JP2016198027A - Method for diagnosing ovarian cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for diagnosing ovarian clear cell adenocarcinoma by a non-invasive method using a free DNA that exists in blood or the like.SOLUTION: A method for diagnosing ovarian clear cell adenocarcinoma includes detecting, in a chromosome DNA acquired from a subject, amplification of at least a part of a region including a specific gene of each on 8th chromosome, on 12th chromosome and on 20th chromosome, deletion of at least a part of a region including a specific gene on 3rd chromosome, deletion of at least a part of a region including a specific gene on 4th chromosome, and/or deletion of at least a part of a specific gene on 22nd chromosome and a region including the gene. A method for diagnosing ovarian cancer includes cutting off a DNA derived from a normal chromosome out of chromosome DNA acquired from a subject by a Cas9-sgRNA composite body, increasing an abundance ratio of a DNA derived from chromosome having mutation related to an onset of ovarian cancer, and detecting the presence of the mutation.SELECTED DRAWING: None

Description

  The present invention relates to a method for diagnosing ovarian cancer, particularly ovarian clear cell adenocarcinoma. More specifically, the presence or absence of ovarian cancer (particularly ovarian clear cell adenocarcinoma) by detecting amplification or deletion in a specific region of a patient-derived chromosome and somatic mutation of a specific gene, Alternatively, a method for diagnosing ovarian cancer (particularly ovarian clear cell adenocarcinoma), including prognostic assessment, is provided.

Epithelial ovarian cancer is the ninth most common cancer in the United States and the 14th most common in Japan, with the number of patients increasing gradually. Epithelial ovarian cancer is mainly composed of four histological subgroups. Serous adenocarcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma and clear cell adenocarcinoma. Among them, clear cell adenocarcinoma is the second most common in North America and Europe, and its prevalence is estimated to be 1-12%, and it is 15-25% in Japan. Furthermore, in Japan, the increase rate of clear cell adenocarcinoma was the highest among the four subgroups in the age group over 50 years in the past 20 years. Patients with clear cell adenocarcinoma are diagnosed at an early stage (I or II) and their tumors are painless, whereas patients with advanced stage (III or IV) usually have a poor prognosis (non-patented) Document 1 and Non-Patent Document 2) are one of the cancers that are highly needed for early detection.
Moreover, according to a recent report, it is clear that endometriosis increases the risk of clear cell adenocarcinoma and endometrial adenocarcinoma (Non-patent Document 3).

In addition to epidemiological differences in histological types of epithelial ovarian cancer, differences in molecular characteristics have also been reported. Mutation of the ARID1A tumor suppressor gene and loss of function of BAF3250a, a protein encoded by ARID1A, have been confirmed in 46% of clear cell adenocarcinoma and 30% of endometrial cancer and endometrial cancer. There is no such report for serous adenocarcinoma. In view of this result, it is suggested that some somatic mutations trigger the transition from endometriosis to clear cell adenocarcinoma or endometrial adenocarcinoma. Using chromosome CGH (Comparative Genomic Hybridization) method, we found that amplification or deletion of a specific region of chromosome is related to prognosis of endometrial cancer. It has also been reported that it is possible to make a prognosis diagnosis (Patent Document 1, Non-Patent Document 4).
In addition, TP53 mutation is relatively low in clear cell adenocarcinoma by genetic analysis or genomic analysis (Non-patent Document 5), but TP53 mutation occurs in almost all tumors in highly advanced serous adenocarcinoma In addition, it has been reported that mutations of NF1, BRCA1, BRCA2, RB1, and CDK12 have occurred as non-patent but statistically significant (Non-patent Document 6).

  Currently, ovarian cancer is diagnosed based on blood tests and diagnostic imaging when ovarian enlargement is confirmed by ultrasonic diagnosis. In blood tests, tumor markers are also confirmed, and CA125 is used as a representative tumor marker. CA125 is positive in about 80% of people with ovarian cancer, but it is also expressed in the peritoneum and endometrium and is also positive in cases of endometriosis. It is a marker that cannot be said to be specific for cancer. In the case of ovarian cancer, CA125 rise is poor in early cancer and clear cell adenocarcinoma, so a more specific tumor for early detection of ovarian cancer or diagnosis of ovarian clear cell adenocarcinoma. A marker is needed.

JP2000-166557

Takano et al., Br J Cancer 94: 1369? 1374 2006 Mizuno et al., J Surg Oncol 94: 138? 143 2006 Pearce et al., Lancet Oncol. 13: 385-394 2012 Okamoto et al., PLoS One. 6; 10 (2): e0116977.doi: 10.1371 / journal.pone.0116977.eCollection 2015. Kuo et al., Am J Pathol. 174: 1597-1601 2009 Bell et al., Nature 474: 609-615 2011

In view of the above circumstances, the present invention is based on the structural change of the chromosome and aims at application of a method for diagnosing the presence or possibility of onset of ovarian clear cell adenocarcinoma, malignancy or prognosis of ovarian clear cell adenocarcinoma .
Furthermore, the present invention also provides a method for diagnosing ovarian cancer by a non-invasive method (a method not using tumor tissue) using free DNA present in blood or the like.

  In order to solve the above-mentioned problems, the present inventors have analyzed the chromosomes (or chromosomal DNA) derived from patients with clear cell adenocarcinoma of the ovary using an array CGH (Comparative Genomic Hybridization) method. It has been revealed for the first time that the amplification or deletion in the chromosomal DNA region has a relationship with the onset, malignancy, prognosis, etc. of ovarian clear cell adenocarcinoma. Furthermore, we have succeeded in confirming the presence or absence of these amplified or deleted regions using blood-derived DNA samples, and diagnosed the presence or absence of ovarian cancer by a non-invasive method with less burden on the living body. Revealed that it is possible.

The inventors collected DNA from tumor tissue from patients with ovarian clear cell adenocarcinoma and used it to significantly amplify or delete in ovarian clear cell adenocarcinoma cells by high resolution array CGH methods. I examined the region on the chromosome.
As a result, in ovarian clear cell adenocarcinoma cells, a region containing ADAM5 gene / ADAM3A gene on chromosome 8 (referred to as “region 1”), a region containing ANK1 gene / MYST3 gene on chromosome 8 (“region 2”) ), A region containing PLAT gene / IKBKB gene / POLB gene / DKK4 gene / VDAC gene / SLC20A2 gene / SMIM19 gene on chromosome 8 (referred to as “region 3”), 20p13.3 on chromosome 12 Amplification of a region containing -13.2 (referred to as “region 4”) and a region containing the ZNF217 gene on chromosome 20 (referred to as “region 5”) were frequently observed.
In addition, amplification in the region containing PLAT gene / IKBKB gene / POLB gene / DKK4 gene / VDAC gene / SLC20A2 gene / SMIM19 gene on chromosome 8 shows progression-free survival in Japanese patients with clear cell adenocarcinoma of the ovary. It was related to shortness. Further, among the above regions 1 to 5, in the case where the amplification region is amplified in two or less regions and the case where the amplification region is amplified over three or more regions, the progression-free survival in the case where the amplification region is amplified over three or more regions It was found that the period (PFS; Progression Free Survival) and the overall survival (OS) were significantly shorter.

In ovarian clear cell adenocarcinoma cells, a region containing BC073807 gene (non-coding region) on chromosome 3 (referred to as “region 6”) and a region containing UGT2B17 gene on chromosome 4 (“region 7”) ), And deletion of the region containing GSTT1 gene / LOC391322 gene on chromosome 22 (referred to as “region 8”) was frequently observed.
Furthermore, a deletion in the region containing BC073807 gene (non-coding RNA) on chromosome 3 was associated with a short progression-free survival in Japanese patients with clear cell adenocarcinoma of the ovary. It was also found that progression-free survival was significantly shorter in endometriosis complications with deletions in the region containing the BC073807 gene (noncoding RNA) on chromosome 3. Furthermore, in a case of a Japanese patient having a deletion in any one of the above regions 6 to 8, the progression-free survival tends to be short, and in any of the above regions 6 or 7, Cases of Japanese patients with deletions tended to have short progression-free survival.

  These results show that the abnormalities in the specific chromosomal region described above serve as an index for diagnosing the presence or possibility of the onset of ovarian clear cell adenocarcinoma, It shows that it is an index for predicting prognosis.

