A probe sequence composition and detection method for tracing the primary lesion of head and neck primary unknown metastatic squamous carcinoma

Abstract

The application establishes a probe sequence combination, a kit, an in vitro non-diagnostic detection system and a use thereof for preparing a diagnostic product based on a head and neck primary unknown metastatic squamous carcinoma primary focus tracing based on targeted DNA methylation sequencing. The position of the primary focus is determined by detecting the methylation modification state of the relevant sequence sites of the head and neck metastatic cancer tissue DNA. The primary tracing can be subdivided into the head and neck subclinical anatomical subareas: tongue, tongue root, oropharynx, hypopharynx, larynx, tonsil, gum and floor of mouth, etc. The tracing accuracy is high, the defects of the prior art such as high cost, time-consuming and low patient acceptance are overcome, and the application has the advantages of high cost performance, fast diagnosis speed, high patient acceptance and excellent clinical popularization prospect.

Description

Technical Field

[0001] This invention belongs to the field of detection, and in particular relates to a probe sequence composition, kit, and detection method for tracing the primary lesion of head and neck unexplained metastatic squamous cell carcinoma. Background Technology

[0002] Carcinoma of unknown primary origin (CUP) refers to a type of cancer that has been pathologically diagnosed as metastatic, but whose primary site remains unclear despite detailed examination and evaluation. Worldwide, CUP is the 6th to 8th most common malignant tumor, and metastatic CUP occurring in the cervical lymph nodes accounts for more than 80% of CUP cases. 70%-80% of cervical lymph node metastases originate from the head and neck region, and more than 80% of head and neck malignancies are squamous cell carcinomas. Squamous cell carcinoma of unknown primary origin (SCCUP) has an average age of onset of 60-75 years and is the type of CUP with the worst prognosis. Therefore, early and accurate diagnosis of SCCUP is currently a key focus and challenge in the field of head and neck cancer.

[0003] Unlike other malignant tumors that easily metastasize to cervical lymph nodes, such as those in the breast, lungs, and stomach, the head and neck region has a complex anatomy and numerous anatomical subregions. For example, cervical lymph nodes can be divided into regions I to VI according to their location, and metastasis patterns vary across different parts of the head and neck (Table 1). Therefore, it is difficult to determine the primary lesion site for lymph node cancer in a specific region of the neck.

[0004] Table 1. Primary lesion sites and corresponding metastatic areas of head and neck squamous cell carcinoma.

[0005] partition Original stove location Zone I oral cavity, oropharynx Zone II oropharynx, larynx, nasal cavity, hypopharynx, parotid gland, nasopharynx Zone III oropharynx, larynx, hypopharynx, thyroid gland, nasopharynx Zone IV larynx, thyroid gland, hypopharynx, esophagus Zone V Nasopharynx, hypopharynx, thyroid gland, oropharynx Zone VI thyroid gland, larynx, hypopharynx, cervical esophagus

[0006] Current diagnostic techniques for SCCUP require invasive biopsy, pathological examination, and CT scans from the head to the neck. For lymph node metastases in zones II and III, MRI and even PET-CT scans are also necessary. Along with clinical examination and imaging assessment, endoscopic examination of the upper gastrointestinal tract is routine, encompassing the nasal cavity, sinuses, nasopharynx, oropharynx, soft and hard palate, base of the tongue, tonsils, posterior pharyngeal wall, uvula, supraglottic, glottic, subglottic, pyriform fossa, postcricoid region, and proximal esophagus. If these examinations still fail to reveal any lesions, diagnostic bilateral tonsillectomy is an option, which can often uncover some occult lesions.

[0007] As can be seen from the above, the current examination process is complex and costly. Furthermore, due to the low sensitivity of CT, it is difficult to detect small primary lesions (longest diameter <1cm). Even with PET-CT, the specificity is only 68%. Patient compliance with bronchoscopy and diagnostic resection is also low, making the procedure inconvenient. Even with detailed clinical examination, imaging evaluation, and surgical procedures, the primary lesion of metastatic squamous cell carcinoma in the neck cannot be detected in more than half of the patients. Treatment for primary lesions of metastatic squamous cell carcinoma in the neck that cannot be detected generally begins with debridement of the metastatic lesions and concurrent chemoradiotherapy. However, radiotherapy has significant side effects; the larger the radiotherapy area, the greater the side effects. Therefore, the selection of an effective radiotherapy area is highly controversial. Therefore, developing new examination methods to identify the primary lesion in these cases where the primary lesion of SCCUP remains undetectable is urgently needed to reduce side effects and treatment costs.

