WO2020096024A1 - Use of vaginal microflora as ovarian cancer biomarker - Google Patents
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- the present invention relates to a method for diagnosing ovarian cancer. Specifically, it relates to a method for diagnosing ovarian cancer based on the flora structure of the vaginal microflora.
- Ovarian cancer is found in about 10,000 women a year in Japan, and the number of patients is increasing. Ovarian cancer is classified into epithelial type, germ cell type, and sex cord interstitial type depending on the place where it occurs, but about 90% or more is epithelial ovarian cancer.
- Epithelial ovarian cancer is mainly composed of four histological subgroups. That is, serous cancer, mucinous cancer, endometrioid cancer and clear cell cancer. Among them, the ratio of clear cell cancer is relatively high, and the morbidity rate is about 15-25% in Japan.
- Ovarian cancer is painless in the early stages (stage I or stage II), has almost no subjective symptoms, and is diagnosed at the first consultation only after a lump or sensation of pressure in the lower abdomen or frequent urination caused by pressure on the bladder. Often. However, in this state, the cancer is often advanced. Patients in the advanced stage (stage III or stage IV) usually have a poor prognosis (Non-patent Document 1 and Non-patent Document 2), and are one of the cancers that require early detection.
- CA125 is used as a typical tumor marker.
- CA125 is positive in about 80% of people with ovarian cancer, but is also expressed in the peritoneum and endometrium, and is also positive in cases such as endometriosis. It is a marker that is not specific to ovarian cancer. Further, in early cancers and clear cell adenocarcinomas, the increase of CA125 is poor, and thus a biomarker with higher specificity is desired for early detection of ovarian cancer.
- the present invention has as a problem to be solved a method for early diagnosis of ovarian cancer.
- the inventors conducted a bacterial flora analysis on a vaginal flora sample collected from a patient who had serous cancer or clear cell carcinoma of the ovary, and the composition of the vaginal flora of an ovarian cancer patient was different from that of a healthy subject. I found that.
- the vaginal flora of healthy people has a simple structure in which the majority of bacteria of the genus Lactobacillus or Prevotella is pre-menopausal, but when it reaches menopause, it is a diverse variety of bacteria with Propionibacterium as the first species. It is known to transition to.
- vaginal flora samples from ovarian cancer patients the majority of the vaginal flora samples showed the same diverse flora structure as those in postmenopausal healthy subjects, regardless of the presence or absence of menopause. That is, it was found for the first time that there was a strong correlation between the presence or absence of ovarian cancer and the vaginal flora structure in premenopausal women. In addition, the high diversity of the vaginal flora was also found in patients with early stage I ovarian cancer. Therefore, the vaginal flora is considered to function as a biomarker that enables early detection of ovarian cancer in premenopausal women. The present invention has been completed based on the above findings.
- the present invention is the following (1) to (8).
- (1) A method of diagnosing the possibility that a premenopausal subject has ovarian cancer, wherein the composition of the vaginal microflora of the subject has a high similarity to that of the ovarian cancer patient.
- (2) The method according to (1) above, wherein the evaluation index of the constitution of the vaginal microflora is any one or more of a diversity index, the number of types of microorganisms or the abundance ratio of specific microorganisms. ..
- the subject has ovarian cancer
- the evaluation index is the number of types of microorganisms, and the number of types of microorganisms in the vaginal microflora of the subject is greater than the number of types of microorganisms in the vaginal microflora of a healthy person before menopause, the subject is The method according to (2) above, wherein diagnosing that the patient may have ovarian cancer.
- the evaluation index is the abundance ratio of genera other than Lactobacillus genus and Prevotella genus, and the abundance ratio of the genus in the vaginal microbiota of the subject is The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer when the proportion is higher than the abundance ratio.
- the evaluation index is the abundance ratio of Lactobacillus genus and / or Prevotella genus, and the abundance ratio of the genus in the vaginal microbiota of the subject is The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer when the ratio is lower than the ratio.
- the evaluation index is the abundance ratio of Propionibacterium acnes, and the abundance ratio of the species in the vaginal microflora of the subject is higher than the abundance ratio of the species in the vaginal microflora derived from a premenopausal healthy subject.
- the method for diagnosing ovarian cancer of the present invention can judge the possibility of ovarian cancer even at an early stage without subjective symptoms. Therefore, by using the diagnostic method of the present invention, it becomes possible to undertake early treatment of ovarian cancer.
- List of analyzed bacteria present in the vaginal flora of subjects (1).
- List of bacteria analyzed in the subject's vaginal flora (2).
- List of analyzed bacteria present in the subject's vaginal flora (3).
- the graph shows the top five genera in existence ratio.
- the cancer grade is also shown. It is a graph which shows the relative abundance of the Lactobacillus genus (A) and the sum of the abundances of the Lactobacillus genus and the Prevotella genus (B) in the vaginal flora of premenopausal subjects. It is a graph which shows the abundance of Propionibacterium genus (A) and Propionibacterium acnes (B) in the vaginal microflora of a premenopausal subject. It is a graph which shows the abundance of Corynebacterium genus in the vaginal microflora of a premenopausal subject.
- FIG. 6 is a graph showing the number of bacterial species present in the vaginal flora of premenopausal subjects at a relative ratio of 0.1% or more.
- FIG. 3 is a graph showing ⁇ -diversity indexes (Simpson index (A) and Shannon index (B)) in vaginal flora of premenopausal subjects.
- the dissimilarity index (weighted UniFrac index) of the vaginal flora of the subject is shown.
- the result of principal coordinate analysis based on the dissimilarity index of vaginal flora is shown.
- the present invention relates to methods of diagnosing whether a pre-menopausal subject has, or is likely to have, ovarian cancer.
- the method is also highly useful as an aid in the definitive diagnosis of ovarian cancer.
- the method for diagnosing the presence or absence of ovarian cancer according to the present invention (hereinafter, also referred to as the “diagnosis method of the present invention”) is a vaginal microbiota (eg, vaginal bacteria) of an ovarian cancer patient.
- the flora, etc.) is based on the fact that the composition of the microorganisms is different from the composition of the vaginal microflora of healthy individuals, and is more diverse.
- the configurations of the vaginal flora and the cervical flora are very similar, and in the description of the present specification, the cervical microflora (particularly the bacterial flora) and the vaginal flora are Microbiota (especially bacterial flora) should be comparable.
- An embodiment of the present invention is a method for diagnosing the possibility that a pre-menopausal subject has ovarian cancer, wherein the composition of the vaginal microflora of the subject is the composition of the vaginal microbiota of the ovarian cancer patient.
- the subject is a method of diagnosing that the subject may have ovarian cancer.
- the “composition” of the vaginal microflora refers to the composition of the microflora, and the state of the microflora characterized by the number of microbes that are elements of the microflora, the number of types of microbes, and the abundance ratio of the microbes.
- the inventors as a result of advancing the analysis focusing on the bacterial flora, have a higher diversity in the composition of the vaginal microflora of patients with ovarian cancer, as compared with the composition of the vaginal microflora of healthy people before menopause, It was clarified that the kinds of microorganisms present are abundant and that the abundance ratio of each microorganism is different in both.
- the type of “microorganism” is not particularly limited, and for example, bacteria, fungi, etc. are particularly preferable, and the microbial flora to be analyzed may be the microbial flora.
- the "type" of the microorganism may be any of phylum, class, order, family, genus and species.
- the composition of the microflora, as described above, for example, the number of microorganisms present, the type of microorganisms, the abundance ratio of microorganisms and the diversity index of the microflora (for example, Simpson index or Shannon index, etc.) are used as indicators. However, it is not limited to these evaluation indicators.
- the composition of the vaginal microflora can be examined by measuring the types of microorganisms present in the microflora, the number of each microorganism, and the like, and carrying out based on the obtained values.
- Microorganisms may be identified by any method, for example, genomic DNA analysis of microorganisms (for example, sequencing of 16S rRNA or a genomic region other than 16S rRNA, PCR using primers specific to a particular microorganism). Analysis, etc.), metabolome analysis to detect microorganism-specific proteins and metabolites, discrimination based on the morphology of the microorganism, discrimination based on the conditions necessary for growth on the medium, biochemical properties (sugar fermentation) Or catalase activity) and the like.
- the vaginal microflora sample of a subject can be collected from the subject's vagina, cervix, etc. by a method such as scraping with absorbent cotton or a brush (cytology etc.).
- a method well known to those skilled in the art for example, a lytic enzyme method.
- the nucleotide sequence of the DNA is analyzed by, for example, a next-generation ultra-high speed sequencer device, etc., and the DNA sequence information of all the microorganisms present in the microbiota is acquired. To do. Then, the composition of the microflora can be clarified based on the obtained DNA sequence information of the microorganism.
- the 16S rRNA method is particularly often used, and the type and amount of bacteria contained in the bacterial flora can be known based on the obtained 16S rRNA sequence information.
- PCoA principal coordinate analysis
- clustering analysis or the like, it is possible to compare the samples of the bacterial flora composition among samples.
- the analysis of the bacterial flora by the 16S rRNA method may be carried out by entrusting it to a company (for example, Fujifilm Wako Pure Chemical Industries, Ltd., Takara Inc., etc.).
- the subject When the diversity index is used as an evaluation index for the composition of the vaginal microbiota, the subject has ovarian cancer when the diversity index of the vaginal microbiota of the subject is close to the diversity index of the ovarian cancer patient. It can be diagnosed as possible. Specifically, when evaluating the diversity of the vaginal flora, the diversity of the vaginal flora in patients with ovarian cancer, for example, when expressed by the Simpson index, often shows a value of 0.7 or more, and the Shannon index. When expressed, it often shows a value of 2.0 or more.
- the composition of the vaginal flora from the subject is the vaginal flora from the ovarian cancer patient. It is possible to diagnose that the subject may be suffering from ovarian cancer due to the high similarity to the constitution of.
- the number of types of microorganisms may be used as an evaluation index for the composition of the vaginal microflora. Comparing the number of bacteria present in the vaginal flora of ovarian cancer patients with the number of bacteria present in the vaginal flora of healthy pre-menopausal persons at the species and genus level, the number of patients with ovarian cancer is higher. ..
- the composition of the vaginal flora from the subject is:
- the composition of the vaginal flora derived from an ovarian cancer patient has high similarity, and the subject can be diagnosed as possibly having ovarian cancer.
- the number of types of microorganisms can be easily calculated from the data obtained by the 16S rRNA method, for example.
- the abundance ratio of a specific microorganism as an evaluation index of the structure of vaginal microflora.
- Analysis of the types of bacteria present in the vaginal flora of ovarian cancer patients and their abundance ratios revealed that the abundance ratios of several types differed from those in the vaginal flora of healthy pre-menopausal subjects. It was That is, when comparing the abundance ratio of specific bacteria present in the vaginal flora, the abundance ratio in the vaginal flora of patients with ovarian cancer is significantly higher than the abundance ratio in the vaginal flora of healthy menopause, or It turned out to be low.