  In addition, the inventors used the CRISPR / Cas system (a system using the sgRNA-Cas9 complex), and the mutation (P p.H1047R) mutation frequently observed in the PIK3CA gene of ovarian clear cell carcinoma patients IK3CA's 1047th histidine was changed to arginine) and was successfully detected from blood samples of ovarian clear cell adenocarcinoma patients and DNA samples derived from cervical and endometrial cytology.

That is, this invention is the following (1)-(11).
(1) Amplification of at least part of the region containing ADAM5 gene and ADAM3A gene on chromosome 8 and amplification of at least part of the region containing ANK1 gene and MYST3 gene on chromosome 8 in chromosomal DNA obtained from the subject , Amplification of at least a part of the region containing PLAT gene on chromosome 8, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene, region containing 20p13.3-13.2 on chromosome 12 At least partial amplification, at least partial amplification of the region containing the ZNF217 gene on chromosome 20, at least partial deletion of the region containing BC073807 gene on chromosome 3, including UGT2B17 gene on chromosome 4 A method for diagnosing ovarian clear cell adenocarcinoma, comprising detecting a deletion of at least a part of a region and / or a deletion of at least a part of a region containing GSTT1 gene and LOC391322 gene on chromosome 22.
(2) In the chromosomal DNA obtained from the subject, detecting the amplification of at least a part of the region containing the PLAT gene, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene on chromosome 8 A diagnostic method for malignancy or prognosis of ovarian clear cell adenocarcinoma.
(3) In chromosomal DNA obtained from a subject, a region containing ADAM5 gene and ADAM3A gene on chromosome 8, a region containing ANK1 gene and MYST3 gene on chromosome 8, PLAT gene on chromosome 8, IKBKB gene, Arbitrarily selected from the group consisting of a region containing POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene, a region containing 20p13.3-13.2 on chromosome 12, and a region containing ZNF217 gene on chromosome 20. A method for diagnosing malignancy or prognosis of ovarian clear cell adenocarcinoma, comprising detecting amplification of at least a part of each of the three regions.
(4) Chromosomal DNA obtained from the subject consists of a region containing BC073807 gene on chromosome 3, a region containing UGT2B17 gene on chromosome 4, and a region containing GSTT1 gene and LOC391322 gene on chromosome 22. A method for diagnosing malignancy or prognosis of ovarian clear cell adenocarcinoma, comprising detecting deletion of at least a part of the one region in one region arbitrarily selected from a group.
(5) The method according to any one of (1) to (4), wherein the detection of the amplification or deletion is performed by detecting a change in the number of DNA copies including the corresponding region.
(6) The method according to (5) above, wherein the change in the DNA copy number is detected by PCR method, FISH method, CGH method, ISH method, quantitative PCR method, digital PCR method or sequencing method. .
(7) The chromosomal DNA obtained from the subject is extracted from the subject's tumor tissue, cervical / intimal cytology sample, cerebrospinal fluid, blood or urine (1) to ( The method according to any one of 6).
(8) cleaving normal chromosomal DNA out of chromosomal DNA obtained from a subject with a Cas9-sgRNA complex, increasing the abundance of chromosomal DNA having a mutation associated with the onset of ovarian cancer, A method for diagnosing ovarian cancer, comprising detecting the presence or absence of a mutation.
(9) The method according to (8) above, wherein the ovarian cancer is ovarian clear cell adenocarcinoma.
(10) The method according to (8) or (9) above, wherein the mutation is a mutation in which the 1047th histidine at the PIK3CA locus is substituted with arginine.
(11) The above (8) to (8), wherein the chromosomal DNA obtained from the subject is collected from a tumor tissue, a cervical / intimal cytology sample, cerebrospinal fluid, blood or urine of the subject. The method according to any one of (10).

  The present invention enables early detection, malignancy or prognosis of ovarian cancer, particularly ovarian clear cell adenocarcinoma. Since it becomes possible to predict the malignancy or prognosis of ovarian cancer such as ovarian clear cell adenocarcinoma, the choice of pre- or post-operative therapy is expanded, and more appropriate treatment can be performed.

  In addition, according to the present invention, mutations significantly observed in ovarian cancer such as clear cell adenocarcinoma can be detected noninvasively and rapidly using blood samples and the like, thereby enabling early detection of ovarian cancer. Become.

The frequency of increase of SCNAs in chromosome 8 is shown. A peak appears on the right side of the central axis of the frequency (%) of increase in the number of chromosome copies. Peaks were confirmed in the ADAM5 / ADAM3A gene region (region 1), ANK1 / MYST3 gene region (region 2) and PLAT / IKBKB / POLB / DKK4 / VDAC / SLC20A2 / SMIM19 gene region (region 3). The frequency of increase of SCNAs in chromosome 12 is shown. A peak was confirmed in the 20p13.3-13.2 region (region 4). The frequency of increase of SCNAs in chromosome 20 is shown. A peak was confirmed in the ZNF217 gene region (region 5). The frequency of deletion of SCNAs in chromosome 3 is shown. A peak was confirmed in the region containing the BC073807 gene (region 6). The frequency of SCNAs deletion in chromosome 4 is shown. A peak was confirmed in the region (region 7) containing the UGT2B17 gene. The frequency of SCNAs deletion in chromosome 22 is shown. A peak was confirmed in the region (region 8) containing the GSTT1 gene and the LOC391322 gene. The relationship between region 3 amplification and progression-free survival is shown. The relationship between the amplification of region 1 to region 5 and progression-free survival is shown. It is the result of having compared the progression-free survival time of the patient who has abnormality in the 3-5 area | region and the patient who has abnormality in the 0-2 area | region in the area | regions 1-5. The relationship between the amplification of region 1 to region 5 and the overall survival time is shown. It is the result of having compared the total survival time of the patient who has abnormality in the 3-5 area | region, and the patient who has abnormality in the 0-2 area | region in the area | regions 1-5. The relationship between the deletion of region 6 and progression-free survival is shown. In endometriosis complication cases, the relationship between region 6 deletion and progression-free survival is shown. In a Japanese endometriosis complication example, the relationship between deletion of region 6 and progression-free survival is shown. The relationship between the deletion of region 6 to region 8 and progression-free survival is shown. Comparison of progression-free survival between patient groups (6-8) with deletions in any one symptom area of regions 6 to 8 and patient groups (none) with no deletions in any region It is the result of having performed. The result of having confirmed the presence or absence of SNP (chr8 41747333) in an ANK1 gene area | region using the DNA prepared from the buffy coat of a Japanese healthy person is shown. The upper figure shows a positive value when the position of the SNP (chr8 41747333) is A, and the lower figure shows a positive value when the position of the SNP is T. The vertical axis shows the measured value of fluorescence derived from FAM (upper figure) or HEX (lower figure). N1, N6, N7, N8, N9, and N10 indicate sample numbers derived from normal subjects. It is the result of having confirmed the presence or absence of amplification of ANK1 gene using the DNA prepared from the buffy coat of the test subject who has SNP in the position of 41747333 of the chromosome 8 among Japanese healthy persons. The vertical axis shows the ratio of the copy number of the allele whose position is 41747333 on chromosome 8 and whose allele is T. It is the result of having confirmed the presence or absence of amplification of ANK1 gene using cfDNA (2.5 ng) of a test subject who has SNP (the position of 41747333 of chromosome 8 is A / T) among Japanese healthy individuals. The vertical axis shows the ratio of the copy number of the allele whose position is 41747333 on chromosome 8 and whose allele is T. The result of having confirmed the presence or absence of SNP (chr8 41747333) in an ANK1 gene area | region using the DNA prepared from the buffy coat of the ovarian clear cell adenocarcinoma patient is shown. The upper figure shows a positive value when the position of the SNP (chr8 41747333) is A, and the lower figure shows a positive value when the position of the SNP is T. The vertical axis shows the measured value of fluorescence derived from FAM (upper figure) or HEX (lower figure). It is the result of having confirmed the presence or absence of amplification of ANK1 gene using the DNA prepared from the buffy coat of the patient who has SNP in the position of 41747333 of chromosome 8 among the ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 41747333 on chromosome 8 and whose allele is T. It is the result of having confirmed the presence or absence of amplification of ANK1 gene using the DNA prepared from the cancer part of the patient who has SNP in the position of 41747333 of chromosome 8 among the ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 41747333 on chromosome 8 and whose allele is T. It is the result of having confirmed the presence or absence of amplification of ANK1 gene using cfDNA (2.5 ng) of a patient who has SNP in the position of 41747333 of the chromosome 8 among ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 41747333 on chromosome 8 and whose allele is T. It is the result of having confirmed the presence or absence of amplification of ANK1 gene using the DNA prepared from the cancer part of the patient who has SNP in the position of 4165823 of chromosome 8 among the ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele where the position of 41589823 on chromosome 8 is G and the allele where T is T. It is the result of having confirmed the presence or absence of amplification of ANK1 gene using cfDNA (2.5 ng) of a patient with SNP in the position of 4165823 of chromosome 8 among patients with ovarian clear cell adenocarcinoma. The vertical axis shows the ratio of the copy number of the allele where the position of 41589823 on chromosome 8 is G and the allele where T is T. It is the result of having confirmed the presence or absence of amplification of MYST3 gene using the DNA prepared from the cancer site of the patient who has SNP in the position of 41850222 of chromosome 8 among the ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 41850222 on chromosome 8 and whose position is C. It is the result of having confirmed the presence or absence of amplification of MYST3 gene using cfDNA (2.5 ng) of a patient who has SNP in the position of 41850222 of the chromosome 8 among the ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 41850222 on chromosome 8 and whose position is C. It is the result of having confirmed the presence or absence of amplification of IKBKB gene using the DNA prepared from the cancer part of the patient who has SNP in the position of 42180716 of chromosome 8 among the ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 4280716 on chromosome 8 and whose allele is T. It is the result of having confirmed the presence or absence of amplification of IKBKB gene using cfDNA (2.5 ng) of a patient who has SNP in the position of 42807716 of chromosome 8 among ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele whose position is 4280716 on chromosome 8 and whose allele is T. It is the result of having confirmed the presence or absence of amplification of ZNF217 gene using the DNA prepared from the cancer part of the patient who has SNP in the position of 52208838 of the chromosome 20 among ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele where the position of 52208838 of chromosome 20 is C and the allele which is G. It is the result of having confirmed the presence or absence of amplification of ZNF217 gene using cfDNA (2.5 ng) of a patient who has SNP in the position of 52208838 of chromosome 20 among ovarian clear cell adenocarcinoma patients. The vertical axis shows the ratio of the copy number of the allele where the position of 52208838 of chromosome 20 is C and the allele which is G. The target sequence used for sgRNA binding to the nearby sequence of the mutation (H1047R) on the PIK3CA gene found in ovarian clear cell adenocarcinoma is shown. As a result of the examination, when the sgRNA containing the Fw2 sequence was used, the wild type sequence was cleaved most efficiently.

