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CN112176065A - Reagents and methods for cervical cancer prognosis or prediction of cervical cancer recurrence or metastasis risk - Google Patents

Reagents and methods for cervical cancer prognosis or prediction of cervical cancer recurrence or metastasis risk Download PDF

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CN112176065A
CN112176065A CN202011117181.8A CN202011117181A CN112176065A CN 112176065 A CN112176065 A CN 112176065A CN 202011117181 A CN202011117181 A CN 202011117181A CN 112176065 A CN112176065 A CN 112176065A
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cervical cancer
prognosis
insertion site
hpv
risk
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沈捷
王伟伟
高建勇
庞伟
张福泉
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Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Abstract

本发明公开用于宫颈癌预后或预测宫颈癌复发或转移风险的试剂和方法。本发明基于纳米孔测序技术对临床样本进行融合基因检测，发现了大量新的HPV整合位点及融合基因，进一步研究发现这些位点可以用于临床HPV感染相关疾病的预后及复发或转移风险评估。

Figure 202011117181

The present invention discloses reagents and methods for cervical cancer prognosis or prediction of cervical cancer recurrence or metastasis risk. The invention performs fusion gene detection on clinical samples based on nanopore sequencing technology, and discovers a large number of new HPV integration sites and fusion genes. Further research finds that these sites can be used for the prognosis of clinical HPV infection-related diseases and the risk assessment of recurrence or metastasis .

Figure 202011117181

Description

Reagents and methods for prognosis of cervical cancer or prediction of risk of cervical cancer recurrence or metastasis
Technical Field
The present invention relates to disease diagnosis, in particular to products and methods for prognosis of cervical cancer or predicting the risk of recurrence or metastasis of cervical cancer.
Background
Cervical cancer, the most common malignancy in gynecology, poses a serious threat to women's health. Human Papillomaviruses (HPV) are closely related to genital epithelial malignancies and precancerous lesions and are the major etiological factors of cervical cancer. It takes about 15 years from the infection with HPV to the development of cervical cancer, during which viral DNA is inserted into the host genome, and is considered to be a key event in the development of cervical cancer. The treatment effect of the cervical precancerous lesion is far better than that of cervical cancer. Therefore, the clinical treatment is guided by checking the HPV infection and the HPV genome integration state, and the method has important significance for preventing and treating the cervical cancer. Especially, in cervical cancer patients with advanced stage and recurrence and metastasis after treatment, the role played by HPV is lack of understanding, and if integration sites of HPV at key positions in genomes of the patients can be found, the integration sites can become key breakthrough points for treating the patients.
Currently, there are HPV detection methods:
1. cytological examination (pap smear, thin-layer liquid-based cells), HPV-infected cervical cells often show characteristic changes, and if these changes are found in the cells, the report suggests HPV infection. HPV infection is often found by cytology and is not sensitive enough, because early HPV infected cells may not show typical morphological changes. The method is greatly influenced by the subjectivity of doctors, needs to be judged by doctors with rich experience, has high false negative and is not easy to popularize.
2. Molecular Detection (DNA)
2.1 hybrid Capture assay (HC-II)
The method can detect 13 high-risk HPV simultaneously, and has high sensitivity and specificity for detecting high-grade cervical lesions. The disadvantage is that no specific HPV type can be determined. .
2.2 real-time fluorescent quantitative PCR technique (Cobas 4800)
The real-time fluorescent quantitative PCR technology adds a fluorescent labeled probe on the basis of the conventional PCR, and different fluorescent dyes label different probes. PCR amplification is run in a completely closed system, avoiding the possibility of contamination and cross-contamination of the amplification products. The method has high sensitivity and strong specificity.
2.3 Gene chip method
The method is based on PCR technology and DNA chip technology, the PCR product is hybridized with HPV specific probe fixed on the DNA chip, and the typing detection of HPV in the sample is carried out through the combination of Cy5-dUTP labeled probe and label sequence. The method can detect 24 HPV subtypes simultaneously, and has the disadvantages of high cost and poor specificity.