- the vaginal flora of ovarian cancer patients are selected, and the abundance ratio of the bacteria in the vaginal flora of the subject is selected.
- the composition of vaginal flora from the subject is similar to that of ovarian cancer patients. Therefore, the subject can be diagnosed as possibly having ovarian cancer. It should be noted that it is appropriate to analyze the vaginal flora of ovarian cancer patients and healthy people before menopause, measure the abundance ratio of bacteria etc. present therein, and obtain the analysis results shown in FIGS. 2 to 10, for example. It is easy for a trader.
- genus other than Lactobacillus genus and Prevotella genus in vaginal flora samples derived from ovarian cancer patients for example, Propionibacterium genus, Staphylococcus genus, Corynebacterium genus, etc. abundance ratio, in the vaginal flora of healthy people before menopause It tended to be significantly higher than the abundance ratio.
- the abundance ratio of genus Lactobacillus and / or Prevotella in the vaginal flora derived from ovarian cancer patients tended to be significantly lower than the abundance ratio in the vaginal flora of healthy men before menopause.
- the abundance ratio is higher than the abundance ratio in the vaginal flora of healthy menopause subjects. If significantly higher, the composition of the vaginal flora from the subject is highly similar to the composition of the vaginal flora from the patient with ovarian cancer, and the subject is diagnosed as possibly having ovarian cancer. can do.
- the vaginal flora derived from the subject is The composition is highly similar to the composition of the vaginal flora derived from an ovarian cancer patient, and the subject can be diagnosed as possibly having ovarian cancer.
- the abundance ratio of Propionibacterium acnes in vaginal flora samples derived from patients with ovarian cancer is significantly higher than the abundance ratio in the vaginal flora of healthy subjects before menopause.
- the abundance ratio of Propionibacterium acnes in the vaginal flora of the premenopausal subject is significantly higher than the abundance ratio in the vaginal flora of the normal premenopausal subject
- the composition of the vaginal flora derived from the subject is It is highly similar to the composition of the vaginal flora derived from cancer patients, and the subject can be diagnosed as possibly having ovarian cancer.
- Figures 7 to 20 show the abundance ratios of each bacterial species at the species level for healthy individuals ( Figures 7 to 13) and ovarian cancer patients ( Figures 14 to 20).
- Figures 2 to 6 list the bacterial species analyzed.
- the lists in FIGS. 2-6 also include levels other than species and genera, namely, phylum, lepidoptera, eye, and family levels. Therefore, by comparing the data shown in FIG. 7 to FIG. 20 with the lists of FIG. 2 to FIG. You can also Needless to say, it can be used as an evaluation index for the composition of the vaginal flora with reference to FIGS. 2 to 6 and FIGS.
- Method 1-2 Analysis of Bacterial Flora
- a specimen for analyzing the bacterial flora a specimen for cervical cytology was used. Specifically, cells of the cervix were collected by cytobrush, and the collected material was stored in fixative. The collected specimen contains bacterial groups in the cervix. Extraction of bacterial DNA was performed using a part of the fixative containing bacteria. The extraction was performed according to the method of Kim et al., Which digests bacterial cell wall with lysozyme and protease (Kim et al., DNA Res. (2013) 20: 241-253. Doi: 10.1093 / dnares / dst006.).
- the extracted DNA was used for analysis of bacterial flora (FIG. 1, S1 to S3). Since the cervical flora has been shown to closely resemble the vaginal flora (Chen et al., Nat. Commun. (2017) 8: 875), the DNA prepared as described above was used. The sample was used as DNA from the vaginal flora.
- Genome analysis step Using the DNA solution obtained as described above, the 16S ribosomal RNA gene contained in the solution is amplified by the PCR method, and then a sequencer (NGS) (Illumina, trade name: Miseq) was used for DNA analysis.
- NGS sequencer
- the analysis results obtained as described above were analyzed using the pipeline analysis tool Qiime. Then, drawing and statistical analysis were performed based on the bacterial species identification results at the species level or genus level.
- the statistical analysis program R was used for drawing and statistical analysis.
- the Weighted Uni Frac index used for principal coordinate analysis was also calculated using Qiime.
- FIG. 21 shows the results summarized for the top 5 genera with the highest abundance ratio. See FIGS. 2 to 6 for species corresponding to each data shown in FIGS. 7 to 20. These results show that the composition of vaginal flora in ovarian cancer patients is diversified. This diversification was observed regardless of grade (1-4) differences.
- FIG. 22 shows the results focused on the subjects before menopause. From FIG.
- vaginal flora of healthy people before menopause has a simple structure in which the bacteria of the genus Lactobacillus or Prevotella occupy the majority, whereas the vaginal flora derived from an ovarian cancer patient is of the genus Propionibacterium . , Staphylococcus spp, Corynebacterium spp, etc. were found to be present in increasing proportions.
- the ratio of the bacteria of the top 5 genera present in the vaginal flora was further examined for premenopausal healthy subjects and ovarian cancer patients.
- the ratio of Lactobacillus genus alone (Fig. 23A) or the total of Lactobacillus genus and Prevotella genus (Fig. 23B) accounted for about 80% or more, whereas in ovarian cancer patients.
- Lactobacillus alone is about 20% or less (Fig. 23A)
- the total abundance of Lactobacillus and Prevotella is about 50% or less (Fig. 23B), which is low.
- Propionibacterium genus Comparable genus level
- Propionibacterium acnes species level comparison
- significantly higher proportions of the vaginal flora of ovarian cancer patients Was found (FIGS. 24A and B).
- the genus Corynebacterium (Fig. 25) and the genus Staphylococcus (Fig. 26) were also significantly higher in the vaginal flora of the ovarian cancer patients.
- the present invention provides a method for diagnosing whether or not a premenopausal subject has ovarian cancer.
- the diagnostic method of the present invention can be sufficiently used for diagnosing ovarian cancer at an early stage, and can be expected to be used in fields such as preventive medicine for ovarian cancer.
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Abstract
The present invention addresses the problem of providing an early diagnosis method for ovarian cancer. More specifically, the present invention pertains to a method for diagnosing ovarian cancer possibility in a premenopausal subject, wherein, when the composition of the vaginal microflora of the subject is highly similar to the composition of the vaginal microflora of an ovarian cancer patient, then the subject is diagnosed as having ovarian cancer at a high possibility.
Description
本発明は、卵巣がんの診断方法に関する。具体的には、膣微生物叢の叢構造に基づく卵巣がんの診断方法に関する。
The present invention relates to a method for diagnosing ovarian cancer. Specifically, it relates to a method for diagnosing ovarian cancer based on the flora structure of the vaginal microflora.
卵巣がんは、日本において、年間約1万人程度の女性に見つかっており、その患者数は増加傾向にある。卵巣がんは、その発生する場所によって上皮性、胚細胞性および性索間質性などの種類が存在するが、約9割以上は、上皮性の卵巣がんである。上皮性卵巣癌は主として4つの組織学的なサブグループから構成されている。すなわち、漿液性癌、粘液性癌、類内膜癌および明細胞癌である。なかでも、明細胞癌の割合が比較的多く、日本では、15-25%程度の罹患率である。
Ovarian cancer is found in about 10,000 women a year in Japan, and the number of patients is increasing. Ovarian cancer is classified into epithelial type, germ cell type, and sex cord interstitial type depending on the place where it occurs, but about 90% or more is epithelial ovarian cancer. Epithelial ovarian cancer is mainly composed of four histological subgroups. That is, serous cancer, mucinous cancer, endometrioid cancer and clear cell cancer. Among them, the ratio of clear cell cancer is relatively high, and the morbidity rate is about 15-25% in Japan.
卵巣がんは、初期の段階（ステージIまたはステージII）では無痛性でほとんど自覚症状がなく、下腹部のしこりや圧迫感、あるいは膀胱の圧迫による頻尿症状などが出て初めて受診により診断されることが多い。しかし、この状態ではすでにがんが進行していることが多い。進行期（ステージIIIまたはステージIV）の患者は、通常、予後が悪く（非特許文献１および非特許文献２）、早期発見の必要性の高いがんの一つである。
Ovarian cancer is painless in the early stages (stage I or stage II), has almost no subjective symptoms, and is diagnosed at the first consultation only after a lump or sensation of pressure in the lower abdomen or frequent urination caused by pressure on the bladder. Often. However, in this state, the cancer is often advanced. Patients in the advanced stage (stage III or stage IV) usually have a poor prognosis (Non-patent Document 1 and Non-patent Document 2), and are one of the cancers that require early detection.
現在、卵巣がんの診断は、超音波診断によって卵巣の腫大が確認された場合、血液検査と画像診断に基づいて行われている。血液検査においては、腫瘍マーカーの確認も行われており、代表的な腫瘍マーカーとしてCA125が用いられている。CA125は、卵巣がんに罹患している者の約8割が陽性を示すが、腹膜や子宮内膜にも発現しており、子宮内膜症などの場合にも陽性を示すため、悪性の卵巣がんに特異的とまでは言えないマーカーである。また、早期のがんや明細胞腺がんにおいては、CA125の上昇が乏しいため、卵巣がんの早期発見のためには、より特異性の高いバイオマーカーが切望されている。
Currently, ovarian cancer is diagnosed based on blood tests and diagnostic imaging if ovarian enlargement is confirmed by ultrasound diagnosis. Tumor markers have been confirmed in blood tests, and CA125 is used as a typical tumor marker. CA125 is positive in about 80% of people with ovarian cancer, but is also expressed in the peritoneum and endometrium, and is also positive in cases such as endometriosis. It is a marker that is not specific to ovarian cancer. Further, in early cancers and clear cell adenocarcinomas, the increase of CA125 is poor, and thus a biomarker with higher specificity is desired for early detection of ovarian cancer.
上記事情に鑑み、本発明は、卵巣がんを早期に診断するための方法を解決課題とする。
In view of the above circumstances, the present invention has as a problem to be solved a method for early diagnosis of ovarian cancer.
発明者らは、卵巣に漿液性癌または明細胞癌を生じた患者から採取した膣細菌叢検体について、細菌叢解析を行い、卵巣がん患者の膣細菌叢の構成は健常者のものと異なっていることを見いだした。
健常者の膣細菌叢は、閉経前においてはLactobacillus属またはPrevotella属の細菌が過半を占める単純な構造をしているが、閉経を迎えるとPropionibacterium属を筆頭種とする多様性に富んだ細菌叢に遷移することが知られている。
卵巣がん患者の膣細菌叢検体では、その大多数で閉経後健常者と同じ多様性に富んだ細菌叢構造が観察され、これは閉経の有無によらなかった。すなわち、閉経前の女性においては卵巣がんの有無と膣細菌叢構造の間に強い相関があることが初めて見いだされた。さらに、高い多様性という膣細菌叢の特性は初期のステージIの卵巣がん患者でも見いだされた。
従って、膣細菌叢は閉経前の女性において、卵巣がんの早期発見を可能とするバイオマーカーとして機能すると考えられる。
本発明は以上の知見に基づいて完成されたものである。 The inventors conducted a bacterial flora analysis on a vaginal flora sample collected from a patient who had serous cancer or clear cell carcinoma of the ovary, and the composition of the vaginal flora of an ovarian cancer patient was different from that of a healthy subject. I found that.
