JP2010066225A - Method and biological marker for blood detection of plurality of cancers utilizing mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of blood detection of a human cancer-carrying patient aiming at a plurality of cancers including pancreatic cancer and ovarian cancer. <P>SOLUTION: The method for detecting and identifying a plurality of cancers by using a single blood sample relative to a human cancer-carrying patient has processes for: (1) acquiring a protein manifestation profile by measuring a mass spectrum of each protein in a blood sample originated in a human cancer-carrying patient carrying each cancer relative to a plurality of kinds of cancers and in a blood sample originated in a non-cancer healthy person; (2) selecting a peak of a mass number (m/z) capable of discriminating each of a specific kind of human cancers from non-cancer, relative to a peak showing the protein manifestation profile in step (1); and (3) measuring a mass spectrum of a protein in a single blood sample originated in the human cancer-carrying patient in which the kind of the cancer is unknown, and detecting and identifying a plurality of human cancers by using a difference of a relative manifestation level with the non-cancer healthy person as an index, relative to the peak of the mass number (m/z) selected in step (2), based on the acquired protein manifestation profile. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a novel blood diagnostic biomarker useful for detection of carcinoma in human cancer-bearing patients having multiple carcinomas including pancreatic cancer and ovarian cancer, and to a blood detection method for carcinoma using mass spectrometry.

  The number of deaths due to malignant neoplasms in Japan is about 300,000 per year, accounting for about one-third of all deaths (one in two in the 60s) and has been consistent since the 1980s. The number is increasing. Cancer is the biggest cause of death in the mid-aged generation, and for Japan, where the declining birthrate and aging population are ahead of other countries, the loss of labor force is an issue that should be addressed urgently nationally. It is done. Furthermore, the increase in the elderly population is thought to lead to an increase in the number of new cases. In fact, this tendency is remarkable, and an estimate that about 850,000 people will suffer from cancer in 2020 is presented. Yes. As described above, drastic countermeasures are considered to be urgent from any viewpoint. Extending healthy life expectancy and improving quality of life are global concerns and aspirations, and it is clear that the provision of better and innovative medical services is essential to achieve these. is there. With the promotion of intensive medical research to date, there are tumors that are already in the medical system so that cure can be sufficiently expected from the incurable disease like leukemia by starting appropriate treatment early. In addition, due to thorough early detection and early treatment of gastric cancer, the number of affected people is still overwhelming, but the number of deaths has been consistently decreasing since the war. That is below that of lung cancer, and the 5-year relative survival rate has improved to about 60%. As described above, the development of an early diagnosis method for cancer is the most important point in future cancer countermeasures, and the present inventors have developed a method for easily performing the diagnosis of the presence of multiple carcinomas on a blood sample. We started the development using blood samples from patients with pancreatic and ovarian cancer and healthy non-cancerous people.

  Pancreatic cancer that occurs in the pancreas, which is a deep organ hidden in the gastrointestinal tract or liver, is difficult to detect early in abdominal CT examinations, and there is no direct examination technique such as a gastrocamera or barium contrast examination. Furthermore, the lack of clinical symptoms also makes early detection difficult, and in many cases, the disease has already progressed to a state where no cure can be expected at the time of diagnosis. Surgical excision is the first choice for curative treatment as a consensus on the current treatment of pancreatic cancer. However, even in cases where surgery is expected to be cured, the 5-year survival rate is about 20%, which is far below the therapeutic results of other cancers. Furthermore, in many cases, the condition has progressed to a state where no cure can be expected at the time of diagnosis (for example, the 5-year survival rate in all pancreatic cancer patients is around 5%). There is almost no improvement in comparison. Thus, pancreatic cancer, whose annual number of cases and deaths are almost equal, is considered to be the most difficult of all human cancer types. However, there is a statistic that the 5-year survival rate is improved to about 60% only in cases that have undergone resection surgery up to Stage II where the tumor diameter is 2 cm or less and is limited in the pancreas. This suggests the importance of early detection and early treatment. In current clinical practice, only 5% of cases are discovered at this stage, only a very small number, and the number of deaths has been increasing in recent years (20 years). About twice as much as before and about 1.5 times compared to 10 years ago.) A radical change in the medical care system is strongly desired, and the development of a new diagnostic method for early pancreatic cancer is supreme. It is a proposition.

  Currently, X-ray CT and ultrasonography are usually used for screening tests aimed at diagnosing pancreatic cancer, and there are many cases of resectable pancreatic cancer found by ultrasonography However, due to the fact that it takes about 30 minutes for each patient and there is a large difference in skill among the persons in charge of ultrasonic examinations, at present, ultrasonic examinations are carried out as general medical examinations in all local governments. It has not been done. In addition, the frequency of detection is very low and is about one case after about 25,000 examinations. Furthermore, detection of extremely early pancreatic cancer is very difficult, and there is a false negative (“no abnormality” at the time of screening). Despite this, pancreatic cancer develops within half a year). As described above, although there is no room for doubt regarding the usefulness of ultrasonic diagnosis in general clinical practice, when the target is considered only for pancreatic cancer diagnosis, the effectiveness is low. From this point of view, it is desired to establish an early detection method that is highly sensitive, minimally invasive, simple and inexpensive and can be widely applied to medical examinations.

  Furthermore, the ovary is deep in the abdomen and there are few subjective symptoms in the early stage of tumor formation. For this reason, 2/3 or more cases visit a hospital only after having metastasized. The most common metastasis to ovarian cancer is peritoneal dissemination, where dislocations spread from the surface of the ovary to the entire peritoneum. Ovarian cancer without metastasis can be cured by surgery alone, but in metastatic cases, all tumors cannot be removed by surgery alone, and postoperative chemotherapy is performed on the remaining tumor. If the diagnosis is a symptomatic visit and an increase in the tumor has already been observed, the presence of the tumor can be distinguished from uterine fibroids with considerable accuracy even by gynecological examination alone. As the secondary examination, image diagnosis such as ultrasound, X-ray CT, and MRI is performed, and the site where the tumor occurred, the internal structure, the presence or absence of metastasis, etc. are examined in detail. As a tumor marker in blood, CA125 is clinically used, and most humans are CA125 positive in metastatic ovarian cancer. However, since the positive rate is low in early cancer, and some young women are mildly positive even without cancer, it can be said that CA125 is insufficient for early detection of ovarian cancer.

  The development of various biotechnology technologies, such as the completion of the human genome project in recent years and the comprehensive expression analysis technology using a microarray / mass spectrometer, has transformed the concept of diagnosis and treatment of cancer in general, including refractory cancer. It is being urged. The current state of proteomics / bioinformatics fusion research is a field that is gaining great expectations because genetic information in the human genome is basically translated into protein, but it uses mature analysis technology called microarray. Compared to genomics research, it is in an early stage, and it is also true that proteomics research with medical applications in mind still has considerable technical difficulties. However, when looking at global trends, research results suggesting that explosive progress and development are already imminent are being reported, such as research groups such as the US Cancer Institute and Vanderbilt University. Have reported high-quality results such as finding the relationship between the clinical pathology of ovarian cancer, prostate cancer and lung cancer and the expression profile (Non-Patent Documents 1 to 6). However, there are no reports of analysis targeting multiple carcinomas such as pancreatic cancer and ovarian cancer at the same time. The reason for this is that, as mentioned above, research groups that can perform high-level acquisition and verification of detailed, high-quality, large-scale proteomics analysis data are still extremely limited globally due to their technical difficulties. And a limited number of institutions with large clinical sample banks with detailed clinical information.

  In addition, the pancreatic cancer marker is described in, for example, Patent Documents 1 to 4, and the ovarian cancer marker is described in, for example, Patent Document 5. Furthermore, a method for screening a cancer marker from a mass spectrum peak is described in Patent Document 6.

JP2007-051880 JP2004-248585 JP 2004-248575 A Special table 2000-502902 Special table 2007-504463 JP2008-076202 EF Petricoin et al., Lancet 2002, 359: 572-577 EF Petricoin et al. Natl. Cancer Inst. 2002, 94: 1576-1578 BL Adam et al., Cancer Res. 2002, 62: 3609-14 D Sidransky et al., J. MoI. Natl. Cancer Inst. 2003, 95: 1711-1717 MJ Campa et al., Cancer Res. 2003, 63: 1652-1656 K Yanagisawa et al., Lancet 2003, 362: 433-439

  Pancreatic cancer and ovarian cancer, which are cancers occurring in deep organs, which are typical intractable cancers, have poor clinical symptoms and currently lack effective testing methods for early diagnosis. There is a need to develop a new molecular testing method that enables early detection of these refractory cancers in a minimally invasive, simple and inexpensive manner.

