Prognosis prediction model for middle-high differentiated esophageal squamous cell carcinoma and construction method and application thereof

Abstract

The application discloses a prognosis prediction model for medium-highly differentiated esophageal squamous cell carcinoma (ESCC) and a construction method and application thereof, and the construction method comprises the following steps: establishing an input module, establishing an analysis module and establishing an output module, and specific contents are described in the body of the application. The application detects whether 12q13.13 amplification occurs in the esophageal squamous cell carcinoma, so as to predict the prognosis of the medium-highly differentiated ESCC. Compared with the medium-highly differentiated ESCC patients without 12q13.13 amplification, the prognosis of the medium-highly differentiated ESCC patients with 12q13.13 amplification is poorer. The application can provide certain help for diagnosis and prognosis evaluation of the medium-highly differentiated esophageal squamous cell carcinoma patients.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and relates to a prognostic prediction model for moderately and well-differentiated esophageal squamous cell carcinoma, its construction method and application; more specifically, it relates to the application of 12q13.13 amplification in assessing the prognosis of moderately and well-differentiated esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal cancer, one of the most common cancers in humans, is the sixth leading cause of cancer death worldwide. It mainly comprises two types: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Adenocarcinoma primarily occurs in Western countries, while in East Asia, over 90% of esophageal cancers are esophageal squamous cell carcinoma. ESCC ranks among the top cancers in both incidence and mortality. Currently, the primary treatment for esophageal squamous cell carcinoma is surgical resection. For earlier benign cases, endoscopic submucosal dissection can be performed. However, the fundamental reason for the poor overall prognosis of esophageal squamous cell carcinoma lies in its early tendency to metastasize to surrounding tissues and lymphatic systems. Even with surgery, long-term survival rates for patients with metastasis are not ideal, with a cumulative recurrence rate of 70%-90% within five years. Current research on biomarkers related to the recurrence and metastasis potential of esophageal cancer is insufficient, leading to delays in clinical diagnosis of recurrence and metastasis. This also results in a lack of specific drug targets for ESCC, all of which severely impact the prognosis of ESCC patients.

[0003] Esophageal squamous cell carcinoma can be divided into two types: poorly differentiated and moderately well differentiated. Moderately well differentiated esophageal squamous cell carcinoma is one of the most common types, accounting for more than half of all esophageal cancers. In China, for example, patients with moderately well differentiated esophageal squamous cell carcinoma account for approximately 40% to 80% of all cases. Factors such as age, sex, diet, and pathological type can affect the degree of differentiation in esophageal squamous cell carcinoma. Moderately well differentiated esophageal squamous cell carcinoma usually occurs in adults over 40 years of age. Some studies have also shown that the proportion of well differentiated esophageal squamous cell carcinoma gradually increases over time, especially in people over 50 years of age. Smoking, alcohol consumption, gastroesophageal reflux, chemical damage to the esophagus, malnutrition, high-temperature cooked foods, smoked foods, oral cancer, and head and neck cancers are all associated with an increased risk of developing moderately well differentiated esophageal squamous cell carcinoma.

[0004] In theory, moderately to well-differentiated tumors have better prognoses. In esophageal cancer, after esophagectomy, the 5-year survival rate for poorly differentiated esophageal squamous cell carcinoma is between 10% and 30%, while the 5-year survival rate for moderately to well-differentiated esophageal squamous cell carcinoma is only about 30% to 50%. Since moderately to well-differentiated tumors account for a relatively large proportion of esophageal cancer patients, the mortality rate of esophageal cancer caused by moderately to well-differentiated tumors cannot be ignored.

[0005] Early-stage moderately to well-differentiated esophageal squamous cell carcinoma has a high cure rate, which decreases as the tumor stage progresses. Postoperative chemotherapy and / or radiotherapy can improve the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma, especially in cases of lymph node metastasis. However, compared to other tumors with targeted therapies, the intervention options are limited, and the overall prognosis is unfavorable. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a prognostic prediction model for moderately to well-differentiated esophageal squamous cell carcinoma, its construction method, and its application. This invention assesses the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma by detecting whether amplification occurs on the long arm of chromosome 12, region 13.13 (12q13.13). Compared to patients with moderately to well-differentiated esophageal squamous cell carcinoma without 12q13.13 amplification, patients with moderately to well-differentiated esophageal squamous cell carcinoma with 12q13.13 amplification have a poorer prognosis. This invention can provide some assistance in the diagnosis and prognostic assessment of patients with moderately to well-differentiated esophageal squamous cell carcinoma.

