Probe composition for detecting salivary gland tumor gene panel and use thereof

Abstract

The application relates to the field of biotechnology, in particular to a probe composition for detecting a salivary gland tumor gene panel and application thereof, and further relates to a library construction method, a sequencing method, a kit and a device for the salivary gland tumor gene panel. The probe composition can capture nucleic acid molecules of multiple genes related to salivary gland tumors, such as AKT1, APC, AR, ARID1A, ATM, AXIN1, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, CDH1, CDK11B, CDKN2A, CDKN2B, CREBBP, CTNNB1, CYLD, EGFR and EP300. The nucleic acid molecules in a sample to be analyzed are captured by using the probe composition, and library construction and sequencing are carried out; and the analysis result of the sequencing data is helpful to improving the diagnosis accuracy of salivary gland tumors.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to probe compositions for detecting salivary gland tumor gene panels and their applications, and further to methods for constructing libraries, sequencing methods, kits and devices for salivary gland tumor gene panels. Background Technology

[0002] Salivary gland tumors (SGTs) are tumors that occur in the salivary glands, originating from major salivary glands (parotid, submandibular, and sublingual glands) or minor salivary glands (distributed in the oral cavity, lips, cheeks, and palate), accounting for 1-6% of all head and neck tumors. SGTs represent a diverse and histologically complex group of tumors, posing significant diagnostic challenges to routine pathological practice. There are numerous types of SGTs; the latest 2021 WHO classification system divides them into 36 different pathological subtypes, including 15 benign tumors such as pleomorphic adenoma, myoepithelioma, Warthin's tumor, eosinophilic cell tumor, sclerosing polycystic adenoma, keratotic cystadenoma, interlobular duct adenoma, and striatal duct adenoma; and 21 malignant tumors such as adenoid cystic carcinoma, mucoepidermoid carcinoma, acinar cell carcinoma, pleomorphic low-grade adenocarcinoma, microsecretory adenocarcinoma, sclerosing microcystic adenocarcinoma, secretory carcinoma, salivary gland duct carcinoma, and mucinous adenocarcinoma (containing multiple subtypes). SGT occurs in a wide range of locations, and different subtypes exhibit distinct behaviors. The growth rate, metastatic pathways, and prognosis of different malignant subtypes vary greatly. At the same time, the morphology and structure of SGT tumor cells are extremely complex and variable, characterized by disordered cell origin, diverse structural patterns, and high diagnostic difficulty.

[0003] Precise pathological classification of SGT is the cornerstone of prognostic assessment and treatment decisions, but significant morphological overlap between subtypes and their rarity often limit diagnostic accuracy. Although the fifth edition of the WHO Classification of Head and Neck Tumors has incorporated genetic molecular characteristics into the core diagnostic criteria for most subtypes, significantly improving the objectivity of the classification system, in practical application, the interpretation of histopathological features of limited biopsy specimens remains subjective, especially in core needle biopsies, atypical lesions, poorly differentiated or metastatic cases.

[0004] The advent of molecular diagnostic technologies, particularly next-generation sequencing (NGS), has profoundly changed the diagnostic landscape for scleroderma-associated gastroenteritis (SGT). The identification of recurrent, tumor-type-specific genetic alterations, such as the CRTC1 / 3-MAML2 fusion in mucoepidermoid carcinoma, the ETV6-NTRK3 fusion in secretory carcinoma, and the MYB / MYBL1-NFIB fusion in adenoid cystic carcinoma, has made molecular testing an indispensable adjunct to pathological diagnosis, alongside conventional morphology and immunohistochemistry. While these molecular advances have significantly improved diagnostic accuracy and revealed potential therapeutic targets, their widespread application in routine clinicopathology remains limited. A key limitation is the reliance on single-gene sequencing, which is often inefficient and consumes valuable, limited tissue resources. Furthermore, although comprehensive commercial gene panels (gene combinations) are available, such as GENESEEQPRIME® (425-gene panel) or FoundationOne CDx, TruSight Oncology 500, and MSK-IMPACT, these pan-cancer platforms are not optimized for SGT. Due to the rarity of SGTs, none of them are specifically designed to cover the full spectrum of tumor type-specific fusion oncogenes necessary for the diagnosis of these malignancies, including but not limited to PLAG1, HMGA2, MYB / MYBL1, PRKD1-3, MSANTD3, and MAML2.

[0005] Therefore, many complex SGT cases continue to face diagnostic uncertainty or misclassification due to the lack of standardized and efficient molecular detection methods, and there is an urgent need to develop a comprehensive diagnostic product that is specifically validated and is both economical and tissue-saving. Summary of the Invention

[0006] Therefore, it is necessary to provide a probe composition for detecting the salivary gland tumor gene panel and its application.

