A system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring recurrence based on saliva genomic characteristics and application

Abstract

This invention provides a system and application for non-invasive diagnosis of oral squamous cell carcinoma (OSCC) and monitoring of recurrence based on salivary genomic characteristics. By performing targeted deep sequencing on tumor DNA derived from an individual's saliva, analyzing its somatic mutation profile, and calculating the tumor mutation burden, the risk of having OSCC or postoperative recurrence is assessed based on the presence of driver gene mutations and / or the elevation of the tumor mutation burden. This invention is the first to discover and verify a strong correlation between salivary TMB and matched tumor tissue TMB, and that salivary TMB is significantly elevated in recurrent patients, independent of lymph node metastasis and clinical stage, indicating that it is a highly specific biomarker for monitoring local recurrence. It has the advantages of high non-invasiveness and accessibility, high sensitivity and fidelity, accurate recurrence monitoring capabilities, and a clear clinical translation pathway, and can be directly translated into clinical testing products or services for early auxiliary diagnosis of OSCC, detection of minimal residual disease after surgery, and recurrence early warning.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method, reagent kit, and system for non-invasive diagnosis, molecular subtyping, and recurrence risk monitoring of oral squamous cell carcinoma using circulating tumor DNA derived from saliva. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck, with a high mortality rate primarily attributed to delayed diagnosis and high postoperative recurrence. Currently, the gold standard for OSCC diagnosis is histopathological biopsy, a method that is invasive, susceptible to sampling bias, and lacks dynamic monitoring capabilities. Liquid biopsy, particularly based on circulating cell-free DNA (cfDNA) detection, offers the possibility of non-invasive tumor monitoring; however, for early-stage or locally recurrent OSCC, plasma cfDNA abundance is low, limiting its sensitivity.

[0003] Saliva, as a bodily fluid that comes into direct contact with oral lesions, can theoretically capture tumor-released DNA more extensively. However, whether salivary DNA can accurately and faithfully reflect the complete genomic map of OSCC tumor tissue, especially its ability to differentiate between primary and recurrent diseases, has previously lacked systematic, high-level genomic validation. Existing technologies mostly focus on detecting mutations in a few known genes, failing to conduct comprehensive comparisons at the levels of mutation spectrum, clonal structure, tumor mutation burden, and functional pathways, thus limiting the credibility and scope of their clinical translation. Summary of the Invention

[0004] This invention addresses the aforementioned problems by providing a system and application for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics. By performing targeted deep sequencing on tumor DNA derived from an individual's saliva, analyzing its somatic mutation profile, and calculating the tumor mutation burden, the risk of the individual having oral squamous cell carcinoma or postoperative recurrence is assessed based on the presence of driver gene mutations and / or whether the tumor mutation burden exceeds a preset threshold.

[0005] This invention is based on deep sequencing and bioinformatics analysis of salivary DNA. By systematically comparing and verifying its genomic characteristics with those of tumor tissue, a reliable diagnostic and monitoring model is established. It is the first to systematically demonstrate the high degree of consistency between salivary DNA and tumor tissue at the mutation profile, functional pathway, and even protein domain levels. Furthermore, it uniquely proposes salivary tumor mutation burden (Salivary TMB) as a specific biomarker for monitoring local recurrence.

[0006] Based on the above research, the technical solution to be protected by this invention is as follows:

[0007] In a first aspect, this invention provides a system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics, comprising a sample processing system, a sequencing system, and an analysis system. The sample processing system is used to automatically process collected saliva and peripheral blood samples and extract genomic DNA from them; the sequencing system is used to sequence the genomic DNA of the samples; the analysis system analyzes the somatic mutation profile and calculates the tumor mutation burden, assessing the individual's risk of having oral squamous cell carcinoma or postoperative recurrence based on the presence of driver gene mutations and / or whether the tumor mutation burden is elevated. Details are as follows:

[0008] (1) Sample processing system

[0009] This includes sterile tubes for holding collected saliva and peripheral blood samples, as well as a DNA extraction kit.

[0010] Saliva samples of 1.5-2 mL were collected from the individuals to be tested in sterile tubes, and peripheral blood samples were collected simultaneously as germline controls. Total DNA was extracted from the saliva samples and genomic DNA was extracted from peripheral blood leukocytes using a universal genomic DNA extraction kit with magnetic beads.