[0007] Overview

[0008] Whole-exome sequencing (WES) uses nucleic acid sequence capture methods to enrich DNA in the exon regions of the genome, then constructs a DNA library by inserting adapters, and finally sequences it using next-generation sequencing (NGS). This method can detect single nucleotide variants (SNVs) and copy number variants (CNVs) in gene exon sequences, and can also assess structural variations such as chromosome arm amplification and deletion based on this information.

[0009] The inventors performed WES analysis on tumor tissues and paired adjacent normal tissue samples from 144 patients with esophageal squamous cell carcinoma, and collected clinicopathological information and follow-up data for over 10 years. They found that amplification of the 13.13 region on the long arm of chromosome 12 was one of the high-frequency mutations in esophageal squamous cell carcinoma in this cohort. At the overall cohort level, 12q13.13 amplification was significantly associated with esophageal squamous cell carcinoma survival. The inventors further compared the relationship between 12q13.13 amplification and survival in patients with poorly differentiated and moderately to well-differentiated esophageal squamous cell carcinoma. The results showed that in poorly differentiated patients, 12q13.13 amplification did not significantly differ from prognosis; however, in moderately to well-differentiated patients, 12q13.13 amplification was significantly associated with esophageal squamous cell carcinoma incidence and survival (e.g., ...). Figure 3 As shown, the five-year overall survival rate for patients with moderately to well-differentiated esophageal squamous cell carcinoma without 12q13.13 amplification is close to 60%, while the ten-year overall survival rate for patients with moderately to well-differentiated esophageal squamous cell carcinoma with 12q13.13 amplification is less than 30%. The results of this invention indicate that, using the detected 12q13.13 amplification in moderately to well-differentiated patients, 12q13.13 amplification can serve as a risk factor for progression and poor prognosis in moderately to well-differentiated esophageal squamous cell carcinoma.

[0010] Detailed Explanation

[0011] To address the aforementioned technical problems, the first aspect of this invention provides a method for constructing a prognostic prediction model for moderately to well-differentiated esophageal squamous cell carcinoma, comprising the following steps:

[0012] (1) Establish an input module, which is used to input gene amplification information of the 13.13 region of the long arm of chromosome 12 of moderately and well differentiated esophageal squamous cell carcinoma samples;

[0013] (2) Establish an analysis module, which is used to analyze whether there is a statistically significant increase in gene amplification information of region 13.13 on the long arm of chromosome 12 of the sample compared with the database;

[0014] (3) Establish an output module. If there is a statistically significant increase, the output module is used to output the result as a poor prognosis for the sample.

[0015] In a preferred embodiment, the criterion for a statistically significant increase is a q value less than 0.1.

[0016] The q-value, or q-value(p-adjusted), is the corrected p-value (q-value = padj = FDR = Correctedp-Value = p-adjusted). It is a multiple hypothesis test performed on the p-value, which can better control the false positive rate.

[0017] The p-value is a statistical significance test indicator.

[0018] The q value described in this invention is preferably calculated using the software GISTIC2.

[0019] In a preferred embodiment, the statistically significant increase criterion of the present invention is a p-value less than 0.05.

[0020] In this invention, the 13.13 segment of the long arm of chromosome 12 is also referred to as 12q.13.13, which is the region corresponding to the 3rd subband, 1st subband, and 3rd subband of region 1 of the long arm of chromosome 12.

[0021] The prognosis described in this invention can be reflected in the survival rate, such as progression-free survival or total survival; preferably, it refers to total survival.

[0022] Overall survival (OS) is the time from when a patient begins treatment until the patient dies from any cause; in other words, how long a patient lives after treatment.

[0023] The poor prognosis described in this invention can be understood in the conventional sense of those skilled in the art, or it can refer to a reduced survival rate, preferably a survival rate of less than 30%, and more preferably a 5-year survival rate of less than 30%.

[0024] The 12q13.13 amplification described in this invention refers to the amplification of any one or more genes or gene fragments occurring on the entire 12q.13.13 sequence, and does not specifically refer to the amplification of a certain sequence or gene on 12q.13.13.

[0025] In some preferred embodiments, the above construction method includes step (1) further comprising performing whole exome sequencing on the sample to obtain gene amplification information of region 13.13 on the long arm of chromosome 12.

[0026] In some preferred embodiments, in the above construction method, the database in step (2) includes a paired 1,000-person gene database, an adjacent normal genome database of esophageal squamous cell carcinoma patients, or a blood cell gene database.