Disclosure of Invention
The invention carries out fusion gene detection on samples of clinically confirmed high-risk HPV infected patients based on a nanopore sequencing technology, discovers a large number of new HPV integration sites and fusion genes for the first time, and further researches and discovers that the sites can be used for clinical cervical cancer prognosis and recurrence or transfer risk assessment. Specifically, the present invention includes the following.
In a first aspect of the invention, there is provided a method for prognosis of cervical cancer or prediction of risk of recurrence or metastasis of cervical cancer, comprising:
(1) providing a biological sample taken from a subject after receiving treatment;
(2) obtaining information of a reference set of insertion sites in the biological sample using a reagent, wherein the reference set of insertion sites consists of a plurality of insertion sites, and each insertion site is a site of insertion of a DNA fragment derived from HPV into a subject's genome, respectively, and each insertion site is located inside or near a gene selected from the group consisting of: RPN, NFATC, RASGRP, FHIT, TRERF, SNTB, EPHA, BIRC, ZFLX, PRKDC, TBX, LHFP, PCDHGB, EIF3, ERBB, DDX, MGMT, MAP3K, MKL, LRP1, KLF, GOLGA, TFE, NRG, CUL, ARAP, KMT2, TCF, STAG, GOLIM, MTOR, PAX, BBX, RAD51, PBX, RSPO, ZNF638, GNAQ, PSMD, NOTCH, ARHGAP, RECQL, PTCH, TFDP, TGIF, CDH, DMD, ACVR1, MACF, ERCC, EPHA, ATP11, MDS, SSX, FLT, XRCC, SMARCA, SMAK, CHD, SPANA 1, NTRK, SOX, LIG, WRN, FANCB, SHBP, PHAK, FAWB 2, MAG, MAGRD, MAG, FARD, FABR, MAGBR, PAPR, FABR, PAPR, FABR, PAPR, FABR, PHR, FABR, PAPR, FABR, PAPR, PHR, FABR, FAR, PAPR, FAR, USP47, LNPEP, CANT1, TERT, DHX36, PABPC 36, MLLT 36, NFATC 36, COPS 36, CDKN1 36, GALC, CD163, ACVR 36, PCSK 36, CNBP, ERG, MAP2K 36, NR2F 36, ROS 36, ERBB2 36, NCOA 36, PAX 36, ITPR 36, ROBO 36, MECOM, 1L 36, CACNA1 36, TOP 36, ATP6AP 36, TRIM 36, NAV 36, ATP2B 36, PIK3R 36, ERC 36, NUP214, ZNF510, NNT, AFKIF 21 36, STAR 36, FAF 36, CARD 36, EML 36, GTF2H 36, DIP 36, CANFT 36, FAN 36, DTX1, ZBTB16, RNF168, PCDH 16, SMC 16, SVEP 16, INPP4 16, JAK 16, CDH 16, RXRRA, MLLT 16, ARFGEF 16, UBE2V 16, BCL11 16, CSF3 16, GATA 16, ARID5 16, RSPO 16, IRS 16, ACKR 16, NEDD4 16, RUNX1T 16, SDHC, PABPC 16, CRRADD 16, RANBP 16, BCL7 16, SCAI, PDGFRA, UVSSA, WT 16, FBXXO 16, PCSK 16, JAZF 16, SMAD 16, AKT 16, SYNE 16, MITF 36NTL, ARN 16, SANTS 16, SANDP 16, SANDC 16, SANDP 16, SANDC 16, SANDP 16, SAND, PLEC, RTF1, APLF, NKX3-1 and APC;
(3) predicting the subject as having a good/low risk prognosis for cervical cancer when the insertion site detected in the biological sample is not in the reference insertion site set; predicting the subject as poor/high risk of prognosis of cervical cancer when an insertion site detected in the biological sample occurs in the set of reference insertion sites.
In certain embodiments, the method for prognosis of cervical cancer or predicting the risk of recurrence or metastasis of cervical cancer according to the invention, wherein the set of reference insertion sites is selected from the sites shown in table 1.