The vaginal flora of healthy people has a simple structure in which the majority of bacteria of the genus Lactobacillus or Prevotella is pre-menopausal, but when it reaches menopause, it is a diverse variety of bacteria with Propionibacterium as the first species. It is known to transition to.
In the vaginal flora samples from ovarian cancer patients, the majority of the vaginal flora samples showed the same diverse flora structure as those in postmenopausal healthy subjects, regardless of the presence or absence of menopause. That is, it was found for the first time that there was a strong correlation between the presence or absence of ovarian cancer and the vaginal flora structure in premenopausal women. In addition, the high diversity of the vaginal flora was also found in patients with early stage I ovarian cancer.
Therefore, the vaginal flora is considered to function as a biomarker that enables early detection of ovarian cancer in premenopausal women.
The present invention has been completed based on the above findings.
健常者の膣細菌叢は、閉経前においてはLactobacillus属またはPrevotella属の細菌が過半を占める単純な構造をしているが、閉経を迎えるとPropionibacterium属を筆頭種とする多様性に富んだ細菌叢に遷移することが知られている。
卵巣がん患者の膣細菌叢検体では、その大多数で閉経後健常者と同じ多様性に富んだ細菌叢構造が観察され、これは閉経の有無によらなかった。すなわち、閉経前の女性においては卵巣がんの有無と膣細菌叢構造の間に強い相関があることが初めて見いだされた。さらに、高い多様性という膣細菌叢の特性は初期のステージIの卵巣がん患者でも見いだされた。
従って、膣細菌叢は閉経前の女性において、卵巣がんの早期発見を可能とするバイオマーカーとして機能すると考えられる。
本発明は以上の知見に基づいて完成されたものである。 The inventors conducted a bacterial flora analysis on a vaginal flora sample collected from a patient who had serous cancer or clear cell carcinoma of the ovary, and the composition of the vaginal flora of an ovarian cancer patient was different from that of a healthy subject. I found that.
The vaginal flora of healthy people has a simple structure in which the majority of bacteria of the genus Lactobacillus or Prevotella is pre-menopausal, but when it reaches menopause, it is a diverse variety of bacteria with Propionibacterium as the first species. It is known to transition to.
In the vaginal flora samples from ovarian cancer patients, the majority of the vaginal flora samples showed the same diverse flora structure as those in postmenopausal healthy subjects, regardless of the presence or absence of menopause. That is, it was found for the first time that there was a strong correlation between the presence or absence of ovarian cancer and the vaginal flora structure in premenopausal women. In addition, the high diversity of the vaginal flora was also found in patients with early stage I ovarian cancer.
Therefore, the vaginal flora is considered to function as a biomarker that enables early detection of ovarian cancer in premenopausal women.
The present invention has been completed based on the above findings.
すなわち、本発明は、以下の（１）～（８）である。
（１）閉経前の被験者が卵巣がんに罹患している可能性を診断する方法であって、被験者の膣微生物叢の構成が、卵巣がん患者の膣微生物叢の構成と類似性が高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する方法。
（２）前記膣微生物叢の構成の評価指標が、多様性指数、微生物の種類数または特定の微生物の存在比率のいずれか１または複数であることを特徴とする上記（１）に記載の方法。
（３）前記評価指標が、多様性指数であって、被験者の膣細菌叢のSimpson指数が0.7以上および／またはShannon指数が2.0以上である場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（４）前記評価指標が、微生物の種類数であって、被験者の膣微生物叢の微生物の種類数が、閉経前の健常者の膣微生物叢の微生物の種類数より多い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（５）前記評価指標が、Lactobacillus属およびPrevotella属以外の属の存在比率であって、被験者の膣微生物叢における当該属の存在比率が、閉経前の健常者由来の膣微生物叢における当該属の存在比率より高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（６）前記Lactobacillus属およびPrevotella属以外の属が、Propionibacterium属、Corynebacterium属および／またはStaphylococcus属であることを特徴とする上記（５）に記載の方法。
（７）前記評価指標が、Lactobacillus属および／またはPrevotella属の存在比率であって、被験者の膣微生物叢における当該属の存在比率が、閉経前の健常者由来の膣微生物叢における当該属の存在比率より低い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（８）前記評価指標が、Propionibacterium acnes の存在比率であって、被験者の膣微生物叢における当該種の存在比率が、閉経前の健常者由来の膣微生物叢における当該種の存在比率より高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。 That is, the present invention is the following (1) to (8).
(1) A method of diagnosing the possibility that a premenopausal subject has ovarian cancer, wherein the composition of the vaginal microflora of the subject has a high similarity to that of the ovarian cancer patient. A method of diagnosing, where the subject may have ovarian cancer.
(2) The method according to (1) above, wherein the evaluation index of the constitution of the vaginal microflora is any one or more of a diversity index, the number of types of microorganisms or the abundance ratio of specific microorganisms. ..
(3) When the evaluation index is a diversity index and the Simpson index of vaginal flora of the subject is 0.7 or more and / or the Shannon index is 2.0 or more, the subject has ovarian cancer The method according to (2) above, wherein the method diagnoses the possibility.
(4) When the evaluation index is the number of types of microorganisms, and the number of types of microorganisms in the vaginal microflora of the subject is greater than the number of types of microorganisms in the vaginal microflora of a healthy person before menopause, the subject is The method according to (2) above, wherein diagnosing that the patient may have ovarian cancer.
(5) The evaluation index is the abundance ratio of genera other than Lactobacillus genus and Prevotella genus, and the abundance ratio of the genus in the vaginal microbiota of the subject is The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer when the proportion is higher than the abundance ratio.
(6) The method according to (5) above, wherein the genera other than the genus Lactobacillus and the genus Prevotella is genus Propionibacterium, genus Corynebacterium and / or Staphylococcus .
(7) The evaluation index is the abundance ratio of Lactobacillus genus and / or Prevotella genus, and the abundance ratio of the genus in the vaginal microbiota of the subject is The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer when the ratio is lower than the ratio.
(8) In the case where the evaluation index is the abundance ratio of Propionibacterium acnes, and the abundance ratio of the species in the vaginal microflora of the subject is higher than the abundance ratio of the species in the vaginal microflora derived from a premenopausal healthy subject. The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer.
（１）閉経前の被験者が卵巣がんに罹患している可能性を診断する方法であって、被験者の膣微生物叢の構成が、卵巣がん患者の膣微生物叢の構成と類似性が高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する方法。
（２）前記膣微生物叢の構成の評価指標が、多様性指数、微生物の種類数または特定の微生物の存在比率のいずれか１または複数であることを特徴とする上記（１）に記載の方法。
（３）前記評価指標が、多様性指数であって、被験者の膣細菌叢のSimpson指数が0.7以上および／またはShannon指数が2.0以上である場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（４）前記評価指標が、微生物の種類数であって、被験者の膣微生物叢の微生物の種類数が、閉経前の健常者の膣微生物叢の微生物の種類数より多い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（５）前記評価指標が、Lactobacillus属およびPrevotella属以外の属の存在比率であって、被験者の膣微生物叢における当該属の存在比率が、閉経前の健常者由来の膣微生物叢における当該属の存在比率より高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（６）前記Lactobacillus属およびPrevotella属以外の属が、Propionibacterium属、Corynebacterium属および／またはStaphylococcus属であることを特徴とする上記（５）に記載の方法。
（７）前記評価指標が、Lactobacillus属および／またはPrevotella属の存在比率であって、被験者の膣微生物叢における当該属の存在比率が、閉経前の健常者由来の膣微生物叢における当該属の存在比率より低い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。
（８）前記評価指標が、Propionibacterium acnes の存在比率であって、被験者の膣微生物叢における当該種の存在比率が、閉経前の健常者由来の膣微生物叢における当該種の存在比率より高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する上記（２）に記載の方法。 That is, the present invention is the following (1) to (8).
(1) A method of diagnosing the possibility that a premenopausal subject has ovarian cancer, wherein the composition of the vaginal microflora of the subject has a high similarity to that of the ovarian cancer patient. A method of diagnosing, where the subject may have ovarian cancer.
(2) The method according to (1) above, wherein the evaluation index of the constitution of the vaginal microflora is any one or more of a diversity index, the number of types of microorganisms or the abundance ratio of specific microorganisms. ..
(3) When the evaluation index is a diversity index and the Simpson index of vaginal flora of the subject is 0.7 or more and / or the Shannon index is 2.0 or more, the subject has ovarian cancer The method according to (2) above, wherein the method diagnoses the possibility.
(4) When the evaluation index is the number of types of microorganisms, and the number of types of microorganisms in the vaginal microflora of the subject is greater than the number of types of microorganisms in the vaginal microflora of a healthy person before menopause, the subject is The method according to (2) above, wherein diagnosing that the patient may have ovarian cancer.
(5) The evaluation index is the abundance ratio of genera other than Lactobacillus genus and Prevotella genus, and the abundance ratio of the genus in the vaginal microbiota of the subject is The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer when the proportion is higher than the abundance ratio.
(6) The method according to (5) above, wherein the genera other than the genus Lactobacillus and the genus Prevotella is genus Propionibacterium, genus Corynebacterium and / or Staphylococcus .
(7) The evaluation index is the abundance ratio of Lactobacillus genus and / or Prevotella genus, and the abundance ratio of the genus in the vaginal microbiota of the subject is The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer when the ratio is lower than the ratio.
(8) In the case where the evaluation index is the abundance ratio of Propionibacterium acnes, and the abundance ratio of the species in the vaginal microflora of the subject is higher than the abundance ratio of the species in the vaginal microflora derived from a premenopausal healthy subject. The method according to (2) above, wherein the subject is diagnosed as possibly having ovarian cancer.
本発明の卵巣がんの診断方法は、自覚症状のない初期の段階においても、卵巣がんの罹患の可能性を判断することができる。そのため、本発明の診断方法を用いることで、卵巣がんの早期治療に着手することが可能になる
The method for diagnosing ovarian cancer of the present invention can judge the possibility of ovarian cancer even at an early stage without subjective symptoms. Therefore, by using the diagnostic method of the present invention, it becomes possible to undertake early treatment of ovarian cancer.