  The first embodiment of the present invention is obtained from a subject (a subject to whom the method of the present invention is applied, such as a patient with ovarian clear cell adenocarcinoma or a person who may have ovarian clear cell adenocarcinoma). In the obtained chromosomal DNA, a region containing ADAM5 gene on chromosome 8 (start position: 39237438 to end position: 39299582 (position according to BED format, the same applies hereinafter) and ADAM3A gene (start position: 39299582 to end position: 39386158) Amplification of at least a part of region 1), region (region 2) containing ANK1 gene (start position: 41640598 to end position: 41777845) and MYST3 gene (start position: 41777845 to end position: 41889042) on chromosome 8 At least partial amplification, PLAT gene on chromosome 8 (start position: 42033118 ~ end position: 42101440), IKBKB gene (start position: 42155030 ~ end position: 42190171), POLB gene (start position: 42195973 ~ end position: 42229331), DK K4 gene (start position: 42231586 to end position: 42234674), VDAC3 gene (start position: 42249279 to end position: 42263455), SLC20A2 gene (start position: 42273980 to end position: 42317561) and SMIM19 gene (start position: 42396298 to Amplification of at least a part of the region up to 42543909 (region 3), including end position 42408140 (in this case, the end position is 42543909 in Table 1, but indicates the end point of the gene existing region), chromosome 12 Amplification of at least part of the region (region 4) containing 20p13.3-13.2 (start position: 9637323 to end position: 9696948) above, ZNF217 gene on chromosome 20 (start position: 52163628 to end position: 52215265) Amplification of at least a part of the region containing (region 5), deletion of at least a part of the region (region 6) containing the BC073807 gene (start position: 162514534 to end position: 162619141) on chromosome 3, chromosome 4 Above UGT2B17 gene (start posit ion: 69456442 to end position: 69483277) and / or deletion of at least part of the region (region 7) and / or GSTT1 gene / LOC391322 gene (start position: 24371205 to end position: 24390254) on chromosome 22 A method for diagnosing ovarian clear cell adenocarcinoma, comprising detecting a deletion of at least a part of a region to be included (region 8).

Here, “amplification of at least a part of the region” means that a part of the chromosomal region of the region 1 to region 5 is amplified, and of course, even if all of the region is amplified. Good. For example, the amplification of at least a part of region 3 means that at least a part of the PLAT gene (42033118-42101440) region is amplified, or at least IKBKB gene / POLB gene / DKK4 gene / VDAC gene / SLC20A2 gene / This is the case when a part of the SMIM19 gene (42155030-42543909) region is amplified. Of course, the entire region 3 region is PLAT gene / IKBKB gene / POLB gene / DKK4 gene / VDAC gene / SLC20A2 gene / The case where the region of the SMIM19 gene (42033118-42543909) is amplified is also included.
The “deletion of at least a part of the region” means that a part of the chromosomal region of the region 6 to region 8 is deleted, and of course, all of the region is deleted. May be.

  According to the first embodiment of the invention, amplification of at least part of region 1, region 2, region 3, region 4 or region 5 or deletion of at least part of region 6, region 7 or region 8 If any one of them is detected, the subject from which the chromosome is derived can be diagnosed as having or possibly having ovarian clear cell adenocarcinoma. Even if at least a part of a plurality of chromosomal regions of the subject is amplified or deleted, or is amplified and deleted, it is determined whether or not the subject is affected by ovarian clear cell adenocarcinoma can do.

In addition, amplification of at least part of the region (region 3) containing PLAT gene / IKBKB gene / POLB gene / DKK4 gene / VDAC gene / SLC20A2 gene / SMIM19 gene on chromosome 8 It was associated with a short progression-free survival (PFS) progression of cancer.
Furthermore, a region containing BC073807 gene on chromosome 3 (region 6), a region containing UGT2B17 gene on chromosome 4 (region 7), and a region containing GSTT1 gene and LOC391322 gene on chromosome 22 (region 8). At least some deletions in a region arbitrarily selected from the group consisting of) were associated with a short prognosis progression-free survival of ovarian clear cell adenocarcinoma in Japanese. In particular, in endometriosis complications in which at least a part of region 6 was deleted and in Japanese endometriosis complications, progression-free survival was significantly shorter.

  Therefore, in the second embodiment of the present invention, a region containing a PLAT gene, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene on chromosome 8 in the chromosomal DNA obtained from the subject (region) 3) At least a part of the amplification, or a region containing BC073807 gene on chromosome 3 (region 6), a region containing UGT2B17 gene on chromosome 4 (region 7), and a GSTT1 gene on chromosome 22 And a method for diagnosing the malignancy or prognosis of ovarian clear cell adenocarcinoma, comprising detecting at least a partial deletion in one region arbitrarily selected from the group consisting of the region comprising the LOC391322 gene (region 8) is there.

Furthermore, regarding the regions 1 to 5, when the cases where the amplification region is amplified in two or less regions and the case where the amplification region is amplified over three or more regions are compared, in the case where the amplification region is amplified over three or more regions, there is no progression Survival and overall survival (OS) was found to be significantly shorter.
Therefore, in the second embodiment of the present invention, in the chromosomal DNA obtained from the subject, the region containing ADAM5 gene and ADAM3A gene on chromosome 8 (region 1), the ANK1 gene and MYST3 gene on chromosome 8 are included. Contained region (region 2), PLAT gene on chromosome 8, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene region (region 3), 20p13.3-13.2 on chromosome 12 At least a part of each selected region in a region containing 3 (region 4) or three or more regions arbitrarily selected from the group consisting of a region containing the ZNF217 gene on chromosome 20 (region 5) A method for diagnosing the grade or prognosis of ovarian clear cell adenocarcinoma comprising detecting amplification is included.