[0008] In recent years, the development of gene sequencing technology has provided new ideas and directions for solving this problem. However, based on existing methods, the primary lesion can only be located to large anatomical sites such as the lung, breast, stomach, and colorectal region. For squamous cell carcinoma originating from the head and neck, the existing methods can only locate the head and neck region, and cannot be further subdivided into anatomical subregions such as the tongue, pharynx, and larynx.

[0009] It is evident that, given the numerous anatomical sites and complex structure of the head and neck region, an urgent issue to be addressed is to trace the primary lesions of unexplained metastatic squamous cell carcinoma in the head and neck region, thus laying the foundation for developing different treatment methods for different primary lesions. Summary of the Invention

[0010] In view of this, the present invention aims to provide a probe composition, device, and kit for tracing the primary lesion of head and neck squamous cell carcinoma, as well as the use of them in the preparation of diagnostic products.

[0011] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0012] The first aspect of the present invention is a probe composition for tracing the primary lesion of head and neck squamous cell carcinoma, wherein the probe composition has at least four detection probe sequences for each target gene CpG site, and the target gene is selected from one or more of the target genes listed in Table 2 of this application specification.

[0013] A second aspect of the invention is a kit for tracing the primary lesion of head and neck squamous cell carcinoma, comprising the probe composition according to claim 1 and bisulfite.

[0014] The first aspect of the present invention is an apparatus for tracing the primary lesion of head and neck squamous cell carcinoma, the apparatus comprising:

[0015] Module 1) DNA extraction module;

[0016] Module 2) Bisulfite treatment module;

[0017] Module 3) Constructing DNA methylation sequencing libraries;

[0018] Module 4) Liquid phase hybridization capture module, wherein the probe composition according to claim 1 is used to perform liquid phase hybridization capture on the DNA methylated library constructed in module 3);

[0019] Module 5) High-throughput sequencing module.

[0020] The application of the probe composition of the first aspect of the present invention, the kit of the second aspect of the present invention, or the device of the third aspect of the present invention in the preparation of products for early diagnosis and source tracing of primary lesions of head and neck tumors; preferably, the primary lesion of the head and neck tumor is selected from the following locations: tongue, base of tongue, oropharynx, hypopharynx, larynx, tonsils, gingiva, or floor of mouth.

[0021] Compared with existing technologies, the detection kit and non-diagnostic detection method described in this invention have the following advantages:

[0022] 1. Utilizing the TCGA database combined with tissue specimens from our clinical research and employing artificial intelligence analysis methods, we obtained a set of target genes for tracing the primary lesions of unexplained metastatic cancers in the head and neck region. By detecting the methylation modifications of this target gene set extracted from pathological sections, we established a novel product, device, and method for predicting the primary lesion of squamous cell upheaval (SCCUP). Using this invention, we were able to distinguish malignant tumors originating from squamous epithelium in different anatomical subregions of the head and neck (oral cavity, oropharynx, larynx, hypopharynx). The results were verified by comparison with known primary lesion tracing results, demonstrating an accuracy rate of 96.15%. This invention provides a new and reliable method for predicting the diagnosis of SCCUP in cervical lymph nodes in the head and neck region, offering a basis for precision cancer treatment and showing broad clinical application prospects.

[0023] 2. This invention provides a probe assembly and apparatus for detecting methylation of CpG sites in target gene sequences and for tracing the primary origin of head and neck unexplained metastatic squamous cell carcinoma. The probe assembly involved in this invention comprises probe sequences targeting 1018 target gene sets with CpG sites, including probe sequences that specifically bind to CpG sites in the target gene sequences after bisulfite treatment.