[0027] The paired 1,000-person genome database can be standard practice in this field, for example, it can be from https: / / www.internationalgenome.org / .

[0028] The construction of the adjacent normal genome of the esophageal squamous cell carcinoma population described in this invention can be carried out in accordance with conventional methods in the field, or the construction of the cancer genome of the esophageal squamous cell carcinoma population can be performed with reference to the embodiments of this invention.

[0029] In some preferred embodiments, step (1) further includes the following steps:

[0030] S1. Establishing a DNA extraction module: The DNA extraction module is used to extract genomic DNA from the sample;

[0031] S2. Establishing a gene library module: The gene library module is used to obtain a gene library obtained by performing ultrasonic treatment to break down genomic DNA, end repair, adapter ligation, hybridization capture, and PCR amplification.

[0032] S3. Establish a sequencing module: The sequencing module is, for example, Illumina HiSeq for whole exome sequencing to obtain sequencing data;

[0033] S4. Establish a data analysis module: The data analysis module performs analysis on the sequencing data obtained by the sequencing module, selecting from the following groups: quality control detection, fragment matching, variant analysis, copy number analysis, and significant mutant gene analysis; and obtains information on copy number variations, single nucleotide variations, and small fragment sequence insertions and deletions;

[0034] S5. Establish a bioinformatics analysis module: The bioinformatics analysis module is used to determine the mutation location and mutation type of the 13.13 region of the long arm of chromosome 12, and to perform biological analysis to screen for amplified gene variations in the 13.13 region of the long arm of chromosome 12.

[0035] In some implementations, in step S1: the DNA extraction module uses a kit to extract genomic DNA from the sample.

[0036] The kit is preferably the QIAamp DNA Mini Kit.

[0037] In some preferred embodiments, step S1 involves extracting genomic DNA from the sample using the QIAamp DNA Mini Kit and its standard operating procedures; quality control of the extracted DNA is performed using 1% agarose gel electrophoresis; and the concentration of the extracted DNA sample is determined by... DNA Assay Kit DNA concentration detection kit, in The test was performed using the 2.0 Fluorimeter nucleic acid detection instrument.

[0038] In some implementations, in step S2, the gene library module uses the Agilent Sure SelectHuman All Exon 50Mb Kit to construct the gene library.

[0039] In some implementations, step S2 involves using a 0.6 μg genomic DNA sample for subsequent library construction.

[0040] In some implementations, step S2, the ultrasonic fragmentation includes breaking the genomic DNA into fragments of 180bp-280bp by ultrasonic treatment, followed by quality control and DNA concentration detection.

[0041] Preferably, in step S2, the DNA sample is fragmented using an ultrasound device (Covaris) so that the molecular weight of the DNA fragments is between 180-280 bp.

[0042] Preferably, in step S2, end repair involves ligating a sequencing adapter A base to the 3' end of the DNA fragment.

[0043] Preferably, the PCR amplification conditions in step S2 are: 95℃ pre-denaturation for 2 min; 98℃ denaturation for 20 s, 70℃ for 5 min, 25-30 cycles; the obtained amplification product is stored at 4℃ for short-term storage or at -20℃ for long-term storage.

[0044] Preferably, in step S2, the PCR product is enriched with biotin magnetic beads.

[0045] Preferably, in step S2, the PCR enrichment product is purified by adding adapter tag sequences via PCR using the AMPure XP system, and quantified using the Agilent high sensitivity DNA assay DNA detection kit in an Agilent Bioanalyzer 2100 system.

[0046] In some implementations, in step S3, the sequencing data is raw sequence data.

[0047] In some implementations, in step S3, the sequencing is performed on an NGS sequencing platform.

[0048] Preferably, in step S3, the sample is reacted in the cBotCluster Generation system using the HiSeq PE Cluster Kit (Illumina); and sequenced in the Illumina HiSeq platform sequencer to obtain 150bp paired-tailed sequence information.

[0049] Preferably, the data obtained in S4 is compared with the database to obtain information on copy number variations, single nucleotide variations, small fragment sequence insertions, and deletions.

[0050] Preferably, in steps S3 to S5, obtaining the raw data also includes a data cleaning step, such as data cleaning according to the Illumina data processing standard process.

[0051] Preferably, in steps S3 to S5, the data after data cleaning also includes steps of data matching and gene annotation, for example, using the human genome reference sequence (UCSC hg19) and performing sequence matching and gene annotation using Burrows-Wheeler Aligner (BWA) software, SAMtools software, or Picard software (http: / / broadinstitute.github.io / picard / ).