As described above, the diagnostic method according to the second embodiment of the present invention can be used as a method for determining the presence or absence of ovarian clear cell adenocarcinoma and the good or bad prognosis when suffering from ovarian clear cell adenocarcinoma. It is. That is, the diagnosis of ovarian clear cell adenocarcinoma of the present invention is not particularly limited. For example, in the presence or absence of ovarian clear cell adenocarcinoma, in a patient suffering from ovarian clear cell adenocarcinoma or in a subject suffering from the disease And determining the prognosis of the ovarian clear cell adenocarcinoma.
Here, the “malignancy” of cancer does not represent a meaning that is particularly different from the meaning of words normally used in the medical field, and is not limited. For example, the prognosis of cancer is good, or It means the state of cancer for judging whether the prognosis is poor. The determination of the grade of malignancy or prognosis determined by the method of diagnosing malignancy according to the present invention includes determination of the excision range by surgical operation of the tumor, preoperative or postoperative adjuvant therapy or medical therapy (chemotherapeutic agents, This is also effective in selecting treatment methods such as the necessity of therapy using molecular target drugs.
In addition, “prognosis” does not represent a meaning that is particularly different from the meaning of words normally used in the medical field, and is not limited. For example, an opinion on an expected medical condition (health condition) It is the future state of illness / wound. In addition, when the disease is cancer or the like, the prognostic evaluation or prognosis includes, for example, cancer grade diagnosis, malignancy diagnosis, prediction of the presence or absence of metastasis in the future, prediction of the deterioration of symptoms after surgery In addition, prediction of long and short progression-free survival can be mentioned. The poor prognosis refers to, for example, a case where there is a possibility that the progression-free survival period or the overall survival period is shortened, the risk of recurrence is increased, and / or the tumor has metastasized to another site.

The method according to the present invention is characterized by detecting at least a partial amplification or deletion in the chromosomal region described above. Here, “at least part” means a continuous sequence of 1 to 10 bp, 10 bp or more, or 15 bp or more, preferably 60 bp or more, more preferably 1 kb or more, and further preferably 100 kb or more.
In the present invention, any method may be used to detect amplification or deletion of a target region of genomic DNA on a chromosome, for example, BAC (bacterial artificial chromosome) clone, PAC (P1-derived artificial chromosome) clone Competitive hybridization method (eg, chromosomal CGH) between a subject-derived DNA and a control DNA (a normal person, ie, a DNA not containing an abnormality of a chromosomal region targeted by the present invention) against a human genome library such as For example), PCR method, FISH (fluorescence in situ hybridization) method, ISH (in situ hybridization) method, quantitative PCR method, digital PCR method or sequencing method (exon sequence etc.).

For example, the CGH (Comparative Genomic Hybridization) method is a simple detection method because it can simultaneously detect chromosome deletion and amplification.
In the array CGH method, a DNA sequence (referred to as a probe) corresponding to a genomic region to be examined for the presence or absence of chromosome deletion or amplification is converted into a solid phase (eg, glass slide, glass, metal or resin plate-like chip). Etc.) is used (array). For example, a BAC clone, a PAC clone, or the like can be used as the probe. The array may be produced by a known method, or a commercially available one may be purchased and used according to the instruction manual.
When the CGH method is used, the subject-derived DNA and the control DNA are labeled with different labeling substances. When a fluorescent dye is used as a label, the DNA can be directly labeled by using a random primer method, a nick translation method, or the like using the DNA as a template.
The labeled DNA is added with about 100 μg of blocking DNA (for example, Cot-1 DNA) in order to block repeated sequences and the like, and labeled DNA is prepared by a well-known method. The conditions for hybridization depend on the situation of the CGH array, but for example, 50% formamide, 2 × SSC (standard citrate buffer), 10% dextran sulfate, 4% SDS, 100 mg / ml yeast tRNA, pH 7 Incubation in a solution such as 0.0 at 60 ° C. for 60 minutes makes it possible to repeatedly block sequences and the like.

  In addition, when detecting at least a partial amplification or deletion in the above chromosomal region, pay attention to SNP (Single Nucleotide Polymorphism) present in the target region, and amplify or lack the region containing the SNP. The loss may be detected by a digital PCR method or a sequencing method (as a method for focusing on SNP, for example, Dhallan et al., Lancet 369: 474-481 2007; Lo et al., Proc. Natl. Acad. Sci. 104: 13116 -13121 2007; Chiu et al., Proc. Natl. Acad. Sci. 105: 20458-20463 2008; Tsui et al., Blood 117: 3684-3691 2011).

  When using each of the methods described above, in addition to the subject-derived DNA, as necessary, DNA derived from normal cells (cells consisting only of chromosomes having no mutation in the DNA region to be examined) is obtained as a control. In this case, the DNA derived from the subject is derived from the subject in addition to the tumor tissue, pathological tissue section, tissue obtained by biopsy (for example, cervical cytology sample, endometrial cytology sample, etc.) The free DNA in cerebrospinal fluid, urine or blood can be isolated and purified by a known general method.

  In another embodiment of the present invention, a region containing ADAM5 gene and ADAM3A gene on chromosome 8 (region 1), a region containing ANK1 gene and MYST3 gene on chromosome 8 (region 2), and number 8 A region containing the PLAT gene, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene on the chromosome (region 3), a region containing 20p13.3-13.2 on chromosome 12 (region 4), and , Amplification of at least a part of one or more chromosomal regions selected from the region containing the ZNF217 gene on chromosome 20 (region 5), or region containing the BC073807 gene on chromosome 3 (region 6), number 4 Detection of at least partial deletion of one or more chromosomal regions selected from the region containing the UGT2B17 gene on the chromosome (region 7) and the region containing the GSTT1 gene and LOC391322 gene on the chromosome 22 (region 8) To exist in that area to A kit comprising one or more probes or primers having a base sequence complementary to the base sequence to be detected, the method for diagnosing ovarian clear cell adenocarcinoma, the method for diagnosing the malignancy of ovarian clear cell adenocarcinoma, etc. A kit for performing the method is provided.

The probe used in the CGH array method may be any nucleic acid or nucleic acid derivative that can be used for detecting the amplification or deletion of the chromosomal region. It can be obtained from human genome information. The length of the probe is not particularly limited, but for example, is preferably 15 bases or more, more preferably 60 bases or more, 100 bases or more.
In addition, the probe used when amplification of the target region is performed using SNP amplification as an index is a probe that anneals to the region including the position of the SNP serving as the index, such as fluorescent labels such as FAM and HEX, TAMRA, etc. Quenchers of may be combined. Furthermore, the probe may contain an artificial base such as 2 ′, 4′-Bridged Nucleic Acid (LNA) as necessary. The length of the probe for detecting amplification using SNP as an index is not particularly limited, but may be, for example, about 5 to 20 bases.

The kit according to the present invention includes a region containing ADAM5 gene and ADAM3A gene on chromosome 8 (region 1), a region containing ANK1 gene and MYST3 gene on chromosome 8 (region 2), and chromosome 8 The region containing PLAT gene, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene (region 3), region containing 20p13.3-13.2 on chromosome 12 (region 4), chromosome 20 Region containing ZNF217 gene (region 5) above, region containing BC073807 gene on chromosome 3 (region 6), region containing UGT2B17 gene on chromosome 4 (region 7), or GSTT1 on chromosome 22 A primer pair for amplifying all or part of the region containing the gene and the LOC391322 gene (region 8) by the PCR method (quantitative PCR method or the like) and detecting the amplification of the region may be included.
Furthermore, the kit of the present invention also includes a primer pair for detecting the target chromosomal region by using a digital PCR method or a sequencing method when SNP is used as an index.

  Furthermore, the kit according to the present invention may include, for example, an array in which the one or more probes are fixed on a support. In addition, a buffer solution, a washing solution, and the like necessary for detection, and a normal human-derived DNA as a control may be appropriately included.