[0024] Terminology definition:

[0025] In this invention, the term "probe composition" (sometimes referred to herein as "composition") refers to a combination of multiple probes, including mixtures of multiple probes, or multiple probes in which each probe exists in a separate, independent form. In some embodiments, the mixture includes, but is not limited to, a solution mixture of multiple probes, or a powder mixture of multiple probes. Examples of solutions include aqueous solutions or organic solutions (e.g., ethanol) or combinations of both. Powder mixtures comprise solid mixtures / mixtures of two probes. In some embodiments, the probe composition exists in a separate, independent form. For example, each probe is fixed / bound to a specific region, such as a substrate, which is regularly aggregated on the substrate to form an array. In some embodiments, each probe in the probe composition exists individually in a specific container (e.g., a vial). In some embodiments, the composition includes at least four probes targeting each target sequence CpG site.

[0026] In this invention, the term "sequencing" refers to targeted DNA methylation sequencing. Currently, the mainstream techniques are liquid phase hybridization capture methylation sequencing and amplicon methylation sequencing, with enrichment strategies of probe hybridization and multiplex PCR, respectively. In a specific embodiment, liquid phase hybridization capture methylation sequencing is used. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 The detection process is as described in Embodiment 2 of the present invention;

[0029] Figure 2 The results of the source tracing analysis using the model of the present invention in Example 2 are shown. Detailed Implementation

[0030] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0031] The present invention will now be described in detail with reference to the embodiments.

[0032] Example 1: Constructing a Source Tracing Diagnostic Model

[0033] 1. Experimental Methods

[0034] Building a source tracing and diagnostic model includes the following steps:

[0035] 1) A methylation source database for head and neck cancers was established. DNA methylation data samples (427 cases) of head and neck squamous cell carcinoma tissues were downloaded from TCGA (The Cancer Genome Atlas), the world's largest cancer research database, and integrated with the DNA methylation data of our existing head and neck squamous cell carcinoma tissue bank (382 cases). The tissue types covered by the samples included the tongue, tongue base, oropharynx, hypopharynx, larynx, tonsils, gingiva, floor of mouth, and lymph nodes. Based on the methylation data of these cancer types, multiple CpG sites were used as features, and cancer type classification was used as a label to establish the aforementioned head and neck squamous cell carcinoma methylation source database.

[0036] 2) A model for predicting the origin of head and neck squamous cell carcinoma was constructed. First, methylation differences of CpG sites were analyzed among cancer groups in different locations using one-way ANOVA. CpG sites for model construction were initially screened based on p-values ​​(<0.01) after multiple-factor testing. Then, using R language and related toolkits, 40% of the samples from each cancer group were randomly selected to form the training set, and the remaining 60% formed the test set. The Boruta feature selection method was used to identify key categorical variables in the training set data, and important and potentially important variables were obtained for classification prediction (a total of 1018). Caret was used for hyperparameter tuning and modeling, and important hyperparameters were determined through 10-fold cross-validation. The model was then built using the training set data and the optimal hyperparameter combination. Finally, the model effect was validated on the test set, resulting in a set of 1018 target genes with the function of tracing the origin of head and neck cancer of unknown primary metastasis.

[0037] 3) Based on the validated prediction model for primary lesions of head and neck squamous cell carcinoma obtained in step 2), probe sequences were designed based on CpG sites of 1018 target gene sequences (listed in Table 2). This probe combination is a probe combination for tracing the primary lesions of head and neck squamous cell carcinoma of unknown origin based on targeted DNA methylation sequencing. The design principle of the probe sequences is as follows: design probe sequences for specific binding to CpG sites of target gene sequences after bisulfite treatment. Each target gene sequence CpG site has at least 4 probe sequences that specifically bind, of which 1 specifically binds to the positive strand of the target gene sequence without CpG methylation, 1 specifically binds to the negative strand of the target CpG without methylation, 1 binds to the positive strand of the target CpG methylated, and 1 specifically binds to the negative strand of the target CpG methylated. The probe combination shown in Table 2 was synthesized and produced by Roche Diagnostics.

[0038] Table 2 lists 1018 target genes and the detection probe sequences for each target gene.

