept that includes “objectively measured and evaluated characteristics” as an index.

  The “mammal” includes any mammal, and includes humans, domestic animals, pet animals, zoo animals, or sports animals such as dogs, cats, cows, pigs, horses, sheep, rabbits. Etc. The mammal is preferably a human. In addition, when the marker, reagent, or kit according to the following embodiment is applied to a mammal, the mammal may be a mammal suspected of suffering from pancreatic cancer.

  In addition, “pancreatic cancer” includes a malignant tumor arising from the pancreas. The stage classification of pancreatic cancer is divided into four stages of progression (stages) based on the TNM classification based on the pancreatic cancer handling regulations of the Japan Pancreas Society.

Pancreatic Cancer Treatment Regulations (10th edition)
Stage I-The size is 2 cm or less and is confined inside the pancreas.
Stage II-Cancer stays inside the pancreas but is more than 2 cm in size or has metastasis to the first group of lymph nodes.
Stage III—Cancer is slightly out of the pancreas but has no lymph node metastasis or is limited to group 1. Or, the cancer remains inside the pancreas, but lymph node metastasis is up to the second group.
Stage IV-Cancer has involved organs around the pancreas or has spread to distant organs.

The T classification is defined as follows.
T classification (local pancreatic extent)
T1: TS1, the infiltrated area is limited in the pancreas T2: TS2-4, the infiltrated area is limited in the pancreas T3: CH, DU, S, RP infiltrate CH (intrapancreatic bile duct), DU (duodenal invasion), S ( Pancreatic anterior tissue-pancreatic capsule, omentum, omentum, and mesocolon), RP (rear pancreatic connective tissue)
T4: Infiltrate any of PV, A, PL, OO PV (portal vein PVp, superior mesenteric vein PVsm, splenic vein PVsp), A (total hepatic artery Ach, superior mesenteric artery Asm, splenic artery Asp, Abdominal artery Ace), PL (extrapancreatic plexus invasion, PLphI pancreatic head plexus part I, PLphII pancreatic head plexus part II, PLsma superior mesenteric artery plexus, PLcha total hepatic artery plexus, PLhdl liver duodenal mesentery Internal plexus, PLspa splenic artery plexus, PLce celiac plexus), OO (inferior vena cava, kidney, renal vein, adrenal gland, stomach, large intestine, spleen)

TS is defined as follows.
TS (tumor size) means the maximum diameter (of the infiltrated portion).
TS1 <= 2.0cm
2.0cm <TS2 <= 4.0cm
4.0cm <TS3 <= 6.0cm
TS4> 6.0cm

On the other hand, the stage classification of pancreatic cancer is divided into the following four stages of progression (stages) based on the UICC rules, and is slightly different from the pancreatic cancer handling rules (10th edition) of the Japan Pancreas Society There are places.
UICC Rules (7th edition)
Stage I-Pancreatic cancer remains inside the pancreas and there is no lymph node metastasis.
Stage II-Pancreatic cancer has spread around the pancreas but does not extend to important blood vessels around the pancreas and has no lymph node metastasis or is limited to the first group.
Stage III-Cancer has spread to important blood vessels around the pancreas, but there are no metastases in distant organs.
Stage IV-There is metastasis away from the pancreas.
In addition, the stage classification of pancreatic cancer and the like in the examples described later are all based on the Pancreatic Cancer Handling Regulations (10th edition) of the Japanese Pancreatic Society, and in this embodiment, the Pancreatic Cancer Handling Regulations (10th edition) of the Japan Pancreatic Society. If the UICC rules differ, the pancreatic cancer handling rules (10th edition) of the Pancreatic Society of Japan shall prevail.

<Diagnostic marker for mRNA alone encoding TERT>
The diagnostic marker according to the present embodiment is a diagnostic marker for diagnosing pancreatic cancer in a mammal. In addition, the diagnostic marker according to this embodiment includes the amount of mRNA encoding TERT in blood or saliva obtained from the mammal.

  Here, “TERT” (telomerase reverse transcriptase) according to the present embodiment is a subunit located at the active center of telomerase, which is a malignant tumor (cancer) -specific antigen (enzyme) expressed in many malignant tumors in mammals. (In particular, human TERT is referred to as hTERT (human telomerase reverse transcriptase)). In this embodiment, a TERT polypeptide gene polymorph or a splicing variant can also be used as TERT.

  In addition, the “mRNA” encoding TERT in the present embodiment does not necessarily need to be full length, and includes mRNA having a chain length and specificity according to each use purpose such as tumor diagnosis. In this embodiment, when quantitatively detecting the mRNA encoding TERT, instead of directly quantifying the mRNA itself, the mRNA is first quantified using reverse transcriptase, and then the cDNA is quantified. Also good.