[0007] In a first aspect, a probe composition for detecting a salivary gland tumor gene panel is provided, the probe composition comprising probes targeting genes in the salivary gland tumor gene panel, the salivary gland tumor gene panel comprising the following genes:

[0008] AKT1、APC、AR、ARID1A、ATM、AXIN1、BAP1、BARD1、BCOR、BRAF、BRCA1、BRCA2、CDH1、 CDK11B、CDKN2A、CDKN2B、CREBBP、CTNNB1、CYLD、EGFR、EP300、FBXW7、FGFR1、FLT3、 FOXA1, HER2, HER4, HMGA2, HRAS, IDH1, IDH2, IGF1R, IGF2, KDM6A, KDR, KMT2C, KRAS, LRFN1, MDM2, MET, MLH1, MSANTD3, MSH2, MUTYH, MYB, MYBL1, MYC, NF1, NOTCH1, NOT CH2、NOTCH3、NR4A3、NRAS、PDGFRA、PIK3CA、PIK3R1、PLAG1、PRKD1、PRKD2、PRKD3、 PRSS1、PTEN、RAD51B、RB1、SETD2、SF3B1、SMARCA2、SMARCA4、SMARCB1、SMO、SPEN、S TK11、TERT、TP53、TPTE2P1、VHL、ABL1、ALK、BCL6、CACNA1B、DEK、ENOX1、ETV5、ETV6 、EWSR1、IRF2BP2、KMT2A、MAML2、NTRK1、NTRK2、NTRK3、NUTM1、RAPGEF6、RET、SS18。

[0009] In an optional embodiment, the probe targets genomic DNA encoding the following genes: AKT1, APC, AR, ARID1A, ATM, AXIN1, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, CDH1, CDK11B, CDKN2A, CDKN2B, CREBBP, CTNNB1, CYLD, EGFR, EP300, FBXW7, FGFR1, FLT3, FOXA1, HER2, HER4, HMGA2, HRAS, IDH1, IDH2, IGF1R, IGF2, KDM6A, KDR, KMT2C, KRA. S, LRFN1, MDM2, MET, MLH1, MSANTD3, MSH2, MUTYH, MYB, MYBL1, MYC, NF1, NOTCH1, NOTCH2, NOTCH3, NR4A3, NRAS, PDGFRA, PIK3CA, PIK3R1, PLA G1, PRKD1, PRKD2, PRKD3, PRSS1, PTEN, RAD51B, RB1, SETD2, SF3B1, SMARCA2, SMARCA4, SMARCB1, SMO, SPEN, STK11, TERT, TP53, TPTE2P1, and VHL;

[0010] Furthermore, the probe targets mRNAs encoding the following genes: ABL1, ALK, BCL6, CACNA1B, CDH1, DEK, EGFR, ENOX1, ETV5, ETV6, EWSR1, HMGA2, IRF2BP2, KMT2A, MAML2, MSANTD3, MYB, MYBL1, NOTCH1, NR4A3, NTRK1, NTRK2, NTRK3, NUTM1, PLAG1, PRKD1, PRKD2, PRKD3, RAPGEF6, RET, and SS18.

[0011] In an optional embodiment, the probe targets the coding region of the target gene, wherein the coding region does not include the coding region of the TERT gene; and the probe targets the promoter region of the TERT gene.

[0012] In an optional embodiment, each probe in the probe composition binds to the target molecule at a length of 100-150 nt independently.

[0013] In an optional embodiment, the nucleotide sequences of the probe-target molecule binding region are shown in SEQ ID NO.1 to SEQ ID NO.4473, or in complementary sequences of SEQ ID NO.1 to SEQ ID NO.4473, respectively.

[0014] Secondly, a method for constructing a library of salivary gland tumor gene panels is provided, the method comprising: capturing nucleic acid molecules in a sample using a probe composition for detecting salivary gland tumor gene panels according to the first aspect.

[0015] In an optional implementation, the library construction method includes first constructing a genomic DNA library and a cDNA library of the subject, and then using the probe composition to capture target nucleic acid molecules from the genomic DNA library and the cDNA library.

[0016] In an optional embodiment, the library construction method includes using probes in the probe composition that target and encode genomic DNA to capture target nucleic acid molecules in the genomic DNA library.

[0017] In an optional embodiment, the library construction method includes using a probe in the probe composition that targets mRNA encoding a gene to capture target nucleic acid molecules in the cDNA library.

[0018] Thirdly, a sequencing method for a salivary gland tumor gene panel is provided, the sequencing method comprising preparing a library using the library construction method of the first aspect, and then sequencing the obtained library.

[0019] In an optional embodiment, the sequencing method further includes variation analysis of the gene sequence in the salivary gland tumor gene panel obtained by sequencing.

[0020] In optional implementations, variant analysis includes, but is not limited to, analyzing at least one of the following variants: single nucleotide variants, insertion / deletion variants, copy number variants, and RNA fusions.

[0021] Fourthly, a kit is provided that comprises: (a), or (a) and (b):

[0022] (a) The probe composition for detecting the salivary gland tumor gene panel in the first aspect; and,

[0023] (b) At least one of the reagents used for library construction and the reagents used for sequencing.

[0024] Fifthly, the probe composition for detecting salivary gland tumor gene panels of the first aspect or the kit of the fourth aspect are provided for use in the preparation of products for the detection of salivary gland tumors.

[0025] In an optional implementation, the salivary gland tumor detection product is used to classify salivary gland tumors.

[0026] In a sixth aspect, an apparatus for detecting salivary gland tumors is provided, the apparatus comprising a sequencing module and an analysis module;

[0027] The sequencing module is used to implement the sequencing method for the salivary gland tumor gene panel in the third aspect;

[0028] The analysis module is used to perform bioinformatics analysis on the data obtained by the sequencing module to obtain data information of the gene panel defined in the first aspect of the sample;

[0029] In an optional embodiment, the device for detecting salivary gland tumors further includes at least one of a nucleic acid extraction module and a library construction module.