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[0161] Example 2: Validation of the source tracing and diagnostic model of the present invention

[0162] 1. Experimental reagents

[0163] Table 3 Reagent List

[0164] name Specification Item number company Rapid DNA Extraction Kit for Paraffin-Embedded Tissue 50 reactions DP330-02 Heavenly Root EZDNAMethylation-GoldKit 50 reactions D5005 ZYMORESEARCH Probe sequence composition kit (including matching reagents) 96 reactions 9063188001 RocheDiagnostics

[0165] 2. Experimental steps:

[0166] 1) Genomic DNA was extracted from paraffin sections of head and neck primary metastatic carcinomas with known primary lesions from Tianjin Cancer Hospital. The primary lesions were: tongue (78 cases), tongue base (54 cases), oropharynx (44 cases), hypopharynx (42 cases), tonsils (35 cases), gingiva (39 cases), and floor of mouth (17 cases).

[0167] 2) Each sample of 200 ng of genomic DNA was subjected to bisulfite treatment using the EZ DNA Methylation-Gold Kit (ZYMO RESEARCH);

[0168] 3) Constructing DNA methylation sequencing libraries

[0169] a. Place the bisulfite-treated DNA sample obtained in step 2) on a PCR instrument, denature at 95°C for 2 minutes, and immediately transfer the PCR tube to ice / ice box and let it stand for 2 minutes.

[0170] b. Prepare the T7 connection reaction mixture. Prepare the reaction mixture according to the sample number in Table 4. After preparation, place it in an ice box / on ice and use it within 20 minutes.

[0171] Table 4: T7 Connection Reaction Mixture

[0172]

[0173] c. Add 4.5 μL of T7 ligation reaction mixture to each sample, carefully and quickly mix with a pipette, centrifuge briefly, and then place on a PCR instrument. Perform the reaction according to the program of 37°C, 15 min; 95°C, 2 min; 4°C, hold. After the reaction is complete, quickly transfer the PCR tube to ice / ice box.

[0174] d. Add 21.5 μL of 2X Synthesis Mix and 1.5 μL of Synthesis Reagent to the reaction solution in c. Mix carefully and quickly by pipetting, centrifuge briefly, and then place on a PCR instrument. Perform the reaction according to the following program: 98 °C, 1 min; 60 °C, 2 min; 68 °C, 5 min; 4 °C, hold.

[0175] e. Equilibrate the DNA purification beads to room temperature beforehand and prepare 80% ethanol. Prepare a 1.5 ml centrifuge tube for each sample. Add 53 μL of magnetic beads to each centrifuge tube, transfer the reaction solution obtained in 4.4 to the corresponding centrifuge tube, perform the purification step, and finally elute with 12 μL of low EDTA TE.

[0176] f. Transfer 10 μL of the elution buffer obtained in step e to a labeled 0.2 mL PCR tube, and add 1.5 μL LowEDTA TE, 2 μL T5 Buffer, 5 μL T5 Adapter and 1.5 μL Ligase Mix. Carefully and quickly mix by pipetting, centrifuge briefly, and then place on a PCR instrument. Perform the reaction at 25 °C for 15 min; then at 4 °C for hold.

[0177] g. Prepare one 1.5ml centrifuge tube for each sample. Add 20ul of nuclease-free water, 40ul of magnetic beads, and 20ul of the reaction solution obtained in step 4.6 to each centrifuge tube for purification. Finally, elute with 12ul of low EDTA TE. Transfer 10ul of the elution buffer to a labeled 0.2ml PCR tube and proceed to the next step or freeze at -20℃.

[0178] h. Add 12.5 μL of 2X Synthesis Mix and 2.5 μL of paired-end index primer (10p) to the sample obtained in g. Carefully and quickly mix using a pipette, then briefly centrifuge and place on a PCR instrument. React according to the following steps (approximate reaction time is 43 minutes): 98℃, 45 s; 98℃, 15 s, 60℃, 30 s, 68℃, 30 s, 18 cycles; 4℃, hold.

[0179] i. Prepare one 1.5ml centrifuge tube for each sample. Add 25ul nuclease-free water, 42.5ul magnetic beads and 25ul reaction solution obtained in step h to each centrifuge tube for purification. Finally, elute with 30ul low EDTA TE to complete the construction of the DNA methylation sequencing library, measure the qubit recording concentration, and store the library at -20℃.