In certain embodiments, the method for prognosis of cervical cancer or predicting the risk of recurrence or metastasis of cervical cancer according to the present invention further comprises the step of detecting the presence or absence of a high frequency insertion site in the biological sample.
In certain embodiments, the method for prognosis of cervical cancer or predicting the risk of recurrence or metastasis of cervical cancer according to the present invention, wherein the high frequency insertion site is located within or near at least one gene selected from the group consisting of FANCC, NRG, CDH, DMD, PLCB, HDAC, MED, BBX, BCL11, RUNX1T, RAD23, SMARCA, DHX, MAFB, IKZF, TP, IFT140, CCDC88, JUN, 1, NTRK, SMAD LRP, NAV, KLF, PGR, MAGI, GOT, KDM6, CNOT, FBXW, ERBB, ACVR2, LAMA, EPHA, ACACA, AR, PLEC, RTF, NBPF, ll, APLF, ZNF521, ZNF638, TBL1XR, WHSC, TOP, NKX-1, nex, PCDH, PTCH, and prptk.
In certain embodiments, the method for prognosis of cervical cancer or prediction of recurrence or metastasis risk of cervical cancer according to the present invention, wherein the HPV is a high-risk HPV comprising HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73, 82.
In a second aspect of the invention, there is provided a primer and/or probe for prognosis of cervical cancer or for predicting the risk of recurrence or metastasis of cervical cancer, wherein the primer and/or probe is capable of complementarily binding to a sequence flanking each insertion site in a reference insertion site set, or is capable of complementarily binding to a sequence comprising a reference insertion site.
In a third aspect of the present invention, there is provided a gene chip, wherein a substrate and primers and/or probes regularly immobilized on the substrate are provided, wherein the primers and/or probes are the primers and/or probes of the second aspect.
In a fourth aspect of the invention, a kit or composition comprises a primer and/or probe according to the second aspect.
Drawings
FIG. 1 shows that primary HPV fusion genes have different characteristic distributions in different regions of the chromosome.
FIG. 2 shows that the recurrent group HPV fusion genes have different characteristic distributions in different regions of the chromosome.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
The invention carries out fusion gene detection on samples clinically confirmed to be high-risk HPV infected patients based on a nanopore sequencing technology, and discovers a large number of new HPV integration sites and fusion genes for the first time. The HPV detection method based on hybridization capture can detect known high-risk HPV types, but can not detect specific HPV types. Although specific HPV types can be identified by using more real-time fluorescent quantitative PCR detection methods, whether HPV is fused with human genome or not cannot be judged. The HPV detection method based on the NGS and the probe capture can simultaneously detect the HPV type and the fusion state, but has long detection period and low fusion detection rate near a human genome repetitive sequence region due to the limitation of NGS read length.
Nanopore sequencing belongs to a third-generation sequencing technology, and is different from the existing HPV detection method in that the nanopore sequencing can detect known high-risk HPV and specific HPV types, and more importantly, the nanopore sequencing reading length is longer and can reach 1K-1Mbp, so that fusion near a human genome repetitive sequence region can be detected. Based on the above, by designing probes for the whole genome of high-risk HPV, a plurality of novel integration sites and fusion genes are discovered for the first time, and important directions are provided for the diagnosis, prognosis and treatment of HPV infection-related diseases, particularly cervical cancer.
The biological sample in the present invention includes a tissue, a cell or a treated material thereof derived from the cervix.
(2) Obtaining or detecting information of a reference insertion site set in the biological sample by using a reagent, wherein the reference insertion site set consists of a plurality of insertion sites, and each insertion site is a site for inserting a DNA fragment derived from Human Papillomavirus (HPV) into a genome of a subject;
(3) predicting the subject as having a good/low risk prognosis for cervical cancer when the insertion site detected in the biological sample is not in the reference insertion site set; predicting the subject as poor/high risk of prognosis of cervical cancer when an insertion site detected in the biological sample occurs in the set of reference insertion sites. Preferably, the prognosis of cervical cancer is predicted to be good for the subject when the number of insertion sites detected in the biological sample accounts for less than 7% of the reference insertion site set, the prognosis of cervical cancer is predicted to be low when the number of insertion sites detected in the biological sample accounts for 7% -20% of the reference insertion site set, the prognosis of cervical cancer is predicted to be poor when the number of insertion sites detected in the biological sample accounts for 7% -20% of the reference insertion site set, and the prognosis of cervical cancer is predicted to be poor when the number of insertion sites detected in the biological sample accounts for more than 20% of the reference insertion site set.