[0030] The probe composition provided in this application can capture gene panels rich in salivary gland tumors. After constructing a sequencing library using the nucleic acid molecules captured by this probe composition, sequencing is performed, demonstrating high accuracy in detecting SNVs (single nucleotide variants), insertion / deletion variants, copy number variations, and fusion variants. It also offers better diagnostic capability for challenging cases and has identified novel variants associated with salivary gland tumors. Using this probe composition for detecting salivary gland tumor gene panels to capture nucleic acid molecules in the sample for sequencing analysis can significantly improve diagnostic accuracy and clarify a considerable number of previously undiagnosed cases. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0032] Figure 1 The results show the consistency of small variant frequencies detected by gene panel analysis in Example 1 with those detected by gene panel analysis in Example 425.

[0033] Figure 2This image shows the results of the reassessment of the original diagnoses of 112 SGT cases in Example 3 and the establishment of new diagnoses through molecular detection. The original pathological diagnosis (left) and the new diagnosis integrating molecular profiling and histopathology (right) are shown. The abbreviations for salivary gland tumor subtypes in the image are as follows: Adenoid cystic carcinoma: ACC; Acinar cell carcinoma: AciCC; Basal cell adenocarcinoma: BCAC; Basal cell adenoma: BCA; Carcinoma in pleomorphic adenoma: Ca ex PA; Diagnostic Challenge Tumors: DCT; Eosinophilic Hyperplasia: ESH; Epithelial-Myoepithelial Carcinoma: EMC; Clear Cell Carcinoma: HCCC; Ductal Carcinoma in Situ: IDC; Myoepithelioma: MEO; Mucoepidermoid Carcinoma: MEC; Myoepithelial Carcinoma: MECA; Microcritate Adenocarcinoma: MCAC; Nasal Cavity and Sinus Nonkeratinizing Squamous Cell Carcinoma: NKSCC; Metastatic Primary Lung Cancer: MPLC; NTRK Rearranged Spindle Cell Tumor: NRSCT; Eosinophilic Adenoma: OCY; Pleomorphic Adenoma: PA; Pleomorphic Adenocarcinoma: PAC; Secretory Carcinoma: SC; Sebaceous Adenoma: SA; Squamous Cell Carcinoma: SCC; Salivary Gland Duct Carcinoma: SDC; Salivary Gland Duct Carcinoma with Rhabdomyomorphic Features: SDC-RF;

[0034] Figure 3 The pathological morphology profile of a reclassified mucoepidermoid carcinoma (MEC) in Example 4 is shown below; A is hematoxylin-eosin staining (magnification 200x), B is immunohistochemical results (magnification 200x), and C is PAS-AB staining results (magnification 200x).

[0035] Figure 4 The pathological morphology profile of a DEK-AFF2 squamous cell tumor reclassified in Example 5 is shown in Figure 5. A is magnified at 20x and B is magnified at 40x.

[0036] Figure 5 This is a schematic diagram of a fusion of a squamous cell tumor reclassified as DEK-AFF2 in Example 5. Detailed Implementation

[0037] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0039] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0040] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0041] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0042] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0043] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0044] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0045] In this application, "Gene Panel" refers to a pre-selected set of genes associated with a specific disease or phenotype, and variations in these genes are detected simultaneously in one go using high-throughput sequencing technology (usually NGS).

[0046] In this application, "genomic DNA library" refers to a library containing the whole genome of a subject, which is a collection of clones derived from the subject's entire chromosome or genome fragments.

[0047] In this application, "genomic cDNA library" refers to a collection of clones containing all the mRNA information of a cell, formed by reverse transcription of all the mRNA of a subject at a certain developmental stage or under specific conditions into cDNA in vitro using reverse transcriptase catalysis.

[0048] In a first aspect, some embodiments provide a probe composition for detecting a salivary gland tumor gene panel, the probe composition comprising probes targeting genes in the salivary gland tumor gene panel, the salivary gland tumor gene panel including genes as shown in Table 1, the genes encoding Table 1 including at least one of genomic DNA and its transcripts, the transcripts of the genes including mRNA transcribed from genomic DNA.

[0049] Table 1. Salivary gland tumor genes Panel

[0050]

[0051]

[0052]

[0053] The information in Table 1 comes from the gene nomenclature database (HUGO Gene Nomenclature Committee).

[0054] In optional embodiments, the probes in the probe composition target genomic DNA encoding the following genes: AKT1, APC, AR, ARID1A, ATM, AXIN1, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, CDH1, CDK11B, CDKN2A, CDKN2B, CREBBP, CTNNB1, CYLD, EGFR, EP300, FBXW7, FGFR1, FLT3, FOXA1, HER2, HER4, HMGA2, HRAS, IDH1, IDH2, IGF1R, IGF2, KDM6A, KDR, KMT2C, KR AS, LRFN1, MDM2, MET, MLH1, MSANTD3, MSH2, MUTYH, MYB, MYBL1, MYC, NF1, NOTCH1, NOTCH2, NOTCH3, NR4A3, NRAS, PDGFRA, PIK3CA, PIK3R1, PLAG1, PRKD1, PRKD2, PRKD3, PRSS1, PTEN, RAD51B, RB1, SETD2, SF3B1, SMARCA2, SMARCA4, SMARCB1, SMO, SPEN, STK11, TERT, TP53, TPTE2P1, and VHL; and,

[0055] Furthermore, the probes in the probe composition target mRNAs encoding the following genes: ABL1, ALK, BCL6, CACNA1B, CDH1, DEK, EGFR, ENOX1, ETV5, ETV6, EWSR1, HMGA2, IRF2BP2, KMT2A, MAML2, MSANTD3, MYB, MYBL1, NOTCH1, NR4A3, NTRK1, NTRK2, NTRK3, NUTM1, PLAG1, PRKD1, PRKD2, PRKD3, RAPGEF6, RET, and SS18.