[0180] 4) Liquid-phase hybridization capture and high-throughput sequencing based on the 1018 probe sequence compositions shown in Table 2 of this invention (the reagents used in this step were purchased from Roche).

[0181] a. Take the DNA methylated library from step 3) and perform liquid phase hybridization capture. Perform liquid phase hybridization capture once for every 4 samples of methylated library. Take 400 ng of each library and add it to a 1.5 ml centrifuge tube. Add 10 μl Bisulfite Capture Enhancer, 1 μl SeqCap HE Universal Oligo and 1 μl SeqCap HE IndexOligo, vortex and briefly centrifuge.

[0182] b. Use a vacuum concentrator to concentrate the mixture obtained in step a at 60°C until the liquid is completely dry.

[0183] c. Add 7.5 μL Hybridization Buffer and 3 μL Component H to the dried sample, vortex for 10 seconds, centrifuge at 16000g for 10 seconds, and then place the centrifuge tube at 95°C for denaturation for 10 minutes.

[0184] d. After centrifuging the sample at 16000g for 10 seconds, transfer it to a 96-well plate and add 4.5ul of the probe composition mixture of the present invention. Mix well by pipetting, cover with sealing film, and place on a PCR instrument at 47°C for 64 hours.

[0185] e. Prepare the elution solution in advance according to the following list based on the number of hybridization reactions.

[0186] Table 5 Hybridization reaction solution

[0187]

[0188] a. For each hybridization reaction, take 100 μL of the hybridization purification magnetic beads that have been equilibrated at room temperature into a 1.5 mL centrifuge tube, place it on a magnetic rack, collect the supernatant, wash twice with 200 μL of 1X Bead Wash Buffer, and finally resuspend in 100 μL of 1X Bead Wash Buffer and transfer to a 0.2 mL PCR tube.

[0189] b. Place the PCR tube on a magnetic rack and wait for it to clarify. Discard the supernatant and transfer the hybridization reaction solution that has been reacting for 64 hours into the PCR tube. Use a pipette to quickly mix the reaction solution and magnetic beads. Place the PCR tube on the PCR instrument and react at 47°C for 45 minutes, vortexing to mix once every 15 minutes.

[0190] c. For each hybridization reaction, transfer 100 μL of 47°C preheated 1X Wash Buffer I to the sample PCR tube, vortex to mix, and then transfer the entire mixture to a labeled 1.5 ml centrifuge tube. Place the tube on a magnetic rack and wait for clarification. Discard the supernatant.

[0191] d. Take 200ul of 1X StringentWash Buffer preheated at 47℃ into a 1.5ml centrifuge tube, quickly pipette and mix well, then incubate at 47℃ for 5 minutes.

[0192] e. Place the 1.5ml centrifuge tube on a magnetic rack and wait for it to clarify. Discard the supernatant, add 200ul of 1X Stringent Wash Buffer preheated at 47°C to the 1.5ml centrifuge tube, quickly pipette and mix well, then incubate at 47°C for 5 minutes.

[0193] f. Place the 1.5ml centrifuge tube on a magnetic rack and wait for it to clarify. Discard the supernatant, add 200ul of 1XWash Buffer I at room temperature, vortex for 2 minutes, and repeat.

[0194] g. Add 200ul of 1X Wash Buffer II, vortex for 1 minute, place the 1.5ml centrifuge tube on a magnetic rack, wait for clarification, and discard the supernatant.

[0195] h. Add 200ul of 1X Wash Buffer III, vortex for 30 seconds, place the 1.5ml centrifuge tube on a magnetic rack, wait for clarification, and discard the supernatant.

[0196] i. Centrifuge for 5 seconds, place the 1.5ml centrifuge tube on a magnetic rack, discard the residual liquid, and add 50ul of nuclease-free water and mix well.

[0197] j. Prepare two PCR tubes for each hybridization reaction. Add 20 μL of the mixture obtained in the previous step, 25 μL of KAPAHiFi HotStart ReadyMix, and 5 μL of PostLM-PCR Oligo 1 & 2, 5 μM to each tube. Mix well by pipetting and place on a PCR instrument and react according to the following program: 98℃, 45 s; 98℃, 15 s, 60℃, 30 s, 72℃, 30 s, 16 cycles; 72℃, 1 min; 4℃, hold.