[0052] Preferably, steps S3 to S5 further include performing site variation analysis and copy number variation analysis, for example, using tools such as Samtools mpileup, bcftools, and GATK's (GATK 4) to perform site variation analysis and copy number variation analysis.

[0053] In some implementations, step S4 includes quality control testing, which includes sequencing depth, coverage uniformity, and contrast ratio analysis.

[0054] Preferably, step (2) also includes result analysis, such as analyzing whether there is a statistically significant increase in gene amplification information of region 13.13 on the long arm of chromosome 12 of the sample compared with the database using GISTIC2 software.

[0055] The second aspect of the present invention provides a predictive model for the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma, which is established by the construction method described in the first aspect of the present invention.

[0056] In a preferred embodiment, the prediction model includes:

[0057] (1) Input module, the input module is used to input gene amplification information of the 13.13 segment of the long arm of chromosome 12 of moderately and well differentiated esophageal squamous cell carcinoma samples;

[0058] (2) Analysis module, which is used to analyze whether there is a statistically significant increase in gene amplification information of region 13.13 on the long arm of chromosome 12 of the sample compared with the database;

[0059] (3) Output module: If there is a statistically significant increase, the output module is used to output the result as a poor prognosis of the sample.

[0060] A third aspect of the present invention provides the use of reagents for amplification of region 13.13 on the long arm of chromosome 12 in the preparation of a prognostic test for moderately differentiated esophageal squamous cell carcinoma samples.

[0061] In a preferred embodiment, the reagent is used to detect the expression level of region 13.13 on the long arm of chromosome 12; the expression level is the protein expression level and / or mRNA transcription level.

[0062] In a preferred embodiment of the present invention, the reagent is a biomolecular reagent that specifically binds to the 13.13 region of the long arm of chromosome 12, or specifically hybridizes with the nucleic acid encoding the 13.13 region of the long arm of chromosome 12.

[0063] In a preferred embodiment of the present invention, the biomolecular reagent is selected from primers, probes, and antibodies.

[0064] In a preferred embodiment of the present invention, the reagent is a reagent used for transcriptome and / or proteome sequencing.

[0065] In a preferred embodiment, the prognosis of the sample is poor when amplification occurs in region 13.13 of the long arm of chromosome 12.

[0066] In a preferred embodiment, the sample may be a patient.

[0067] In a preferred embodiment, sequencing technology is used to detect whether amplification has occurred in region 13.13 of the long arm of chromosome 12 in the sample.

[0068] In a preferred implementation, whole exome sequencing technology is used.

[0069] The fourth aspect of this invention provides a method for assessing the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma in a subject using a predictive model. The method determines the subject's prognosis by detecting whether amplification occurs on the 13.13 region of the long arm of chromosome 12 in the sample. When amplification occurs on the 13.13 region of the long arm of chromosome 12, the subject's prognosis is poor. The predictive model is established by the construction method described in the first aspect of this invention, or is a predictive model described in the second aspect of this invention.

[0070] In a preferred embodiment, sequencing technology is used to detect whether amplification has occurred in region 13.13 of the long arm of chromosome 12 in the sample.

[0071] In a preferred implementation, whole exome sequencing technology is used.

[0072] In a preferred embodiment, the method is not for therapeutic or diagnostic purposes and is applicable to scenarios such as laboratory research and environmental monitoring.

[0073] The fifth aspect of the present invention provides the application of a prediction model established by the construction method described in one aspect of the present invention or a prediction model described in the second aspect of the present invention in assessing the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma in subjects.

[0074] Preferably, in the application, the prognosis of the subject is determined by detecting whether amplification occurs on the 13.13 region of the long arm of chromosome 12 in the sample. When amplification occurs on the 13.13 region of the long arm of chromosome 12, the prognosis of the subject is poor.

[0075] The fifth aspect of the present invention provides a method for treating moderately or well-differentiated esophageal squamous cell carcinoma, which involves administering to a patient in need a therapeutically effective amount of an agent that inhibits or activates genes on the long arm of chromosome 12, segment 13.13.

[0076] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0077] The reagents and raw materials used in this invention are all commercially available.