[0011] (2) Sequencing system

[0012] The sequencing system uses a customized targeted sequencing panel for OSCC to construct libraries and capture hybridizations from saliva DNA. The panel covers all or part of the exon regions of no less than 42 high-frequency mutant genes in OSCC. The saliva DNA is then sequenced at an average sequencing depth of no less than 500×.

[0013] Preferably, the 42 high-frequency OSCC mutant genes are selected from the following genes: TP53, TTN, FAT1, CSMD3, MUC16, CDKN2A, SYNE1, LRP1B, NOTCH1, PIK3CA, PCLO, KMT2D, DNAH5, NSD1, USH2A, FLG, AHNAK, RYR2, CASP8, COL11A1, FAM135B, PKHD1L1, FAT3, DST, PLEC, XIRP2, DMD, SI, HUWE1, MUC17, PAPPA2, RELN, COL22A1, PCDH15, APOB, FAT4, PCDH11X, AHNAK2, CUBN, MUC5B, NAV3, DNAH8

[0014] (3) Analysis System

[0015] It consists of the following modules:

[0016] (3-1) Sequencing data preprocessing module,

[0017] The quality-controlled raw sequencing data was aligned to the hg38 reference genome, and duplication labeling, local realignment, and base quality recalibration were performed.

[0018] In a preferred embodiment of the present invention, FastQC is used for raw data quality control; BWA-MEM is used to align sequencing data to the hg38 reference genome; and GATK tools are used for repeat labeling, local realignment, and base quality recalibration.

[0019] By aligning sequencing data with the hg38 reference genome, the location of sequencing reads on the genome can be determined, providing a basis for subsequent SNP, Indel, SV and other variant detection. It also helps to estimate allele-specific expression or copy number variations and discover known or unknown insertions / deletions. At the same time, it supports downstream analysis processes, such as variant correction, local reassembly, base quality fraction recalibration (BQSR) and other GATK best practice steps, all of which require hg38 alignment results.

[0020] (3-2) Sequencing data filtering module

[0021] Using paired germline DNA as a control, somatic single nucleotide variants and insertions / deletions are called up to identify somatic single nucleotide variants, and common germline variants in dbSNP are filtered based on preset filtering criteria.

[0022] Preferably, the individual's own peripheral blood leukocyte DNA sequencing data is used as the paired germline DNA; Mutect2 is used to retrieve somatic single nucleotide variants and insertions / deletions using the paired germline DNA as a control.

[0023] The filtering criteria are as follows: mutations with sequencing depth ≥10×, variant allele frequency ≥4%, and strand bias <90% are retained.

[0024] (3-3) Feature Extraction and Analysis Module

[0025] Somatic mutation profiles were extracted from saliva, including mutation types and a list of high-frequency mutated genes. The similarity between the saliva mutation profile and the mutation profile of typical OSCC tumor tissue was calculated. Based on somatic mutations, the tumor mutation burden (TMB) in saliva was calculated. At the same time, the core signaling pathways affected by saliva mutations were analyzed, and the cosine similarity between their enrichment profile and the pathway enrichment profile in tumor tissue was calculated.

[0026] In a preferred embodiment of the present invention, the salivary tumor mutational burden (TMB) is defined as follows: the number of nonsynonymous cell mutations detected per megabase pair in the target region.

[0027] In current clinical and research practices, TMB calculation is mainly divided into tissue TMB and blood TMB. Saliva, due to its extremely low tumor DNA content and significant interference, is generally not used as a sample source for direct TMB calculation. In this invention, through post-sequencing data processing and filtering, false positives and interfering mutations were successfully eliminated. The results showed that the calculated saliva TMB was strongly positively correlated with tumor tissue TMB (Spearman's ρ = 0.99, P < 0.001), and can be fully used for recurrence risk assessment.

[0028] In a preferred embodiment of the present invention, the core signaling pathways affected by saliva mutations include TP53, RTK-RAS, Hippo, and NOTCH pathways. Validation of 54 pairs of rigorously screened high-quality versions showed a high degree of consistency between the mutation profiles and high-frequency genes (TP53, FAT1, etc.) of saliva and tumor tissue, with a cosine similarity of 0.97 in the enrichment profile of the signaling pathways.