  In such a situation, the present inventors can dramatically improve sensitivity and accuracy by introducing a test method for analyzing a plurality of proteins rather than a test method using a single gene or protein. We believe that there are multiple useful biomarkers that can distinguish between human pancreatic and ovarian cancer-bearing blood samples and non-cancerous healthy human blood samples, and a single measurement of multiple cancer-bearing patients. The purpose of this study was to construct a diagnostic algorithm and test method that could be discriminated.

  The purpose is to obtain a useful index for accurately discriminating blood samples from patients with pancreatic cancer and non-cancerous healthy subjects, and proteomic profiles in blood samples from patients with cancer are clinically meaningful tools. To prove this, we used MALDI MS to analyze protein expression profiles in 160 blood samples including 80 pancreatic cancer patient plasma and 80 non-cancerous healthy human plasma as training groups Thus, it was possible to select a (protein) peak that accurately distinguishes a blood sample of a pancreatic cancer patient from a blood sample of a non-cancerous healthy person. Furthermore, the present inventors applied a discrimination method based on the proteomic profile of a cancer-bearing patient blood sample and a non-cancerous healthy human blood sample to an independent data set obtained from the blinded group, and in the diagnosis of cancer presence We succeeded in verifying the usefulness.

  With the aim of obtaining a useful index for accurately discriminating between blood samples of ovarian cancer patients and blood samples of non-cancerous healthy subjects, the present inventors have used MALDI MS, Analyzing protein expression profiles in 127 blood samples including 63 ovarian cancer patient plasmas and 64 non-cancerous normal human plasmas as a training group to accurately distinguish ovarian cancer patient blood samples from non-cancerous healthy human blood samples The (protein) peak could be selected.

  Furthermore, the present inventors applied a discrimination method based on the proteomic profile of a cancer-bearing patient blood sample and a non-cancerous healthy human blood sample to an independent data set obtained from the blinded group, and in the diagnosis of cancer presence We succeeded in verifying the usefulness.

  Furthermore, bioinformatics analysis was advanced, and a plurality of carcinomas including the above-mentioned two carcinomas could be distinguished from healthy individuals by single sampling and measurement.

Therefore, in summary, the present invention has the following features.
(1) A method for detecting and identifying a plurality of carcinomas using a single blood sample in a human cancer-bearing patient having a plurality of carcinomas, comprising the following (a) to (c):
(A) A protein expression profile is obtained by measuring mass spectra of proteins in a blood sample derived from a human cancer-bearing patient having a specific cancer and a blood sample derived from a non-cancerous healthy person for a plurality of specific types of cancer. Step,
(B) selecting a peak having a mass number (m / z) capable of distinguishing each of the specific types of human cancer from non-cancer for the peak indicating the protein expression profile in the step (a);
(C) The mass spectrum of the protein in a single blood sample derived from a human cancer-bearing patient whose type of cancer is unknown is measured, and the peak indicating the obtained protein expression profile is measured with a non-cancerous healthy person. The step of detecting and identifying a plurality of human cancers using as an index the difference in the relative expression level of the protein corresponding to the peak of the mass number (m / z) selected in the step (2). Method.

  (2) The method according to (1) above, wherein the plurality of carcinomas include pancreatic cancer and ovarian cancer.

  (3) The mass number (m / z) of the selected peak that can distinguish the pancreatic cancer from the non-cancer is 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 The method according to (2) above, which is one or more selected from the group consisting of ± 2%, 13761.49 ± 2%, 14145.18 ± 2%, and 17250.82 ± 2%.

  (4) The mass numbers (m / z) of the selected peaks that can distinguish the ovarian cancer from non-cancer are 4471.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140. The method according to (2) above, which is one or more selected from the group consisting of 222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2%, and 13935.1 ± 2%.

  (5) The method according to any one of (1) to (4), further comprising identifying a protein having the mass number of the selected peak.

  (6) The method according to (5), further comprising determining a gene encoding the protein based on the identified protein.

  (7) Use of an antibody against a protein selected from the group consisting of proteins having the mass number (m / z) or a variant thereof, or a polynucleotide encoding the protein or the variant or a polynucleotide complementary thereto. And the method in any one of said (1)-(6) which measures the difference of the said expression level.

  (8) A method for detecting the above-mentioned carcinoma using a single blood sample in a human cancer-bearing patient having pancreatic cancer, ovarian cancer or both, and derived from the first cancer-bearing patient having pancreatic cancer or ovarian cancer And obtaining a protein expression profile by measuring mass spectra of the blood sample from the second non-cancer healthy person and the blood sample from the first cancer-bearing patient with respect to the peak indicating the protein expression profile Selecting a peak of a mass number (m / z) that can be distinguished from a blood sample from the second group of healthy non-cancerous individuals, and between the first cancer-bearing patient and the second non-cancerous healthy person Detecting cancer in the blood sample of the human cancer-bearing patient using as an index the difference in the relative expression level of the protein corresponding to the selected mass number (m / z) peak between so Each of the proteins corresponding to the selected peaks is 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2 for pancreatic cancer. %, 14145.18 ± 2% and 17250.82 ± 2% selected from the group consisting of one or more mass numbers (m / z), and for ovarian cancer, 4470.801 ± 2%, 6940 1 or 2 selected from the group consisting of .418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2%, and 13935.1 ± 2% Each of the above-mentioned methods having the above mass number (m / z).

  (9) The mass number (m / z) of the peak in the mass spectrum measurement is 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± Hematological diagnosis of human pancreatic cancer patients comprising at least one antibody or fragment thereof to each of a protein or variant thereof selected from the group consisting of 2%, 14145.18 ± 2% and 17250.82 ± 2% Composition for doing.

  (10) The mass number (m / z) of the peak in the mass spectrum measurement is 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± Polynucleotides encoding proteins that are selected from the group consisting of 2%, 14145.18 ± 2% and 17250.82 ± 2%, polynucleotides complementary thereto, variants thereof, stringent to them A composition for blood diagnosis of a human pancreatic cancer patient, comprising at least one polynucleotide selected from the group consisting of a polynucleotide that hybridizes under conditions or a fragment thereof containing 15 or more consecutive bases.

  (11) The composition according to (9) or (10) above, wherein the composition is in the form of a kit or a microarray.

  (12) The mass number (m / z) of the peak in the mass spectrum measurement is 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± The blood of a human ovarian cancer patient comprising at least one antibody or fragment thereof to each of a protein or variant thereof selected from the group consisting of 2%, 13879.6 ± 2% and 13935.1 ± 2% A composition for making a diagnosis.

  (13) The mass number (m / z) of the peak in the mass spectrum measurement is 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± Polynucleotides encoding proteins that are selected from the group consisting of 2%, 13879.6 ± 2% and 13935.1 ± 2%, polynucleotides complementary thereto, variants thereof, stringent to them A composition for blood diagnosis of a human ovarian cancer patient, comprising at least one polynucleotide selected from the group consisting of a polynucleotide that hybridizes under conditions or a fragment thereof containing 15 or more consecutive bases.

  (14) The composition according to (12) or (13) above, wherein the composition is in the form of a kit or a microarray.

  According to the present invention, it becomes possible to accurately and accurately predict patients with pancreatic cancer and ovarian cancer who had difficulty in early detection and had great problems in clinical settings. It provides an exceptional effect on current cancer practice that leads to a decrease in mortality.

Definitions The terms used in this specification have the following meanings.

  As used herein, “early” with respect to cancer refers to stages where the stage of cancer progression is I and II.

  As used herein, “sample” refers to blood collected from a human. “Blood” as used herein shall refer to either whole blood, serum or plasma.

  In the present invention, relative to the expression level of the protein or nucleic acid marker of the present invention between a blood sample derived from a human patient having a plurality of carcinomas including pancreatic cancer and ovarian cancer and a blood sample derived from a non-cancerous healthy person. There are significant differences.