[0056] In an optional embodiment, the probe in the probe composition targets the coding region of the target gene, which does not include the coding region of the TERT gene; and the probe targets the promoter region of the TERT gene (chr5: 1295104~1296120).

[0057] In an optional embodiment, the length of the region of each probe that binds to the target gene is independently 100 to 150 nt, for example, but not limited to 100 nt, 110 nt, 120 nt, 130 nt, 140 nt or 150 nt, preferably 120 nt.

[0058] In an optional embodiment, the nucleotide sequences of the probes in the probe composition are as shown in SEQ ID NO.1 to SEQ ID NO.4473, or as shown in the complementary sequences of SEQ ID NO.1 to SEQ ID NO.4473. Optionally, in the probe composition, a portion of the probe's nucleotide sequence is a portion of the sequence shown in SEQ ID NO.1 to SEQ ID NO.4473, and the remaining probe sequence is a complementary sequence to the remaining portion of the sequence shown in SEQ ID NO.1 to SEQ ID NO.4473; or, optionally, all the probes in the probe composition have nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.4473; or, optionally, all the probes in the probe composition have nucleotide sequences as shown in the complementary sequences of SEQ ID NO.1 to SEQ ID NO.4473.

[0059] In an optional embodiment, the nucleotide sequences of the probes in the probe composition that target and encode genomic DNA are shown in SEQ ID NO.1 to SEQ ID NO.3356, respectively.

[0060] In an optional embodiment, the nucleotide sequences of the probes targeting the mRNA encoding the gene in the probe composition are shown in SEQ ID NO.3357 to SEQ ID NO.4473, respectively.

[0061] The probe composition provided in the first aspect can capture the salivary gland tumor gene panel shown in Table 1. Analysis and verification show that the salivary gland tumor gene panel has a 98% consistency in variant sites (SNV and InDel), and a 100% consistency in copy number variation and fusion, indicating that the probe composition has high accuracy in detecting the salivary gland tumor gene panel.

[0062] Secondly, in some embodiments, a method for constructing a library of a salivary gland tumor gene panel is provided, the method comprising: capturing nucleic acid molecules in a sample using a probe composition of the first aspect.

[0063] In optional embodiments, the samples include, but are not limited to, nucleic acid molecules directly extracted from the subject, i.e., raw nucleic acid material or fragments of such raw nucleic acid material obtained directly from the organism without complex processing such as in vitro amplification or library construction; or nucleic acid molecules obtained after in vitro processing of the subject's raw nucleic acid molecules, examples of which include, but are not limited to, nucleic acid amplification, reverse transcription, addition of adapter sequences or addition of tag sequences; and at least one of the following methods for constructing a standardized library suitable for a high-throughput sequencing platform from the subject's raw nucleic acid molecules.

[0064] In an optional implementation, nucleic acid molecules include DNA and RNA.

[0065] In an optional implementation, the library construction method further includes at least one step such as nucleic acid extraction, nucleic acid quality control, nucleic acid fragmentation, addition of adapter sequences, nucleic acid fragment purification, and library enrichment.

[0066] In an optional implementation, the library construction method includes first constructing a genomic DNA library and a cDNA library of the subject, and then using the probe composition to capture target nucleic acid molecules from the genomic DNA library and the cDNA library.

[0067] In an optional embodiment, the library construction method includes using probes in the probe composition that target and encode genomic DNA to capture target nucleic acid molecules in a genomic DNA library.

[0068] In an optional embodiment, the library construction method includes using a probe in the probe composition that targets mRNA encoding a gene to capture target nucleic acid molecules in a cDNA library.

[0069] Thirdly, some embodiments provide a sequencing method for a salivary gland tumor gene panel, which includes preparing a library using the library construction method of the second aspect, and then sequencing the obtained library.

[0070] In an optional implementation, sequencing may include next-generation sequencing.

[0071] In an optional implementation, the sequencing method further includes variation analysis of genes in the salivary gland tumor gene panel obtained from sequencing.

[0072] In optional implementations, variant analysis includes, but is not limited to, analyzing at least one of the following variants: SNV (single nucleotide variant), InDel (insertion / deletion variant), copy number variant, and RNA fusion.

[0073] Fourthly, some embodiments provide a kit comprising: (a), or (a) and (b):

[0074] (a) A first aspect probe composition;

[0075] (b) At least one of the reagents used for library construction and the reagents used for sequencing.

[0076] In an optional implementation, the kit is a library construction kit or a sequencing kit.

[0077] In an optional implementation, the kit is used to implement the library construction method of the salivary gland tumor gene panel in the second aspect.

[0078] In optional embodiments, the reagents used for library construction include, but are not limited to, at least one of fragmentation enzymes, DNA polymerases, reverse transcriptases, buffer systems, adapters, indexes / barcodes, primers, and magnetic beads.

[0079] In an optional implementation, the kit is used to implement a sequencing method for salivary gland tumor gene panels, which is a third aspect.