  Further, the blood in the present embodiment may be collected from any part of a mammal's living body, and includes blood collected from either arterial blood or venous blood. In this embodiment, when quantitatively detecting mRNA encoding TERT, instead of quantitatively detecting mRNA encoding TERT directly in blood, serum is separated from blood and separated from serum. The mRNA encoding TERT may be detected quantitatively.

  In the diagnostic marker according to the present embodiment, the amount of mRNA encoding TERT in blood or saliva is a suitable indicator for diagnosis of pancreatic cancer, as shown in the examples described later. When diagnosing pancreatic cancer in mammals such as, blood or saliva can be easily and accurately diagnosed without performing a biopsy or the like.

  Conventionally, an ultrasonic endoscopic fine needle aspiration biopsy (hereinafter referred to as EUS-FNA) for a pancreatic mass is difficult unless it is a doctor who has specialized training and has a skilled technique, and is actively performed in a clinic or a community hospital. Not done. On the other hand, the diagnostic marker according to the present embodiment can be easily used in a clinic or a city hospital as long as there is an apparatus such as a real-time RT-PCR described later. Therefore, if there is a patient suspected of having pancreatic cancer, it is possible to detect pancreatic cancer after quantitatively detecting TERT-encoding mRNA at a clinic or community hospital using a device such as real-time RT-PCR described later. Only high-quality patients can be introduced to advanced general hospitals such as university hospitals, and a definitive diagnosis can be made upon receiving EUS-FNA by a skilled doctor.

  The diagnostic marker according to the present embodiment is particularly useful when the stage classification of pancreatic cancer is TS1 or TS2. Since the maximum diameter of pancreatic cancer (invasion part) is TS1 <= 2.0 cm and 2.0 cm <TS2 <= 4.0 cm, if pancreatic cancer is staged TS1 or TS2, the discovery of pancreatic cancer is It was very difficult. Therefore, if pancreatic cancer can be found at a stage where the size of pancreatic cancer is small, mRNA that encodes TERT can be quantitatively detected using a device such as real-time RT-PCR, which will be described later, and a patient with high possibility of pancreatic cancer can be found. This is preferable because the possibility of healing by surgery, chemotherapy, radiotherapy, etc. increases.

<Diagnostic kit for diagnostic marker of mRNA alone encoding TERT>
The diagnostic kit according to the present embodiment is a diagnostic kit for diagnosing pancreatic cancer in a mammal. Moreover, the diagnostic kit according to the present embodiment has a TERT detection reagent for quantitatively detecting mRNA encoding TERT in blood or saliva obtained from a mammal.

  As the TERT detection reagent, any reagent can be used as long as it can quantitatively detect mRNA encoding TERT. For example, it can be hybridized to mRNA encoding TERT (or specific. One set of primers). The “primer” in the present embodiment includes a nucleic acid that is hybridized with a template in the reverse transcription reaction or nucleic acid amplification reaction and is required to start the reverse transcription reaction or nucleic acid amplification reaction. In a reverse transcription reaction or a nucleic acid amplification reaction, a primer is preferably used so that it can hybridize with a target template and can carry out a subsequent reaction, preferably a specific product (in chain length or sequence). It is preferably designed so that it contains its template-specific sequence. Moreover, although a general primer is not restricted to this, Usually, it is preferable that it is designed so that it may have a chain length of 15 bases to 100 bases, preferably 15 bases to 35 bases.

  Such a primer can be used by those skilled in the art by using well-known common knowledge and programs such as BLAST and databases such as the ratio of primer GC (guanine / cytosine) and melting temperature. It can be easily designed. Various primer design software may be used, for example, Primer Express ™ (Perkin Elmer, Applied Biosystems) or Primer Explorer (Fujitsu Ltd .; for LAMP method). In this case, the 3 ′ region of the primer is preferably complementary to the template strand, but a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ side.

  Further, for example, when the ICAN method is used as a nucleic acid amplification method, the primer may contain not only DNA but also RNA, or may be composed only of RNA as necessary. Further, depending on the nucleic acid amplification method / nucleic acid detection method used, the primer may further include various functional sequences such as an RNA polymerase promoter sequence, a stem loop structure site, and a restriction enzyme sequence. Particularly suitable primers for this embodiment will be described later.

  In the present embodiment, “specifically hybridizes” means that another nucleic acid binds complementarily to a certain nucleic acid through hydrogen bonding or the like, and the nucleic acid to be used as a comparative control is under the same conditions. Includes a state that does not combine. It does not necessarily have to “do not bind to all other nucleic acids”, and may have specificity depending on the purpose of use. For example, in detection of TERT gene mRNA, a nucleic acid that binds negligibly weakly to other mRNAs contained in a sample can be said to be a "specifically hybridizing" nucleic acid.

  Hybridization conditions can be selected according to the intended use of the nucleic acid. For example, if it is a nucleic acid as a primer used for PCR, it is selected so that it may hybridize on the conditions at the time of annealing in PCR. Preferably, those that hybridize under stringent hybridization conditions are selected.