  As used herein, “variant” includes, for example, a mutant caused by biological events such as mutation, polymorphism, alternative splicing, degeneracy of the genetic code, and the like. The mutant includes a mutant of a protein marker related to the present invention or a mutant of a gene encoding the protein marker. Such a variant is a variant having one or more, preferably one or several substitutions, deletions, additions, insertions, etc. on the sequence of the protein marker or nucleic acid marker, the sequence or a part thereof. It includes variants having a sequence identity of 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, 99% or more. Here, “several” usually means an integer of 10 or less, preferably 2 to 5. Identity (%) can also be determined using known BLAST or FASTA programs with or without gaps introduced (S. F. Altschul et al., J. Mol. Bio. 215: 403). -410, 1990). In general, in the alignment of two sequences, there was a match for the total number of amino acid residues or the total number of bases when the most amino acids or bases are aligned so that they overlap (including gaps, if any) Identity (%) can be calculated as a percentage of the number of amino acid residues or bases. Furthermore, amino acid mutations in proteins include conservative or non-conservative amino acid mutations. Here, a conservative amino acid mutation is a substitution mutation between amino acids having similar properties in terms of structure (aromatic group, branch, etc.), charge (basic, acidic, neutral), polarity or hydrophobicity. Point to.

  “Stringent conditions” as used herein are not limited to the following, but nucleotide sequences having at least 90%, preferably at least 95%, more preferably at least 98% identity hybridize to each other: Such hybridization and washing conditions are meant. An example of such conditions is 1 to 6 × SSC, hybridization at room temperature to 40 ° C., followed by washing at 0.1 to 1 × SSC, 0.1% SDS, 50 to 65 ° C. Here, 1 × SSC is 150 mM sodium chloride and 15 mM sodium citrate aqueous solution (pH 7.2). For hybridization, see F.C. M.M. Ausbel et al., Short Protocols in Molecular Biology (3rd edition) A Compendium of Methods Current Protocols in Molecular Biology, 1995, John Wisley. (United States).

Detailed Description In the following, the present invention will be described in more detail.

1. Cancer-related marker In the human cancer-related marker of the present invention, at least (i) a significant difference (ie, increase or decrease) in the expression level of the marker is observed in the blood sample of pancreatic cancer patients as compared to the blood sample of a non-cancerous healthy person. A marker that enables detection of pancreatic cancer, and (ii) a significant difference in the expression level of the marker in a blood sample of an ovarian cancer patient compared to a non-cancerous healthy human blood sample (ie, , Increase or decrease), and thereby markers that make it possible to detect ovarian cancer.

  The marker measures the mass spectrum of the blood sample, compares the spectral pattern between the blood samples, selects peaks that can be distinguished from each other (ie, has a difference in expression level), and if necessary, selects those peaks. Identifies the protein or peptide corresponding to the peak, and is a reliable marker based on these procedures to confirm the detection of multiple carcinomas, including pancreatic cancer, ovarian cancer, etc., in blood samples from many cancer patients And was found by confirming the reproducibility of the judgment. Here, identification of protein or peptide (including sequencing) can be performed by a known technique such as mass fingerprinting or sequence tagging.

  Any mass spectrometer can be used for mass spectrum measurement as long as it is used for protein identification. Examples of such mass spectrometers include surface enhanced laser desorption / ionization MS devices, matrix-assisted laser desorption / ionization MS devices (eg, MALDI MS), and the like. A particularly preferred mass spectrometer in the present invention is MALDI MS. Specific analysis conditions using this apparatus are as follows.

  The blood sample is divided into 5 microliters and diluted 10 times with 50% acetonitrile / 0.1% trifluoroacetic acid solution. Separate 5 microliters of 10-fold diluted blood sample and mix with 20 microliters of sinapinic acid at a concentration of 30 mg / ml dissolved in 50% acetonitrile / 0.1% trifluoroacetic acid solution. A matrix mixed blood sample (0.5 microliters) is dropped into 6 wells on the sample stage and dried. A protein expression profile is obtained from the dropped blood sample on the sample stage using mass spectrometry (for example, model 4800 manufactured by Applied Biosystems). The analysis conditions of the instrument at this time are acceleration voltage; 25,000V, focus mass; 10,000, analysis range; 2,000 to 20,000 daltons.

  By the above method, the present inventors have found a peak that makes it possible to discriminate both samples when measuring mass spectra of a human pancreatic cancer patient blood sample and a non-cancer healthy human blood sample. Such a peak corresponds to a protein or a fragment peptide thereof as a marker of the present invention. Seven peaks are found that make it possible to distinguish blood samples from pancreatic cancer patients and blood samples from healthy non-cancerous people (see FIGS. 1 to 7 for pancreatic cancer patients, and FIGS. 8 to 14 for non-cancerous healthy people), which correspond to these peaks. The mass numbers of the proteins to be performed are 8562.262 ± 2%, 8684.438 ± 2%, 8765.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2%, 14145.18 ± 2% and 17250 .82 ± 2%. Here, “± 2%” means an error range due to differences in apparatus, measurement conditions, and the like.

  Furthermore, the present inventors have found a peak that makes it possible to discriminate both samples when measuring mass spectra of a human ovarian cancer patient blood sample and a non-cancerous healthy human blood sample by the same method as described above. Seven peaks that make it possible to discriminate between the blood sample of the ovarian cancer patient and the blood sample of a non-cancerous healthy person are found (see FIGS. 15 to 21 for ovarian cancer patients and FIGS. 22 to 28 for non-cancerous healthy persons). The mass number of the protein corresponding to these peaks is 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, 13879. 6 ± 2% and 13935.1 ± 2%. Here, “± 2%” has the same meaning as described above.

  In the present invention, in addition to the above protein, a polynucleotide encoding the protein or a polynucleotide complementary thereto is also included as a cancer-related marker. If a protein is identified, the primary structure (sequence) of the gene or DNA encoding the protein can be determined based on the sequence by a search means such as BLAST or FESTA search. Specific examples of such polynucleotides have peak mass numbers in the mass spectrum of 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761. Proteins with 49 ± 2%, 14145.18 ± 2% and 17250.82 ± 2%, or mass numbers 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140. Polynucleotides encoding proteins that are 222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2%, polynucleotides complementary thereto, or variants thereof are included. Such variants include those produced in vivo as a result of biological events such as mutations, polymorphisms, alternative splicing. In practice, the nucleic acid marker is detected at the nucleic acid or protein level as a transcript or translation product.

  In the case of a transcript, it is detected as mRNA, cRNA or cDNA. Total RNA is extracted from cells or tissues by a known method, poly A (+) RNA or mRNA is prepared by an oligo dT cellulose column method, and if necessary, cDNA and cRNA are further synthesized from the mRNA.

  In the case of a translation product, it is detected as a protein or fragment thereof encoded by the gene or variant thereof.

2. Cancer-related marker detection composition A. Composition for Detecting Pancreatic Cancer The present invention, as described above, obtains a protein expression profile in each sample by measuring mass spectra of a human pancreatic cancer patient blood sample and a non-cancerous healthy human blood sample, Selecting a peak capable of discriminating between the blood sample of a pancreatic cancer patient and a blood sample of a non-cancerous healthy person, and corresponding to the selected peak between the blood sample of the pancreatic cancer patient and a blood sample of a non-cancerous healthy person Detecting pancreatic cancer in a human pancreatic cancer blood sample using the difference in the relative expression level of the protein as an index. Here, the protein corresponding to the selected peak has a mass number (m / z) of 8562.262 ± 2% and 8684.4438 ± 2 in the mass spectra shown in FIGS. 1 to 7 and FIGS. %, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2%, 14145.18 ± 2% and 17250.82 ± 2%.

  The expression level of at least one protein marker corresponding to the peak or a variant thereof in a blood sample derived from a patient with pancreatic cancer or a blood sample of a non-cancerous healthy person, or the level of a polynucleotide encoding the protein or the variant , Using a probe corresponding to the marker, and detecting pancreatic cancer in a cancer patient using a significant level difference of the protein or polynucleotide as an index between a blood sample of a pancreatic cancer patient and a blood sample of a non-cancerous healthy person Including doing.

  The probe used in the present invention is not particularly limited as long as the marker can be detected, but is usually a polynucleotide or an antibody. Thus, a composition comprising such a polynucleotide or antibody can be used to detect a patient's pancreatic cancer in vitro.