In the present invention, a reference insertion site set refers to a set consisting of a plurality of reference insertion sites. Wherein the reference insertion site refers to a site where a DNA fragment derived from Human Papillomavirus (HPV) is inserted into the genome of the subject. In general, the reference insertion site set includes 50 or more, preferably 100 or more, more preferably 150 or more, and further preferably 200 or more sites. On the other hand, the reference insertion site set of the present invention contains 500 or less, preferably 450 or less, and more preferably 400 or less sites. If the reference insertion site is passed, the detection result may be less accurate. If the number of reference insertion sites is too large, the detection cost is liable to increase, and the reliability of the detection result may deteriorate.
In the present invention, the reference insertion site is generally located within or in the vicinity of a specific gene in the genome of the subject, particularly in the 5' regulatory region of the gene, such as a promoter region or the like. The invention discovers that the HPV DNA fragment inserted into the gene or nearby is more easy to cause the recurrence or the metastasis of the cervical cancer. Genes closely related to the recurrence of cervical cancer are preferred.
In the present invention, the subject is generally a human, preferably a patient suspected of having cervical cancer or a patient diagnosed with cervical cancer. The biological sample of the present invention is a tissue or a cell derived from the cervix or a processed product thereof. The treatment substance comprises a tissue or cell disruption or lysis solution or extract, particularly a DNA extract.
The kit of the present invention comprises primers and/or probes for prognosis of cervical cancer or for predicting the risk of recurrence or metastasis of cervical cancer. These primers and/or probes can complementarily bind to sequences flanking each insertion site in the reference set of insertion sites, or the probes can complementarily bind to sequences comprising the reference insertion sites.
In addition to the primer set or probe set described above, the kit of the present invention may include precautions relating to the regulatory manufacture, use or sale of the diagnostic kit in a form prescribed by a governmental agency. In addition, the kits of the invention may be provided with detailed instructions for use, storage, and troubleshooting. The kit may optionally also be provided in a suitable device, preferably for robotic handling in a high throughput setting.
In certain embodiments, the components (e.g., probe set) of the kits of the invention can be provided as a dry powder. When the reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically comprise at least one vial, test tube, flask, bottle, syringe, and/or other container means, optionally in which the solvent is placed in equal portions. The kit may further comprise means for a second container comprising a sterile, pharmaceutically acceptable buffer and/or other solvent.
In certain embodiments, the components of the kits of the invention may be provided in the form of a solution, e.g., an aqueous solution. The concentrations or contents of these ingredients, in the case of being present in aqueous solution, are readily determinable by the person skilled in the art as a function of the various requirements. For example, for storage purposes, the concentration of the probe may be present in a higher form, for example, and when in an operating state or in use, the concentration may be reduced to the operating concentration by, for example, diluting the higher concentration solution.
The kit of the present invention may further comprise other reagents or ingredients. For example, DNA polymerase, dNTPs of various types and ions such as Mg, required for carrying out PCR2+And the like. These additional agents or components are known to those skilled in the art and are readily known from publications such as molecular cloning, a laboratory manual, fourth edition, cold spring harbor, and the like.
Where more than one component is present in a kit, the kit will also typically comprise a second, third or other additional container into which additional components may be separately placed. In addition, combinations of various components may be included in the container.
Kits of the invention may also include components that retain or maintain DNA, such as agents that are resistant to nucleic acid degradation. Such components may be, for example, nucleases either without RNase or with protection against RNase. Any of the compositions or reagents described herein can be a component of a kit.
Examples
1. Experimental Material
Clinically, a liquid-based thin-layer cell test sample (TCT) of a patient infected with high-risk HPV is confirmed.