  In addition, “stringent conditions” in the present embodiment are conditions including a denaturing agent such as formamide during hybridization, for example, 50% (v / v) formamide and 0.1% bovine serum albumin / 0 at 42 ° C. .1% Ficoll / 0.1% polyvinyl pyrrolidone / 50 mM sodium phosphate buffer pH 6.5, 750 mM sodium chloride, 75 mM sodium citrate and those appropriately modified may be mentioned. Not limited to. The temperature of the hybridization conditions can be determined according to the nucleic acid amplification reaction or hybridization reaction to be used. For example, 42 ° C, 43 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C, 68 ° C, 70 ° C, 72 ° C.

  In addition, the “nucleic acid” in the present embodiment refers to a nucleic acid (including both RNA and DNA) composed of naturally occurring bases, sugars, and intersugar bonds, and oligomers thereof (for example, about 2 to 100 bases). ) And polymers (for example, 100 bases or more). The nucleic acid in the present embodiment includes a non-naturally occurring monomer that functions similarly, a monomer labeled with a fluorescent molecule or a radioisotope, or an oligomer or polymer containing these.

  In the present embodiment, as the “nucleic acid amplification” method, any method may be used as long as it is a known method for amplifying a target nucleic acid, but as a method using a primer and a primer set, for example, PCR method, LAMP method or other nucleic acid concerted nucleic acid amplification methods are used. In particular, when amplifying RNA, a RT-PCR method (Kinetic RT) in which cDNA is synthesized using RNA as a template by reverse transcriptase and simultaneously or subsequently, a nucleic acid product derived from a target RNA is amplified by a thermostable DNA polymerase. -PCR method, etc.), RT-LAMP method or other nucleic acid concerted RNA amplification methods are used. Furthermore, as a nucleic acid amplification method, in addition to the above LAMP method, NASBA method, TMA method, 3SR method, ICAN method, TRC method and the like can be used. These methods can be carried out in accordance with the commonly used uses.

  In the diagnostic kit according to the present embodiment, the one set of primers may be a set of primers for use in real-time RT-PCR using a fluorescent dye. Here, as a method of RT-PCR (reverse transcription reaction and nucleic acid amplification reaction), for example, One-Step RT-PCR can also be used. One-Step RT-PCR is RT-PCR that allows RT-PCR to be performed quickly and easily in one step from RT incubation to PCR cycling without opening and closing tubes or adding reagents. Various kits and protocols for One-Step RT-PCR are available in the art, including methods (eg, QIAGEN OneStep RT-PCRMix, KAPA BIOSSYSTEMS KAPA SYBR FAST Universal One-Step qRT-PCR Kit Etc.) can be selected and implemented as appropriate.

  By using real-time PCR, it is possible to carry out the detection process simply, in real time, and quantitatively in one step or in a short number of steps. As the real-time RT-PCR, for example, various fluorescent PCR-based techniques can be used. Examples of the fluorescent PCR-based technology include an intercalator method using various fluorescent nucleic acid labeling agents such as SYBR Green (for example, Light Cycler (registered trademark: Roche), ABI Prizm 7700 Sequence Detection System (registered trademark) ( Perkin Elmer, Applied Biosystems, Inc.), TaqMan probe method for monitoring amplification in real time using 5 ′ exonuclease activity of Taq DNA polymerase enzyme, RNase activity of RNase H enzyme and dedicated chimeric RNA probe For example, the cycling probe method is not limited to this.

  In this embodiment, any method may be used as a method for detecting an amplification product as long as it is a known method for detecting a nucleic acid. For example, the amplified nucleic acid may be detected by electrophoresis or may be detected using a hybridization method using a nucleic acid probe bound with a detectable label. Alternatively, an intercalator fluorescent dye (SYBR Green or the like) that is advantageous in terms of secondary contamination or the like may be used.

  The detection method of the amplified nucleic acid is not limited to the above. For example, in the LAMP method, it may be detected by using white turbidity due to by-product magnesium pyrophosphate as an index, and each nucleic acid may be detected even if the amplified nucleic acid is not directly measured. Any known amplification product detection method applicable to the amplification method can be used.

  In addition, the quantification method in real-time RT-PCR using a fluorescent dye may be an absolute quantification method for calculating the copy number, or a relative quantification method for examining expression with a relative value. Absolute quantification is a method of serially diluting the DNA of the target product whose concentration is known in advance, performing real-time RT-PCR using the dilution series, writing a calibration curve, and applying the Ct value of a sample with unknown concentration to the calibration curve. The number is calculated. On the other hand, the relative quantification method is a method in which a target gene and a reference gene are analyzed simultaneously and compared to the reference gene to compare how much the target gene is expressed.