本発明は、閉経前の被験者が卵巣がんに罹患しているかどうか、または、罹患している可能性があるかどうかを診断する方法に関するものである。また、本方法は、卵巣がんであることの確定診断の補助としての利用性も高い。
本発明にかかる卵巣がん罹患の有無、または罹患の可能性の有無を診断する方法（以下「本発明の診断方法」とも記載する）は、卵巣がん患者の膣微生物叢（例えば、膣細菌叢など）の微生物の構成が、健常者の膣微生物叢の構成とは異なり、より多様であることに基づいている。なお、膣細菌叢と子宮頸部細菌叢の構成は酷似していることが知られており、本明細書の記載においては、子宮頸部由来の微生物叢（特に、細菌叢）と膣由来の微生物叢（特に細菌叢）は同等であるものとする。 The present invention relates to methods of diagnosing whether a pre-menopausal subject has, or is likely to have, ovarian cancer. The method is also highly useful as an aid in the definitive diagnosis of ovarian cancer.
The method for diagnosing the presence or absence of ovarian cancer according to the present invention (hereinafter, also referred to as the “diagnosis method of the present invention”) is a vaginal microbiota (eg, vaginal bacteria) of an ovarian cancer patient. The flora, etc.) is based on the fact that the composition of the microorganisms is different from the composition of the vaginal microflora of healthy individuals, and is more diverse. It is known that the configurations of the vaginal flora and the cervical flora are very similar, and in the description of the present specification, the cervical microflora (particularly the bacterial flora) and the vaginal flora are Microbiota (especially bacterial flora) should be comparable.
本発明にかかる卵巣がん罹患の有無、または罹患の可能性の有無を診断する方法（以下「本発明の診断方法」とも記載する）は、卵巣がん患者の膣微生物叢（例えば、膣細菌叢など）の微生物の構成が、健常者の膣微生物叢の構成とは異なり、より多様であることに基づいている。なお、膣細菌叢と子宮頸部細菌叢の構成は酷似していることが知られており、本明細書の記載においては、子宮頸部由来の微生物叢（特に、細菌叢）と膣由来の微生物叢（特に細菌叢）は同等であるものとする。 The present invention relates to methods of diagnosing whether a pre-menopausal subject has, or is likely to have, ovarian cancer. The method is also highly useful as an aid in the definitive diagnosis of ovarian cancer.
The method for diagnosing the presence or absence of ovarian cancer according to the present invention (hereinafter, also referred to as the “diagnosis method of the present invention”) is a vaginal microbiota (eg, vaginal bacteria) of an ovarian cancer patient. The flora, etc.) is based on the fact that the composition of the microorganisms is different from the composition of the vaginal microflora of healthy individuals, and is more diverse. It is known that the configurations of the vaginal flora and the cervical flora are very similar, and in the description of the present specification, the cervical microflora (particularly the bacterial flora) and the vaginal flora are Microbiota (especially bacterial flora) should be comparable.
本発明の実施形態は、閉経前の被験者が卵巣がんに罹患している可能性を診断する方法であって、被験者の膣微生物叢の構成が、卵巣がん患者の膣微生物叢の構成と類似性が高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する方法である。
ここで、膣微生物叢の「構成」とは、微生物叢の組成のことで、微生物叢の要素である微生物の数、微生物の種類の多寡、微生物の存在比率などによって特徴づけられる微生物叢の状態のことで、特に限定はしないが、例えば、微生物の数、微生物の種類数、微生物叢の多様性および各微生物の存在比率などによって評価することができる。発明者らは、細菌叢に着目して解析を進めた結果、卵巣がん患者の膣微生物叢の構成は、閉経前の健常者の膣微生物叢の構成と比較して、多様性が高く、存在する微生物の種類が豊富であり、また、各微生物の存在比率が両者で異なることを明らかにした。 An embodiment of the present invention is a method for diagnosing the possibility that a pre-menopausal subject has ovarian cancer, wherein the composition of the vaginal microflora of the subject is the composition of the vaginal microbiota of the ovarian cancer patient. When the similarity is high, the subject is a method of diagnosing that the subject may have ovarian cancer.
Here, the “composition” of the vaginal microflora refers to the composition of the microflora, and the state of the microflora characterized by the number of microbes that are elements of the microflora, the number of types of microbes, and the abundance ratio of the microbes. Therefore, although not particularly limited, it can be evaluated by, for example, the number of microorganisms, the number of types of microorganisms, the diversity of microflora, the abundance ratio of each microorganism, and the like. The inventors, as a result of advancing the analysis focusing on the bacterial flora, have a higher diversity in the composition of the vaginal microflora of patients with ovarian cancer, as compared with the composition of the vaginal microflora of healthy people before menopause, It was clarified that the kinds of microorganisms present are abundant and that the abundance ratio of each microorganism is different in both.
ここで、膣微生物叢の「構成」とは、微生物叢の組成のことで、微生物叢の要素である微生物の数、微生物の種類の多寡、微生物の存在比率などによって特徴づけられる微生物叢の状態のことで、特に限定はしないが、例えば、微生物の数、微生物の種類数、微生物叢の多様性および各微生物の存在比率などによって評価することができる。発明者らは、細菌叢に着目して解析を進めた結果、卵巣がん患者の膣微生物叢の構成は、閉経前の健常者の膣微生物叢の構成と比較して、多様性が高く、存在する微生物の種類が豊富であり、また、各微生物の存在比率が両者で異なることを明らかにした。 An embodiment of the present invention is a method for diagnosing the possibility that a pre-menopausal subject has ovarian cancer, wherein the composition of the vaginal microflora of the subject is the composition of the vaginal microbiota of the ovarian cancer patient. When the similarity is high, the subject is a method of diagnosing that the subject may have ovarian cancer.
Here, the “composition” of the vaginal microflora refers to the composition of the microflora, and the state of the microflora characterized by the number of microbes that are elements of the microflora, the number of types of microbes, and the abundance ratio of the microbes. Therefore, although not particularly limited, it can be evaluated by, for example, the number of microorganisms, the number of types of microorganisms, the diversity of microflora, the abundance ratio of each microorganism, and the like. The inventors, as a result of advancing the analysis focusing on the bacterial flora, have a higher diversity in the composition of the vaginal microflora of patients with ovarian cancer, as compared with the composition of the vaginal microflora of healthy people before menopause, It was clarified that the kinds of microorganisms present are abundant and that the abundance ratio of each microorganism is different in both.
本発明の実施形態において、「微生物」の種類は特に限定されず、例えば、細菌や真菌などのことで、特に細菌が好ましく、解析対象の微生物叢が細菌叢であってもよい。また、微生物叢の構成について言及する場合、微生物の「種類」とは、門、綱、目、科、属および種のいずれに関するものであってもよい。
また、微生物叢の構成は、上述したように、例えば、存在する微生物の数、微生物の種類、微生物の存在比率および微生物叢の多様性指数（例えば、Simpson指数やShannon指数など）などを指標にして評価することができるが、これらの評価指標に限定されるものではない。 In the embodiment of the present invention, the type of “microorganism” is not particularly limited, and for example, bacteria, fungi, etc. are particularly preferable, and the microbial flora to be analyzed may be the microbial flora. When referring to the composition of the microbiota, the "type" of the microorganism may be any of phylum, class, order, family, genus and species.
The composition of the microflora, as described above, for example, the number of microorganisms present, the type of microorganisms, the abundance ratio of microorganisms and the diversity index of the microflora (for example, Simpson index or Shannon index, etc.) are used as indicators. However, it is not limited to these evaluation indicators.
また、微生物叢の構成は、上述したように、例えば、存在する微生物の数、微生物の種類、微生物の存在比率および微生物叢の多様性指数（例えば、Simpson指数やShannon指数など）などを指標にして評価することができるが、これらの評価指標に限定されるものではない。 In the embodiment of the present invention, the type of “microorganism” is not particularly limited, and for example, bacteria, fungi, etc. are particularly preferable, and the microbial flora to be analyzed may be the microbial flora. When referring to the composition of the microbiota, the "type" of the microorganism may be any of phylum, class, order, family, genus and species.
The composition of the microflora, as described above, for example, the number of microorganisms present, the type of microorganisms, the abundance ratio of microorganisms and the diversity index of the microflora (for example, Simpson index or Shannon index, etc.) are used as indicators. However, it is not limited to these evaluation indicators.
膣微生物叢の構成を調べるには、微生物叢に存在する微生物の種類および各微生物の数などを測定し、得られた値に基づいて実施することができる。微生物の同定は、いかなる方法を用いて行ってもよく、例えば、微生物のゲノムDNA解析（例えば、16S rRNAまたは16S rRNA以外のゲノム領域の配列解読、特定の微生物に特異的なプライマーを用いたPCRにより解析など）、微生物特異的なタンパク質や代謝産物を検出するメタボローム解析、微生物の形態に基づく判別、培地上での増殖に必要な条件に基づく判別、微生物が示す生化学的性質（糖発酵性やカタラーゼ活性など）に基づく判別などにより実施することができる。詳細は、例えば、Microbiology, 8th ed. （著者Jacquelyn G. Black、出版John Wiley & Sons Singapore Pte. Ltd.）などを参照のこと。
被験者（または、卵巣がん患者および健常者）の膣微生物叢検体は、被験者の膣や子宮頸部等から、脱脂綿やブラシなどでこすり取る方法（細胞診等）などにより採取することができる。膣微生物叢検体中の微生物叢の構成を調べるために、例えば、上述のDNA解析を行う場合、検体からのDNAの抽出は、当業者において周知な方法、例えば、溶菌酵素法などにより実施することができる。この場合、膣微生物叢検体に含まれるDNAを可能な限りすべて抽出することが望ましい。微生物叢に存在するDNAを網羅的に抽出した後、DNAの塩基配列を、例えば、次世代型超高速シークエンサー装置等で解析して、当該微生物叢に存在するすべての微生物のDNA配列情報を取得する。そして、得られた微生物のDNA配列情報に基づいて微生物叢の構成を明らかにすることができる。 The composition of the vaginal microflora can be examined by measuring the types of microorganisms present in the microflora, the number of each microorganism, and the like, and carrying out based on the obtained values. Microorganisms may be identified by any method, for example, genomic DNA analysis of microorganisms (for example, sequencing of 16S rRNA or a genomic region other than 16S rRNA, PCR using primers specific to a particular microorganism). Analysis, etc.), metabolome analysis to detect microorganism-specific proteins and metabolites, discrimination based on the morphology of the microorganism, discrimination based on the conditions necessary for growth on the medium, biochemical properties (sugar fermentation) Or catalase activity) and the like. For details, see, for example, Microbiology, 8 th ed. (Author Jacquelyn G. Black, published by John Wiley & Sons Singapore Pte. Ltd.).