B. Composition for detecting ovarian cancer The present invention also provides a protein expression profile in each sample by measuring a mass spectrum of a human ovarian cancer patient blood sample and a non-cancerous healthy human blood sample, as described above, Selecting a peak capable of discriminating between the blood sample of the ovarian cancer patient and the blood sample of a non-cancerous healthy person for the peak indicating the protein expression profile, and the selection between the blood sample of the ovarian cancer patient and the blood sample of a non-cancerous healthy person Detecting ovarian cancer in a human ovarian cancer blood sample using as an index the difference in the relative expression level of the protein corresponding to the peak produced. Here, the protein corresponding to the selected peak has mass numbers (m / z) of 4470.801 ± 2% and 6940.418 ± 2% in the mass spectra shown in FIGS. 876.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2%.

  Expression level of at least one protein marker corresponding to the peak or a variant thereof in a blood sample of ovarian cancer patient derived from the patient or a blood sample of a non-cancerous healthy person, or a polynucleotide encoding the protein or a variant thereof Is measured using a probe corresponding to the marker, and a significant level difference of the protein or polynucleotide between an ovarian cancer patient blood sample and a non-cancerous healthy human blood sample is used as an index. Detecting.

  The probe used in the present invention is not particularly limited as long as the marker can be detected, but is usually a polynucleotide or an antibody. Accordingly, compositions comprising such polynucleotides or antibodies can be used to detect ovarian cancer in a patient in vitro.

2.1 Antibody Probe In the present invention, an antibody can be used as a probe for detecting a cancer-related marker.

The antibodies that can be used in the present invention are divided into the following groups.
(1) Mass numbers (m / z) 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± in the mass spectra shown in FIGS. 1 to 7 and FIGS. 8 to 14 2%, 13761.49 ± 2%, 14145.18 ± 2% and 17250.82 ± 2%, or mass numbers 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± To a protein or fragment thereof selected from the group consisting of 2%, 9140.222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2% and variants thereof An antibody or fragment thereof. These antibodies or fragments thereof are used to measure the difference in the expression level of a protein marker that discriminates between a blood sample of a pancreatic cancer patient and a blood sample of a non-cancerous healthy person.

(2) Mass numbers (m / z) 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± in the mass spectra shown in FIGS. An antibody or fragment thereof against a protein or fragment thereof selected from the group consisting of a protein having 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2% and variants thereof. These antibodies or fragments thereof are used to measure differences in the expression levels of protein markers that distinguish ovarian cancer patient blood samples from non-cancerous healthy human blood samples.

  In each of the above groups, the composition can comprise at least 1, preferably at least 2, more preferably 3 to all, for example 4 to all, 5 to all probes.

  Variants are generally 80% or more, 85% or more, preferably 90% or more, 93% or more, more preferably 95% or more, 98% or more, 99% or more at the amino acid level with the complete or partial sequence of the protein. It has sequence identity.

  Further, the protein may be derivatized, and may include, for example, chemically modified derivatives such as glycosylation, phosphorylation, sulfation, alkylation, acylation and the like.

The antibodies used in the present invention include polyclonal antibodies, monoclonal antibodies, anti-peptide antibodies, single chain antibodies, chimeric antibodies and the like. Antibody fragments include Fab, (Fab ′) ₂ , Fc, Fc ′, Fd, Fv, and the like. These antibody fragments can be obtained, for example, by limited degradation of the antibody with a protease such as papain or pepsin.

  Each protein or fragment thereof is synthesized using protein synthesis or gene recombination techniques, and the resulting protein or fragment thereof is used as an antigen to immunize animals such as rabbits, mice, rats, horses, cows, goats, and sheep. And antibodies against those antigens are produced and purified.

  Polyclonal antibodies are obtained by immunizing the above animals subcutaneously with about 10 to 300 μg of the antigen, followed by additional immunization after about 2 weeks, blood collection about 3 weeks to 1 month after the first immunization, The IgG component can be produced by a method including separation using ammonium sulfate fractionation and ion exchange chromatography. In order to increase specificity, the obtained IgG is bound to a column made by binding the target protein to a carrier such as cellulose or agarose, and then eluted with a high salt buffer for dialysis or ultrafiltration. A specific polyclonal antibody can be obtained by desalting by a method such as The antibody titer can be measured by a conventional immunoassay, for example, an enzyme immunoassay (EIA, ELISA), a radioimmunoassay (RIA), a fluorescent antibody method, or the like.

A monoclonal antibody can be prepared, for example, by the following method.
The target protein or fragment thereof is administered subcutaneously to mice or rats (for example, Balb / c mice) in the same manner as polyclonal antibody production, and booster immunization is performed about 2 to 4 times at intervals of 1 to 4 weeks. When the antibody titer reaches a peak, the antigen is injected intravenously or intraperitoneally to obtain the final immunization. Two to five days later, antibody-producing cells (eg spleen cells or lymph node cells) are collected. The antibody-producing cells are then fused to a myeloma cell line (preferably a hypoxanthine / guanine / phosphoribosyl transferase (HGPRT) deficient cell line) to generate hybridoma cells, and HAT (hypoxanthine, aminopterin, thymidine) selection is performed. Do. In cell fusion, antibody-producing cells and myeloma cell lines are mixed at a ratio of about 1: 1 to 20: 1 in animal cell culture medium such as serum-free DMEM, RPMI-1640 medium, and the like. It is carried out in the presence of a cell fusion promoter such as Confirmation of the target antibody can be performed by the immunoassay described above. Furthermore, in order to proliferate the hybridoma, the hybridoma is administered into the abdominal cavity of the mouse, and after the hybridoma is proliferated, ascites is collected after 1 to 2 weeks. Antibody purification can be performed by appropriately combining methods such as ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and gel chromatography.

  Anti-peptide antibodies are antibodies against linear peptides on the surface of proteins and can increase immunological specificity. Such peptides are described, for example, in the estimation of hydrophilic-hydrophobic regions of Kyte-Doolittle et al. (J. Mol. Biol. 157: 105-132, 1982), Emini et al. (J. Viol. 55: 836-839, 1985), the probability of being located on the surface of a specific peptide site on a protein molecule, the degree of protein chain folding, such as Chou-Fasman et al. (Adv. Enzymol. Relat. Area Mol. Biol. 47: 45-148, 1978). It can be estimated using alpha helix, beta sheet, secondary structure prediction of protein displaying turn, etc. alone or in combination. The estimated peptide can then be synthesized using a peptide synthesizer.

  Target protein synthesis can be obtained from the cell or culture supernatant by incorporating a cDNA clone into an expression vector and culturing a prokaryotic or eukaryotic host cell transformed or transfected with the vector. A commercially available expression vector can be used. Host cells include prokaryotic cells such as bacteria (eg, E. coli, Bacillus subtilis, Pseudomonas bacteria), yeast (eg, Saccharomyces, Pichia, etc.), insect cells (eg, Sf cells), mammalian cells (eg, CHO, COS, BHK, HEK293, etc.). The vector is composed of a plasmid, cosmid, phage, virus, etc. and may contain DNA encoding the target protein, promoter, enhancer if necessary, polyadenylation signal, ribosome binding site, replication origin, terminator, selection marker, etc. it can. In order to facilitate protein purification, a labeled peptide, for example, a DNA sequence encoding a 6 to 10 residue histidine tag, FLAG, GFP protein, or the like may be included. The gene recombination techniques are described in Sambrook et al. (Above), Ausbel et al. (Above), and the techniques described therein can be used for the present invention.

  The target protein obtained as described above is appropriately combined with gel filtration, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, isoelectric focusing, electrophoresis, ultrafiltration, salting out, dialysis, etc. And can be purified.

  The sequence of the target protein or fragment thereof can be obtained by accessing NCBI HomePage (USA) based on GenBank or UniGene accession numbers.

  According to an embodiment of the present invention, the composition may be in the form of a kit or microarray, i.e., the probe is included in the form of a kit or microarray.

  In the case of a kit, antibodies capable of detecting the total number of markers from one or more of each protein marker in the two groups can be packaged individually or in combination of two or more in a suitable container.

The antibody is a polyclonal antibody, a monoclonal antibody, an anti-peptide antibody or the like as prepared by the above method, but is not limited thereto. The type of antibody may be any type, class, subclass, and includes, for example, IgG, IgM, IgE, IgD, IgA, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2. Antibody fragments include Fab, (Fab ′) ₂ , Fc, Fd, Fv, and the like.