  In the diagnostic kit according to this embodiment, when the mammal is a human, the TERT is preferably hTERT. In addition, SEQ ID: 1 and SEQ ID: 2, SEQ ID: 1 and SEQ ID: 2 have been demonstrated that the above-described one set of primers can accurately quantify the amount of mRNA encoding hTERT in human blood or saliva in Examples described later. It is preferably composed of any combination of ID: 3 and SEQ ID: 4, SEQ ID: 5 and SEQ ID: 6, SEQ ID: 7 and SEQ ID: 8.

  SEQ ID NOs: 1 (gttcttccaa acttgctgat gaaat; TERT_NM_198253.2 from 3049-3071) and 2 (gtgcaccaac atctacaaga tcc; TERT_NM_198253.2 from 3118-3142) are suitable primers for this embodiment, and SEQ ID NO: 3 (Atttcatcag caagtttgga agaac; TERT_NM_198253.2 from 3049-3071) and 4 (ggatcttgta gatgttggtg cac; TERT_NM_198253.2 from 3118-3142) are the sequences (or complementary sequences) on the mRNA of the TERT gene corresponding to these primers ).

Further, SEQ ID NOs: 5 (cggaagagtg tctggagcaa; derived from 1787-1806 of TERT_NM_198253.2) and 6 (ggatgaagcg gagtctgga; derived from 1913-1931 of TERT_NM_198253.2) are some members of the present inventor in Patent Document 1. SEQ ID NOs: 7 (from ttgctccaga cactcttccg; 1787-1806 from TERT_NM_198253.2) and 8 (from tccagactcc gcttcatcc; 1913-1931 from TERT_NM_198253.2) are suitable primers for this disclosed embodiment The sequence (or complementary sequence) on the mRNA of the TERT gene corresponding to. These primers were designed using the sequence of the TERT gene (sequence which is “TERT_NM_198253.2” on the gene database).

  By carrying out reverse transcription reaction and nucleic acid amplification using primers for these regions capable of effective reverse transcription reaction, it became possible to carry out accurate and stable reverse transcription reaction and nucleic acid amplification reaction. Furthermore, these primers are extremely effective in detecting evidence of the presence of pancreatic cancer from blood or saliva in the early stage of pancreatic cancer (TS1 or TS2) due to their high sensitivity.

  In the diagnostic kit according to the present embodiment, as a set of primers, SEQ ID: 1 and SEQ ID: 2, SEQ ID: 3 and SEQ ID: 4, SEQ ID: 5 and SEQ ID: 6, SEQ. Not only the combination of ID: 7 and SEQ ID: 8, but also the identity between the base sequences of these combinations is 80%, 85%, 90%, 95% or 98%, respectively. You may use 1 set of primers which consist of the combination more than (100% or less). Alternatively, as a set of primers, a combination in which substitution, deletion, or insertion of 1 base, 2 bases, 3 bases, 4 bases, 5 bases, or several bases is made to the base sequence of any of the above combinations, respectively. (As in the definition of identity, the substitution of RNA and DNA having the same base is not included in the substitution, deletion, or insertion here).

  “Identity” in this embodiment includes the relationship between two or more nucleic acids determined by comparing the sequences, and the degree of sequence identity as determined by matching between the nucleic acid base sequences including. Parameters relating to “identity” can be easily calculated by known methods. At this time, DNA and RNA having the same base (thin and uracin are also considered to be the same) have very similar binding characteristics, and therefore may be defined as the same nucleobase. Alignments for the purpose of determining identity are publicly available in various ways within the skill of the artisan, such as BLAST, BLAST2SEQ or ALIGN provided by NCBI (National Center for Biotechnology Information) Although it can be achieved by using computer software, a value calculated using standard initial parameters in BLAST2SEQ can be conveniently used.

  Nucleic acid amplification using the primer set of the present embodiment is performed using the TERT gene mRNA as a template, and the antisense primer (SEQ ID: 2, SEQ ID: 4, SEQ ID: 6, SSEQ ID: 8) among the above primer sets. Is synthesized by a nucleic acid amplification method carried out in a chain with the above-mentioned primer set and an appropriate DNA polymerase activity and / or RNA polymerase activity. The amplification product obtained by the above nucleic acid amplification method can be detected by a known nucleic acid detection method.

  Each primer may contain various additional types depending on the purpose, but considering its effectiveness as a primer, its length is at least 15 bases, or 18 bases, or 20 bases. That's it. The upper limit of the length is not particularly limited, but the upper limit is 100 bases or less and 80 bases or less in consideration of the efficiency reduction of the nucleic acid amplification reaction due to the primer being too long and the primer length necessary for each amplification reaction. , 60 bases or less, or 50 bases or less.

  In the diagnostic kit of the present embodiment, since the amount of mRNA encoding TERT serves as an index for diagnosis of pancreatic cancer, when the expression level of mRNA of the TERT gene is high, it is diagnosed that suspicion of pancreatic cancer is high. The cut-off value) can be appropriately set through comparison of cases by doctors in the field.