[0029] (3-4) Risk Analysis Module

[0030] If at least one somatic mutation of a high-frequency OSCC gene is detected in a saliva sample, oral squamous cell carcinoma is determined to be present; when the calculated saliva TMB value is greater than a preset recurrence risk threshold, postoperative recurrence risk is determined to be present.

[0031] In a preferred embodiment of the present invention, the high-frequency mutation genes of OSCC include the above 42 genes, including TP53, FAT1, CDKN2A, PIK3CA, NOTCH1, etc.

[0032] The relapse risk threshold was obtained by analyzing salivary TMB data from a validation cohort including patients with primary and recurrent OSCC. It was determined through a retrospective analysis of OSCC patient cohorts with known clinical outcomes, and used to differentiate between primary and recurrent disease. Salivary TMB was significantly higher in the relapse group compared to that of the primary and recurrent patients.

[0033] (3-5) Input display module, used to receive raw sequencing data of saliva samples and display the analysis results of the risk analysis module; such as generating a standardized report containing a mutation list, TMB value, risk assessment conclusion and clinical recommendations.

[0034] (3-6) The storage module is used to store the received raw sequencing data and its corresponding basic patient information, the analysis results of each module, the preset filtering criteria, and the recurrence risk threshold. By storing relevant data during the analysis process in real time, it provides conditions for subsequent reverse lookup.

[0035] (3-7) Communication module, used for communication connection between the analysis system and terminal devices or other databases. The analysis system in this invention can be installed on a computer, sequencing equipment, or other devices. During the analysis process, it communicates with other devices or analysis tools through the communication module, and transmits the detection results to remote devices for viewing.

[0036] Furthermore, the present invention provides a non-transitory computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the functions of the analysis system as described above.

[0037] In a second aspect, the present invention provides a kit for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence, comprising reagents for sample collection and standardization, and deep sequencing reagents. The reagents for sample collection and standardization include sterile tubes for holding saliva and peripheral blood samples respectively, and a universal genomic DNA extraction reagent; the deep sequencing reagents are customized targeted sequencing panels for OSCC.

[0038] Preferably, the universal genomic DNA extraction reagent is a magnetic bead-based universal genomic DNA extraction kit; the customized targeted sequencing panel for OSCC covers all or part of the exon regions of no less than 42 high-frequency mutant genes in OSCC.

[0039] In a third aspect, this invention provides a method for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics. The method utilizes the aforementioned reagent kit for detection and the aforementioned system for data processing and analysis, comprising the following steps:

[0040] (1) Sample collection and standardization: 1.5-2 mL of saliva sample from the individual to be tested was collected in a sterile tube, and peripheral blood sample was collected simultaneously as a germline control; total DNA was extracted from the saliva sample using magnetic beads, and genomic DNA was extracted from peripheral blood leukocytes.

[0041] (2) Targeted deep sequencing and bioinformatics analysis:

[0042] a) Use a customized targeted sequencing panel for OSCC to construct and capture a library of salivary DNA, the panel covering all or part of the exon regions of no less than 42 high-frequency mutant genes in OSCC; and perform sequencing of salivary DNA at an average sequencing depth of no less than 500×.

[0043] b) The quality-controlled raw sequencing data was aligned to the hg38 reference genome, and duplication labeling, local realignment, and base quality recalibration were performed. Using the individual's own peripheral blood leukocyte DNA as a germline control and paired germline DNA as a control, somatic single nucleotide variants (SNVs) and insertions / deletions were retrieved to identify somatic SNVs, and common germline variants in dbSNPs were filtered based on preset filtering criteria.

[0044] The preferred approach for analyzing saliva sequencing data is a bioinformatics workflow that includes the following steps:

[0045] i. Use BWA-MEM to align sequencing reads to the GRCh38 / hg38 reference genome;

[0046] ii. Use the GATK toolkit to perform duplicate labeling, local realignment, and base mass recalibration;

[0047] iii. Use Mutect2 to call up somatic single nucleotide variants and insertions / deletions using paired germline DNA as a control;

[0048] iv. Apply filtering criteria: retain mutations with sequencing depth ≥10×, variant allele frequency ≥4% and ≥0.5%, and strand bias <90%, and filter common germline variations in dbSNP;

[0049] v. Use ANNOVAR to annotate features.

[0050] 3) Multidimensional genomic feature extraction and validation:

[0051] a) Mutation spectrum analysis: Extract the somatic mutation spectrum from saliva, including mutation types and a list of high-frequency mutated genes; calculate the similarity between the saliva mutation spectrum and the mutation spectrum of typical OSCC tumor tissue.