2.2 Polynucleotide Probes Polynucleotide probes according to the present invention are divided into the following groups.
(1) Mass numbers (m / z) 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± in the mass spectra shown in FIGS. 1 to 7 and FIGS. 8 to 14 A polynucleotide encoding a protein selected from the group consisting of 2%, 13761.49 ± 2%, 14145.18 ± 2% and 17250.82 ± 2%, and variants thereof, Nucleotides, polynucleotides that hybridize to them under stringent conditions, or fragments thereof comprising at least 15 bases. These polynucleotides, complementary polynucleotides or fragments are used to measure the difference in transcription level corresponding to the expression of a protein marker that discriminates between a blood sample of a pancreatic cancer patient and a blood sample of a non-cancerous healthy person.

(2) Mass numbers (m / z) in the mass spectra shown in FIGS. 15 to 21 and FIGS. 22 to 28 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± A polynucleotide encoding a protein selected from the group consisting of proteins having 2%, 94222.677 ± 2%, 13879.6 ± 2%, and 13935.1 ± 2%, and variants thereof; Nucleotides, polynucleotides that hybridize to them under stringent conditions, or fragments thereof comprising at least 15 bases. These polynucleotides, complementary polynucleotides or fragments are used to measure the difference in transcription levels corresponding to the expression of protein markers that discriminate between ovarian cancer patient blood samples and non-cancerous healthy human blood samples.

  In each of the above groups, the composition can comprise at least 1, preferably at least 2, more preferably 3 to all, for example 4 to all, 5 to all probes.

  The cancer-related markers according to the present invention can be divided into two groups as follows according to the target carcinoma. That is, group 1 is useful for discriminating blood samples from pancreatic cancer patients, while group 2 is useful for discriminating blood samples from ovarian cancer patients.

  In the present invention, the mutant as a probe has one or more, preferably one or several, mutations such as substitution, deletion, addition, insertion, etc. in the nucleotide sequence of the group 1 or group 2 polynucleotide. Variants, and variants comprising a sequence having the identity of usually 80% or more, 85% or more, preferably 90% or more, more preferably 95% or more and 98% or more, etc. are included.

  Furthermore, in the present invention, a polynucleotide that hybridizes under stringent conditions hybridizes to the above-mentioned group 1 or group 2 polynucleotide or a complementary polynucleotide thereof under hybridization conditions as defined above. If it is possible, there is no particular limitation. Such polynucleotides allow the detection of the target nucleic acid, as some patient individuals may have genes that have been altered by biological events such as mutations, polymorphisms, alternative splicing.

  Furthermore, in the present invention, the fragment of the polynucleotide has a size of 15 bases to less than the total bases. The fragment has any number of bases within this range, for example, 20 bases or more, 30 bases or more, 50 bases or more, 70 bases or more, 100 bases or more, 150 bases or more, 200 bases or more, 250 bases or more.

  The polynucleotide probe used in the present invention can be synthesized by a conventional chemical DNA synthesis technique or a gene recombination technique.

  If the polynucleotide is a DNA molecule of about 100 bases or less, it can be synthesized using an automatic DNA synthesizer (for example, Applied Biosystems, USA) using the phosphoramidite method.

  Alternatively, the polynucleotide can be produced by cDNA cloning. After obtaining total RNA from the target cancer tissue and obtaining poly A (+) RNA by oligo dT cellulose column treatment, a cDNA library was prepared by reverse transcription-polymerase chain reaction (RT-PCR) method. A probe (15 or more, preferably 30 or more, more preferably 50 to 100 or more bases) prepared in advance based on a base sequence that can be obtained from a rally by a sequence search registered in a data bank such as GenBank or UniGene. CDNA clones can be obtained by hybridization. The obtained clone is incorporated into an expression vector such as a commercially available one, introduced into an appropriate host cell such as Escherichia coli or Bacillus subtilis, and propagated, or based on a sequence registered in the data bank. Amplification can be performed by a polymerase chain reaction (PCR) using a primer (generally 15 to 30 bases, preferably 17 to 25 bases) prepared in advance and using the cDNA clone as a template. For specific procedures and reagents for cDNA cloning and PCR, commercially available kits, devices, and reagents can be used. For example, Sambrook J et al., Molecular Cloning A Laboratory Manual, 1989, Cold Spring Harbor Laboratory. Press (USA); M.M. Ausbel et al., Short Protocols in Molecular Biology (3rd edition) A Compendium of Methods Current Protocols in Molecular Biology, 1995, John Wisley. (United States).

  The composition of the present invention may be in the form of a mixture comprising at least one of antibodies or polynucleotide probes for detecting pancreatic cancer and ovarian cancer associated markers of the present invention, or individual or The probe may be in the form of a so-called kit packaged in a container, or may be in the form of a microarray.

  The kit may further contain reagents for performing hybridization, such as a buffer, a reverse transcriptase, a labeled secondary antibody, and the like.

  In the case of a microarray, the array is a DNA microarray (also referred to as a DNA chip), a tissue array, or a protein microarray.

  Each of these microarrays is bound with the above-mentioned polynucleotide as a probe or the above-mentioned antibody or fragment thereof. That is, on the surface of the array, a polynucleotide capable of detecting the nucleic acid marker or transcription or translation product, a polynucleotide capable of hybridizing with the nucleic acid marker or a variant thereof, or a protein encoded by the gene, or An antibody or fragment thereof that can specifically bind to the mutant or derivative is bound as a probe.

  As the substrate of the array, glass or polymer is used, and poly L-lysine, silane or densified amino groups are introduced on the surface thereof. In addition, the polynucleotide or antibody is bound to the substrate by a spot method or an ink jet method.

  Hybridization is also possible as an alternative to the array, and includes DNA-DNA hybridization, DNA-RNA hybridization, RNA-RNA hybridization, dot blot, and the like.

3. As described above, the present invention is a method for detecting and identifying a plurality of carcinomas using a single blood sample in a human cancer-bearing patient having a plurality of carcinomas, comprising the following (1) to ( 3):
(1) obtaining a protein expression profile by measuring mass spectra of proteins in a blood sample derived from a human cancer-bearing patient having each cancer and a blood sample derived from a non-cancerous healthy person for a plurality of types of cancer;
(2) a step of selecting a peak having a mass number (m / z) capable of distinguishing each of the specific types of human cancer from non-cancer for the peak indicating the protein expression profile in the step (1);
(3) The mass spectrum of the protein in a single blood sample derived from a human cancer-bearing patient whose type of cancer is unknown is measured, and the peak indicating the obtained protein expression profile is measured with a non-cancerous healthy person. And detecting and identifying a plurality of human cancers using as an index the difference in the relative expression level of the protein corresponding to the peak of the mass number (m / z) selected in the above step (2). provide.

  According to an embodiment of the present invention, the plurality of carcinomas include pancreatic cancer and ovarian cancer.

  Selected peak mass numbers (m / z) that can distinguish pancreatic cancer from non-cancer are 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 1 or 2 or more, preferably all, selected from the group consisting of 13761.49 ± 2%, 14145.18 ± 2% and 17250.82 ± 2%.

  The mass numbers (m / z) of selected peaks that can distinguish ovarian cancer from non-cancer are 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± One or more, preferably all, selected from the group consisting of 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2%.

  The expression level is relative to the protein in the blood sample between the blood sample from a cancer-bearing patient with a particular carcinoma and the blood sample from a healthy non-cancerous person at the transcriptional or translational product level. It means the difference (ie increase or decrease) when comparing expression levels.

  By identifying the above-mentioned protein showing a difference in expression level, carcinomas such as pancreatic cancer and ovarian cancer can be detected in vitro.

  Cancers that can be identified in the present invention include pancreatic cancer, ovarian cancer, gastric cancer, lung cancer, liver cancer, colon cancer, rectal cancer, brain cancer, renal cancer, esophageal cancer, prostate cancer, myeloma, leukemia, lymphoma, and the like. Other cancers, preferably those arising in deep organs, are included without limitation. By the method of the present invention, the presence of two or more, three or more, four or more of these cancers can be detected from a single blood sample of a cancer-bearing patient.

  According to the present invention, the difference in expression level corresponds to each protein on the mass spectrum by measuring the presence or amount of the protein or its corresponding polynucleotide (mRNA, cDNA, cRNA, etc.) as a marker. Can be determined based on the intensity of the peak to be.

Hereinafter, these two different methods will be described in detail.
3.1 Method by Polynucleotide In order to detect the protein marker according to the present invention, the above-mentioned polynucleotide hybridized with the nucleic acid encoding each protein marker is used. The number of markers to be detected is 1 or 2 or more per group, preferably 2 or more, more preferably 3 to the total number, for example, 4 to the total number, 5 or more to the total number. The greater the number of markers to be detected, the better the prediction accuracy.