In tumor diagnosis, for example, if the presence of one tumor cell in 1 ml of blood can be detected, the total circulating blood is comparable to 3000 to 4000 cells, which is the tumor engraftment. This is comparable to the number of tumor cells that can be engrafted in animal experiments. Assuming that 3000 to 4000 circulating cells are 0.01% of the total cells in the tumor, the tumor at that stage contains only about 4 × 10 ⁷ cells, Tumors containing large numbers of cells are difficult to detect by any current method.

  Therefore, when tumor cells flow out at an early stage of a tumor (cancer), if a highly sensitive detection method can be developed, even an early stage tumor (cancer) can be detected. Thus, for example, the diagnostic kit according to this embodiment is effective when tumor cells flow into the blood or saliva with some association with tumor size and is a quantitative test for assessing tumor burden. It is also effective.

  In addition, when fighting outflowed tumor cells and immune cells in the body, RNA may be detected as a variety of DNA, proteins, RNA, etc. in tumor cells and immune cells flow into the blood. Thus, detection of tumor specific RNA may also reflect the earliest event of metastasis.

  Therefore, the diagnostic kit according to this embodiment capable of stable detection with very high sensitivity is not only useful as a diagnostic kit for normal pancreatic cancer, but particularly in the early stage (TS1 or TS2) of pancreatic cancer. It is even more useful in the diagnosis of This diagnostic kit can detect evidence of the presence of cancer cells in blood or saliva in the early stage of pancreatic cancer (TS1 or TS2), thus detecting cancer-related genes in cancer tissue tumor formation and metastasized tissue after metastasis It is also possible to detect the presence or absence of cancer cells more accurately than in the case of doing so.

<Diagnostic marker combining mRNA encoding TERT and CA19-9>
In addition to the amount of mRNA encoding TERT in blood or saliva obtained from a mammal, the diagnostic kit according to this embodiment further comprises the amount of CA19-9 in blood or saliva obtained from the mammal. It is preferable to have.

  Here, “CA19-9” (carbohydrate antigen 19-9) according to the present embodiment is a glycan recognized by monoclonal antibody NS19-9 produced in 1979 by Koprowski et al. Using colon cancer culture strain SW1116 as an immunizing antigen. Contains antigen. The determination site of this antigen is sialyl lacto-N-fucopentaose II, which is a sialyl Lea antigen obtained by sializing the sugar chain of Lewis blood group Lewis A (Lea).

  CA19-9 is present in salivary glands, bile ducts, bronchial glands and the like in normal tissues. Since it shows a high positive rate in gastrointestinal cancer, especially pancreatic cancer, gallbladder cancer, bile duct cancer, and colon cancer, it is effective as a diagnostic aid for these cancers, as a treatment progress, and as a monitor for recurrence. However, as shown in Non-Patent Document 7, even when combined with other diagnostic markers (TSGF, CA242), the sensitivity as a biomarker of pancreatic cancer is only 77%. Since the sensitivity in the first stage and the second stage is as low as 40.0% and 58.3%, there is room for improvement in practical use.

  In contrast, in the present embodiment, an index obtained by combining the amount of mRNA encoding TERT and the amount of CA19-9 is shown as a comprehensive index for diagnosis of pancreatic cancer. Specifically, in this embodiment, when the amount of mRNA encoding TERT is not less than a predetermined cut-off value or the amount of CA19-9 is not less than a predetermined cut-off value, the mammal is treated with pancreatic cancer. It is preferable to determine that there is a suspicion (positive as a comprehensive index). When determined in this way, depending on the setting of a predetermined cut-off value, for example, as shown in the examples described later, the sensitivity as a biomarker for pancreatic cancer is as high as 100.0%. Since the sensitivity in the first stage and the second stage is as high as 100.0% and 100.0%, it is suitable for practical use. In addition, the comprehensive index for diagnosis of pancreatic cancer is not limited to the above criteria, and for example, the amount of mRNA encoding TERT is not less than a predetermined cutoff value, and the amount of CA19-9 is predetermined. If it is greater than or equal to the cut-off value, it may be determined that the mammal is suspected of having pancreatic cancer. Further, the amount of mRNA encoding TERT and the amount of CA19-9 are normalized and added together with a predetermined weight, and a total index is calculated. The total index is equal to or greater than a predetermined cut-off value. In some cases, it may be determined that the mammal is suspected of having pancreatic cancer.

<Diagnostic Kit for Diagnostic Marker Combining mRNA Encoding TERT and CA19-9>
The diagnostic kit according to the present embodiment is a diagnostic kit for diagnosing pancreatic cancer in a mammal. Moreover, the diagnostic kit according to the present embodiment includes CA19 in blood obtained from mammals in addition to a TERT detection reagent for quantitatively detecting mRNA encoding TERT in blood obtained from mammals. It further has a CA19-9 detection reagent for quantitatively detecting -9.