[0080] In optional embodiments, the reagents used for sequencing include, but are not limited to, at least one of sequencing primers, DNA polymerase, dNTPs, and buffer systems.

[0081] Fifthly, some embodiments provide the use of the probe composition of the first aspect or the kit of the fourth aspect in the preparation of products for the detection of salivary gland tumors.

[0082] In an optional embodiment, the salivary gland tumor detection product contains a probe composition of the first aspect. By using the probe composition of the first aspect to capture nucleic acid molecules in the sample to be analyzed, nucleic acid molecules encoding each gene in the salivary gland tumor gene panel in the sample to be analyzed are obtained for subsequent sequencing steps to obtain variation information of the salivary gland tumor gene panel in the sample to be analyzed, thereby performing salivary gland tumor detection on the sample to be analyzed.

[0083] In an optional implementation, the salivary gland tumor detection product contains a fourth aspect kit, which uses the kit to extract nucleic acid molecules of each gene in the salivary gland tumor gene panel in the sample to be analyzed, and to perform at least one step in subsequent library construction or sequencing, so as to obtain the variation information of the salivary gland tumor gene panel in the sample to be analyzed, and then perform salivary gland tumor detection on the sample to be analyzed.

[0084] In an optional implementation, the salivary gland tumor detection product is used for the classification of salivary gland tumors.

[0085] Sixthly, some embodiments provide an apparatus for detecting salivary gland tumors, the apparatus including a sequencing module and an analysis module;

[0086] The sequencing module is used to implement the sequencing method for the third aspect of salivary gland tumor genes panel;

[0087] The analysis module is used to perform bioinformatics analysis on the data obtained by the sequencing module to obtain data information of the gene panels defined in the first aspect of the sample.

[0088] In an optional implementation, the data information of the gene panel includes the variation information of the genes in the gene panel.

[0089] In optional implementations, the variation information includes, but is not limited to, variation information of at least one of the following: single nucleotide variants, insertion / deletion variants, copy number variants, and RNA fusions.

[0090] In an optional implementation, the sequencing module includes a gene sequencer.

[0091] In an optional implementation, the analysis module may be stored in memory or embedded in the operating system (OS) of the device for detecting salivary gland tumors, either as software or firmware. The device for detecting salivary gland tumors may also optionally include a storage module storing data, program code, etc., required to execute the module.

[0092] In an optional embodiment, the device for detecting salivary gland tumors further includes at least one of a nucleic acid extraction module and a library construction module.

[0093] An apparatus for detecting salivary gland tumors may include a memory, a processor, a bus, and a communication interface, which are electrically connected directly or indirectly to enable data transmission or interaction. For example, these components may be electrically connected to each other via one or more buses or signal lines. The processor may process information and / or data related to target identification to perform one or more functions described in this application.

[0094] In practical applications, this device for detecting salivary gland tumors can be a server or a cloud platform.

[0095] The following are some examples.

[0096] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0097] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0098] Example 1

[0099] Determination of the composition of salivary gland tumor gene panel and probe:

[0100] This embodiment identified genes relevant to the biology and clinical practice of salivary gland tumors through a comprehensive review of literature, guidelines, and databases. Biomarkers relevant to differential diagnosis in routine diagnostic practice were also included, such as DEK-AFF2 fusions in non-keratinizing carcinomas of the nasal cavity and sinuses, SMARCB1 / SMARCA4 / SMARCA2 mutations in SWI / SNF-deficient carcinomas, and IDH1 / IDH2 mutations in undifferentiated carcinomas of the nasal cavity and sinuses. Furthermore, gene markers related to clinical trials or drugs in development were also included. The reference genome was hg19. A total of 3356 DNA capture probes were identified, containing the genes shown in Table 1, with a capture region of 350 kb (probe sequences shown in SEQ ID NO. 1–SEQ ID NO. 3356); and 1117 RNA capture probes, with a capture region of 134 kb (probe sequences shown in SEQ ID NO. 3357–SEQ ID NO. 4473). The total capture region was 484 kb.

[0101] DNA Panel Design: The 76 target molecules listed in Table 1 are DNA coding regions (CDS) and TERT promoter regions (chr5: 1295104-1296120). A combined, deduplicated 1× DNA probe design is used (for low-GC regions, a 2× probe design is used by default). Target molecules that are DNA genes include: AKT1, APC, AR, ARID1A, ATM, AXIN1, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, CDH1, CDK11B, CDKN2A, CDKN2B, CREBBP, CTNNB1, CYLD, EGFR, EP300, FBXW7, FGFR1, FLT3, FOXA1, HER2, HER4, HMGA2, H... RAS, IDH1, IDH2, IGF1R, IGF2, KDM6A, KDR, KMT2C, KRAS, LRFN1, MDM2, MET, MLH1, MSANTD3, MSH2, MUTYH, MYB, MYBL1, MYC, NF1, NOTCH1, NOTCH2, NOTCH3, NR4A3, NRAS, PD GFRA, PIK3CA, PIK3R1, PLAG1, PRKD1, PRKD2, PRKD3, PRSS1, PTEN, RAD51B, RB1, SETD2, SF3B1, SMARCA2, SMARCA4, SMARCB1, SMO, SPEN, STK11, TERT, TP53, TPTE2P1, and VHL.