The vaginal microflora sample of a subject (or an ovarian cancer patient and a healthy person) can be collected from the subject's vagina, cervix, etc. by a method such as scraping with absorbent cotton or a brush (cytology etc.). In order to examine the composition of the microflora in the vaginal microflora sample, for example, when performing the above-mentioned DNA analysis, extraction of DNA from the sample should be performed by a method well known to those skilled in the art, for example, a lytic enzyme method. You can In this case, it is desirable to extract all the DNA contained in the vaginal microflora sample as much as possible. After comprehensively extracting the DNA present in the microflora, the nucleotide sequence of the DNA is analyzed by, for example, a next-generation ultra-high speed sequencer device, etc., and the DNA sequence information of all the microorganisms present in the microbiota is acquired. To do. Then, the composition of the microflora can be clarified based on the obtained DNA sequence information of the microorganism.
被験者（または、卵巣がん患者および健常者）の膣微生物叢検体は、被験者の膣や子宮頸部等から、脱脂綿やブラシなどでこすり取る方法（細胞診等）などにより採取することができる。膣微生物叢検体中の微生物叢の構成を調べるために、例えば、上述のDNA解析を行う場合、検体からのDNAの抽出は、当業者において周知な方法、例えば、溶菌酵素法などにより実施することができる。この場合、膣微生物叢検体に含まれるDNAを可能な限りすべて抽出することが望ましい。微生物叢に存在するDNAを網羅的に抽出した後、DNAの塩基配列を、例えば、次世代型超高速シークエンサー装置等で解析して、当該微生物叢に存在するすべての微生物のDNA配列情報を取得する。そして、得られた微生物のDNA配列情報に基づいて微生物叢の構成を明らかにすることができる。 The composition of the vaginal microflora can be examined by measuring the types of microorganisms present in the microflora, the number of each microorganism, and the like, and carrying out based on the obtained values. Microorganisms may be identified by any method, for example, genomic DNA analysis of microorganisms (for example, sequencing of 16S rRNA or a genomic region other than 16S rRNA, PCR using primers specific to a particular microorganism). Analysis, etc.), metabolome analysis to detect microorganism-specific proteins and metabolites, discrimination based on the morphology of the microorganism, discrimination based on the conditions necessary for growth on the medium, biochemical properties (sugar fermentation) Or catalase activity) and the like. For details, see, for example, Microbiology, 8 th ed. (Author Jacquelyn G. Black, published by John Wiley & Sons Singapore Pte. Ltd.).
The vaginal microflora sample of a subject (or an ovarian cancer patient and a healthy person) can be collected from the subject's vagina, cervix, etc. by a method such as scraping with absorbent cotton or a brush (cytology etc.). In order to examine the composition of the microflora in the vaginal microflora sample, for example, when performing the above-mentioned DNA analysis, extraction of DNA from the sample should be performed by a method well known to those skilled in the art, for example, a lytic enzyme method. You can In this case, it is desirable to extract all the DNA contained in the vaginal microflora sample as much as possible. After comprehensively extracting the DNA present in the microflora, the nucleotide sequence of the DNA is analyzed by, for example, a next-generation ultra-high speed sequencer device, etc., and the DNA sequence information of all the microorganisms present in the microbiota is acquired. To do. Then, the composition of the microflora can be clarified based on the obtained DNA sequence information of the microorganism.
細菌叢の解析においては、特に、16S rRNA法がよく用いられており、取得した16S rRNA配列情報に基づいて細菌叢に含まれる細菌の種類や量を知ることができる。得られた結果をさらに主座標分析（Principal Coordinates Analysis：PCoA）やクラスタリング解析などで解析することにより細菌叢の構成の検体間での比較を行うことができる。
細菌叢の16S rRNA法による解析は、企業等（例えば、富士フイルム和光純薬株式会社、Takara Incなど）に委託して実施してもよい。 In the analysis of bacterial flora, the 16S rRNA method is particularly often used, and the type and amount of bacteria contained in the bacterial flora can be known based on the obtained 16S rRNA sequence information. By further analyzing the obtained results by principal coordinate analysis (PCoA), clustering analysis, or the like, it is possible to compare the samples of the bacterial flora composition among samples.
The analysis of the bacterial flora by the 16S rRNA method may be carried out by entrusting it to a company (for example, Fujifilm Wako Pure Chemical Industries, Ltd., Takara Inc., etc.).
細菌叢の16S rRNA法による解析は、企業等（例えば、富士フイルム和光純薬株式会社、Takara Incなど）に委託して実施してもよい。 In the analysis of bacterial flora, the 16S rRNA method is particularly often used, and the type and amount of bacteria contained in the bacterial flora can be known based on the obtained 16S rRNA sequence information. By further analyzing the obtained results by principal coordinate analysis (PCoA), clustering analysis, or the like, it is possible to compare the samples of the bacterial flora composition among samples.
The analysis of the bacterial flora by the 16S rRNA method may be carried out by entrusting it to a company (for example, Fujifilm Wako Pure Chemical Industries, Ltd., Takara Inc., etc.).
膣微生物叢の構成の評価指標として多様性指数を用いる場合、被験者の膣微生物叢の多様性指数が、卵巣がん患者の多様性指数に近い場合に、当該被験者は卵巣がんに罹患している可能性があると診断することができる。具体的には、膣細菌叢の多様性を評価する場合、卵巣がん患者の膣細菌叢の多様性を、例えば、Simpson指数で表すと、多くの場合0.7以上の値を示し、Shannon指数で表すと、多くの場合2.0以上の値を示す。従って、閉経前の被験者由来の膣細菌叢のSimpson指数が0.7以上のとき、または、Shannon指数が2.0以上のときに、被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。
When the diversity index is used as an evaluation index for the composition of the vaginal microbiota, the subject has ovarian cancer when the diversity index of the vaginal microbiota of the subject is close to the diversity index of the ovarian cancer patient. It can be diagnosed as possible. Specifically, when evaluating the diversity of the vaginal flora, the diversity of the vaginal flora in patients with ovarian cancer, for example, when expressed by the Simpson index, often shows a value of 0.7 or more, and the Shannon index. When expressed, it often shows a value of 2.0 or more. Therefore, when the Simpson index of the vaginal flora from the premenopausal subject is 0.7 or more, or when the Shannon index is 2.0 or more, the composition of the vaginal flora from the subject is the vaginal flora from the ovarian cancer patient. It is possible to diagnose that the subject may be suffering from ovarian cancer due to the high similarity to the constitution of.
膣微生物叢の構成の評価指標として、微生物の種類数を用いてもよい。卵巣がん患者の膣細菌叢に存在する細菌の種類数を、種および属レベルで閉経前の健常者の膣細菌叢に存在する細菌の種類数を比較すると、卵巣がん患者の方が多い。従って、閉経前の被験者の膣細菌物叢の細菌の種類数が、閉経前の健常者の膣細菌叢の微生物の種類数より有意に多い場合に、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。
微生物の種類数は、例えば、16S rRNA法で取得したデータより容易に算定することができる。 The number of types of microorganisms may be used as an evaluation index for the composition of the vaginal microflora. Comparing the number of bacteria present in the vaginal flora of ovarian cancer patients with the number of bacteria present in the vaginal flora of healthy pre-menopausal persons at the species and genus level, the number of patients with ovarian cancer is higher. .. Therefore, when the number of types of bacteria in the vaginal flora of the pre-menopausal subject is significantly larger than the number of types of microorganisms in the vaginal flora of the pre-menopausal healthy subject, the composition of the vaginal flora from the subject is: The composition of the vaginal flora derived from an ovarian cancer patient has high similarity, and the subject can be diagnosed as possibly having ovarian cancer.
The number of types of microorganisms can be easily calculated from the data obtained by the 16S rRNA method, for example.
微生物の種類数は、例えば、16S rRNA法で取得したデータより容易に算定することができる。 The number of types of microorganisms may be used as an evaluation index for the composition of the vaginal microflora. Comparing the number of bacteria present in the vaginal flora of ovarian cancer patients with the number of bacteria present in the vaginal flora of healthy pre-menopausal persons at the species and genus level, the number of patients with ovarian cancer is higher. .. Therefore, when the number of types of bacteria in the vaginal flora of the pre-menopausal subject is significantly larger than the number of types of microorganisms in the vaginal flora of the pre-menopausal healthy subject, the composition of the vaginal flora from the subject is: The composition of the vaginal flora derived from an ovarian cancer patient has high similarity, and the subject can be diagnosed as possibly having ovarian cancer.
The number of types of microorganisms can be easily calculated from the data obtained by the 16S rRNA method, for example.
また、膣微生物叢の構成の評価指標として、特定の微生物の存在比率を用いてもよい。卵巣がん患者の膣細菌叢に存在する細菌の種類とその存在比率を解析すると、いくつかの種類の存在比率が、閉経前の健常者の膣細菌叢における存在比率と異なることが明らかになった。すなわち、膣細菌叢に存在する特定の細菌の存在比率を比較すると、卵巣がん患者の膣細菌叢における存在比率が、閉経前の健常者の膣細菌叢における存在比率よりも有意に高い、または、低いことが分かった。従って、閉経前の健常者の膣細菌叢と比較して、卵巣がん患者の膣細菌叢における存在比率が高い、または、低い細菌を選択し、被験者の膣細菌叢における当該細菌の存在比率を測定し、閉経前の健常者の膣細菌叢における存在比率よりも高い、または、低い場合には、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。
なお、卵巣がん患者と閉経前の健常者の膣細菌叢を解析し、そこに存在する細菌等の存在比率を測定し、例えば、図２～図１０に示す解析結果を得ることは、当業者であれば容易である。 Moreover, you may use the abundance ratio of a specific microorganism as an evaluation index of the structure of vaginal microflora. Analysis of the types of bacteria present in the vaginal flora of ovarian cancer patients and their abundance ratios revealed that the abundance ratios of several types differed from those in the vaginal flora of healthy pre-menopausal subjects. It was That is, when comparing the abundance ratio of specific bacteria present in the vaginal flora, the abundance ratio in the vaginal flora of patients with ovarian cancer is significantly higher than the abundance ratio in the vaginal flora of healthy menopause, or It turned out to be low. Therefore, compared with the vaginal flora of healthy people before menopause, bacteria with a high or low abundance in the vaginal flora of ovarian cancer patients are selected, and the abundance ratio of the bacteria in the vaginal flora of the subject is selected. When measured and higher or lower than the prevalence of vaginal flora in healthy premenopausal subjects, the composition of vaginal flora from the subject is similar to that of ovarian cancer patients. Therefore, the subject can be diagnosed as possibly having ovarian cancer.
It should be noted that it is appropriate to analyze the vaginal flora of ovarian cancer patients and healthy people before menopause, measure the abundance ratio of bacteria etc. present therein, and obtain the analysis results shown in FIGS. 2 to 10, for example. It is easy for a trader.