[0052] b) Tumor mutation burden calculation: Based on somatic mutations, the salivary tumor mutation burden is calculated and defined as the number of non-synonymous somatic mutations detected in the target region per megabase pair.

[0053] c) Functional consistency verification (optional but preferred): Analyze the core signaling pathways affected by salivary mutations (such as TP53, RTK-RAS, Hippo, NOTCH pathways) and calculate the cosine similarity between their enrichment profiles and the enrichment profiles of tumor tissue pathways.

[0054] 4) Comprehensive interpretation and risk assessment:

[0055] a) Positive diagnostic indication: If at least one somatic mutation from a gene list including TP53, FAT1, CDKN2A, PIK3CA, and NOTCH1 is detected in a saliva sample, it suggests the presence of oral squamous cell carcinoma.

[0056] b) Relapse risk stratification: Comparing the calculated salivary TMB values ​​of patients with relapse and those with primary disease revealed that salivary TMB was significantly elevated in patients with relapse (P = 0.0385), but not related to lymph node metastasis or clinical stage, indicating that it is a highly specific biomarker for monitoring local relapse.

[0057] The role and effect of invention

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0059] 1. High non-invasiveness and accessibility: Only saliva needs to be collected, which is completely non-invasive, resulting in high patient compliance and facilitating repeated sampling and dynamic monitoring.

[0060] 2. High sensitivity and fidelity: Based on deep targeted sequencing, this invention verifies that salivary ctDNA can replicate key genomic features of tumor tissue with high fidelity (cosine similarity up to 0.97), including mutation profiles, protein domain variation patterns and core oncogenic pathways (such as TP53 and RTK-RAS pathways) activity, with a detection rate as high as 92.7%.

[0061] 3. Precise recurrence monitoring capability: This invention is the first to discover and verify that salivary tumor mutational burden is highly correlated with TMB in matched tumor tissue (Spearman's ρ = 0.99), and salivary TMB is significantly elevated in recurrent patients, but is not related to lymph node metastasis or clinical stage, indicating that it is a highly specific biomarker for monitoring local recurrence.

[0062] 4. Clear clinical translation path: The method of this invention is based on rigorous self-controlled clinical research with solid data, and can be directly translated into clinical testing products or services for early auxiliary diagnosis of OSCC, detection of minimal residual lesions after surgery, and early warning of recurrence. Attached Figure Description

[0063] Figure 1 : Schematic diagram of the screening process of this invention.

[0064] Figure 2 Overall Flowchart of the Method of this Invention

[0065] Figure 3 This example demonstrates a high degree of consistency between the somatic mutation landscape of tumor tissue (A) and saliva sample (B).

[0066] Figure 4This example demonstrates the high degree of consistency between tumor tissue and saliva samples in the mutation patterns of key driver genes (TP53, PIK3CA) protein domains.

[0067] Figure 5 Example: Cosine similarity matrix of oncogenic pathway enrichment spectrum between tumor tissue and saliva sample, where A is the pathway mutation frequency distribution of tumor sample; B is the pathway mutation frequency distribution of saliva sample; C is the 2×2 cosine similarity matrix of tumor sample and saliva sample; D is the spider diagram of pathway mutation frequency distribution of tumor sample and saliva sample.

[0068] Figure 6 Box plot showing the comparison of salivary tumor mutation burden between primary (A) and recurrent OSCC (B) patients.

[0069] Figure 7 : A scatter plot showing the correlation between salivary TMB and tumor tissue TMB.

[0070] Figure 8 : Block diagram of a non-invasive diagnostic system for oral squamous cell carcinoma and a system for monitoring recurrence based on salivary genomic characteristics. Detailed Implementation

[0071] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0072] Example 1: Method Establishment and Verification

[0073] 1. Research Subjects and Sample Collection

[0074] Patients with a confirmed pathological diagnosis of oral squamous cell carcinoma (OSCC) who visited the Department of Oral and Maxillofacial Head and Neck Oncology and the Department of Oral Mucosal Diseases at the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine between May and October 2023 were screened and enrolled. Paired samples were collected from these patients before treatment: tumor tissue, saliva, and peripheral blood. (Reference) Figure 1 The specific screening process includes the following steps:

[0075] Inclusion criteria: OSCC patients; surgical biopsy pathological diagnosis of OSCC; age 18–75 years; no history of surgery, cryotherapy, laser therapy, radiotherapy, or chemotherapy; able to sign informed consent form and voluntarily participate in the trial.