The third embodiment of the present invention is the presence of a DNA derived from a chromosome having a mutation associated with the onset of ovarian cancer by cleaving a normal chromosome-derived DNA in a sample obtained from a subject with a Cas9-sgRNA complex. It is a method for diagnosing ovarian cancer, comprising increasing the ratio and detecting the presence or absence of the mutation.
Here, the “mutation associated with the development of ovarian cancer” is a chromosomal DNA derived from a tumor site of a patient of ovarian cancer (for example, serous adenocarcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma and clear cell adenocarcinoma), It is a mutation observed frequently. The term “mutation is frequent” means that the mutation is observed in 5% or more, 10% or more, more preferably 30% or more of ovarian cancer patients. The mutation associated with the incidence of ovarian cancer may be a known mutation or a newly discovered mutation. Examples of known mutations include, for example, ovarian clear cell gland ARID1A gene mutation (frequency: 40-57%), PIK3CA gene mutation (frequency: 33%), PPP2RIA gene mutation (frequency: 7%), KRAS gene mutation (frequency) ; 5%).
“Mutation” is not particularly limited, and examples thereof include substitution, insertion, deletion, and addition.
“Normal chromosome-derived DNA” refers to chromosome-derived DNA that does not contain a mutation for detection purposes. For example, when focusing on mutations present on the PIK3CA gene as mutations related to ovarian clear cell adenocarcinoma, all chromosomal DNA that does not have the mutation of interest is referred to as “normal” Chromosome-derived DNA ". That is, even if it has a mutation that is not focused on, if it does not have a focused mutation, it is “normal chromosome-derived DNA” here. In the present specification, DNA fragments amplified by a PCR method using chromosome-derived DNA as a template are also included in “chromosome-derived DNA”.

A technique called CRISPR / Cas system is a technique that cleaves DNA in a site-specific manner using a complex of Cas9 and sgRNA, and is used for genome editing and the like. For details, see, for example, Mali et al., Science 339: 823-826 2013, Sapranauskas et al., Nucleic Acids Res., 2011, 1-8 doi: 10. 1093 / nar / gkr606, Wang et al., Cell 153: 910-918 2013 Jinek et al., Science 337: 816-821 2012.
Here, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a DNA sequence containing short repeat sequences, and is found in bacteria and archaea. In the vicinity of CRISPR, there is a group of genes encoding Cas (CRIPR-associated) protein family. Foreign DNA that has invaded bacteria and archaea from the outside is fragmented by the Cas protein family. Cas9 in the Cas protein family is a nuclease that recognizes a sequence called PAM (Proto-spacer adjacent motif) on foreign DNA, cuts the upstream, and inserts it into the CRISPR sequence of the host (such as bacteria). Cas9 alone does not show cleavage activity and is activated by interaction with a single-stranded guide RNA (sgRNA) involved in recognition of the target site (site on the DNA to be cleaved). The sgRNA has a sequence complementary to the target site, and the Cas9 and sgRNA complex uses this complementary portion to bind to the target site and cleave the target portion. In the region of 10 to 12 bases from the PAM sequence, if even a single base has a mismatch with sgRNA, the cleavage activity by Cas9 disappears.
The inventors thought that only normal chromosome-derived DNA can be selectively cleaved by utilizing the DNA cleaving activity of the sgRNA-Cas9 complex. The PAM sequence differs depending on the Cas9 used, but in the case of Cas9 derived from Streptococcus pyogenes, the PAM sequence is NGG, and if there is a sequence of two guanines, the sgRNA-Cas9 complex is located upstream. Can be cut with the body. In other words, if you find the NGG sequence near the target region (including the site to be cleaved) of the normal chromosome-derived DNA that you want to cleave, and make an appropriate sgRNA, you can cleave the normal chromosome-derived DNA. .
If a normal DNA sequence in the region where the target mutation is present is targeted and sgRNA is prepared and treated with the sgRNA-Cas9 complex, only normal chromosome-derived DNA is cleaved at the site where the mutation occurs, and the mutation is Since the chromosome-derived DNA remains without being cut, it is possible to increase the abundance ratio of the chromosome-derived DNA having the mutation in the sample.

In this embodiment, a method for diagnosing the presence or absence of ovarian cancer or the possibility of ovarian cancer using free DNA (cfDNA) present in blood or the like is provided. The free DNA present in the blood contains many DNAs derived from normal cells in addition to DNA derived from tumor sites, and detecting mutations present in a small amount of DNA derived from tumor sites is quantitative. It has been difficult with conventional technology.
In such a situation, the present inventors, using the CRISPR / Cas system, specifically cleaved normal cell-free DNA having no mutation in the Cas9 and sgRNA complex, in a sample of DNA having the mutation. It has been found that the detection sensitivity can be remarkably increased by increasing the abundance ratio.

The sgRNA may be prepared by itself or may be prepared using a commercially available kit (for example, Clontech). As for Cas9, a commercially available product may be purchased and used. Samples derived from subjects treated with the sgRNA-Cas9 complex have an increased proportion of DNA containing mutations for detection purposes (ie, chromosomal DNA derived from tumor sites or DNA fragments amplified using the chromosomal DNA as a template). Using this sample, the presence or absence of mutation can be confirmed by, for example, PCR, FISH, CGH, ISH, quantitative PCR, serial PCR, or sequencing.
As a result, if a mutation associated with the onset of the ovarian cancer that is the test subject is detected, it can be determined that the subject subject to the test is affected by or may be affected by the ovarian cancer. . For example, a known somatic mutation in clear cell adenocarcinoma of the ovary, p.H1047R of the gene PIK3CA on chromosome 3 (mutation in which the 1047th amino acid of the PIK3CA gene is changed from histidine to arginine) on the subject's chromosome The subject can be diagnosed as having or possibly having ovarian clear cell adenocarcinoma.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

1. Experimental method 1-1. Subject The study for detecting somatic copy number alterations (SCNAs) associated with ovarian clear cell adenocarcinoma according to the present invention was conducted by the Biomedical Research Ethics Committee of Jikei University, Clinical Trial Committee. It was approved by the association. Surgical samples from patients diagnosed with ovarian clear cell adenocarcinoma include Jikei University (Tokyo, Japan), Niigata University Graduate School of Medical and Dental Sciences (Niigata, Japan), Tottori University School of Medicine (Tottori, Japan), Provided by Kyoto University Graduate School of Medicine (Kyoto, Japan), Korea Sungkyunkwan University School of Medicine (Seoul, Korea), Charite University Hospital (Berlin, Germany). Data from the Affymetrix Mapping Array (Affymetrix) was obtained from the Peter McCarran Cancer Center (Melbourne, Australia) and used to confirm findings regarding national differences.

1-2. Samples All tumor samples were obtained surgically with the patient's informed consent. All samples were collected as frozen material. A cryostat section containing 80% or more of cancer cells was microdissected (Okamoto et al., Clin Cancer Res. 11: 6030-6039 2005), and DNA was extracted.

1-3. Array comparative genomic hybridization
The human genome CGH 244A Oligo Microarray Kit (Agilent Technologie) was used to detect somatic copy number changes. Cleavage, labeling and hybridization of chromosomal DNA were performed according to the protocol attached to the kit. Specifically, tumor sample-derived DNA (2 μg) and control DNA (2 μg) were cleaved with Rsa I and Alu I, labeled with Cy5-dUTP and Cy3-dUTP, respectively, and mixed. The DNA mixture was hybridized to a human genome CGH 244K microarray. After hybridization, the slide glass was scanned with a DNA microarray scanner (Agilent Technologies). Data was extracted from the scan image using Feature Extraction software, version 10.7.3.1 (Agilent Technologies).

1-4. Statistical processing Abnormal chromosomal permeability and abnormal DNA copy number on the genome were expressed using the Aberration Detection Method 2 quality-weighted interval score algorithm of Agilent Genomic Workbench 7.0 (Agilent Technologie). The position of the gene was searched by National Center for Biotechnology Information (NCBI) build 36 (hg 19).
The change in the detected copy number was filtered according to the conditions. The change in the number of chromosomes at a locus when the change is in a region where 3 or more adjacent oligonucleotides on the chromosome are continuous, Log2Ratio ≧ 0.9 for amplification and Log2Ratio ≧ 1.5 for deletion Changes were considered significant.
The progression-free survival (PFS; progression-free survival) and overall survival (OS) were compared by somatic copy number change by log rank test using Kaplan-Meier method. All statistical analyzes were performed using StatView-J 5.0. Statistical significance was considered when P <0.05.