[0078] The positive and progressive effects of this invention are as follows: This invention predicts the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma by detecting whether 12q13.13 amplification occurs. Compared to patients with moderately to well-differentiated esophageal squamous cell carcinoma without 12q13.13 amplification, patients with moderately to well-differentiated esophageal squamous cell carcinoma exhibiting 12q13.13 amplification have a poorer prognosis. This invention can provide some assistance in the diagnosis, treatment, and medication of patients with moderately to well-differentiated esophageal squamous cell carcinoma. Attached Figure Description

[0079] Figure 1 The differential analysis of chromosomal arm events between moderately and well-differentiated esophageal squamous cell carcinoma and poorly differentiated esophageal squamous cell carcinoma showed that 12q13.13 amplification had a significant difference in the association with survival between the two populations. The p-values ​​on the vertical axis represent the p-values ​​of chromosomal amplification information between the two populations, and Amp in the figure represents esophageal squamous cell carcinoma patients.

[0080] Figure 2 The overall survival (OS) results for the sample revealed that patients with differentiated esophageal squamous cell carcinoma who had 12q13.13 amplification (Amp) had significantly worse survival than those who did not have 12q13.13 amplification (other).

[0081] Figure 3 For two populations with independent overall survival (OS) results, in patients with poorly differentiated esophageal squamous cell carcinoma, there was no significant difference in survival between patients with 12q13.13 amplification (Amp) and those without; in patients with moderately or well-differentiated esophageal squamous cell carcinoma, there was a significant difference in survival between patients with 12q13.13 amplification (Amp) and those without.

[0082] Figure 4 The ROC analysis results are shown for well-differentiated and poorly differentiated esophageal squamous cell carcinoma patients. Detailed Implementation

[0083] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0084] Example 1: Differential analysis of arm events in moderately and well-differentiated esophageal squamous cell carcinoma and poorly differentiated esophageal squamous cell carcinoma showed that 12q13.13 amplification was significantly associated with survival.

[0085] Study subjects: 144 patients who underwent surgery for esophageal squamous cell carcinoma (88 cases of moderately to well-differentiated esophageal squamous cell carcinoma and 56 cases of poorly differentiated esophageal squamous cell carcinoma).

[0086] Sample collection: Surgically removed tumor tissue samples.

[0087] Assessing the prognosis of esophageal squamous cell carcinoma using whole-exome sequencing includes the following steps:

[0088] S1. Genomic DNA extraction from esophageal squamous cell carcinoma sections and adjacent or blood cells: Genomic DNA was extracted using kits and standard DNA extraction methods;

[0089] Genomic DNA was extracted from the samples using the QIAamp DNA Mini Kit and its standard operating procedures. Quality control of the extracted DNA was performed using 1% agarose gel electrophoresis. The concentration of the extracted DNA sample was determined using [the appropriate method / method]. DNA Assay Kit DNA concentration detection kit, in The test was performed using the 2.0 Fluorimeter nucleic acid detection instrument.

[0090] S2. Gene library construction: Genomic DNA is broken by sonication, end repair, adapter ligation, hybridization capture, and PCR amplification;

[0091] Genomic DNA samples of 0.6 μg were fragmented using a Covaris sonicator to achieve DNA fragment molecular weights ranging from 180 to 280 bp. Library construction was performed using the Agilent Sure Select Human All Exon kit following standard operating procedures. End repair involved ligating sequencing adapter A bases to the 3' end of the DNA fragments. PCR amplification conditions were: 95°C pre-denaturation for 2 min; 98°C denaturation for 20 s; 70°C for 5 min; 25–30 cycles. The amplified products were stored briefly at 4°C. PCR products were enriched with biotin beads. The enriched products were purified using the AMPureXP system after PCR addition of adapter tag sequences, and quantified using the Agilent High Sensitivity DNA Assay kit on an Agilent Bioanalyzer 2100 system.

[0092] S3. Sequencing: High-throughput sequencing uses NGS sequencing platforms, including but not limited to Illumina HiSeq, for whole exome sequencing.

[0093] The samples were reacted using the HiSeq PE Cluster Kit (Illumina) in the cBot ClusterGeneration system; sequencing was performed on the Illumina HiSeq platform sequencer to obtain 150bp paired-tailed sequence information.

[0094] S4. Data Analysis: Perform quality control checks, read mapping, variant calling, copy number analysis (CNA), and analysis of significant mutated genes on the data.

[0095] S5. Screening: The obtained data are compared with the adjacent genome or blood cell gene database of esophageal squamous cell carcinoma patients to obtain copy number variations, single nucleotide variations, small fragment sequence insertions and deletions;

[0096] S6. Results Bioinformatics Analysis: Determine the mutation location and mutation type of the corresponding gene fragment, and perform biological analysis.