2. Primary sequencing platform and reagents
A sequencing platform: multi-model nanopore sequencer
The main reagents are as follows: PCR Barcoding Kit (SQK-PBK004)
3. Probe design Synthesis
A mixed probe is designed and synthesized according to the clear 18 HPV (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73, 82) genomes issued by CFDA as the "HPV nucleic acid detection and genotyping, reagent technical examination guide".
4. Experimental methods
4.1 genomic DNA extraction
DNA of TCT samples of 27 clinically confirmed patients with high-risk HPV infection was extracted. The method refers to a micro sample genome DNA extraction kit of Tiangen biology company.
4.2 disruption of genomic DNA
Disruption of genomic DNA to the major band 1-5Kb using a covaris ultrasonicator
4.3 library construction
Library construction was performed using a nanopore library construction kit with Barcode.
4.4HPV genomic DNA Capture
The 18 high-risk probes capture HPV genome DNA in a sample to be detected, the process refers to xGen Lockdown Reagents (IDT), and the hybridization temperature and time are optimized to obtain a longer HPV-containing sequence segment and a higher virus sequence proportion.
4.5 sequencing
Sequencing using a nanopore platform.
5. Results of the experiment
5.1 statistical analysis of fusion genes
The detection method based on probe capture and third generation nanopore sequencing designed by the invention detects that the integration of HPV genes exists in 390 genes or nearby genes in 36 samples, and the specific genes are as follows: RPN, NFATC, RASGRP, FHIT, TRERF, SNTB, EPHA, BIRC, ZFLX, PRKDC, TBX, LHFP, PCDHGB, EIF3, ERBB, DDX, MGMT, MAP3K, MKL, LRP1, KLF, GOLGA, TFE, NRG, CUL, ARAP, KMT2, TCF, STAG, GOLIM, MTOR, PAX, BBX, RAD51, PBX, RSPO, ZNF638, GNAQ, PSMD, NOTCH, ARHGAP, RECQL, PTCH, TFDP, TGIF, CDH, DMD, ACVR1, MACF, ERCC, EPHA, ATP11, MDS, SSX, FLT, XRCC, SMARCA, SMAK, CHD, SPANA 1, NTRK, SOX, LIG, WRN, FANCB, SHBP, PHAK, FAWB 2, MAG, MAGRD, MAG, FARD, FABR, MAGBR, PAPR, FABR, PAPR, FABR, PAPR, FABR, PHR, FABR, PAPR, FABR, PAPR, PHR, FABR, FAR, PAPR, FAR, USP47, LNPEP, CANT1, TERT, DHX36, PABPC 36, MLLT 36, NFATC 36, COPS 36, CDKN1 36, GALC, CD163, ACVR 36, PCSK 36, CNBP, ERG, MAP2K 36, NR2F 36, ROS 36, ERBB2 36, NCOA 36, PAX 36, ITPR 36, ROBO 36, MECOM, 1L 36, CACNA1 36, TOP 36, ATP6AP 36, TRIM 36, NAV 36, ATP2B 36, PIK3R 36, ERC 36, NUP214, ZNF510, NNT, AFKIF 21 36, STAR 36, FAF 36, CARD 36, EML 36, GTF2H 36, DIP 36, CANFT 36, FAN 36, DTX1, ZBTB16, RNF168, PCDH 16, SMC 16, SVEP 16, INPP4 16, JAK 16, CDH 16, RXRRA, MLLT 16, ARFGEF 16, UBE2V 16, BCL11 16, CSF3 16, GATA 16, ARID5 16, RSPO 16, IRS 16, ACKR 16, NEDD4 16, RUNX1T 16, SDHC, PABPC 16, CRRADD 16, RANBP 16, BCL7 16, SCAI, PDGFRA, UVSSA, WT 16, FBXXO 16, PCSK 16, JAZF 16, SMAD 16, AKT 16, SYNE 16, MITF 36NTL, ARN 16, SANTS 16, SANDP 16, SANDC 16, SANDP 16, SANDC 16, SANDP 16, SAND, PLEC, RTF1, APLF, NKX3-1 and APC.