1-5. Confirmation experiment of chromosomal DNA amplification using SNP First, we obtained information on SNP (Single Nucleotide Polymorphism) related to the human genome from the International Hapmap Project (Hapmap). In examining the genotype, we selected Japanese who had a large proportion of heterozygous types. In this example, in order to confirm the amplification of the ANK1 gene of chromosome 8, the SNP (rs1532940) of Position 41747333 existing between exon 1 and exon 2 of the ANK1 gene was selected. There are three types of genotypes related to this SNP: AA, AT, and TT. According to Hapmap, AA is 46.7%, AT 53.3%, TT 0%, and the ratio of AT is larger than AA and TT. I was able to confirm.
The test for identifying the genotype of the SNP was performed by digital PCR (BioRad apparatus: QX200 ^™ Droplet Digital ^™ PCR system). First, probes using artificial bases were prepared for each allele (probe for allele that is A; fluorescent FAM CA + C + C + A + GTGCT: probe for allele that is T (SEQ ID NO: 1)) Fluorescent HEX CA + C + C + T + GTGCT, + indicates artificial base (LNA; locked nucleic acid. Quencher uses 3IABLFQ in any probe) (SEQ ID NO: 2). The difference between the mismatch and extra match Tm values can be increased by more than 10 ° C, and more accurate detection is possible.The region centered around the position where this probe anneals is amplified even when DNA is fragmented. As possible, the following primers were prepared so that the amplicon (PCR amplification product) was 100 bp or less.
Forward; 5'-GCTTAGATACTTGCAAGCTTTATTG-3 '(SEQ ID NO: 3)
Reverse; 5'-GCAAAGTTAGGAAGCCCTTT-3 '(SEQ ID NO: 4)
In this case, the amplicon is 88 bp.
For the buffy coat (a layer of leukocytes generated by centrifuging blood) or the DNA recovered from the normal site, the ANK1 gene genotype was determined by digital PCR. Here, the fluorescence of A: FAM and T: HEX is measured, and whether the amplitude (Amplitude) derived from the PCR product is 0 or 100 (%) for FAM (A / A if 100), HEX Whether the result is 0 or 100 for T (T / T if 100) or 50 or 50 for FAM and HEX (A / T if 50 or 50) It was confirmed.

As a result of confirmation, DNA derived from cancer tissue of a subject found to have SNP (that is, a subject from which a DNA sample with A: T = 50: 50) or free DNA (cfDNA) prepared from peripheral blood Was used for digital PCR analysis. Amplification of ANK1 gene region with SNP is judged as amplification when the ratio of the copy number of the allele that is A and the copy number of the allele that is T deviates from 1. Can do.
Similar judgments can be made for other SNPs.

In addition to the SNP (rs1532940) of Position 41747333, the presence or absence of amplification of the target gene was confirmed using the SNPs shown in Table 1. The probe and primer sequences used in the detection of the genotype of each SNP are summarized in Table 1.

1-6. Detection of mutations present in the PIK3CA gene using the CRISPR / Cas system First, sgRNA that recognizes the PAM sequence was prepared near the 1047th mutation site (PIK3CA H1047R; histidine was replaced by arginine) on the PIK3CA gene. The length and base sequence of the PAM sequence vary depending on the bacterioma, for example, NGG for Streptococcus pyogenes and NGGNG or NNAGAAW for Streptococcus thermophilus. The PAM sequence in this example is -NGG-.
FIG. 29 shows a sequence (SEQ ID NO: 25) in the vicinity of the site where the H1047R mutation is present. Two PAM sequences exist in this region, and 20 bases (Fw1 (SEQ ID NO: 26) and Fw2 (SEQ ID NO: 27)) and 18 bases (Fw3 (SEQ ID NO: 28) and Fw4 (sequence) adjacent to each sequence are present. No. 29)) was used as a target sequence to produce sgRNA for each (using Guide-it ^™ sgRNA InVitro Transcription and Screening Systems from Clontech Laboratories, Inc.). Among the four sgRNAs prepared here, when sgRNA containing Fw2 was used, the difference in the cleavage efficiency between the mutant type sequence and the wild type sequence was large, so that this sgRNA was used in the experiments.
The sgRNA was prepared according to the instruction manual of Guide-it ^™ sgRNA InVitro Transcription and Screening Systems of Clontech Laboratories, Inc. The Forward PCR primers used for the production of sgRNA are shown below.
Forward ； 5'-gcggcctctaatacgactcactatagggcaaatgaatgatgcacatcagttttagagctagaaatagca-3 '
(SEQ ID NO: 30)
Reverse: Use the one included in Guide-it ^™ sgRNA InVitro Transcription and Screening Systems.

A sample containing the DNA to be examined was prepared as follows.
1. The blood of the subject was collected with a BCT blood collection tube (cell-Free DNA BCT ^™ , Streck).
2. The collected blood was centrifuged at 1,600 g for 20 minutes at room temperature to collect the supernatant (plasma), and further centrifuged at 16,000 g for 10 minutes at room temperature to collect the supernatant.
3. The collected supernatant (plasma) was stored at −80 ° C.
4). The cfDNA was recovered from the thawed plasma using QIAamp Circulating Nucleic Acid (QIAGEN).
5. The recovered cfDNA was quantified with a Qubit (registered trademark) 2.0 Fluorometer (Qubit ^™ dsDNA HS Assay Kit).
6). The obtained cfDNA (2.5 ng) was pre-amplified by digital PCR using the following primers.
Forward; 5'-actgagcaagaggctttgga-3 '(SEQ ID NO: 31)
Reverse; 5'-gcatgctgtttaattgtgtgg-3 '(SEQ ID NO: 32)
7). The PCR product is extracted by the chloroform method from the droplet produced by the digital PCR method (the reaction solution containing the PCR product that is present in the microcompartment when PCR is performed by dividing the microcompartment), and MinElute (QIAGEN CfDNA amplification products were purified by the company.
8). Using the amplification product of cfDNA purified in 7 above, using Clontech Laboratories, Inc.'s Guide-it ^™ sgRNA InVitro Transcription and Screening Systems, DNA without the H1047R mutation was added to CRISPR / Cas Cutting was performed using the system.

The presence or absence of H1041R mutation was confirmed in the DNA sample after cleavage by digital PCR.
Probes for detecting a site having no mutation are as follows.
C + CA TG + A + T + GT G + CA (SEQ ID NO: 33) (+ is an artificial base)
In addition, detection probes for sites having mutations are as follows.
C + CA TG + A + C + GT GCA (SEQ ID NO: 34) (+ is an artificial base)
The primers for amplification are as follows.
Forward; 5'-TTGTGTGGAAGATCCAATCCA-3 '(SEQ ID NO: 35)
Reverse; 5'-GAGCAAGAGGCTTTGGAGTAT-3 '(SEQ ID NO: 36)
In this case, the amplicon is 99 bp.

2. Result 2-1. Correlation between ovarian clear cell adenocarcinoma and amplification of regions 1 to 5 When the adjacent oligonucleotide on the chromosome is a continuous region of 3 or more and the copy number change is Log2Ratio ≧ 0.9, the gene Amplification of the number of chromosomes at the locus was considered significant, and regions 1-5 were judged to be significantly amplified.
The frequency of somatic copy number amplification in regions 1 to 5 is shown in FIG. 1 (region 1 to region 3), FIG. 2 (region 4) and FIG. 3 (region 5). The peak portion corresponds to the amplified region. For region 3, amplification of PLAT gene (42033118-42101440) region, amplification of IKBKB gene / POLB gene / DKK4 gene / VDAC gene / SLC20A2 gene / SMIM19 gene region (42155030-42543909), and PLAT gene-SMIM19 gene (42033118) It was possible to confirm the correlation between the amplification of the region (˜42408140) (all regions of region 3) and ovarian clear cell adenocarcinoma.
Table 2 summarizes the positions of the regions 1 to 5 on the chromosome and the number of confirmed cases.

2-2. Correlation between ovarian clear cell adenocarcinoma and region 6-8 deletion When the adjacent oligonucleotide on the chromosome is a continuous region of 3 or more and the copy number change is Log2Ratio ≧ 1.5 Chromosomal deletion at the locus was considered significant, and regions 6-8 were judged to be significantly amplified.
The frequency of deletion of the somatic copy number in the regions 6 to 8 is shown in FIG. 4 (region 6), FIG. 5 (region 7) and FIG. 6 (region 8). The peak portion corresponds to the deleted region.
Table 3 summarizes the positions of the regions 6 to 8 on the chromosome and the number of confirmed cases.