  As the CA19-9 detection reagent, any reagent can be used as long as it is capable of quantitatively detecting CA19-9 (allyl lacto-N-fucopentaose II which is the antigen determination site thereof). A monoclonal antibody or a polyclonal antibody that specifically binds to CA19-9 may be included. A typical example of such an anti-CA19-9 antibody is monoclonal antibody NS19-9 produced in 1979 by Koprowski et al. Using colon cancer culture strain SW1116 as an immunizing antigen, but is not particularly limited, and other monoclonal antibodies Alternatively, a polyclonal antibody may be used.

  Specifically, the anti-CA19-9 antibody is obtained by using a sugar chain containing allyl lacto-N-fucopentaose II, which is the antigen-determining site of CA19-9, in an animal such as a rabbit, rat, or mouse (or its sugar) based on a conventional method. A method for producing a polyclonal antibody by immunizing a substance containing a chain), and, for example, a sugar chain containing allyl lacto-N-fucopentaose II, which is an antigen determining site of CA19-9 (or its sugar chain) It may be a monoclonal antibody produced by a hybridoma having an autonomous proliferation ability obtained by cell fusion of an antibody-producing cell and a myeloma cell immunized in advance with a substance containing the same. Furthermore, as long as it has antigen-binding activity, it may be a recombinant antibody or a fragment of these antibodies (for example, F (ab ′) 2, Fab region, etc.). Antibody fragments are obtained by decomposing and purifying antibodies with proteolytic enzymes. These antibodies may be used alone or in combination of two or more. Moreover, the anti-CA19-9 antibody can use what is marketed without a restriction | limiting.

  The CA19-9 detection reagent is not limited to an antibody, and a lectin capable of quantitatively detecting CA19-9 (allyl lacto-N-fucopentaose II, which is an antigen determination site) may be used.

In order to facilitate the detection of CA19-9, it is preferable to label a CA19-9 detection reagent (such as an anti-CA19-9 antibody) in advance. As a labeling substance of anti-CA19-9 antibody, based on the detection method, radioisotopes such as ³² P, ¹³¹ I, ³⁵ S, ⁴⁵ Ca, ³ H, ¹⁴ C, Cy3, Cy5, rhodamine, fluorescein, dansyl, Fluorescent materials such as fluorescamine, coumarin, europium, naphthylamine, luminescent materials such as luciferin, isoluminol, luminol, bis (2,4,6-trifluorophenyl) oxalate, ultraviolet absorbing materials such as phenol, naphthol, anthracene, Enzymes such as alkaline phosphatase, β-galactosidase, luciferase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, acetylcholinesterase, malate dehydrogenase, 4-amino-2,2,6,6 Tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl-α- (3,5-di-t-butyl) -4-Oxo-2,5-cyclohexadiene-1-ylidene) -p-tolyloxyl spin labeling agent, gold colloid, silver colloid, platinum colloid and the like can be used.

This CA19-9 detection reagent can measure CA19-9 by, for example, a chemiluminescent enzyme immunoassay using the following two-step sandwich method.
(1) The initially added biotin-conjugated anti-CA19-9 monoclonal antibody reacts specifically with CA19-9 in the sample and then binds to the added streptavidin-conjugated magnetic particles.
(2) After removing the unreacted solution, the subsequently added ALP-labeled anti-CA19-9 monoclonal antibody reacts specifically with CA19-9 on the magnetic particles.
(3) After removing the unreacted solution, the added luminescent substrate CDP-Star is decomposed by ALP on the magnetic particles, and the intensity of the generated luminescence is measured. Since the emission intensity increases reflecting the CA19-9 concentration in the sample, the CA19-9 concentration in the sample can be determined by measuring a sample containing a known concentration of CA19-9 in advance and preparing a calibration curve. Can be sought.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

  For example, in the above-described embodiment, a diagnostic kit for a diagnostic marker that combines mRNA encoding TERT and CA19-9 has been described. This diagnostic kit includes a detection reagent for mRNA encoding TERT alone, CA19 In addition to the detection reagent of -9 alone, it may further have a detection reagent for a diagnostic marker (Span-1, CEA, DUPAN2, TSGF, etc.) useful for improving the accuracy of diagnosis of pancreatic cancer.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

<Example 1>
The clinical significance of hTERT mRNA as a biomarker of pancreatic cancer was examined as follows for a suspected pancreatic cancer in Tottori University Hospital.

  Blood was collected from 72 cases (45 cases of pancreatic cancer and 27 cases of non-pancreatic cancer) suspected of pancreatic cancer experienced at Tottori University Hospital from December 2011 to September 2012, and the serum was subjected to the following experiment. The average patient age was 70.1 years (36 to 87 years).