[0076] Exclusion criteria: Patients with a pathological diagnosis of non-OSCC; age under 18 years or over 75 years; history of other systemic malignancies within the past five years; patients who have undergone surgery, laser therapy, cryotherapy, radiotherapy, or chemotherapy; other conditions that make them unsuitable for participation in the trial, such as pregnancy or lactation.

[0077] 2. Whole-exome sequencing and bioinformatics analysis

[0078] See the method flowchart. Figure 2 Whole exome sequencing (WES, 100×) was performed on tumor tissue. Targeted deep sequencing (500×) was performed on saliva DNA using a custom 600-gene panel. Peripheral blood leukocyte DNA was used as a germline control. Specifically, the above sequencing steps were achieved through the following process:

[0079] 1) Sample processing and DNA extraction: Obtain oral mucosal tissue samples from the patient and immediately place them in liquid nitrogen for rapid freezing for half an hour before DNA extraction. Follow the manufacturer's instructions to ensure a total DNA volume of 500 ng and a concentration ≥20 ng / μL.

[0080] A saliva sample of 1.5-2 mL was collected from the patient in a sterile tube, and a peripheral blood sample was collected simultaneously as a germline control. Total DNA was extracted from the saliva sample using a magnetic bead method, and genomic DNA was extracted from the peripheral blood leukocytes.

[0081] 2) Library construction and sequencing:

[0082] For tumor tissue DNA, the following method is used:

[0083] ① A universal genomic DNA extraction kit using magnetic beads was used to capture probes in the whole exon regions of genes; ② The library quality was evaluated using Qubit 2.0 and Agilent 2100.

[0084] ③ Sequencing was performed on the NovaSeq 6000 platform using paired-end 150bp reads, with each sample generating 30GB of raw data.

[0085] For saliva samples and peripheral blood leukocyte DNA, the following methods were used:

[0086] A customized targeted sequencing panel for OSCC was used to construct libraries and capture hybridizations of salivary DNA. The panel covered all or part of the exon regions of no less than 42 high-frequency mutant genes in OSCC. The salivary DNA was then sequenced at an average sequencing depth of no less than 500×.

[0087] 3) Somatic cell mutation detection:

[0088] For tumor tissue NDA, the following methods were used for detection and bioinformatics analysis:

[0089] ① Use FastQC for raw data quality control;

[0090] ② The sequencing data was aligned to the hg38 reference genome using BWA-MEM;

[0091] ③ VarScan is used to identify somatic single nucleotide mutations (SNVs) and finally obtain mutation detection results.

[0092] For saliva samples and peripheral blood leukocyte DNA, a bioinformatics workflow including the following steps is preferred for analyzing saliva sequencing data:

[0093] i. Use BWA-MEM to align sequencing reads to the GRCh38 / hg38 reference genome;

[0094] ii. Use the GATK toolkit to perform duplicate labeling, local realignment, and base mass recalibration;

[0095] iii. Use Mutect2 to call up somatic single nucleotide variants and insertions / deletions using paired germline DNA as a control;

[0096] iv. Apply filtering criteria: retain mutations with sequencing depth ≥10×, variant allele frequency ≥4% and ≥0.5%, and strand bias <90%, and filter common germline variations in dbSNP;

[0097] v. Use ANNOVAR to annotate features.

[0098] 4) Results and threshold determination:

[0099] Sensitivity and fidelity: Somatic mutations were detected in saliva in 51 out of 54 high-quality samples (92.7%). Mutation profiles of saliva and tumor tissue (…) Figure 3 High-frequency genes (TP53, FAT1, etc.) Figure 4 The signal pathway enrichment spectrum cosine similarity is highly consistent, reaching 0.97. Figure 5 ).

[0100] Discovery of salivary TMB: Calculation of salivary TMB revealed a very strong positive correlation with tumor tissue TMB (Spearman's ρ = 0.99, P < 0.001). Figure 7 Furthermore, salivary TMB was significantly elevated in patients with relapse, but was not related to lymph node metastasis or clinical stage, indicating that it is a highly specific biomarker for monitoring local relapse.