Among the above genes, some sites are present in the initial treatment cases, and these sites are located at or near the following genes: RPN, NFATC, RASGRP, FHIT, TRTRRF, SNTB, EPHA, BIRC, ZFLX, PRKDC, TBX, LHFP, PCDHGB, EIF3, ERBB, DDX, MGMT, MAP3K, MKL, LRP1, KLF, GOLGA, TFE, NRG, CUL, ARHGAP, KMT2, TCF, STAG, GOLIM, MTOR, PAX, BBX, RAD51, PBX, RSPO, ZNF638, GNAQ, PSMD, NOTCH, ARHGAP, RECQL, PTCH, TFDP, TGIF, CDH, DMD, FHIT, ACVR1, MACF, ERCC, EPHA, TBX, ATP11, MDS, SSX, FLT, XRCC, SMARCA, CHTP, DDX, DDNA 1, DDNF, SHFB, SHRB, FLRB, PHAK, FLRB, PHAK, FBG, FARB, PHBR, FARB, PHBR, FARB, PHBR, PSK, PHBR, PSR, PSBR, PSR, BCG, PSBR, PSR, PSBR, BCG, PSBR, PS, ALK, TACC3, SPEN, ETV1, PRDM1, WNT5A, IREB2, GPSM2, RPN 2, CLTC, EIF4G 2, WHSC 2, AHNAK, TACC 2, PSMD 2, EPHA2, LMAN 2, MED2, USP 2, LNPEP, CANT 2, SMARCA2, TERT, FGFR2, DHX 2, PABPC 2, ARHGAP2, ARLT 36LT 2, TACC 2, FGFR2, NRG 2, NFATC2, CARFIT 2 2, COPS2, CDKN 12, GALC, ERCC 2, AFCACNVR 2, ACNNPSATCP 2, PCSSBP 36BP 2, EPTC 2, EP.
These clinical cases were further closely followed and samples of recurrent cases were collected, and genes closely related to cervical cancer recurrence were found as follows: LAMA2, TACC 2, PTPRK, FGFR2, ERC 2, PIK 32, DEK, TSHR, ALK, FXR 2, IDH2, FHIT, TGFBR2, LAMA2, DMD, AXIN2, BMPR 12, NR4A2, ARID 12, SRGAP 2, NTRK2, ATP6AP2, ADAM 2, PCCB, GOLIM 2, RANBP 2, MAF BRWD 2, FAM46 2, TAOK 2, CREBP, SSX2, FHIT, KALRN, EPHA2, SMAD2, XRC 2, XRBCL 2, DLG 2, SMURF2, SMUB 2, MEDG 2, PSR 2, PBAT 2, PSLR 2, PHR 2, TFSC 2, TFAS 2, TFR 2, TFP 2, TFR 2, TFS 2, TFR 2, TFP 2, TFR 2, TFP 2, TFR 2, TFP 2, TFR 36, LRIG, ALK, ETV, ARID1, ROBO, IRS, ACKR, NEDD4, GRIN2, PRDM, EBF, RUNX1T, SDHC, GPHN, ADCY, TP, LAMA, AMER, PABPC, CRTC, RANBP, CUL, BCL7, PBX, SCAI, PDGFRA, UVSSA, TREF, WT, FBXO, PCSK, NAVZF, SMAD, AKT, NTC, SYNE, MITF, ARNTL, SMURF, RADD, DDX, DDD, CHD, ITSN, RB, ATP6AP, XP1, KLF, MAGI, CASC, FCRL, PDE5, MYCBP, FRG, NEDD4, CRIP, JAS, FBN, ZFP, NRG, SND, PDCD1, FGFR, LRPR 51, RABP, SACK 4, PSRBFBVR, FBVR, FB5, PSBR, PSNBR, FBRB, PSRB, TFP, PSRB, TFRB, TFC, TFRB, TFC, TFRB, TFG, TFC, TFRB, TFC, TFRB, TFC, TFRB, TFC, TFRB, TFC, TFRB, TFC, TFRB, FOXP1, ACVR1, HLA-B, DLG1, TP63, MECOM, ZNF608, AFF3, LRP1B, KLF4, ETNK1, RGS3, ARHGAP29, EPAS1, CLASP2, RSPO2, PTPRT, 2, PRDM 2, IRF2, STAU2, NFIB, KIF 52, EPC 2, SENAVP 2, HMGN2P 2, MAF PTCH 2, SMCHD 2, TGFBR2, PCSK 2, BMPR 12, KIF 12 2, ACVR2 2, SANFR 2, NTRK2, FAF 2, SLC34A2, SEMA4 2, NRG 2, ERBMBB 2, TCF 2, RMI2, PRACAK 2, PARD 2, PADDK 2, EPC 2, EPAK 2, EPC 2, EPTC 2, EPTC 2, EPTC 2, EPTC 2, EPTC 2, EPTC 2.