  In conducting this study, informed consent was obtained from the patient, and the study protocol (plan) was in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of Tottori University. The test was conducted.

  In this example, first, blood of a subject (patient) was collected, and then a sample containing RNA was obtained from the blood. More specifically, lymphocytes were reduced by performing three-stage centrifugation (800 × g, 1000 × g, 1500 × g) for 10 minutes at 10 ° C. on blood collected from patients (about 1-2 mL). Serum was obtained and used as a test sample. The effective removal of lymphocytes was confirmed by examining the expression levels of CD2, CD3, CD8, CD19, CD22 and CD68 using β2-microglobulin as a control. Subsequently, RNA was extracted from serum using a deoxyribonuclease treatment by a conventionally known method. RNA extracted from 200 μL of serum was dissolved in 200 μL of nuclease-free water.

  Next, real-time RT-PCR was performed on the obtained sample using the KAPA BIOSSYSTEMS KAPA SYBR FAST Universal One-Step qRT-PCR Kit according to the following protocol. Real-time RT-PCR was performed using Primer F (hTERT F) with SEQ ID: 1 and Primer R (hTERT R) with SEQ ID: 2. The result of this real-time RT-PCR is shown in FIG. Β2-microglobulin RNA was used as a PCR control sample. A control calibration curve is shown in FIG.

  In addition, using a cancer antigen 19-9 kit access GI monitor manufactured by Beckman Coulter, Inc., the obtained sample was subjected to a chemiluminescent enzyme immunoassay according to the protocol attached to the kit, and a CA19-9 quantitative test (kit) (Cut-off value: <35 U / ml).

  In addition, a commercially available kit was used for the obtained sample, and a quantitative test of DUPAN2, CEA, and Span-1 was performed (the recommended value of the kit was adopted as a cut-off value) according to the protocol attached to the kit.

  As a result, the ability to diagnose pancreatic cancer by hTERT mRNA has a sensitivity of 97.8% (44/45) and a specificity of 65.4% (17) when the cutoff value is set to 903 (copy / serum 10 μl) in ROC curve analysis. / 26), positive predictive value 83.0% (44/53), negative predictive value 94.4% (17/18), correct diagnosis rate 83.0% (44/53) It was. The results are summarized in Table 1 below.

  Moreover, the tumor diameter in pancreatic cancer by hTERT mRNA and the sensitivity by T factor are TS1 / TS2 / TS3 (no TS4 cases), 90.0% (9/10) / 100% (26/26) / 100% (9 / 9), 100% (2/2) / 100% (3/3) / 100% (9/9) /96.8% (30/31) at T1 / T2 / T3 / T4, TS1 cases and Good sensitivity was confirmed even in resectable cases such as T1 / T2 cases confined to the pancreas. The results are shown in Table 2 below.

  Moreover, the pancreatic cancer diagnostic ability by CA19-9 has a sensitivity of 75.6% (34/45) and a specificity of 92.6% when the cut-off value of CA19-9 is set to 35 U / ml as described in the kit ( 25/27), positive predictive value 94.4% (34/36), negative predictive value 69.4% (25/36), correct diagnosis rate 81.9% (59/72), room for improvement It was a certain result. The results are summarized in Table 1 above.

  Moreover, pancreatic cancer diagnostic ability by CA19-9 is the tumor diameter in pancreatic cancer, and the sensitivity by T factor is TS1 / TS2 / TS3 (there is no TS4 case) 90% (9/10) / 100% (26/26) / 100 % (9/9), T1 / T2 / T3 / T4, and good sensitivity was confirmed even in resectable cases such as TS1 cases and T1 / T2 cases confined to the pancreas. The results are shown in Table 3 below.

  On the other hand, the pancreatic cancer diagnostic ability by hTERT mRNA and CA19-9 was set to 903 (copy / serum 10 μl) as the cut-off value of hTERT mRNA by ROC curve analysis, and CA19-9 as described in the kit. The cut-off value was set to 35 U / ml, and as shown in Table 4 below, when either hTERT mRNA or CA19-9 was equal to or higher than the cut-off value, it was determined to be positive as a comprehensive index. The sensitivity was 100.0% (45/45).

  Moreover, the overall pancreatic cancer diagnostic ability by hTERT mRNA and CA19-9 is as follows: tumor diameter in pancreatic cancer, sensitivity by T factor is TS1 / TS2 / TS3 (no TS4 cases), 100.0% (10/10) / 100% (26/26) / 100% (9/9), 100% (2/2) / 100% (3/3) / 100% (9/9) / 96 at T1 / T2 / T3 / T4. 8% (31/31), a good sensitivity was confirmed even in resectable cases such as TS1 cases and T1 / T2 cases localized in the pancreas. The results are shown in Table 4 below.