なお、卵巣がん患者と閉経前の健常者の膣細菌叢を解析し、そこに存在する細菌等の存在比率を測定し、例えば、図２～図１０に示す解析結果を得ることは、当業者であれば容易である。 Moreover, you may use the abundance ratio of a specific microorganism as an evaluation index of the structure of vaginal microflora. Analysis of the types of bacteria present in the vaginal flora of ovarian cancer patients and their abundance ratios revealed that the abundance ratios of several types differed from those in the vaginal flora of healthy pre-menopausal subjects. It was That is, when comparing the abundance ratio of specific bacteria present in the vaginal flora, the abundance ratio in the vaginal flora of patients with ovarian cancer is significantly higher than the abundance ratio in the vaginal flora of healthy menopause, or It turned out to be low. Therefore, compared with the vaginal flora of healthy people before menopause, bacteria with a high or low abundance in the vaginal flora of ovarian cancer patients are selected, and the abundance ratio of the bacteria in the vaginal flora of the subject is selected. When measured and higher or lower than the prevalence of vaginal flora in healthy premenopausal subjects, the composition of vaginal flora from the subject is similar to that of ovarian cancer patients. Therefore, the subject can be diagnosed as possibly having ovarian cancer.
It should be noted that it is appropriate to analyze the vaginal flora of ovarian cancer patients and healthy people before menopause, measure the abundance ratio of bacteria etc. present therein, and obtain the analysis results shown in FIGS. 2 to 10, for example. It is easy for a trader.
例えば、卵巣がん患者由来の膣細菌叢検体中におけるLactobacillus属およびPrevotella属以外の属、例えば、Propionibacterium属、Staphylococcus属、Corynebacterium属などの存在比率は、閉経前の健常者の膣細菌叢における存在比率よりも有意に高い傾向にあった。また、卵巣がん患者由来の膣細菌叢におけるLactobacillus属および／またはPrevotella属の存在比率が閉経前の健常者の膣細菌叢における存在比率よりも有意に低い傾向にあった。
従って、閉経前の被験者の膣細菌叢におけるLactobacillus属およびPrevotella属以外の属、例えば、Propionibacterium属、Staphylococcus属、Corynebacterium属などの存在比率が、閉経前の健常者の膣細菌叢における存在比率よりも有意に高い場合、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。あるいは、閉経前の被験者の膣細菌叢におけるLactobacillus属および／またはPrevotella属の存在比率が、閉経前の健常者の膣細菌叢における存在比率よりも有意に低い場合、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。 For example, genus other than Lactobacillus genus and Prevotella genus in vaginal flora samples derived from ovarian cancer patients, for example, Propionibacterium genus, Staphylococcus genus, Corynebacterium genus, etc. abundance ratio, in the vaginal flora of healthy people before menopause It tended to be significantly higher than the abundance ratio. In addition, the abundance ratio of genus Lactobacillus and / or Prevotella in the vaginal flora derived from ovarian cancer patients tended to be significantly lower than the abundance ratio in the vaginal flora of healthy men before menopause.
Therefore, genera other than Lactobacillus and Prevotella in the vaginal flora of premenopausal subjects, for example, Propionibacterium genus, Staphylococcus genus, Corynebacterium genus, the abundance ratio is higher than the abundance ratio in the vaginal flora of healthy menopause subjects. If significantly higher, the composition of the vaginal flora from the subject is highly similar to the composition of the vaginal flora from the patient with ovarian cancer, and the subject is diagnosed as possibly having ovarian cancer. can do. Alternatively, if the prevalence of Lactobacillus and / or Prevotella in the vaginal flora of a premenopausal subject is significantly lower than the prevalence of the vaginal flora of a premenopausal normal subject, the vaginal flora derived from the subject is The composition is highly similar to the composition of the vaginal flora derived from an ovarian cancer patient, and the subject can be diagnosed as possibly having ovarian cancer.
従って、閉経前の被験者の膣細菌叢におけるLactobacillus属およびPrevotella属以外の属、例えば、Propionibacterium属、Staphylococcus属、Corynebacterium属などの存在比率が、閉経前の健常者の膣細菌叢における存在比率よりも有意に高い場合、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。あるいは、閉経前の被験者の膣細菌叢におけるLactobacillus属および／またはPrevotella属の存在比率が、閉経前の健常者の膣細菌叢における存在比率よりも有意に低い場合、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来の膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。 For example, genus other than Lactobacillus genus and Prevotella genus in vaginal flora samples derived from ovarian cancer patients, for example, Propionibacterium genus, Staphylococcus genus, Corynebacterium genus, etc. abundance ratio, in the vaginal flora of healthy people before menopause It tended to be significantly higher than the abundance ratio. In addition, the abundance ratio of genus Lactobacillus and / or Prevotella in the vaginal flora derived from ovarian cancer patients tended to be significantly lower than the abundance ratio in the vaginal flora of healthy men before menopause.
Therefore, genera other than Lactobacillus and Prevotella in the vaginal flora of premenopausal subjects, for example, Propionibacterium genus, Staphylococcus genus, Corynebacterium genus, the abundance ratio is higher than the abundance ratio in the vaginal flora of healthy menopause subjects. If significantly higher, the composition of the vaginal flora from the subject is highly similar to the composition of the vaginal flora from the patient with ovarian cancer, and the subject is diagnosed as possibly having ovarian cancer. can do. Alternatively, if the prevalence of Lactobacillus and / or Prevotella in the vaginal flora of a premenopausal subject is significantly lower than the prevalence of the vaginal flora of a premenopausal normal subject, the vaginal flora derived from the subject is The composition is highly similar to the composition of the vaginal flora derived from an ovarian cancer patient, and the subject can be diagnosed as possibly having ovarian cancer.
また、種レベルで同様の解析を行うと、例えば、卵巣がん患者由来の膣細菌叢検体中におけるPropionibacterium acnes の存在比率は、閉経前の健常者の膣細菌叢における存在比率よりも有意に高い傾向にあった。
従って、閉経前の被験者の膣細菌叢におけるPropionibacterium acnesなどの存在比率が閉経前の健常者の膣細菌叢における存在比率よりも有意に高い場合、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来のー膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。 In addition, when a similar analysis is performed at the species level, for example, the abundance ratio of Propionibacterium acnes in vaginal flora samples derived from patients with ovarian cancer is significantly higher than the abundance ratio in the vaginal flora of healthy subjects before menopause. There was a tendency.
Therefore, when the abundance ratio of Propionibacterium acnes in the vaginal flora of the premenopausal subject is significantly higher than the abundance ratio in the vaginal flora of the normal premenopausal subject, the composition of the vaginal flora derived from the subject is It is highly similar to the composition of the vaginal flora derived from cancer patients, and the subject can be diagnosed as possibly having ovarian cancer.
従って、閉経前の被験者の膣細菌叢におけるPropionibacterium acnesなどの存在比率が閉経前の健常者の膣細菌叢における存在比率よりも有意に高い場合、当該被験者由来の膣細菌叢の構成は、卵巣がん患者由来のー膣細菌叢の構成と類似性が高く、当該被験者は卵巣がんに罹患している可能性があると診断することができる。 In addition, when a similar analysis is performed at the species level, for example, the abundance ratio of Propionibacterium acnes in vaginal flora samples derived from patients with ovarian cancer is significantly higher than the abundance ratio in the vaginal flora of healthy subjects before menopause. There was a tendency.
Therefore, when the abundance ratio of Propionibacterium acnes in the vaginal flora of the premenopausal subject is significantly higher than the abundance ratio in the vaginal flora of the normal premenopausal subject, the composition of the vaginal flora derived from the subject is It is highly similar to the composition of the vaginal flora derived from cancer patients, and the subject can be diagnosed as possibly having ovarian cancer.
図７～２０に、健常者（図７～図１３）および卵巣がん患者（図１４～図２０）について、種レベルでの各細菌種の存在比率をまとめた。図２～図６には解析した細菌種のリストを挙げる。図２～図６のリストには、種および属以外のレベル、すなわち門、綱、目、科の各レベルも記載してある。そのため、図７～図２０に示したデータと図２～図６のリストを照らし合わせることで、種および属以外のレベルの存在比率を算出し、膣細菌叢の構成の評価指標として使用することもできる。なお、本文中に例示した以外の種や属の存在比率に関し、図２～図６および図７～図２０を参照して膣細菌叢の構成の評価指標として使用することができることは言うまでもない。
Figures 7 to 20 show the abundance ratios of each bacterial species at the species level for healthy individuals (Figures 7 to 13) and ovarian cancer patients (Figures 14 to 20). Figures 2 to 6 list the bacterial species analyzed. The lists in FIGS. 2-6 also include levels other than species and genera, namely, phylum, lepidoptera, eye, and family levels. Therefore, by comparing the data shown in FIG. 7 to FIG. 20 with the lists of FIG. 2 to FIG. You can also Needless to say, it can be used as an evaluation index for the composition of the vaginal flora with reference to FIGS. 2 to 6 and FIGS.
本明細書が英語に翻訳されて、単数形の「a」、「an」および「the」の単語が含まれる場合、文脈から明らかにそうでないことが示されていない限り、単数のみならず複数のものも含むものとする。
以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 Where the specification is translated into English and includes the singular forms of the words "a", "an" and "the", the singular as well as the plural unless the context clearly indicates otherwise. It includes the thing of.
The present invention will be further described below with reference to examples, but the examples are merely examples of the embodiments of the present invention and do not limit the scope of the present invention.
以下に実施例を示してさらに本発明の説明を行うが、本実施例は、あくまでも本発明の実施形態の例示にすぎず、本発明の範囲を限定するものではない。 Where the specification is translated into English and includes the singular forms of the words "a", "an" and "the", the singular as well as the plural unless the context clearly indicates otherwise. It includes the thing of.
The present invention will be further described below with reference to examples, but the examples are merely examples of the embodiments of the present invention and do not limit the scope of the present invention.
１．方法
１－２．細菌叢の解析
細菌叢の解析のための検体は、子宮頸部細胞診用の検体を用いた。具体的には、子宮頸部の細胞を cytobrushで採取し、採取物は固定液の中に保存した。採取した検体には子宮頸部の細菌群が含まれている。細菌を含む固定液の一部を用いて細菌DNAの抽出を行なった。抽出はリゾチームやプロテアーゼにより細菌細胞壁を消化するKimらの方法に従って行った（Kimら,DNA Res. (2013) 20:241-253. doi: 10.1093/dnares/dst006.）。抽出されたDNAを、細菌叢の解析に用いた（図１、S1～S3）。
なお、子宮頸部の細菌叢は、膣内細菌叢と酷似していることが示されていることから（Chenら, Nat. Commun. (2017) 8:875）、上記のように調製したDNAサンプルを膣細菌叢由来のDNAとして用いた。 1. Method 1-2. Analysis of Bacterial Flora As a specimen for analyzing the bacterial flora, a specimen for cervical cytology was used. Specifically, cells of the cervix were collected by cytobrush, and the collected material was stored in fixative. The collected specimen contains bacterial groups in the cervix. Extraction of bacterial DNA was performed using a part of the fixative containing bacteria. The extraction was performed according to the method of Kim et al., Which digests bacterial cell wall with lysozyme and protease (Kim et al., DNA Res. (2013) 20: 241-253. Doi: 10.1093 / dnares / dst006.). The extracted DNA was used for analysis of bacterial flora (FIG. 1, S1 to S3).