The following table 1 lists information of gene fusion sites detected using the method of the present invention. These site information are closely related to recurrent metastasis of cervical cancer.
TABLE 1 recurrent group insertion cancer-associated Gene site information
Figure BDA0002730687660000151
Figure BDA0002730687660000161
Figure BDA0002730687660000171
Figure BDA0002730687660000181
Figure BDA0002730687660000191
Figure BDA0002730687660000201
Figure BDA0002730687660000211
Figure BDA0002730687660000221
Figure BDA0002730687660000231
Figure BDA0002730687660000241
Figure BDA0002730687660000251
Figure BDA0002730687660000261
Figure BDA0002730687660000271
Figure BDA0002730687660000281
Figure BDA0002730687660000291
Figure BDA0002730687660000301
Figure BDA0002730687660000311
Figure BDA0002730687660000321
Figure BDA0002730687660000331
Figure BDA0002730687660000341
Figure BDA0002730687660000351
Figure BDA0002730687660000361
Figure BDA0002730687660000371
Figure BDA0002730687660000381
Figure BDA0002730687660000391
Figure BDA0002730687660000401
Figure BDA0002730687660000411
Figure BDA0002730687660000421
Figure BDA0002730687660000431
Figure BDA0002730687660000441
Figure BDA0002730687660000451
Figure BDA0002730687660000461
Figure BDA0002730687660000471
Figure BDA0002730687660000481
Figure BDA0002730687660000491
Figure BDA0002730687660000501
Figure BDA0002730687660000511
Figure BDA0002730687660000521
Figure BDA0002730687660000531
Figure BDA0002730687660000541
Figure BDA0002730687660000551
Figure BDA0002730687660000561
Figure BDA0002730687660000571
Further analysis shows that 1459 sites intersected with the relapse population in the initial treatment population and 70 genes closely related to cervical cancer. The list of genes associated with poor prognosis is shown in table 2.
TABLE 2
Figure BDA0002730687660000581
FIGS. 1 and 2 show that primary and recurrent HPV fusion genes have different characteristic distributions in different regions of the chromosome.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims (8)

1. A method for prognosis of cervical cancer or predicting the risk of recurrence or metastasis of cervical cancer, comprising:
(1) providing a biological sample taken from a subject after receiving treatment;
(2) obtaining information of a reference set of insertion sites in the biological sample using a reagent, wherein the reference set of insertion sites consists of a plurality of insertion sites, and each insertion site is a site of insertion of a DNA fragment derived from HPV into a subject's genome, respectively, and each insertion site is located inside or near a gene selected from the group consisting of:
RPN, NFATC, RASGRP, FHIT, TRERF, SNTB, EPHA, BIRC, ZFLX, PRKDC, TBX, LHFP, PCDHGB, EIF3, ERBB, DDX, MGMT, MAP3K, MKL, LRP1, KLF, GOLGA, TFE, NRG, CUL, ARAP, KMT2, TCF, STAG, GOLIM, MTOR, PAX, BBX, RAD51, PBX, RSPO, ZNF638, GNAQ, PSMD, NOTCH, ARHGAP, RECQL, PTCH, TFDP, TGIF, CDH, DMD, ACVR1, MACF, ERCC, EPHA, ATP11, MDS, SSX, FLT, XRCC, SMARCA, SMAK, CHD, SPANA 1, NTRK, SOX, LIG, WRN, FANCB, SHBP, PHAK, FAWB 2, MAG, MAGRD, MAG, FARD, FABR, MAGBR, PAPR, FABR, PAPR, FABR, PAPR, FABR, PHR, FABR, PAPR, FABR, PAPR, PHR, FABR, FAR, PAPR, FAR, USP47, LNPEP, CANT1, TERT, DHX36, PABPC 36, MLLT 36, NFATC 36, COPS 36, CDKN1 36, GALC, CD163, ACVR 36, PCSK 36, CNBP, ERG, MAP2K 36, NR2F 36, ROS 36, ERBB2 36, NCOA 36, PAX 36, ITPR 36, ROBO 36, MECOM, 1L 36, CACNA1 36, TOP 36, ATP6AP 36, TRIM 36, NAV 36, ATP2B 36, PIK3R 36, ERC 36, NUP214, ZNF510, NNT, AFKIF 21 36, STAR 36, FAF 36, CARD 36, EML 36, GTF2H 36, DIP 36, CANFT 36, FAN 36, DTX1, ZBTB16, RNF168, PCDH 16, SMC 16, SVEP 16, INPP4 16, JAK 16, CDH 16, RXRRA, MLLT 16, ARFGEF 16, UBE2V 16, BCL11 16, CSF3 16, GATA 16, ARID5 16, RSPO 16, IRS 16, ACKR 16, NEDD4 16, RUNX1T 16, SDHC, PABPC 16, CRRADD 16, RANBP 16, BCL7 16, SCAI, PDGFRA, UVSSA, WT 16, FBXXO 16, PCSK 16, JAZF 16, SMAD 16, AKT 16, SYNE 16, MITF 36NTL, ARN 16, SANTS 16, SANDP 16, SANDC 16, SANDP 16, SANDC 16, SANDP 16, SAND, PLEC, RTF1, APLF, NKX3-1 and APC;
(3) predicting the subject as having a good/low risk prognosis for cervical cancer when the insertion site detected in the biological sample is not in the reference insertion site set; predicting the subject as poor/high risk of prognosis of cervical cancer when an insertion site detected in the biological sample occurs in the set of reference insertion sites.
2. The method for the prognosis of cervical cancer or the prediction of the risk of recurrence or metastasis of cervical cancer according to claim 1, wherein the set of reference insertion sites are selected from the sites shown in table 1.
3. The method of claim 1 or 2, further comprising the step of detecting the presence of a high frequency insertion site in the biological sample.
4. The method of claim 3, wherein the high frequency insertion site is located within or near at least one gene selected from the group consisting of FANCC, NRG, CDH, DMD, PLCB, HDAC, MED, BBX, BCL11, RUNX1T, RAD23, SMARCA, DHX, MAFB, IKZF, TP, IFT140, CCDC88, JUN, LRP1, NTRK, SMAD, NAV, KLF, PGR, MAGI, GOT, KDM6, CNOT, FBXW, ERBB, ACVR2, LAMA, EPHA, ACACACACACCA, AR, PLEC, RTF, NBPF, NELL, APLF, ZNF521, ZNF638, WHTBL 1XR, WHSC, TOP, NKX-1, APC, PCDH, PTCH, and PTPRK.
5. The method according to claim 1 or 2, wherein the HPV is a high risk HPV comprising HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73 and 82.
6. A primer and/or probe for prognosis of cervical cancer or for predicting the risk of recurrence or metastasis of cervical cancer, comprising the primer and/or the probe capable of complementary binding to a sequence flanking each insertion site in a reference set of insertion sites, or the probe capable of complementary binding to a sequence comprising a reference insertion site.
7. A gene chip comprising a substrate and primers and/or probes immobilized on the substrate, wherein the primers and/or probes are the primers and/or probes according to claim 6.
8. A kit or composition comprising the primer and/or probe of claim 6.
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