[0097] Raw data was acquired and cleaned according to Illumina's standard data processing workflow. After cleaning, the data was used to perform sequence matching and gene annotation using the Human Genome Reference Sequence (UCSC hg19) with Burrows-Wheeler Aligner (BWA), SAMtools, and Picard software (http: / / broadinstitute.github.io / picard / ). Site variation analysis and copy number variation analysis were performed using tools such as Samtools mpileup, bcftools, and GATK's (GATK 4). GISTIC2 software was used to analyze whether the gene amplification information of region 13.13 on the long arm of chromosome 12 of the samples showed a statistically significant increase compared to adjacent normal genomes or blood cell gene databases of esophageal squamous cell carcinoma patients; specifically, a q-value less than 0.1 was calculated to determine the copy number variation in region 13.13 on the long arm of chromosome 12.

[0098] The results are as follows Figure 1 As shown, the association between 2q13.13 amplification and survival differs significantly between the two populations.

[0099] Overall survival (OS) was determined for both populations, and the results are shown below. Figure 2 This indicates that the survival probability of samples amplified by 12q13.13 is significantly reduced, meaning that differentiated esophageal squamous cell carcinoma patients with 12q13.13 amplification (Amp) have significantly worse survival than patients without 12q13.13 amplification (other).

[0100] Example 2: Survival analysis showed no significant difference in prognosis between patients with 12q13.13 amplification and those without amplification in poorly differentiated esophageal squamous cell carcinoma.

[0101] Study subjects: 56 cases of poorly differentiated esophageal squamous cell carcinoma.

[0102] Sample collection: Surgically removed tumor tissue samples.

[0103] Assessing the prognosis of esophageal epithelial squamous cell carcinoma using whole-exome sequencing includes the following steps:

[0104] S1. Extraction of genomic and adjacent normal genomic DNA from poorly differentiated esophageal squamous cell carcinoma sections: Genomic DNA was extracted using kits and standard DNA extraction methods;

[0105] Genomic DNA was extracted from the samples using the QIAamp DNA Mini Kit and its standard operating procedures. Quality control of the extracted DNA was performed using 1% agarose gel electrophoresis. The concentration of the extracted DNA sample was determined using [the appropriate method / method]. DNA Assay Kit DNA concentration detection kit, in The test was performed using the 2.0 Fluorimeter nucleic acid detection instrument.

[0106] S2. Gene library construction: Genomic DNA is broken by sonication, end repair, adapter ligation, hybridization capture, and PCR amplification;

[0107] Genomic DNA samples of 0.6 μg were fragmented using a Covaris sonicator to achieve DNA fragment molecular weights ranging from 180 to 280 bp. Library construction was performed using the Agilent SureSelect Human All Exon kit following standard operating procedures. End repair involved ligating sequencing adapter A bases to the 3' end of the DNA fragments. PCR amplification conditions were: 95°C pre-denaturation for 2 min; 98°C denaturation for 20 s; 70°C for 5 min; 25–30 cycles. The amplified products were stored briefly at 4°C. PCR products were enriched with biotin beads. The enriched products were purified using the AMPureXP system after PCR addition of adapter tag sequences and quantified using the Agilent High Sensitivity DNA Assay kit on an Agilent Bioanalyzer 2100 system.

[0108] S3. Sequencing: High-throughput sequencing uses NGS sequencing platforms, including but not limited to Illumina HiSeq, for whole exome sequencing.

[0109] The samples were reacted using the HiSeq PE Cluster Kit (Illumina) in the cBot ClusterGeneration system; sequencing was performed on the Illumina HiSeq platform sequencer to obtain 150bp paired-tailed sequence information.

[0110] S4. Data Analysis: Perform quality control checks, read mapping, variant calling, copy number analysis (CNA), and analysis of significant mutated genes on the data.

[0111] S5. Screening: Compare the obtained data with the database to obtain copy number variations, single nucleotide variations, small fragment sequence insertions and deletions;

[0112] S6. Results Bioinformatics Analysis: Determine the mutation location and mutation type of the corresponding gene fragment, and perform biological analysis.

[0113] Raw data was acquired and cleaned according to Illumina's standard data processing workflow. After cleaning, the data was used to perform sequence matching and gene annotation using the Human Genome Reference Sequence (UCSC hg19) with Burrows-Wheeler Aligner (BWA), SAMtools, and Picard software (http: / / broadinstitute.github.io / picard / ). Site variation analysis and copy number variation analysis were performed using tools such as Samtools mpileup, bcftools, and GATK's (GATK 4). GISTIC2 software was used to analyze whether the gene amplification information of region 13.13 on the long arm of chromosome 12 of the sample showed a statistically significant increase compared to the database, i.e., whether the q-value was less than 0.1, to determine the copy number variation in region 13.13 on the long arm of chromosome 12.