Since the cervical flora has been shown to closely resemble the vaginal flora (Chen et al., Nat. Commun. (2017) 8: 875), the DNA prepared as described above was used. The sample was used as DNA from the vaginal flora.
１－２．細菌叢の解析
細菌叢の解析のための検体は、子宮頸部細胞診用の検体を用いた。具体的には、子宮頸部の細胞を cytobrushで採取し、採取物は固定液の中に保存した。採取した検体には子宮頸部の細菌群が含まれている。細菌を含む固定液の一部を用いて細菌DNAの抽出を行なった。抽出はリゾチームやプロテアーゼにより細菌細胞壁を消化するKimらの方法に従って行った（Kimら,DNA Res. (2013) 20:241-253. doi: 10.1093/dnares/dst006.）。抽出されたDNAを、細菌叢の解析に用いた（図１、S1～S3）。
なお、子宮頸部の細菌叢は、膣内細菌叢と酷似していることが示されていることから（Chenら, Nat. Commun. (2017) 8:875）、上記のように調製したDNAサンプルを膣細菌叢由来のDNAとして用いた。 1. Method 1-2. Analysis of Bacterial Flora As a specimen for analyzing the bacterial flora, a specimen for cervical cytology was used. Specifically, cells of the cervix were collected by cytobrush, and the collected material was stored in fixative. The collected specimen contains bacterial groups in the cervix. Extraction of bacterial DNA was performed using a part of the fixative containing bacteria. The extraction was performed according to the method of Kim et al., Which digests bacterial cell wall with lysozyme and protease (Kim et al., DNA Res. (2013) 20: 241-253. Doi: 10.1093 / dnares / dst006.). The extracted DNA was used for analysis of bacterial flora (FIG. 1, S1 to S3).
Since the cervical flora has been shown to closely resemble the vaginal flora (Chen et al., Nat. Commun. (2017) 8: 875), the DNA prepared as described above was used. The sample was used as DNA from the vaginal flora.
１－２．ゲノム解析工程
上記のようにして得られたDNA溶液を用い、溶液内に含まれる16SリボゾームRNA遺伝子を、PCR法を用いて増幅した後、シークエンサー（NGS）（Illumina社製、商品名：Miseq）を用いてDNA解析を行った。
以上のようにして得られた解析結果を、パイプライン解析ツールQiimeを用いて解析した。そして、種レベルないし属レベルでの菌種同定結果に基づいて、作図および統計解析を行った。作図および統計解析には、統計解析プログラムRを使用した。主座標解析に用いるWeighted UniFrac指数の算出もQiimeで行った。 1-2. Genome analysis step Using the DNA solution obtained as described above, the 16S ribosomal RNA gene contained in the solution is amplified by the PCR method, and then a sequencer (NGS) (Illumina, trade name: Miseq) Was used for DNA analysis.
The analysis results obtained as described above were analyzed using the pipeline analysis tool Qiime. Then, drawing and statistical analysis were performed based on the bacterial species identification results at the species level or genus level. The statistical analysis program R was used for drawing and statistical analysis. The Weighted Uni Frac index used for principal coordinate analysis was also calculated using Qiime.
上記のようにして得られたDNA溶液を用い、溶液内に含まれる16SリボゾームRNA遺伝子を、PCR法を用いて増幅した後、シークエンサー（NGS）（Illumina社製、商品名：Miseq）を用いてDNA解析を行った。
以上のようにして得られた解析結果を、パイプライン解析ツールQiimeを用いて解析した。そして、種レベルないし属レベルでの菌種同定結果に基づいて、作図および統計解析を行った。作図および統計解析には、統計解析プログラムRを使用した。主座標解析に用いるWeighted UniFrac指数の算出もQiimeで行った。 1-2. Genome analysis step Using the DNA solution obtained as described above, the 16S ribosomal RNA gene contained in the solution is amplified by the PCR method, and then a sequencer (NGS) (Illumina, trade name: Miseq) Was used for DNA analysis.
The analysis results obtained as described above were analyzed using the pipeline analysis tool Qiime. Then, drawing and statistical analysis were performed based on the bacterial species identification results at the species level or genus level. The statistical analysis program R was used for drawing and statistical analysis. The Weighted Uni Frac index used for principal coordinate analysis was also calculated using Qiime.
２．結果
２－１．膣細菌叢に存在する細菌の存在比率に関する検討
健常者（図７～図１３）、漿液性卵巣癌患者および明細胞卵巣癌患者（図１４～図２０）由来の検体の細菌叢を解析し、存在比率の上位5属についてまとめた結果を図２１に示す。図７～図２０に示す各データに対応する種については、図２～図６を参照のこと。この結果から、卵巣がん患者の膣細菌叢の構成が多様化していることがわかかる。この多様化は、グレード（1-4）の違いによらず認められた。次に、閉経前の被験者に絞った結果を図２２に示す。 図２２から、閉経前の健常者の膣細菌叢は、Lactobacillus属またはPrevotella属の細菌が過半を占める単純な構造をしているのに対し、卵巣がん患者由来の膣細菌叢は、Propionibacterium属、Staphylococcus属、Corynebacterium属などの存在比率が増加していることがわかった。 2. Result 2-1. Examination Regarding the Presence Ratio of Bacteria Present in the Vaginal Microbiota Analyzes of the microbiota of specimens from healthy subjects (FIGS. 7 to 13), serous ovarian cancer patients and clear cell ovarian cancer patients (FIGS. 14 to 20), FIG. 21 shows the results summarized for the top 5 genera with the highest abundance ratio. See FIGS. 2 to 6 for species corresponding to each data shown in FIGS. 7 to 20. These results show that the composition of vaginal flora in ovarian cancer patients is diversified. This diversification was observed regardless of grade (1-4) differences. Next, FIG. 22 shows the results focused on the subjects before menopause. From FIG. 22, it can be seen that the vaginal flora of healthy people before menopause has a simple structure in which the bacteria of the genus Lactobacillus or Prevotella occupy the majority, whereas the vaginal flora derived from an ovarian cancer patient is of the genus Propionibacterium . , Staphylococcus spp, Corynebacterium spp, etc. were found to be present in increasing proportions.
２－１．膣細菌叢に存在する細菌の存在比率に関する検討
健常者（図７～図１３）、漿液性卵巣癌患者および明細胞卵巣癌患者（図１４～図２０）由来の検体の細菌叢を解析し、存在比率の上位5属についてまとめた結果を図２１に示す。図７～図２０に示す各データに対応する種については、図２～図６を参照のこと。この結果から、卵巣がん患者の膣細菌叢の構成が多様化していることがわかかる。この多様化は、グレード（1-4）の違いによらず認められた。次に、閉経前の被験者に絞った結果を図２２に示す。 図２２から、閉経前の健常者の膣細菌叢は、Lactobacillus属またはPrevotella属の細菌が過半を占める単純な構造をしているのに対し、卵巣がん患者由来の膣細菌叢は、Propionibacterium属、Staphylococcus属、Corynebacterium属などの存在比率が増加していることがわかった。 2. Result 2-1. Examination Regarding the Presence Ratio of Bacteria Present in the Vaginal Microbiota Analyzes of the microbiota of specimens from healthy subjects (FIGS. 7 to 13), serous ovarian cancer patients and clear cell ovarian cancer patients (FIGS. 14 to 20), FIG. 21 shows the results summarized for the top 5 genera with the highest abundance ratio. See FIGS. 2 to 6 for species corresponding to each data shown in FIGS. 7 to 20. These results show that the composition of vaginal flora in ovarian cancer patients is diversified. This diversification was observed regardless of grade (1-4) differences. Next, FIG. 22 shows the results focused on the subjects before menopause. From FIG. 22, it can be seen that the vaginal flora of healthy people before menopause has a simple structure in which the bacteria of the genus Lactobacillus or Prevotella occupy the majority, whereas the vaginal flora derived from an ovarian cancer patient is of the genus Propionibacterium . , Staphylococcus spp, Corynebacterium spp, etc. were found to be present in increasing proportions.
上記結果を踏まえて、膣細菌叢に存在する上位5属の細菌の存在比率について、閉経前の健常者および卵巣がん患者について、さらに検討を加えた。
閉経前の健常者に膣細菌叢では、Lactobacillus属単独（図２３Ａ）またはLactobacillus属およびPrevotella属の合計（図２３Ｂ）の存在比率が約80%以上を占めるのに対し、卵巣がん患者では、Lactobacillus属単独の比率は約20%以下（図２３Ａ）、Lactobacillus属およびPrevotella属の合計の存在比率についても、約50%以下（図２３Ｂ）と低い存在率を示すことがわかった。 Based on the above results, the ratio of the bacteria of the top 5 genera present in the vaginal flora was further examined for premenopausal healthy subjects and ovarian cancer patients.
In the normal vaginal flora of premenopausal healthy people, the ratio of Lactobacillus genus alone (Fig. 23A) or the total of Lactobacillus genus and Prevotella genus (Fig. 23B) accounted for about 80% or more, whereas in ovarian cancer patients. , Lactobacillus alone is about 20% or less (Fig. 23A), and the total abundance of Lactobacillus and Prevotella is about 50% or less (Fig. 23B), which is low.
閉経前の健常者に膣細菌叢では、Lactobacillus属単独（図２３Ａ）またはLactobacillus属およびPrevotella属の合計（図２３Ｂ）の存在比率が約80%以上を占めるのに対し、卵巣がん患者では、Lactobacillus属単独の比率は約20%以下（図２３Ａ）、Lactobacillus属およびPrevotella属の合計の存在比率についても、約50%以下（図２３Ｂ）と低い存在率を示すことがわかった。 Based on the above results, the ratio of the bacteria of the top 5 genera present in the vaginal flora was further examined for premenopausal healthy subjects and ovarian cancer patients.
In the normal vaginal flora of premenopausal healthy people, the ratio of Lactobacillus genus alone (Fig. 23A) or the total of Lactobacillus genus and Prevotella genus (Fig. 23B) accounted for about 80% or more, whereas in ovarian cancer patients. , Lactobacillus alone is about 20% or less (Fig. 23A), and the total abundance of Lactobacillus and Prevotella is about 50% or less (Fig. 23B), which is low.