  Hybridization can be performed by a microarray method, a blotting method such as Northern or Southern blotting, Northern or Southern hybridization method, in situ hybridization method, quantitative RT-PCR method or the like. Preferred hybridization methods are microarray, quantitative RT-PCR or blotting. Examples of preferred microarrays are DNA microarrays and protein microarrays.

  In the DNA microarray method, a DNA microarray in which nucleic acid probes that hybridize with the marker gene group (1 to the total number) are bound to a substrate is prepared and used.

  Any kind of substrate can be used as the DNA microarray as long as the nucleic acid probe can be immobilized. The solid phase includes, for example, glass, a polymer, and a spacer or a crosslinker including a reactive group for covalently binding a nucleic acid can be introduced. Since such chips are commercially available, it is desirable to use them.

  The solid phase immobilization of the nucleic acid probe is not particularly limited, but a general method such as a method of spotting DNA using a high-density dispenser called a spotter or an arrayer, an ink jet method of ejecting droplets from a nozzle, or the like This method can be used.

  A nucleic acid obtained by labeling a nucleic acid such as DNA or RNA in a blood sample, cDNA or cRNA derived therefrom with a fluorescent substance such as Cy dye (Cr3 or Cy5) is hybridized with a probe on a DNA microarray. The fluorescence intensity is read using a reading device by laser scanning, and the data is analyzed by a computer.

In blotting, the nucleic acid probe of the present invention is labeled with a radioisotope (for example, ³² P and ³⁵ S) or a fluorescent substance (rhodamine derivative, fluorescamine derivative, Cy dye, etc.) and then transferred to a polymer membrane such as nylon. Hybridization is performed with nucleic acid such as DNA or RNA in a blood sample, cDNA or cRNA derived therefrom. The signal is detected using a radiation detector or a fluorescence detector and its intensity is measured.

  In the quantitative RT-PCR method, PCR is performed by annealing primers with cDNA so that target gene regions can be amplified using cDNA prepared from RNA in biological specimens as a template. Is detected. Detect and quantify target genes by pre-labeling primers with radioisotopes or fluorescent materials, or by electrophoresis of PCR products on agarose gel and staining double-stranded DNA with ethidium bromide can do.

PCR conditions are, for example, denaturation: 92 to 94 ° C. for 30 to 60 seconds; annealing: 50 to 55 ° C. for 30 to 60 seconds; extension: 68 to 72 ° C. for 30 to 60 seconds, and 30 to 40 cycles of reaction. including. Reverse transcriptase, commercially available enzymes, for example SuperScript (TM) III (Invitrogen, USA), AMV Reverse Transcriptase (Promega, ^{USA), M-MLV (RNaseH -} ) ( Takara Shuzo, Kyoto) can be used such as .

  In the present invention, the hybridization may be any of DNA-DNA hybridization, DNA-RNA hybridization, and RNA-RNA hybridization.

  Stringent conditions for hybridization are as described above. As for hybridization conditions and methods, reference can be made, for example, to Ausubel et al., Current Protocols in Molecular Biology, 1995, John Wiley and Sons, US).

3.2 Method using antibody The above-described method for measuring a protein marker is an immunological method.

  The antibody produced as described above can be used for detection of a target protein or fragment thereof in a biological specimen.

  Detect or quantify a large number of target proteins at a time by creating a protein microarray in which a large number of antibodies are bound on a microarray substrate, or by spotting a large number of antibodies in a dot pattern on a filter such as a PVDF membrane It becomes possible. Or, conventional immunological measurement methods such as enzyme immunoassay (ELISA, EIA), fluorescent antibody method, radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetric method, latex agglutination, latex turbidimetric The target protein or a fragment thereof in a biological specimen can be detected or quantified by a method, a hemagglutination reaction, a particle agglutination reaction, a Western blot method, or the like.

  When the reaction is performed on a solid phase, solid phase carriers include films (filters) of polymers such as polystyrene, polycarbonate, and polyethylene, plates, tubes, strips, and particles such as latex and magnetic materials. The solid phase can be physically or chemically performed. For chemical bonding, for example, the solid phase can be treated with a reagent such as a succinimidation reagent or cyanogen bromide to introduce a functional group that reacts with the amino group of the protein into the solid phase.

For detection of the target, the antibody may be labeled, or a labeled secondary antibody may be used. Examples of labels include enzymes such as horseradish peroxidase and alkaline phosphatase, fluorescent materials such as fluorescein, rhodamine and derivatives thereof, luminescent materials such as luciferase and luminol, and radioactive isotopes such as ³² P, ¹²⁵ I and ³⁵ S Is included. Labeling includes, for example, glutaraldehyde method, maleimide method, pyridyl disulfide method, chloramine T method, Bolton Hunter method and the like.

3.3 Method by Mass Spectrum The present invention further provides a method for detecting carcinomas such as pancreatic cancer and ovarian cancer from mass spectrum data.

  These methods include samples obtained from untreated blood samples from human pancreatic cancer patients, ovarian cancer patients, or subjects suspected of having pancreatic cancer and ovarian cancer, or by anticoagulation pretreatment, such as EDTA-Na treatment. Is applied to a mass spectrometer under the same conditions as the above mass spectrum measurement conditions, and a mass spectrum (for example, m / z: in the range of 2,000 to 20,000) is taken, and a designated mass number (m / Z) based on the intensity of the peak, including determining the expression level of the corresponding protein and determining the difference between the expression level of the sample and the expression level of the control sample. Alternatively, the method of the present invention comprises comparing the peak pattern (or profile) of the analyte mass spectrum with that of a standard sample, a positive sample or a negative sample.

  In the detection of pancreatic cancer, for example, if a peak pattern in the case of pancreatic cancer as shown in FIGS. 1 to 7 is obtained, it indicates that the sample is derived from a patient with pancreatic cancer, while the peak pattern in the case of non-cancer (see FIG. If 8-14) is obtained, it indicates that the sample is a non-cancerous healthy person. Alternatively, based on the difference in expression level, proteins that are highly expressed in blood samples of pancreatic cancer patients (mass number 8562.262 ± 2%, 8684.438 ± 2%, 87655.079 ± 2% in FIGS. 1 to 7, 9423. 513 ± 2%, 13761.49 ± 2%, 14145.18 ± 2% and 17250.82 ± 2% peaks), or proteins highly expressed in non-cancer healthy human blood samples (mass 8562 in FIGS. 8-14) .262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2%, 14145.18 ± 2%, and 17250.82 ± 2%) Can be determined.

  In the discrimination between ovarian cancer patient blood samples and non-cancerous healthy human blood samples, as described above, based on the difference in peak pattern or expression level (mass number 4470.801 ± of FIGS. 15 to 21 and FIGS. 22 to 28). Determined by peaks at 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2% .) Can be discriminated.

  A systematic and exhaustive cluster analysis of the relative expression levels of each protein determined as described above for a large number of cases reveals that pancreatic cancer patients and non-cancerous healthy persons, or ovarian cancer patients and non-cancerous healthy persons. It can be seen that and are clearly distinguished.

  The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited by these examples.

(Materials and experiments)
1. Survey target Aichi Prefecture has collected a series of blood samples, including 136 cases of pancreatic cancer and 62 cases of ovarian cancer, which were examined at Aichi Cancer Center Hospital (Nagoya) between January 2001 and October 2005. Selected from the file of the Center for Epidemiology and Prevention at Nagoya Research Institute (Nagoya). Pancreatic and ovarian cancer blood samples were randomly assigned to either the teacher or the validation data set for teacher-validation model analysis. Furthermore, pancreatic cancer patient blood samples obtained from April 2003 to December 2007 at Nagoya University Hospital and Joint Research Hospital (Nagoya) were donated as blood samples for verification. All of these were obtained after obtaining the necessary approval from the Ethics Review Board and stored at −80 ° C. until measurement. Informed consent has been obtained from both cancer-bearing patients and healthy non-cancerous individuals.