[0114] The results are as follows Figure 3 As shown, the poor prognosis associated with 12q13.13 amplification did not differ significantly in patients with poorly differentiated esophageal squamous cell carcinoma (p<1×10⁻⁶). 3 ).

[0115] Example 3: Survival analysis showed that patients with 12q13.13 amplification in moderately to well-differentiated esophageal squamous cell carcinoma had a significantly worse prognosis.

[0116] Study subjects: 88 patients with moderately to well-differentiated esophageal squamous cell carcinoma who underwent surgical treatment.

[0117] Sample collection: Surgically removed tumor tissue samples.

[0118] Assessing the prognosis of moderately to well-differentiated esophageal squamous cell carcinoma using whole-exome sequencing includes the following steps:

[0119] S1. Genomic and adjacent normal genomic DNA extraction from moderately to well-differentiated esophageal squamous cell carcinoma sections: Genomic DNA was extracted using kits and standard DNA extraction methods;

[0120] Genomic DNA was extracted from the samples using the QIAamp DNA Mini Kit and its standard operating procedures. Quality control of the extracted DNA was performed using 1% agarose gel electrophoresis. The concentration of the extracted DNA sample was determined using [the appropriate method / method]. DNA Assay Kit DNA concentration detection kit, in The test was performed using the 2.0 Fluorimeter nucleic acid detection instrument.

[0121] S2. Gene library construction: Genomic DNA is broken by sonication, end repair, adapter ligation, hybridization capture, and PCR amplification;

[0122] Genomic DNA samples of 0.6 μg were fragmented using a Covaris sonicator to achieve DNA fragment molecular weights ranging from 180 to 280 bp. Library construction was performed using the Agilent SureSelect Human All Exon kit following standard operating procedures. End repair involved ligating sequencing adapter A bases to the 3' end of the DNA fragments. PCR amplification conditions were: 95°C pre-denaturation for 2 min; 98°C denaturation for 20 s; 70°C for 5 min; 25–30 cycles. The amplified products were stored briefly at 4°C. PCR products were enriched with biotin beads. The enriched products were purified using the AMPureXP system after PCR addition of adapter tag sequences and quantified using the Agilent High Sensitivity DNA Assay kit on an Agilent Bioanalyzer 2100 system.

[0123] S3. Sequencing: High-throughput sequencing uses NGS sequencing platforms, including but not limited to Illumina HiSeq, for whole exome sequencing.

[0124] The samples were reacted using the HiSeq PE Cluster Kit (Illumina) in the cBot ClusterGeneration system; sequencing was performed on the Illumina HiSeq platform sequencer to obtain 150bp paired-tailed sequence information.

[0125] S4. Data Analysis: Perform quality control checks, read mapping, variant calling, copy number analysis (CNA), and analysis of significant mutated genes on the data.

[0126] S5. Screening: Compare the obtained data with the database to obtain copy number variations, single nucleotide variations, small fragment sequence insertions and deletions;

[0127] S6. Results Bioinformatics Analysis: Determine the mutation location and mutation type of the corresponding gene fragment, and perform biological analysis.

[0128] Raw data was acquired and cleaned according to Illumina's standard data processing workflow. After cleaning, the data was used to perform sequence matching and gene annotation using the human genome reference sequence (UCSC hg19) via Burrows-Wheeler Aligner (BWA), SAMtools, and Picard software (http: / / broadinstitute.github.io / picard / ). Site variation analysis and copy number variation analysis were performed using tools such as Samtools mpileup, bcftools, and GATK's (GATK 4) to obtain the copy number variation information for region 13.13 on the long arm of chromosome 12.

[0129] The results showed that among 88 patients who underwent surgery for moderately to well-differentiated esophageal squamous cell carcinoma, 39 cases had 12q13.13 amplification, while 49 cases did not (e.g., ...). Figure 2 (As shown).

[0130] Statistical analysis based on the ten-year follow-up information of the above cases showed that patients with moderately to well-differentiated esophageal squamous cell carcinoma who developed 12q13.13 amplification (5-year survival rate less than 30%) had significantly worse survival than patients with moderately to well-differentiated esophageal squamous cell carcinoma who did not develop 12q13.13 amplification (5-year survival rate close to 60%). Figure 3 (As shown).