<Example 2>
Pancreatic cancer patients with different tumor diameters (TS1: 10 cases, TS3: 9 cases) were comprehensively diagnosed with hTERT mRNA and CA19-9. The diagnostic ability of pancreatic cancer diagnosis by hTERT mRNA at this time was performed by setting the cut-off value to 924.5 (copy / serum 10 μl) by ROC curve analysis. The ROC curve is shown in FIG. Other diagnostic conditions (conditions such as blood sampling and quantitative tests) were the same as in Example 1. As a result of diagnosing 19 cases, the sensitivity was 100%, which was a very good result (Table 5).

<Consideration of results>
From the experimental results of Examples 1 and 2 above, it was shown that measurement of serum hTERT mRNA is useful for diagnosis of early pancreatic cancer and surpasses current tumor markers for pancreatic cancer. Moreover, it was shown that the combination of serum hTERT mRNA measurement and serum CA19-9 measurement is useful for diagnosis of early pancreatic cancer and far surpasses current tumor markers for pancreatic cancer. That is, if either hTERT mRNA or CA19-9 is equal to or higher than the cut-off value, it is possible to make the sensitivity almost 100% even in the first stage and the second stage of pancreatic cancer by making it “positive”. Therefore, even in a medical institution where it is difficult to implement EUS-FNA such as a clinic and a community hospital, patients with pancreatic cancer are hardly overlooked. Therefore, clinics and community hospitals can introduce patients suspected of having pancreatic cancer to advanced medical institutions such as university hospitals with almost no oversight, and can eliminate false positive patients by performing EUS-FNA at advanced medical institutions. It becomes possible to treat patients with pancreatic cancer in the first stage and the second stage of pancreatic cancer at advanced medical institutions.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

  For example, in the above example, the cut-off value was set to 903 (copy / serum 10 μl) in the ROC curve analysis, and the cut-off value of CA19-9 was set to 35 U / ml as described in the kit. However, the cut-off value can be appropriately adjusted according to the purpose. Specifically, those skilled in the art can easily understand that these cut-off values can be optimized by gathering more cases.

  Moreover, in the said Example, although hTERT mRNA in serum was measured, it is not necessarily limited, You may measure hTERT mRNA in saliva according to the objective. The present inventors have confirmed that hTERT mRNA can be detected by performing real-time RT-PCR on hTERT mRNA using RNA extracted from saliva. Therefore, it can be easily understood by those skilled in the art that not only hTERT mRNA in serum but also hTERT mRNA in saliva can be used as a tumor marker for pancreatic cancer.

Claims (10)

A diagnostic kit for diagnosing pancreatic cancer in a mammal,
A TERT detection reagent for quantitatively detecting mRNA encoding TERT in blood or saliva obtained from the mammal;
The amount of mRNA encoding the TERT serves as an index for diagnosis of pancreatic cancer.
Diagnostic kit. The diagnostic kit according to claim 1,
The stage classification of the pancreatic cancer is TS1 or TS2.
Diagnostic kit. The diagnostic kit according to claim 1 or 2,
The TERT detection reagent comprises a set of primers capable of hybridizing to mRNA encoding TERT;
Diagnostic kit. The diagnostic kit according to claim 3,
The mammal is a human;
The TERT is hTERT;
The set of primers is
(1) SEQ ID: 1 and SEQ ID: 2, SEQ ID: 3 and SEQ ID: 4, SEQ ID: 5 and SEQ ID: 6, any combination of SEQ ID: 7 and SEQ ID: 8, or ( 2) A combination having a base sequence identity of 80% or more with any combination of (1),
Consisting of either
Diagnostic kit. The diagnostic kit according to claim 4,
The set of primers is a set of primers for use in real-time RT-PCR using a fluorescent dye.
Diagnostic kit. In the diagnostic kit according to any one of claims 1 to 5,
A CA19-9 detection reagent for quantitatively detecting CA19-9 in blood or saliva obtained from the mammal;
An overall index including the amount of mRNA encoding TERT and the amount of CA19-9 is shown as an index for diagnosis of pancreatic cancer.
Diagnostic kit. The diagnostic kit according to claim 6,
The CA19-9 detection reagent comprises a monoclonal antibody that specifically binds to CA19-9;
Diagnostic kit. A test method for testing pancreatic cancer in a mammal,
Using the diagnostic kit according to claim 1 to 7 to obtain an indicator for diagnosis of pancreatic cancer in blood or saliva obtained from the mammal,
Inspection method. A diagnostic marker for diagnosing pancreatic cancer in a mammal,
Comprising the amount of mRNA encoding hTERT in blood or saliva obtained from said mammal,
Diagnostic marker. The diagnostic marker according to claim 9,
Further comprising the amount of CA19-9 in blood or saliva obtained from said mammal,
An overall index including the amount of mRNA encoding TERT and the amount of CA19-9 is shown as an index for diagnosis of pancreatic cancer.
Diagnostic marker.