2. Proteomic analysis Blood samples are divided into 5 microliters and diluted 10-fold with 50% acetonitrile / 0.1% trifluoroacetic acid solution. Separate 5 microliters of 10-fold diluted blood sample and mix with 20 microliters of sinapinic acid at a concentration of 30 mg / ml dissolved in 50% acetonitrile / 0.1% trifluoroacetic acid solution. A matrix mixed blood sample (0.5 microliters) is dropped into 6 wells on the sample stage and dried. A protein expression profile is obtained from the dropped blood sample on the sample stage using mass spectrometry (Applied Biosystems, 4800). The analysis conditions of the instrument at this time are acceleration voltage; 25,000V, focus mass; 10,000, analysis range; 2,000 to 20,000 daltons. Peak detection and assignment from each spectrum is performed using MarkerView software (Applied Biosystems). As a result, in pancreatic cancer patient discrimination biomarker search, seven unique signals are generated in a supervised data group, and ovarian cancer patients In the search for discriminating biomarkers, seven unique signals were generated. The spectra were normalized to each other by the maximum sum of the total number of ions. The maximum total number was divided by the sum of each spectrum to obtain a normalization rate of each spectrum, and each normalization rate was used to multiply each data point for each spectrum. The accuracy and reproducibility of MALDI MS analysis using human material has been demonstrated by previous studies (eg, J Natl Cancer Inst 2003, 95: 1711-7; Cancer Res 2003, 63: 1652-6).

3. Statistical analysis Protein profiles obtained by MALDI MS are analyzed using the SAM method (significant analysis of microarray method; calculating false discovery rate (FDR) considering multiple tests), providing information on cancer or non-cancer. The signal obtained was selected. The cut-off was FDR <1%. Signals that met this selection criteria were used for the following analyses. The inventors used a weighted voting algorithm, which is a well-established linear discriminating technique as supervised classification. In the weighted voting algorithm, the weight value for each signal is calculated as a signal-to-noise (S / N) ratio. In order to strongly influence the prediction accuracy, as the number of signals has been found in various microarray studies, we have performed a 10-fold cross-validation procedure with 10,000 random iterations (10− (fold cross-validation) was performed to optimize the number of signals used in the prediction model.

(result)
(1) Proteomics MS signals from blood samples from human pancreatic cancer patients and blood samples from healthy non-cancerous humans The present inventors are in 160 blood samples from 80 pancreatic cancer patients and 80 non-cancerous healthy individuals belonging to the teacher group. Protein expression profiles were analyzed using MALDI MS and 7 different MS signals were obtained. Representative examples of spectra from human pancreatic cancer patient blood samples are shown in FIGS. Furthermore, the present inventors searched for a proteomic profile capable of accurately discriminating a pancreatic cancer patient blood sample from a non-cancer healthy human blood sample, and as a result, the pancreatic cancer patient blood sample (FIGS. 1-7) and non- Seven MS signals (mass number (m / z) 8562.262, 8684.4438, 87655.079, 9423.513, which significantly recognize the difference in expression between blood samples of healthy humans (FIGS. 8 to 14), 13761.49, 14145.18 and 17250.82) were selected (FDR <0.01).

(2) Proteomic MS signals from blood samples of human ovarian cancer patients and blood samples of non-cancerous healthy individuals We express protein expression in 127 samples of blood samples from 63 ovarian cancer patients and 64 non-cancerous healthy people Profiles were analyzed using MALDI MS and 7 different MS signals were obtained. Representative examples of spectra from human ovarian cancer patient blood samples are shown in FIGS. The present inventors further searched for a proteomic profile capable of accurately discriminating ovarian cancer patient blood samples from non-cancer healthy human blood samples, and as a result, ovarian cancer patient blood samples (FIGS. 15 to 21) and Seven MS signals (mass number (m / z) 4470.801, 6940.418, 8764.14, 9140.222) that show significant difference in expression between non-cancerous healthy human blood samples (FIGS. 22-28) , 94222.677, 13879.6 and 13935.1) were selected (FDR <0.01).

(3) Confirmation of availability of pancreatic cancer or ovarian cancer diagnostic methods by using independent blind datasets To evaluate the availability of proteomics analysis using MALDI MS, Effectiveness is extremely important. Therefore, we apply a proteomic profile constructed based on a teacher data set to independent MS signal data sets obtained from 32 pancreatic cancer patient blood samples and 32 non-cancerous healthy blood samples. did. We applied a proteomic profile that distinguishes pancreatic cancer or ovarian cancer blood samples from non-cancerous healthy human blood samples to a validation data set, confirming clear distinction in a systematic and exhaustive cluster analysis. The cluster analysis pattern reflects a strong correlation between the proteomic profile and cancer-bearing / non-cancer cases, with a sensitivity of 75.0% (60/80) and 93.75% (75/80) in the pancreatic cancer discrimination model. (FIG. 29), and the ovarian cancer discrimination model shows 74.2% (46/62) sensitivity and 80.2% (101/126) specificity. (FIG. 30). Here, sensitivity means a positive determination rate for cancer patients, and specificity means a positive determination rate for healthy individuals.

  Furthermore, in order to evaluate proteomics analysis using MALDI MS, measurement was performed using an independent verification blood sample (37 cases of pancreatic cancer, 11 cases of healthy individuals) acquired at another facility (Nagoya University Hospital). went. As a result, it was confirmed that a sensitivity of 81.1% and a specificity of 100% were obtained as described above. Furthermore, 3 cases (15.8%) of cases in which the CA19-9 value, which is widely used as an existing pancreatic cancer tumor marker, was normal among patients diagnosed with pancreatic cancer by this measurement method were observed.

  The above results indicate that the above-described test method of the present invention not only shows the accuracy and specificity of presence diagnosis using human pancreatic cancer blood samples based on proteomic profiles, but also is effective for early cancer detection. And that it can be used complementarily to the CA19-9 test, which is an existing screening method.

(Discussion)
The present inventors performed proteomic profile analysis using MALDI using a very small amount (several microliters) of blood sample, and the protein expression profile is highly accurate for early diagnosis of pancreatic cancer and ovarian cancer-bearing patients. Proved that it contains enough information to make possible. The classification factors found by the present inventors are, firstly, seven different peaks selected from a teacher group that is useful for distinguishing between a blood sample of a pancreatic cancer patient and a blood sample of a non-cancerous healthy person, Since these were applicable to independent validation data sets, the reproducibility of the direct protein expression profile of blood samples by MALDI MS was supported. Furthermore, the results of this analysis were also confirmed in studies using completely independent sample sets obtained at other institutions, and in cases where the existing tumor marker CA19-9 was negative. , Confirmed to be useful.

  Secondly, there are seven different peaks selected from a group of teachers that are useful in distinguishing between blood samples of ovarian cancer patients and non-cancerous healthy individuals, but these also apply to independent validation data sets. The ability to support the reproducibility of the direct protein expression profile of blood samples by MALDI MS.

  In conclusion, the present inventors applied MALDI MS directly to untreated blood samples and obtained pancreatic cancer as well as patient blood samples and non-cancerous healthy human blood samples in both the teacher group and the independent verification group. We found a cancer-related protein expression profile that can be distinguished. This analysis method is believed to be used in the future for clinical management of pancreatic and ovarian cancers that are difficult to diagnose.

  According to the present invention, human pancreatic cancer can be detected with high accuracy. In addition, human ovarian cancer patients can be detected with high accuracy. For this reason, we think that it can be effectively utilized as an early diagnosis / treatment introduction or follow-up indicator after treatment for patients with pancreatic cancer and ovarian cancer. Thus, the present invention can make a great contribution in cancer medicine.