[0102] RNA Panel Design: 1×RNA probes were designed for the 31 genes listed in Table 1 based on the CDS sequences of all transcripts. Genes whose target molecules are RNA include: ABL1, ALK, BCL6, CACNA1B, CDH1, DEK, EGFR, ENOX1, ETV5, ETV6, EWSR1, HMGA2, IRF2BP2, KMT2A, MAML2, MSANTD3, MYB, MYBL1, NOTCH1, NR4A3, NTRK1, NTRK2, NTRK3, NUTM1, PLAG1, PRKD1, PRKD2, PRKD3, RAPGEF6, RET, and SS18.

[0103] Based on the salivary gland tumor gene panel in Table 1 and the above target molecule classification, a probe composition for capturing target nucleic acid molecules was designed. The probes were synthesized by Nanoda (Nanjing) Biotechnology Co., Ltd., and the probe sequences are shown in SEQ ID NO.1~SEQ ID NO.4473.

[0104] Example 2

[0105] Performance confirmation of the salivary gland tumor gene panel and probe composition:

[0106] I. Experimental Methods:

[0107] 1. Patients and Samples:

[0108] A total of 119 cases were included, with the primary cohort consisting of 112 salivary gland tumors, supplemented by 7 cases of other non-head and neck tumors (e.g., synovial sarcoma, breast cancer) to validate the performance of the gene panel and probe composition of Example 1. These cases had previously been confirmed by NGS (next-generation sequencing) to carry characteristic genetic alterations. All cases were drawn from pathology archives of four tertiary hospitals over an eight-year period. The salivary gland cohort (n=112) was divided into 49 cases with a confirmed diagnosis and 63 cases with a challenging diagnosis. All salivary gland cases underwent comprehensive diagnostic testing, including histopathological evaluation, immunohistochemistry, and auxiliary molecular testing (e.g., FISH (fluorescence in situ hybridization) or the 425-gene NGS panel). The original diagnosis was confirmed by two expert pathologists.

[0109] All paraffin-embedded tumor tissues from 119 cases were subjected to targeted sequencing using the gene panel and probe combination provided in Example 1. Clinical and pathological data were collected from medical records, supplemented by telephone follow-ups as needed.

[0110] Based on the consistency between molecular findings and routine pathology, salivary gland cases were divided into four groups:

[0111] Group 1 (Molecular confirmation): Sequencing analysis results of gene panel and probe combination in Example 1 were completely consistent with the diagnosis of conventional methods;

[0112] Group 2 (Molecular Precise Definition): Sequencing analysis of gene panel and probe combinations from Example 1 resolved diagnostic challenges that were initially considered unclassifiable or provisional.

[0113] Group 3 (Molecular Revision): Sequencing analysis using the gene panel and probe combination from Example 1 led to a significant revision of the original diagnosis;

[0114] Group 4 (Molecular unresolved): Despite molecular typing, the cases could not be classified using sequencing analysis of the gene panel and probe combination from Example 1.

[0115] This embodiment was approved by the institutional review committee and informed consent was obtained from all participants.

[0116] 2. Library Construction:

[0117] (1) Nucleic acid extraction and quality control:

[0118] DNA and RNA were extracted from archived FFPE samples (Formalin-Fixed Paraffin-Embedded samples) using an FFPE nucleic acid extraction kit (DNA using GF3132-LM; RNA using GF3132-R-LM; Bluescape, China). Quantification was then performed using a Qubit 4.0 (Thermo Fisher Scientific, Waltham, MA) and dsDNA HS detection kit. A minimum tumor content of 20% and a minimum total of 20 ng DNA and 100 ng RNA were required for downstream library construction and sequencing. Libraries were constructed and sequenced using the gene panel from Example 1 and the probe combinations shown in SEQ ID NO. 1–SEQ ID NO. 4473. Single nucleotide variant, insertion / deletion, and fusion transcript detection analyses were performed.

[0119] (2) Pre-library construction: The isolated DNA / RNA samples were used to construct pre-libraries using the Rapid Plus DNA Lib Prep Kit V2 (RK20271, Abclonal) and the Fast RNA-seq Lib Prep Kit V2 (RK20306; Abclonal, China), respectively. Refer to the manufacturer's instructions for details. Unique Dual Index primers for Illumina (RK21624, Abclonal) were used for pre-library amplification. The appropriate number of PCR cycles was selected based on the amount of nucleic acid input; a library yield of 500–1500 ng was considered reasonable.

[0120] (3) Hybrid capture:

[0121] Preparation: Remove hybridization capture buffer 1 and hybridization capture buffer 2 from NadPrep® Hybrid Capture Reagents (NadPrep® Biotechnology) and thaw them at room temperature. Specific hybridization library mixing methods are shown in Table 2.

[0122] Table 2

[0123]

[0124] According to Table 2, mix each component in a 0.2 / 1.5 mL low-adsorption centrifuge tube, vortex, and centrifuge briefly. Place the centrifuge tube in a vacuum concentrator preheated to 60°C to dry. After all liquid has evaporated and completely dried, seal the centrifuge tube for later use. Take out the probe composition provided in Example 1 and thaw it naturally on ice. Prepare the hybridization reaction solution according to Table 3, mix it thoroughly with a pipette, and add it to the bottom of the vacuum-concentrated and dried centrifuge tube. Gently pipette and mix 15-20 times, centrifuge briefly, and incubate at 25°C for 5-10 min. Vortex the hybridization reaction mixture, centrifuge briefly, and transfer the entire 17 µL of hybridization reaction mixture from the centrifuge tube to a new 0.2 mL PCR tube. Centrifuge briefly and place in a PCR instrument. The hybridization program is shown in Table 4.