[0101] Establishment of relapse risk threshold: Comparing salivary TMB in primary (n=34) and relapsed (n=21) patients, the relapse group had a significantly higher TMB (P = 0.0385). Figure 6 Based on this, the risk of recurrence in patients can be predicted by comparing thresholds.

[0102] Example 2 System Construction

[0103] See Figure 8 The system 100 provided in this embodiment for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic features includes a sample processing system 1, a sequencing system 2, and an analysis system 3.

[0104] The sample processing system 1 is used to automatically process collected saliva and peripheral blood samples and extract genomic DNA from them, including sterile tubes for holding the collected saliva and peripheral blood samples and a DNA extraction kit.

[0105] Saliva samples of 1.5-2 mL were collected from the individuals to be tested in sterile tubes, and peripheral blood samples were collected simultaneously as germline controls. Total DNA was extracted from the saliva samples and genomic DNA was extracted from peripheral blood leukocytes using a universal genomic DNA extraction kit with magnetic beads.

[0106] Sequencing system 2 is used to sequence the genomic DNA of the sample; specifically, a customized targeted sequencing panel for OSCC is used to construct a library and capture hybridization of saliva DNA, the panel covering all or part of the exon regions of no less than 42 high-frequency mutant genes of OSCC; and the saliva DNA is sequenced at an average sequencing depth of no less than 500×.

[0107] The analysis system 3 includes a sequencing data preprocessing module 31, a sequencing data filtering module 32, a feature extraction and analysis module 33, a risk analysis module 34, an input display module 35, a storage module 36, and a communication module 37.

[0108] Sequencing data preprocessing module 31 uses FastQC for raw data quality control; BWA-MEM is used to align the sequencing data to the hg38 reference genome; and GATK tools are used for repeat labeling, local realignment, and base quality recalibration.

[0109] The sequencing data filtering module 32 uses the individual's own peripheral blood leukocyte DNA sequencing data as paired germline DNA, and uses Mutect2 to retrieve somatic single nucleotide variants and insertions / deletions using the paired germline DNA as a control. It filters common germline variants in dbSNPs based on the following preset filtering criteria: retaining mutations with sequencing depth ≥10×, variant allele frequency ≥4%, and strand bias <90%.

[0110] The feature extraction and analysis module 33 extracts the somatic mutation spectrum in saliva, including mutation types and a list of high-frequency mutated genes, and calculates the similarity between the saliva mutation spectrum and the mutation spectrum of typical OSCC tumor tissue. Based on somatic mutations, it calculates the tumor mutation burden (TMB) in saliva, analyzes the core signaling pathways affected by saliva mutations, and calculates the cosine similarity between its enrichment spectrum and the pathway enrichment spectrum of tumor tissue.

[0111] The risk analysis module 34 panning for whether a patient has oral squamous cell carcinoma (OSCC) and the risk of recurrence after treatment is based on the following rules: if a somatic mutation of at least one high-frequency OSCC gene (such as TP53, FAT1, CDKN2A, PIK3CA, NOTCH1) is detected in a saliva sample, oral squamous cell carcinoma is determined to be present; when the calculated saliva TMB value is greater than the preset recurrence risk threshold, the risk of postoperative recurrence is determined to be present.

[0112] The input display module 35 is used to receive the raw sequencing data of the saliva sample and display the analysis results of the risk analysis module, such as generating a standardized report containing a mutation list, TMB value, risk assessment conclusion and clinical recommendations.

[0113] Storage module 36 stores the received raw sequencing data and its corresponding patient basic information, analysis results from each module, preset filtering criteria, and recurrence risk thresholds. By storing relevant data during the analysis process in real time, it provides conditions for subsequent reverse analysis.

[0114] The communication module 37 is used for communication connections between the analysis system and terminal devices or other databases. The analysis system of this invention can be installed on devices such as computers and sequencing equipment. During the analysis process, it communicates with other devices or analysis tools through the communication module, and simultaneously transmits the detection results to remote devices for viewing.