[0131] Prognostic assessment was performed on esophageal squamous cell carcinoma patients with a follow-up period of 80 months. During this follow-up period, death occurring after 24 months was considered a poor long-term prognosis, while no death occurring after 24 months was considered a good prognosis. Using the 12q13.13 copy number as a biomarker, the predictive specificity and sensitivity for moderately and well-differentiated patients were approximately 70% (AUC = 0.7), while in poorly differentiated patients, the AUC of the above classification method was close to 0.5, which was not statistically significant (see results). Figure 4 ).

[0132] References

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[0134] Wild, CP, et al., World Cancer Report: Cancer Research for Cancer Prevention. 2020, Lyon, France: International Agency for Research on Cancer (IARC).

[0135] Pennathur,A.,et al.,Oesophageal carcinoma.Lancet, 2013.381(9864):p.400-12.

[0136] Cancer Genome Atlas Research,N.et al.Integrated genomic characterization of oesophageal carcinoma.Nature 541,169-175,doi:10.1038 / nature20805(2017).

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[0138] Cui,Y.et al.Whole-genome sequencing of 508patients identifies keymolecular features associated with poor prognosis in esophageal squamous cellcarcinoma.Cell research 30,902-913,doi:10.1038 / s41422-020-0333-6(2020).

[0139] The above embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a prognosis prediction model of moderately-highly differentiated esophageal squamous cell carcinoma, characterized in that, The method comprises the following steps: (1) establishing an input module for inputting gene amplification information of the 12q13.13 segment of the sample of the moderately-highly differentiated esophageal squamous cell carcinoma; (2) establishing an analysis module for analyzing whether the gene amplification information of the 12q13.13 segment of the sample has a statistically significant increase compared with the database; (3) establishing an output module for outputting a result that the sample has a poor prognosis if there is a statistically significant increase.

2. The construction method of claim 1, wherein, The judgment standard of the statistically significant increase is that the q value is less than 0.

1.

3. The construction method of claim 1, wherein, Step (1) further comprises performing whole exome sequencing technology on the sample to obtain the gene amplification information of the 12q13.13 segment; And / or, the database in step (2) comprises a paired thousand human gene database, an esophageal squamous cell carcinoma population paracancer genome, or a blood cell gene database.

4. The construction method of claim 3, wherein, Step (1) further comprises the following steps: S1, establishing a DNA extraction module for extracting genomic DNA of the sample; S2, establishing a gene library module for obtaining a gene library obtained by ultrasonic treatment breaking, end repair, adapter ligation, hybrid capture, and PCR amplification of the genomic DNA; S3, establishing a sequencing module for performing whole exome sequencing to obtain sequencing data; S4, establishing a data analysis module for performing analysis selected from the following group on the sequencing data obtained by the sequencing module: quality control detection, fragment matching, variation analysis, copy number analysis, and significant mutation gene analysis; and obtaining information of copy number variation, single nucleotide variation, and small fragment sequence insertion and deletion; S5, establishing a bioinformatics analysis module for determining the mutation position and mutation type of the 12q13.13 segment, and performing biological analysis to screen the amplification gene variation of the 12q13.13 segment.

5. The construction method of claim 4, wherein, The sequencing module is an Illumina HiSeq.

6. The construction method according to claim 4, wherein In step S1, the DNA extraction module extracts genomic DNA of the sample using a kit; and / or In step S2, the gene library module uses an Agilent Sure Select Human All Exon 50Mb Kit to construct a gene library; and / or In step S2, the ultrasonic treatment breaking includes breaking the genomic DNA into 180bp-280bp fragments by ultrasonic treatment, and performing quality control and DNA concentration detection.

7. The construction method of claim 6, wherein, The kit is a QIAamp DNA Mini Kit.

8. The construction method of claim 6, wherein, In step S3, the sequencing data is raw sequence data; and / or In step S4, further comprising the steps of data cleaning, data matching, and gene annotation.

9. A prognosis prediction model of moderately-highly differentiated esophageal squamous cell carcinoma, characterized by, Established by the construction method of any one of claims 1-8.

10. Use of a reagent for detecting amplification of the 13.13 segment of the long arm of chromosome 12 in the preparation of a prognostic test agent for samples of moderately-highly differentiated esophageal squamous cell carcinoma.

11. Use of a prognostic model according to claim 9 for assessing the prognosis of a subject with moderately-highly differentiated esophageal squamous cell carcinoma.

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