[0125] Table 3

[0126]

[0127] Table 4

[0128]

[0129] 3. Document cleansing

[0130] Use the NadPrep® Hybrid Capture Reagents kit and its Streptavidin Beads to elute and purify the library. Refer to the kit instructions for specific procedures.

[0131] 4. PCR amplification:

[0132] Thaw NadPrep® 2×HiFi PCR Master Mix and NadPrep® Amplification PrimerMix on ice, gently mix thoroughly using a pipette or vortex mixer, and centrifuge briefly before use. Prepare the reaction system according to Table 5.

[0133] Table 5

[0134]

[0135] Place the PCR tubes into the PCR instrument and start the following program according to Table 6, setting the hot cap temperature to 105℃.

[0136] Table 6

[0137]

[0138] Table 7. Number of cycles

[0139]

[0140] 5. Library purification and quantification: The final library was purified using Clean-NGS magnetic beads (CNGS-0050, CleanNA).

[0141] 6. Sequencing and data analysis:

[0142] Both DNA and RNA libraries were constructed and then sequenced, totaling approximately 1 GB. The expected average exon coverage depth was set to >500× to identify single nucleotide variants, insertions / deletions, and copy number variations. A minimum of 200M reads was set for gene fusion analysis. All variants obtained from sequencing analysis were manually reviewed.

[0143] (1) Mutation identification: Base identification was performed using bcl2fastq V.2.16.0.10 (Illumina) to generate sequence reads in FASTQ format. Trimmomatic software was used. [1] Quality control was performed. High-quality paired-end reads were aligned to the reference human genome (hg19) using the Burrows-Wheeler alignment tool. [2]The alignment was performed, followed by PCR deduplication (Picard; http: / / broadinstitute.github.io / picard / ) and local re-alignment around insertions and deletions (GATK3). VarScan2 was used. [3] Single nucleotide variants (SNVs) and insertions / deletions were identified. All SNVs / insertions / deletions were annotated using ANNOVAR and further filtered based on the following criteria: (1) the variant supporting reads must be mapped to double strands, (2) the strand bias must not exceed 10%, and (3) the population frequency reported in the 1000 Genomes or ExAC databases must not exceed 1%.

[0144] (2) Copy number analysis: Copy number variation (CNV) analysis was performed using the CNVkit tool. [4] The analysis process begins with generating an unbiased reference copy number baseline from control samples. Sequencing reads are binned and corrected for systematic biases such as GC content and target size at read depth. The log2 copy number ratio is calculated by comparing the read depth of the processed test samples to the reference baseline. Subsequently, the circular bisection (CBS) algorithm is used to segment the data based on the comparison values ​​to identify genomic regions with consistent copy number variations. Finally, the identified fragments are annotated, and their copy number status (e.g., deletion, duplication) is determined based on a set absolute log2 ratio threshold.

[0145] (3) RNA fusion analysis: FusionCatcher was used. [5] Fusion analysis was performed on the RNA data. A blacklist was designed for filtering based on previous fusion detection in clinical samples. Furthermore, connection reads and cross-pair reads identified by FusionCatcher were filtered, and stratified according to fusion hotspots. Additionally, the KM algorithm was employed. [6] This was used to detect intragenic fusions (internal gene variations). All identified fusions were manually verified in Integrated Genomics Viewer (Broad Institute, Cambridge, USA) 6.

[0146] II. Verification Results:

[0147] 1. Accuracy:

[0148] To verify the sequencing capability of the gene panel and probe composition of Example 1, this experiment tested 48 tumor FFPE specimens, covering various SGT subtypes. First, the accuracy was tested by blinding the consistency between the results obtained from the gene panel assay of Example 1 and those obtained from alternative clinical diagnostic techniques, including FISH, IHC, and the 425-gene panel (NGS method) (GENESEEQPRIME®) assay generated by a CAP-certified institution.

[0149] Accuracy validation was performed on 80 variants suitable for both the gene panel used in the examples and orthogonal methods, including 55 minor variants (33 SNVs and 22 InDels), 7 focal copy number alterations, and 18 fusions. High consistency was observed across platforms, as shown in Table 1. The consistency for minor variants was 98%; the consistency of the 7 copy number alterations involving FGFR1, ERBB2, MDM2, HMGA2, and TP53 genes was 100% compared with the 425 gene panel and IHC; orthogonal validation (NGS / FISH / IHC) of the 18 fusions showed 100% consistency.

[0150] Table 8. Accuracy assessment data of gene panel analysis in Example 1

[0151]

[0152]

[0153]

[0154] Furthermore, strong consistency was observed between the expected and measured allele frequencies for all small variant sites (SNV and InDel). Figure 1 This demonstrates the comparable quantitative performance between the two methods.

[0155] These results demonstrate that NGS detection based on the gene panel and probe composition of Example 1 is a robust and comprehensive diagnostic tool. It not only significantly expands the range of detectable genes associated with salivary gland tumors but also maintains accuracy comparable to existing clinical testing methods.