[0115] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above specific embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. An analytical system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics, characterized in that, Analysis of sequencing data from saliva samples, including: The sequencing data preprocessing module aligns the quality-controlled raw sequencing data to the hg38 reference genome and performs duplicate labeling, local realignment, and base quality recalibration. The sequencing data filtering module uses paired germline DNA as a control, calls up somatic single nucleotide variants and insertions / deletions, identifies somatic single nucleotide variants, and filters common germline variants in dbSNP based on preset filtering criteria. The feature extraction and analysis module extracts the somatic mutation spectrum in saliva, calculates the similarity between the saliva mutation spectrum and the mutation spectrum of typical OSCC tumor tissue, and calculates the tumor mutation burden (TMB) in saliva based on somatic mutations. At the same time, it analyzes the core signaling pathways affected by saliva mutations and calculates the cosine similarity between its enrichment spectrum and the pathway enrichment spectrum of tumor tissue. The risk analysis module determines the presence of oral squamous cell carcinoma if at least one somatic mutation of a high-frequency OSCC gene is detected in a saliva sample; and determines the risk of postoperative recurrence if the calculated saliva TMB value is greater than a preset recurrence risk threshold.

2. An analytical system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics, characterized in that: It also includes an input display module, a storage module, and a communication module. The input display module is used to receive the raw sequencing data of the saliva sample and display the analysis results of the risk analysis module; The storage module is used to store the received raw sequencing data and its corresponding patient basic information, the analysis results of each module, preset filtering criteria and recurrence risk thresholds; The communication module is used for communication connections between the analysis system and terminal devices or other databases.

3. An analytical system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics, characterized in that: in, The sequencing data preprocessing module uses FastQC for raw data quality control; BWA-MEM is used to align the sequencing data to the hg38 reference genome; and GATK tools are used for repeat labeling, local re-alignment, and base quality recalibration. In the sequencing data filtering module, the individual's own peripheral blood leukocyte DNA sequencing data is used as the paired germline DNA. The filtering criteria are as follows: mutations with sequencing depth ≥10×, variant allele frequency ≥4%, and strand bias <90% are retained.

4. An analytical system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics, characterized in that: in, The somatic mutation spectrum in the saliva includes at least the mutation type and a list of high-frequency mutated genes. The salivary tumor mutation burden (TMB) is defined as the number of non-synonymous somatic mutations detected per megabase pair in the target region. The core signal pathways include at least the TP53, RTK-RAS, Hippo, and NOTCH pathways; The recurrence risk threshold was obtained by analyzing salivary TMB data from a validation cohort that included patients with primary and recurrent OSCC.

5. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that, When executed by a processor, the computer program performs the functions of the system as described in any one of claims 1 to 4.

6. A system for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence based on salivary genomic characteristics, characterized in that, It includes a sample processing system, a sequencing system, and an analysis system, wherein the data analysis system is the analysis system described in any one of claims 1 to 4.

7. The system according to claim 6, characterized in that, The sample processing system uses magnetic beads to extract total DNA from saliva samples and genomic DNA from peripheral blood leukocytes. The sequencing system uses a customized targeted sequencing panel for OSCC to construct libraries and capture hybridizations from saliva DNA. The panel covers all or part of the exon regions of no less than 42 high-frequency mutant genes in OSCC. The saliva DNA is then sequenced at an average sequencing depth of no less than 500×.

8. The system according to claim 7, characterized in that, The 42 frequently mutated genes in OSCC are as follows: TP53, TTN, FAT1, CSMD3, MUC16, CDKN2A, SYNE1, LRP1B, NOTCH1, PIK3CA, PCLO, KMT2D, DNAH5, NSD1, USH2A, FLG, AHNAK, RYR2, CASP8, COL11A1, FAM135B, PKHD1L1, FAT3, DST, PLEC, XIRP2, DMD, SI, HUWE1, MUC17, PAPPA2, RELN, COL22A1, PCDH15, APOB, FAT4, PCDH11X, AHNAK2, CUBN, MUC5B, NAV3, DNAH8.

9. A kit for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence, characterized in that, This includes reagents for sample collection and standardization, as well as deep sequencing reagents. The reagents used for sample collection and standardization include sterile tubes for holding saliva samples and peripheral blood samples, respectively, and a universal genomic DNA extraction reagent; The deep sequencing reagent is a customized targeted sequencing panel from OSCC.

10. The kit for non-invasive diagnosis of oral squamous cell carcinoma and monitoring of recurrence according to claim 9, characterized in that, in, The universal genomic DNA extraction reagent is a magnetic bead method universal genomic DNA extraction kit; The customized targeted sequencing panel for OSCC covers all or part of the exon regions of no less than 42 high-frequency mutant genes in OSCC.

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