The peak of mass number (m / z) 8562.262 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. The peak of the mass number (m / z) 8684.4438 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. The peak of mass number (m / z) 87655.079 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. The peak of mass number (m / z) 9423.513 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. The peak of mass number (m / z) 13761.49 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. The peak of the mass number (m / z) 14145.18 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. The peak of mass number (m / z) 17250.82 in the mass spectrum from a human pancreatic cancer patient blood sample is shown. 2 shows a peak of mass number (m / z) 8562.262 corresponding to the peak of FIG. 1 in a mass spectrum from a human non-cancer patient blood sample. The peak of mass number (m / z) 8684.438 corresponding to the peak of FIG. 2 in the mass spectrum from a human non-cancer patient blood sample is shown. The peak of mass number (m / z) 87655.079 corresponding to the peak of FIG. 3 in the mass spectrum from a human non-cancer patient blood sample is shown. The mass spectrum (m / z) 9423.513 peak corresponding to the peak of FIG. 4 in the mass spectrum from a human non-cancer patient blood sample is shown. FIG. 6 shows a peak of mass number (m / z) 13761.49 corresponding to the peak of FIG. 5 in a mass spectrum from a human non-cancer patient blood sample. The peak of mass number (m / z) 14145.18 corresponding to the peak of FIG. 6 in the mass spectrum from a human non-cancer patient blood sample is shown. The mass spectrum (m / z) 17250.82 peak corresponding to the peak of FIG. 7 in the mass spectrum from a human non-cancer patient blood sample is shown. The peak of mass number (m / z) 4470.801 in the mass spectrum from a human ovarian cancer patient blood sample is shown. The peak of mass number (m / z) 6940.418 in the mass spectrum from a human ovarian cancer patient blood sample is shown. The peak of mass number (m / z) 8764.14 in the mass spectrum from a human ovarian cancer patient blood sample is shown. The peak of mass number (m / z) 9140.222 in the mass spectrum from a human ovarian cancer patient blood sample is shown. The peak of mass number (m / z) 94222.677 in the mass spectrum from a human ovarian cancer patient blood sample is shown. The peak of mass number (m / z) 138799.6 in the mass spectrum from a human ovarian cancer patient blood sample is shown. The peak of mass number (m / z) 13935.1 in the mass spectrum from a human ovarian cancer patient blood sample is shown. In the mass spectrum from a human non-cancer patient blood sample, the peak of mass number (m / z) 4470.801 corresponding to the peak of FIG. 15 is shown. FIG. 17 shows a peak of mass number (m / z) 6940.418 corresponding to the peak of FIG. 16 in a mass spectrum from a human non-cancer patient blood sample. FIG. 18 shows a peak of mass number (m / z) 8764.14 corresponding to the peak of FIG. 17 in a mass spectrum from a human non-cancer patient blood sample. In the mass spectrum from a human non-cancer patient blood sample, the peak of mass number (m / z) 9140.222 corresponding to the peak of FIG. 18 is shown. The mass spectrum (m / z) 94222.677 corresponding to the peak of FIG. 19 in the mass spectrum from a human non-cancer patient blood sample is shown. FIG. 21 shows a peak of mass number (m / z) 13879.6 corresponding to the peak of FIG. 20 in a mass spectrum from a human non-cancer patient blood sample. The mass spectrum (m / z) 13935.1 peak corresponding to the peak of FIG. 21 in the mass spectrum from a human non-cancer patient blood sample is shown. Mass spectrum mass number (m / z) found from human pancreatic cancer patients 7 consisting of 8562.262, 8684.438, 8765.079, 943.513, 13761.49, 14145.18 and 17250.82 The ROC curve (FIG. 29A) of the pancreatic cancer discrimination model created from the specific peak is shown. Using this discriminant model and determining whether (i) is cut off and (ii) is cut off, the result of determining whether the patient is a pancreatic cancer patient or a non-cancer patient. Sometimes higher sensitivity (sensitivity) and specificity (specificity). Moreover, the result (FIG. 29B) which determined the pancreatic cancer patient and the non-cancer healthy person using this discrimination model is shown, respectively. Mass spectrum mass numbers (m / z) found from human ovarian cancer patients 7 consisting of 4470.801, 6940.418, 8764.14, 9140.222, 9422.677, 13879.6 and 13935.1 The ROC curve (FIG. 30A) of the ovarian cancer discrimination model created from the specific peak is shown. In the figure, an arrow indicates a cutoff as a criterion. Moreover, the result (FIG. 30B) which determined the ovarian cancer patient and the non-cancer healthy person using this discrimination model is shown, respectively.

Claims (14)

A method for detecting and identifying a plurality of carcinomas using a single blood sample in a human cancer-bearing patient having a plurality of carcinomas, comprising the following (1) to (3):
(1) A protein expression profile is obtained by measuring mass spectra of proteins in a blood sample derived from a human cancer-bearing patient having a specific cancer and a blood sample derived from a non-cancerous healthy person for a plurality of specific types of cancer. Step,
(2) selecting a peak having a mass number (m / z) capable of distinguishing each of the specific types of human cancer from non-cancer for the peak indicating the protein expression profile in the step (1);
(3) The mass spectrum of the protein in a single blood sample derived from a human cancer-bearing patient whose type of cancer is unknown is measured, and the peak indicating the obtained protein expression profile is measured with a non-cancerous healthy person. The step of detecting and identifying a plurality of human cancers using as an index the difference in the relative expression level of the protein corresponding to the peak of the mass number (m / z) selected in the step (2). Method.   The method of claim 1, wherein the plurality of carcinomas include pancreatic cancer and ovarian cancer.   Selected peak mass numbers (m / z) distinguishing pancreatic cancer from non-cancer are 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2% 3, 13761.49 ± 2%, 14145.18 ± 2%, and 17250.82 ± 2%.   Selected peak mass numbers (m / z) that can distinguish the ovarian cancer from non-cancer are 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2 3. The method of claim 2, wherein the method is one or more selected from the group consisting of:%, 94222.677 ± 2%, 13879.6 ± 2%, and 13935.1 ± 2%.   5. The method of any one of claims 1-4, further comprising identifying a protein having the selected peak mass number.   6. The method of claim 5, further comprising determining a gene encoding it based on the identified protein.   Using an antibody against a protein selected from the group consisting of proteins having the mass number (m / z) or a variant thereof, or a polynucleotide encoding the protein or the variant or a polynucleotide complementary thereto, The method according to claim 1, wherein the difference in the expression level is measured.   A method of detecting a carcinoma using a single blood sample in a human cancer-bearing patient having pancreatic cancer, ovarian cancer or both, wherein the blood sample is derived from a first cancer-bearing patient having pancreatic cancer or ovarian cancer And measuring a mass spectrum of a blood sample derived from a second healthy non-cancerous person to obtain a protein expression profile, and the blood sample from the first cancer-bearing patient with respect to the peak indicating the protein expression profile and the second Selecting a peak of a mass number (m / z) that can be distinguished from a blood sample from a group of healthy non-cancerous individuals, and between the first cancer-bearing patient and a second non-cancerous healthy person Detecting cancer in the blood sample of the human cancer-bearing patient, using as an index the difference in the relative expression level of the protein corresponding to the selected peak of mass number (m / z), wherein Selection Each of the proteins corresponding to the generated peaks for pancreatic cancer is 8562.262 ± 2%, 8684.438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2%, One or more mass numbers (m / z) selected from the group consisting of 14145.18 ± 2% and 17250.82 ± 2%, and for ovarian cancer, 4470.801 ± 2%, 6940.418 One or more selected from the group consisting of ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, 13879.6 ± 2% and 13935.1 ± 2% Said method, characterized in that each has a mass number (m / z).   The mass number (m / z) of the peak in the mass spectrum measurement is 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2%, To perform blood diagnosis of human pancreatic cancer patients comprising at least one antibody or fragment thereof to each of a protein or a variant thereof selected from the group consisting of 14145.18 ± 2% and 17250.82 ± 2% Composition.   The mass number (m / z) of the peak in the mass spectrum measurement is 8562.262 ± 2%, 8684.4438 ± 2%, 87655.079 ± 2%, 9423.513 ± 2%, 13761.49 ± 2%, A polynucleotide encoding a protein that is selected from the group consisting of 14145.18 ± 2% and 17250.82 ± 2%, polynucleotides complementary thereto, variants thereof, and under stringent conditions thereto A composition for blood diagnosis of a human pancreatic cancer patient, comprising at least one polynucleotide selected from the group consisting of a hybridizing polynucleotide or a fragment thereof containing 15 or more consecutive bases.   11. A composition according to claim 9 or 10, wherein the composition is in the form of a kit or a microarray.   The mass number (m / z) of the peak in the mass spectrum measurement is 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, Hematologic diagnosis of human ovarian cancer patients comprising at least one antibody or fragment thereof to each of a protein or variant thereof selected from the group consisting of 13879.6 ± 2% and 13935.1 ± 2% Composition for.   The mass number (m / z) of the peak in the mass spectrum measurement is 4470.801 ± 2%, 6940.418 ± 2%, 8764.14 ± 2%, 9140.222 ± 2%, 94222.677 ± 2%, Polynucleotides encoding proteins that are selected from the group consisting of 13879.6 ± 2% and 13935.1 ± 2%, polynucleotides complementary thereto, variants thereof, under stringent conditions thereto A composition for blood diagnosis of human ovarian cancer patients, comprising at least one polynucleotide selected from the group consisting of a hybridizing polynucleotide or a fragment thereof comprising 15 or more consecutive bases.   14. A composition according to claim 12 or 13, wherein the composition is in the form of a kit or a microarray.

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