Next, the relationship with the patient's survival time was examined.
Amplification in region 3 (PLAT gene to SMIM19 gene region) was significantly associated with shortening progression-free survival (PFS) (FIG. 7). Moreover, in the case where amplification has occurred in three or more regions among regions 1 to 5, both progression-free survival (FIG. 8) and overall survival (FIG. 9) may be significantly shortened. It was understood that the prognosis was poor.

  The group with deletion in region 6 in Japanese tended to have a short progression-free period (FIG. 10). Moreover, in the endometrial complication example (FIG. 11) or the Japanese endometrial complication example (FIG. 12), the group with deletion in region 6 had a significantly shorter progression-free period. Furthermore, the group with deletion in one of regions 1 to 3 in Japanese tended to have a short progression-free period (FIG. 13).

2-3. Confirmation of amplification of ANK1 gene region using free DNA in blood 2-3-1. Use of SNP (chromosome 8 41747333, rs1532940) on ANK1 gene (1)
Next, it was examined whether or not amplification of the regions 1 to 5 could be confirmed using free DNA (cfDNA) present in blood.
Below, the result of having confirmed the presence or absence of amplification of the ANK1 gene of the area | region 2 using the DNA sample derived from a Japanese is shown.
First, using the DNA prepared from the buffy coat of healthy individuals (Buffy coat, a layer of leukocytes generated by centrifuging blood), the ANP1 gene SNP (chr8 41747333, rs1532940) genotype is A / A, It was confirmed which type was A / T. The upper diagram in FIG. 14 shows a positive value when the position of the SNP (chr8 41747333, rs1532940) of the ANK1 gene is A, and the lower diagram shows a positive value when it is T. Of 6 healthy individuals, 3 were found to have SNPs (N6, N7, N8). In addition, using three DNA samples derived from the buffy coat of healthy individuals with SNP, the ratio of the copy number of the ANK1 gene where the position of 41747333 of chromosome 8 is A and the copy number of T was all found. Both samples were 1: 1, and amplification of the ANK1 gene was not confirmed (FIG. 15).
Next, cfDNA was prepared from the blood of three healthy individuals who had this SNP in the ANK1 gene, and the presence or absence of amplification of the ANK1 gene was confirmed as described above. The copy number ratio was 1, and even when cfDNA was used, amplification of the ANK1 gene was not confirmed (FIG. 16).

Based on the results of analysis of the presence or absence of amplification of ANK1 gene using DNA derived from healthy individuals by digital PCR, the presence or absence of amplification of ANK1 gene was confirmed using DNA derived from patients with clear cell adenocarcinoma of the ovary.
First, DNA was collected from the buffy coat of ovarian clear cell adenocarcinoma patients (17 cases), and the presence or absence of SNP was confirmed in the same manner as in the case of healthy subjects. As a result, SNP was confirmed in 7 cases (FIG. 17). And, for the DNA derived from the buffy coat of 7 cases where SNP was confirmed, the ratio of the copy number of the ANK1 allele where the position of 41747333 of chromosome 8 is A and the copy number of T was found to be almost 1. It was also confirmed that there was (FIG. 18).
Next, DNA was prepared from cancer sites of subjects (7 cases) that were confirmed to retain SNP, and the presence or absence of amplification of the ANK1 gene was confirmed. Using the prepared DNA sample, when digital PCR analysis was performed, the position of 41747333 was T, that is, the ratio of the A / T and T / A allele copy numbers was ≧ 1.3, Three cases were judged to be amplified (case 15 (15T) = 0.131, case 16 (16T) = 0.09, case 17 (17T) = 1.35) (FIG. 19).

For 3 cases in which ANK1 gene amplification was confirmed by digital PCR analysis using DNA samples prepared from cancer sites, peripheral blood was collected from subjects and free DNA (cfDNA; cell free DNA) prepared from peripheral blood was collected. The presence or absence of amplification of the ANK1 gene was confirmed.
As a result of digital PCR analysis, the ratio of ANK1 allele copy number in each case was A / T = 0.789 in case 15, 1.19 in case 16, and 1.05 in case 17. That is, regarding case 15, amplification of ANK1 gene could be confirmed using free blood derived from peripheral blood (FIG. 20).
As a result of digital PCR analysis using DNA derived from cancer sites in cases other than these 3 cases, cfDNA was also used for cases where the allele copy number ratio (A / T and T / A) was ≦ 1.3. A similar analysis revealed that cfDNA for cancer site-derived DNA with A / T copy number ratios of 1.05, 0.933, and 1.03, that is, no amplification of the ANk1 gene was confirmed. When using, the results were 1.02, 0.982, and 1.09, respectively, and even when cfDNA was used, amplification of the ANK1 gene was not confirmed.

  In addition, other SNPs on the ANK1 gene (alleles that are G and alleles that are T) (chr8 41658923, rs1579274), SNPs on the MYST gene (alleles that are C and alleles that are G) (chr8 41850222) , Rs11779404), SNPs on the IKBKB gene (alleles that are C and T) (chr8 421807016, rs10958713) and SNPs on the ZNF217 gene (alleles that are C and G) (chr20 52208838) , Rs1075390), the presence or absence of amplification of each gene was confirmed using the patient's cfDNA (2.5 ng) (Table 1).

2-3-2. Use of SNP (chromosome 8 41658923, rs1579274) on ANK1 gene (2)
Next, the presence or absence of amplification of the ANK1 gene was examined using other SNPs present in the ANK1 gene (chr8 41658923, rs1579274).
DNA was prepared from cancer sites of subjects (11 cases) that were confirmed to retain SNP using a DNA sample derived from buffy coat, and the presence or absence of amplification of ANK1 gene was confirmed. Digital PCR analysis using the prepared DNA sample revealed that the position of chromosome 8 41658923 was amplified with a ratio of G / T and T / G allele copy number ≧ 1.3. There were 6 cases (case 1 (1T) = 0.151, case 6 (6T) = 1.69, case 15 (15T) = 0.144), case 16 (16T) = 7.87, case 18 (18T) = 1.4, Case 19 (19T) = 0.565) (FIG. 21).

For 6 cases in which amplification of ANK1 gene was confirmed by DNA PCR analysis using DNA samples prepared from cancer sites, peripheral blood was collected from subjects and free DNA (cfDNA; cell free DNA) prepared from peripheral blood was collected. The presence or absence of amplification of the ANK1 gene was confirmed.
As a result of digital PCR analysis, the ratio of ANK1 allele copy number in each case was G / T = 0.854 in case 1, 0.671 in case 15, and 0.786 in case 19, and peripheral blood for these cases. The amplification of ANK1 gene could be confirmed using the free DNA derived from the origin (FIG. 22).

2-2-3. Use of SNP (chromosome 8 41850222, rs11779404) on MYST3 gene
Using the SNP (chr8 41850222, rs11779404) present in the MYST3 gene, the presence or absence of amplification of the MYST3 gene was examined.
DNA was prepared from cancer sites of subjects (7 cases) that were confirmed to retain SNP using DNA samples derived from buffy coat, and the presence or absence of amplification of the MYST3 gene was confirmed. Digital PCR analysis was performed using the prepared DNA sample, and it was determined that the position of chromosome 8 41850222 was amplified because the copy number ratio of the C / G and G / C alleles was ≧ 1.3. There were 2 cases (Case 15 (15T) = 0.119, Case 16 (16T) = 8.51) (FIG. 23).

For two cases in which amplification of MYST3 gene was confirmed by DNA PCR analysis using DNA samples prepared from cancer sites, peripheral blood was collected from subjects and free DNA (cfDNA; cell free DNA) prepared from peripheral blood was collected. Using this, the presence or absence of amplification of the MYST3 gene was confirmed.
As a result of digital PCR analysis, the copy number ratio of the MYST3 allele in each case was C / G = 0.82 in case 15, but could not be determined in case 16. When SNP (chromosome 8 41850222, rs11779404) on the MYST3 gene was used and free DNA derived from peripheral blood derived from a patient was used, amplification of MYST3 could be confirmed in one case (FIG. 24).