また、Propionibacterium属についても同様の比較を行ったところ、Propionibacterium属（属レベルの比較）およびPropionibacterium acnes（種レベルの比較）いずれにおいても、卵巣がん患者の膣細菌叢における存在比率が有意に高いことがわかった（図２４ＡおよびＢ）。さらに、Corynebacterium属（図２５）およびStaphylococcus属（図２６）についても、卵巣がん患者の膣細菌叢における存在比率が有意に高かった。
When it was similar comparison for Propionibacterium genus (Comparison genus level) Propionibacterium spp. And Propionibacterium acnes (species level comparison) In any, significantly higher proportions of the vaginal flora of ovarian cancer patients Was found (FIGS. 24A and B). Furthermore, the genus Corynebacterium (Fig. 25) and the genus Staphylococcus (Fig. 26) were also significantly higher in the vaginal flora of the ovarian cancer patients.
２－２．膣細菌叢に存在する細菌種の数に関する検討
閉経前の健常者および卵巣がん患者に由来する膣細菌叢に0.1%以上存在する細菌種の数を比較した。その結果、閉経前健常者においては、5種程度であるのに対し、卵巣がん患者においては、40種程度と顕著に多いことが分かった（図２７）。 2-2. Study on the number of bacterial species present in vaginal flora The number of bacterial species present in 0.1% or more in vaginal flora derived from premenopausal healthy subjects and ovarian cancer patients was compared. As a result, it was found that the number of pre-menopausal healthy subjects was about 5 types, whereas that of ovarian cancer patients was about 40 types (Fig. 27).
閉経前の健常者および卵巣がん患者に由来する膣細菌叢に0.1%以上存在する細菌種の数を比較した。その結果、閉経前健常者においては、5種程度であるのに対し、卵巣がん患者においては、40種程度と顕著に多いことが分かった（図２７）。 2-2. Study on the number of bacterial species present in vaginal flora The number of bacterial species present in 0.1% or more in vaginal flora derived from premenopausal healthy subjects and ovarian cancer patients was compared. As a result, it was found that the number of pre-menopausal healthy subjects was about 5 types, whereas that of ovarian cancer patients was about 40 types (Fig. 27).
２－３．膣細菌叢に存在する細菌の多様性に関する検討
閉経前の健常者および卵巣がん患者に由来する膣細菌叢の多様性を比較した。健常者および卵巣がん患者各々のSimpson指数を算出し、グラフ化したところ健常者では0.6以下であったのに対し、卵巣がん患者では、0.7以上であった（図２８Ａ）。また、健常者および卵巣がん患者各々のShannon指数を算出し、グラフ化したところ健常者では1.0以下であったのに対し、卵巣がん患者では、2.2以上であった（図２８Ｂ）。以上のように、卵巣がん患者の膣細菌叢の方が、多様性が高く、両者の膣細菌叢の構成が大きく異なることがわかった。 2-3. Study on the diversity of bacteria present in the vaginal flora We compared the diversity of vaginal flora derived from premenopausal healthy subjects and ovarian cancer patients. When the Simpson index of each of the healthy subjects and the ovarian cancer patients was calculated and graphed, it was 0.6 or less in the healthy subjects and 0.7 or more in the ovarian cancer patients (FIG. 28A). In addition, when the Shannon index of each of the healthy subjects and the ovarian cancer patients was calculated and graphed, it was 1.0 or less in the healthy subjects and 2.2 or more in the ovarian cancer patients (FIG. 28B). As described above, it was found that the vaginal flora of patients with ovarian cancer is more diverse and the composition of the vaginal flora of both patients is significantly different.
閉経前の健常者および卵巣がん患者に由来する膣細菌叢の多様性を比較した。健常者および卵巣がん患者各々のSimpson指数を算出し、グラフ化したところ健常者では0.6以下であったのに対し、卵巣がん患者では、0.7以上であった（図２８Ａ）。また、健常者および卵巣がん患者各々のShannon指数を算出し、グラフ化したところ健常者では1.0以下であったのに対し、卵巣がん患者では、2.2以上であった（図２８Ｂ）。以上のように、卵巣がん患者の膣細菌叢の方が、多様性が高く、両者の膣細菌叢の構成が大きく異なることがわかった。 2-3. Study on the diversity of bacteria present in the vaginal flora We compared the diversity of vaginal flora derived from premenopausal healthy subjects and ovarian cancer patients. When the Simpson index of each of the healthy subjects and the ovarian cancer patients was calculated and graphed, it was 0.6 or less in the healthy subjects and 0.7 or more in the ovarian cancer patients (FIG. 28A). In addition, when the Shannon index of each of the healthy subjects and the ovarian cancer patients was calculated and graphed, it was 1.0 or less in the healthy subjects and 2.2 or more in the ovarian cancer patients (FIG. 28B). As described above, it was found that the vaginal flora of patients with ovarian cancer is more diverse and the composition of the vaginal flora of both patients is significantly different.
２－４．膣細菌叢の組成類似性解析
非類似度指数 weighted UniFrac （図２９）に基づく主座標解析を行った。閉経前卵巣がん患者の膣細菌叢は閉経前健常者のそれとは異なる場所に固まってプロットされ、細菌叢の構造が異なっていることが可視化された。従って、この主座標解析からも、閉経前の健常者（図３０○）と卵巣がん患者（図３０●）とでは、その膣細菌叢の構成が大きく異なることが明らかとなった。 2-4. Compositional similarity analysis of vaginal flora Main coordinate analysis based on dissimilarity index weighted UniFrac (Fig. 29) was performed. The vaginal flora of premenopausal ovarian cancer patients was plotted in a different location than that of healthy premenopausal individuals, and it was visualized that the structure of the flora was different. Therefore, also from this main coordinate analysis, it was revealed that the constitution of the vaginal microflora was significantly different between the premenopausal normal person (Fig. 30 ○) and the ovarian cancer patient (Fig. 30 ●).
非類似度指数 weighted UniFrac （図２９）に基づく主座標解析を行った。閉経前卵巣がん患者の膣細菌叢は閉経前健常者のそれとは異なる場所に固まってプロットされ、細菌叢の構造が異なっていることが可視化された。従って、この主座標解析からも、閉経前の健常者（図３０○）と卵巣がん患者（図３０●）とでは、その膣細菌叢の構成が大きく異なることが明らかとなった。 2-4. Compositional similarity analysis of vaginal flora Main coordinate analysis based on dissimilarity index weighted UniFrac (Fig. 29) was performed. The vaginal flora of premenopausal ovarian cancer patients was plotted in a different location than that of healthy premenopausal individuals, and it was visualized that the structure of the flora was different. Therefore, also from this main coordinate analysis, it was revealed that the constitution of the vaginal microflora was significantly different between the premenopausal normal person (Fig. 30 ○) and the ovarian cancer patient (Fig. 30 ●).
本発明は、閉経前の被験者が卵巣がんに罹患しているかどうかまたは罹患している可能性を診断する方法を提供する。本発明の診断方法は、初期のステージの卵巣がんの診断にも使用することが十分に可能であり、卵巣がんの予防医学等の分野における利用が期待できる。
The present invention provides a method for diagnosing whether or not a premenopausal subject has ovarian cancer. The diagnostic method of the present invention can be sufficiently used for diagnosing ovarian cancer at an early stage, and can be expected to be used in fields such as preventive medicine for ovarian cancer.
Claims (8)
- 閉経前の被験者が卵巣がんに罹患している可能性を診断する方法であって、被験者の膣微生物叢の構成が、卵巣がん患者の膣微生物叢の構成と類似性が高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する方法。 A method of diagnosing a premenopausal subject's likelihood of having ovarian cancer, wherein the composition of the vaginal microbiota of the subject is highly similar to that of an ovarian cancer patient, A method of diagnosing that the subject may have ovarian cancer.
- 前記膣微生物叢の構成の評価指標が、多様性指数、微生物の種類数または特定の微生物の存在比率のいずれか１または複数であることを特徴とする請求項１に記載の方法。 The method according to claim 1, wherein the evaluation index of the composition of the vaginal microflora is any one or more of a diversity index, the number of types of microorganisms, and the abundance ratio of specific microorganisms.
- 前記評価指標が、多様性指数であって、被験者の膣細菌叢のSimpson指数が0.7以上および／またはShannon指数が2.0以上である場合に、当該被験者は卵巣がんに罹患している可能性があると診断する請求項２に記載の方法。 If the evaluation index is a diversity index and the Simpson index of the vaginal flora of the subject is 0.7 or more and / or the Shannon index is 2.0 or more, the subject may have ovarian cancer. The method according to claim 2, wherein the diagnosis is made.
- 前記評価指標が、微生物の種類数であって、被験者の膣微生物叢の微生物の種類数が、閉経前の健常者の膣微生物叢の微生物の種類数より多い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する請求項２に記載の方法。 Where the evaluation index is the number of types of microorganisms, the number of types of microorganisms of the vaginal microflora of the subject is greater than the number of types of microorganisms of the vaginal microflora of healthy people before menopause, the subject has ovarian cancer. 3. The method according to claim 2, wherein the subject is diagnosed as having a disease.
- 前記評価指標が、Lactobacillus属およびPrevotella属以外の属の存在比率であって、被験者の膣微生物叢における当該属の存在比率が、閉経前の健常者由来の膣微生物叢における当該属の存在比率より高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する請求項２に記載の方法。 The evaluation index is the abundance ratio of genus other than Lactobacillus genus and Prevotella genus, the abundance ratio of the genus in the vaginal microflora of the subject, than the abundance ratio of the genus in the vaginal microflora from healthy people before menopause The method according to claim 2, wherein the subject is diagnosed as having a possibility of having ovarian cancer when it is high.
- 前記Lactobacillus属およびPrevotella属以外の属が、Propionibacterium属、Corynebacterium属および／またはStaphylococcus属であることを特徴とする請求項５に記載の方法。 The method according to claim 5, wherein the genera other than Lactobacillus and Prevotella are Propionibacterium , Corynebacterium and / or Staphylococcus .
- 前記評価指標が、Lactobacillus属および／またはPrevotella属の存在比率であって、被験者の膣微生物叢における当該属の存在比率が、閉経前の健常者由来の膣微生物叢における当該属の存在比率より低い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する請求項２に記載の方法。 The evaluation index is the abundance ratio of the Lactobacillus genus and / or Prevotella genus, and the abundance ratio of the genus in the vaginal microflora of the subject is lower than the abundance ratio of the genus in the vaginal microflora derived from the premenopausal healthy subject. The method according to claim 2, wherein the subject is diagnosed as possibly having ovarian cancer.
- 前記評価指標が、Propionibacterium acnes の存在比率であって、被験者の膣微生物叢における当該種の存在比率が、閉経前の健常者由来の膣微生物叢における当該種の存在比率より高い場合に、当該被験者は卵巣がんに罹患している可能性があると診断する請求項２に記載の方法。 Where the evaluation index is the abundance ratio of Propionibacterium acnes and the abundance ratio of the species in the vaginal microbiota of the subject is higher than the abundance ratio of the species in the vaginal microbiota derived from a pre-menopausal healthy subject, the subject The method according to claim 2, wherein the patient is diagnosed as possibly having ovarian cancer.
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