[0198] k. Place the PCR tubes on a magnetic rack, transfer the supernatant from the two PCR tubes corresponding to each hybridization reaction into the same 1.5 ml centrifuge tube, purify with 180 μL DNA purification magnetic beads, and elute with 52 μL LOW EDTA TE. This completes the liquid-phase hybridization capture of the probe sequence composition based on this invention. The concentration is measured using a qubit and recorded. The library is then stored at -20°C.

[0199] 1. Perform high-throughput sequencing on the DNA methylated library captured by liquid-phase hybridization in the previous step.

[0200] 5) The primary lesion was traced based on sequencing data from multiple head and neck primary unexplained metastatic cancer tissue samples.

[0201] a. Perform bioinformatics analysis on the sequencing data of each sample, including: data quality filtering, data alignment, and calculation of methylation rate at target CpG sites.

[0202] b. Based on the methylation rate of the target CpG site and the head and neck squamous cell carcinoma primary lesion prediction model, the primary lesion site of each sample is predicted.

[0203] c. Based on the predicted primary lesion location for each metastatic tissue sample, compare it with the known primary lesion location for each sample to calculate the prediction accuracy of primary cancer at different locations. The calculation formula is as follows:

[0204]

[0205] 3. Experimental Results:

[0206] Figure 2 The invention demonstrates the detection of DNA methylation in metastatic cancer tissue samples using the probe combination described in Table 2, and the primary origin index calculated based on a random forest prediction model. The horizontal axis represents the primary cancer type in different locations, and the vertical axis represents the probability of the sample having the primary cancer type in different locations, calculated based on the random forest prediction model. The results of primary cancer origin tracing based on the probes of the invention are compared with the origin tracing results obtained by diagnosing experimental samples using classical methods. The accuracy is recorded in Table 6. Based on the content shown in Table 6, it can be seen that the accuracy of tracing primary cancer in different locations of metastatic cancer using the probe set of the invention can reach more than 96.15%, which is superior to existing diagnostic technologies in terms of diagnostic accuracy.

[0207] Table 6 shows the source tracing accuracy of the primary lesions predicted using the probe combination of the present invention compared with the known primary lesions in the samples.

[0208] Table 6. Accuracy of tracing the origin of primary cancer in different sites of metastatic cancer.

[0209] Primary cancer Sample size Correct predictions accuracy Tongue cancer 78 75 96.15％ tongue base cancer 54 48 88.89％ oropharyngeal cancer 44 40 90.91％ hypopharyngeal cancer 42 39 92.86％ Tonsillitis 35 32 91.43％ Gingival cancer 39 36 92.31％ floor of mouth cancer 17 14 82.35％ total 309 284 91.91％

[0210] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probe composition for tracing the primary lesion of head and neck squamous cell carcinoma, characterized in that, The probe composition has at least four detection probe sequences for each target gene CpG site, and the target gene is a combination of target genes described in Table 2 of this application specification; the probe composition is a combination of probes described in Table 2 of this application specification.

2. A reagent kit for tracing the primary lesion of head and neck squamous cell carcinoma, characterized in that, It comprises the probe composition according to claim 1 and bisulfite.

3. A device for tracing the primary lesion of head and neck squamous cell carcinoma, characterized in that, The device includes: Module 1) DNA extraction module; Module 2) Bisulfite treatment module; Module 3) Constructing DNA methylation sequencing libraries; Module 4) Liquid phase hybridization capture module, wherein module 4) uses the probe composition according to claim 1 to perform liquid phase hybridization capture on the DNA methylation library constructed in module 3; Module 5) High-throughput sequencing module.

4. The use of the probe composition according to claim 1, the kit according to claim 2, or the device according to claim 3 in the preparation of a product for tracing the primary lesion of head and neck squamous cell carcinoma; wherein the primary lesion of head and neck squamous cell carcinoma is selected from the following locations: tongue, base of tongue, oropharynx, hypopharynx, larynx, tonsils, gingiva, or floor of mouth.

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