2-2-4. Use of SNP (chromosome 8 42180716, rs10958713) on IKBKB gene
Using the SNP (chr8 42180716, rs10958713) present in the IKBKB gene, the presence or absence of amplification of the IKBKB gene was examined.
DNA was prepared from cancer sites of subjects (7 cases) confirmed to retain SNP using a DNA sample derived from buffy coat, and the presence or absence of amplification of the IKBKB gene was confirmed. Digital PCR analysis using the prepared DNA sample revealed that the position of chromosome 8 42180716 was amplified with a ratio of C / T and T / C allele copy numbers of ≧ 1.3. There were 4 cases (Case 5 (5T) = 0.563, Case 15 (15T) = 7.95, Case 16 (16T) = 6.89, Case 17 (17T) = 1.33) (FIG. 25).

For 4 cases in which amplification of IKBKB gene was confirmed by DNA PCR analysis using DNA samples prepared from cancer sites, peripheral blood was collected from subjects and free DNA (cfDNA; cell free DNA) prepared from peripheral blood was collected. The presence or absence of amplification of the IKBKB gene was confirmed.
As a result of digital PCR analysis, the ratio of IKBKB allele copy number in each case was C / T = 1.72 in case 15, and in this case, IKBKB gene amplification was achieved by using peripheral blood-derived free DNA. Could be confirmed (FIG. 26).

2-2-5. Use of SNP (chromosome 20 52208838, rs1075390) on the ZNF217 gene
Using SNP (chr8 52208838, rs1075390) present in the ZNF217 gene, the presence or absence of amplification of the ZNF217 gene was examined.
DNA was prepared from cancer sites of subjects (9 cases) confirmed to retain SNP using a DNA sample derived from buffy coat, and the presence or absence of amplification of the ZNF217 gene was confirmed. Digital PCR analysis using the prepared DNA sample revealed that the position of chromosome 20 52208838 was amplified with a ratio of C / G and G / C allele copy number ≧ 1.3. There were 4 cases (Case 5 (5T) = 2.04, Case 8 (8T) = 0.747, Case 17 (17T) = 0.721, Case 21 (21T) = 2.12) (FIG. 27).

For 4 cases in which amplification of the ZNF217 gene was confirmed by digital PCR analysis using DNA samples prepared from cancer sites, peripheral blood was collected from subjects and free DNA (cfDNA; cell free DNA) prepared from peripheral blood was collected. The presence or absence of amplification of the ZNF217 gene was confirmed.
As a result of digital PCR analysis, the ZNF217 allele copy number ratio in each case was C / G = 0.742 in case 8, and in this case, ZNF217 gene was amplified by using free DNA derived from peripheral blood. Was confirmed (FIG. 28).

  From the above results, even if free DNA prepared from peripheral blood or the like is used as a sample, the presence or absence of amplification of a desired chromosomal DNA region (for example, regions 1 to 5 in the present invention) can be confirmed. It became clear that it was possible. In addition, when using free DNA as a sample, it is considered that detection with high sensitivity becomes possible by increasing the content of DNA derived from a cancer site.

2-3. Results of detection of mutations present in the PIK3CA gene Table 1 shows the results of applying the CRISPR / Cas system using a complex of sgRNA and Cas9 to detect the mutation of H1047R on the PIK3CA gene frequently observed in ovarian clear cell adenocarcinoma 3 shows.
A study was conducted using cfDNA derived from 4 cases having the PIK3CA H1047R mutation. Mutation was detected using a digital PCR method.
When mutation is detected by digital PCR using cfDNA 5ng without pre-amplification in Table 4, cfDNA 2.5ng is pre-amplified and mutation is detected by digital PCR using the amplified product , Pre-amplification of cfDNA 2.5 ng, cleave the amplified product with normal sequence using the CRISPR / Cas system for the amplified product, and then show the mutation abundance ratio when the mutation is detected by digital PCR It was.
When the CRISPR / Cas system was used, the pre-amplification annealing time was examined under two conditions: 60 seconds and 30 seconds. Each shows the results of two studies.

For example, in the first examination of case OC17, when mutation is detected using cfDNA 5ng as it is and when mutation is detected after pre-amping cfDNA 2.5ng, the abundance ratio of mutation in the sample is Compared to 0.391% and 0.29%, when the DNA derived from the normal sequence was cleaved with the CRISP / Cas system, the mutation ratio was 3.29% at 60 seconds of annealing and 1.58% at 30 seconds of annealing. Rose to. Similar results were obtained in other cases.
Based on the above results, when detecting mutations peculiar to ovarian cancer and diagnosing ovarian cancer, or the possibility of morbidity, mutations are detected using free DNA (cfDNA) present in the blood, etc. CRISPR / Cas system, i.e., sgRNA-Cas9 complex is used to cleave a DNA fragment derived from a normal chromosome in the sequence region where the mutation occurs, and It has been shown that the abundance can be increased. Thus, by increasing the abundance ratio of mutant DNA in a cfDNA-derived sample, non-invasive and highly sensitive diagnosis can be performed.

  By using the method for diagnosing ovarian clear cell adenocarcinoma of the present invention, it becomes possible to easily diagnose the presence or absence of ovarian clear cell adenocarcinoma and the prognosis thereof. Therefore, the present invention is expected to be used in the diagnosis of ovarian clear cell adenocarcinoma and further in the therapeutic field.

Claims (11)

  In the chromosomal DNA obtained from the subject, amplification of at least part of the region containing ADAM5 gene and ADAM3A gene on chromosome 8, amplification of at least part of the region containing ANK1 gene and MYST3 gene on chromosome 8, number 8 Amplification of at least part of the region containing PLAT gene, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene on chromosome 12, and at least part of region containing 20p13.3-13.2 on chromosome 12 Amplification of at least part of the region containing the ZNF217 gene on chromosome 20, deletion of at least part of the region containing BC073807 gene on chromosome 3, at least of the region containing UGT2B17 gene on chromosome 4 A method for diagnosing ovarian clear cell adenocarcinoma, comprising detecting a partial deletion and / or a deletion of at least a part of a region containing GSTT1 gene and LOC391322 gene on chromosome 22.   In the chromosomal DNA obtained from the subject, the ovary comprises detecting amplification of at least a part of the region containing PLAT gene, IKBKB gene, POLB gene, DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene on chromosome 8 A method for diagnosing malignancy or prognosis of clear cell adenocarcinoma.   In the chromosomal DNA obtained from the subject, the region containing ADAM5 gene and ADAM3A gene on chromosome 8, the region containing ANK1 gene and MYST3 gene on chromosome 8, PLAT gene on chromosome 8, IKBKB gene, POLB gene, Arbitrarily selected from the group consisting of a region containing the DKK4 gene, VDAC gene, SLC20A2 gene and SMIM19 gene, a region containing 20p13.3-13.2 on chromosome 12, and a region containing the ZNF217 gene on chromosome 20. A method for diagnosing malignancy or prognosis of ovarian clear cell adenocarcinoma, comprising detecting amplification of at least a part of each of the three regions.   In the chromosomal DNA obtained from the subject, arbitrary from the group consisting of the region containing BC073807 gene on chromosome 3, the region containing UGT2B17 gene on chromosome 4, and the region containing GSTT1 gene and LOC391322 gene on chromosome 22 A method for diagnosing the malignancy or prognosis of ovarian clear cell adenocarcinoma, comprising detecting a deletion of at least a part of the one region in one region selected in (1).   The method according to any one of claims 1 to 4, wherein the detection of the amplification or deletion is performed by detecting a change in the number of DNA copies including the region of interest.   6. The method according to claim 5, wherein the change in the DNA copy number is detected by a PCR method, FISH method, CGH method, ISH method, quantitative PCR method, digital PCR method or sequencing method.   The chromosomal DNA obtained from the subject is extracted from the tumor tissue, cervical / intimal cytodiagnosis sample, cerebrospinal fluid, blood or urine of the subject. The method described.   Among chromosomal DNA obtained from subjects, normal chromosome-derived DNA is cleaved with a Cas9-sgRNA complex to increase the abundance of chromosome-derived DNA having a mutation associated with the onset of ovarian cancer, and the presence or absence of the mutation A method for diagnosing ovarian cancer, comprising detecting   9. The method of claim 8, wherein the ovarian cancer is ovarian clear cell adenocarcinoma.   The method according to claim 8 or 9, wherein the mutation is a mutation in which the 1047th histidine in the PIK3CA locus is substituted with arginine.   The chromosomal DNA obtained from the subject is collected from a tumor tissue, a cervical / intimal cytology sample, cerebrospinal fluid, blood or urine of the subject. The method described in 1.