[0156] 2. Limit of detection:

[0157] To evaluate the sensitivity of the gene panel and probe composition developed in Example 1, this experiment used the standard DNA CA0820, whose variant allele frequencies were confirmed by digital PCR. This standard contains six cancer hotspot minor variants and one CNV amplification. The method is briefly described below: a library was constructed using 50 ng of CA0820 DNA, hybridized with the panel probe for capture, then amplified using adapter primers to generate the final library, and sequenced on the NextSeq 550 platform. Sequencing was performed in triplicate, with an average depth >2000× for each replicate. The resulting data was then downsampling to an average depth of 2000×. The detection of all loci was examined, and the sensitivity was calculated. The results are shown in Table 2. All three replicates consistently detected the expected frequency of 2% for SNVs, and InDel and MET (CN:5) were also consistently detected. In summary, the panel has a detection limit of 2% for minor variants and a copy number detection limit of 5 copies.

[0158] Table 9. Comparison of three replicate results with expected results in DNA detection limit assessment experiment

[0159]

[0160] The detection sensitivity of RNA fusions was assessed using the reference RNA standard RNA-mixV4. This standard contains nine fusion transcripts targeted by the gene panel in Example 1.

[0161] A library was constructed using 100 ng of standard RNA, hybridized with a panel probe for capture, and then amplified using adapter primers to generate the final library, which was then sequenced on a NextSeq 550 platform. This was repeated three times, and the data were downsampled to 200 MB for fusion analysis. The results showed that the gene panel and probe combination developed in Example 1 could stably detect all fusions at a 100 ng RNA input, with a detection limit as low as 3.66 copies / ng (Table 10).

[0162] Table 10. Comparison of three replicate results of the RNA detection limit assessment experiment with the expected results.

[0163]

[0164] 3. Precision:

[0165] In addition to determining the analytical accuracy and detection limit relative to alternative technologies, this embodiment also evaluates precision by assessing the intra-batch reproducibility and inter-batch reproducibility of the panel probe composition for DNA / RNA detection. For DNA precision assessment, this experiment analyzed small variants (SNVs and InDel) using a sample of cancer in pleomorphic adenoma, processed in two independent batches: Batch 1: three technical replicates; Batch 2: single run (no replicates). All variants were pre-confirmed using GENESEEQPRIME® (425-gene Panel). This sample contained six genes with high and low VAF variants, enabling comprehensive precision assessment. For variants ≥1% VAF (Table 11), the concordance was 100%, CV < 5%. Insertions, deletions, and SNVs with less than 1% VAF exhibited random detection failures due to sampling limitations.

[0166] Table 11. Results of three replicates of the DNA precision assessment experiment

[0167]

[0168] This experiment used an ACC sample carrying a pre-confirmed MYB-NFIB fusion (FISH validated) to evaluate the precision of fusion detection. The fusion was consistently detected in all four replicates, with intra-batch VAF heterogeneity (CV < 5%) and inter-batch VAF consistency (CV < 10%) (Table 5).

[0169] Table 12. Results of three replicates of the RNA precision assessment experiment

[0170]

[0171] In summary, these validations demonstrate that the gene panel of Example 1 and the probe composition provided in Example 1 exhibit robust reproducibility (inter-batch CV < 10%) and repeatability (intra-batch CV < 5%).

[0172] Example 3

[0173] Clinical application evaluation of the impact of molecular testing on overall diagnosis:

[0174] This embodiment utilizes the gene panel and probe composition developed in Example 1 to assist in the diagnosis of 112 salivary gland tumors. Key findings include... Figure 2 As shown.

[0175] Diagnostic confirmation group: In 43 routine cases, molecular subtyping confirmed the initial pathological diagnosis in all cases;

[0176] Difficult Case Analysis Group: Among 63 cases with challenging initial diagnoses, molecular subtyping enabled clear classification in 83% (52 cases); limited biopsy sampling, ambiguous morphology, and overlapping immunophenotypes were the main diagnostic challenges, with adenoid cystic carcinoma (ACC) and secretory carcinoma (SC) being the most challenging to diagnose.

[0177] Diagnostic Revision Group: In 5 cases, molecular findings led to significant diagnostic revisions;

[0178] Legacy challenge group: There are still 11 cases (10% of all cases) that cannot be classified even after molecular evaluation.

[0179] Example 4

[0180] The following sections will detail the genetic findings and representative case examples, demonstrating the value of the gene panel and probe composition developed in Example 1 in enhancing molecular typing and aiding diagnosis in challenging cases.

[0181] Figure 3 This is a case of a revised diagnosis: A female patient had a long history of recurrent parotid gland tumors, with several changes in diagnosis at different hospitals and at different times (previously diagnosed as adenoid cystic carcinoma, low-grade malignant tumor). The most recent visit showed atypical morphology of the recurrent lesions, which was misleading. The lesions were characterized by solid nests of tumor cells with pale to translucent cytoplasm, located in a collagenous stroma. Figure 3 (A). Immunohistochemistry showed that the tumor was entirely composed of p63-positive basal-like / myoepithelial cells. Figure 3 (B), while PAS-AB staining did not clearly identify mucus cells (B). Figure 3 The patient (C) was initially diagnosed with clear cell carcinoma. However, after testing with the gene panel and probe composition of Example 1, following the method of Example 2, revealed a CRTC1-MAML2 gene fusion, leading to a revised diagnosis of intermediate-grade mucoepidermoid carcinoma. This case clearly demonstrates the indispensability of molecular testing when histological presentation is deceptive.

[0182] Example 5

[0183] Figure 4Here is another case of a revised diagnosis: The patient was a 56-year-old woman who underwent endoscopic resection of a nasal and sinus mass in November 2020 (initial pathology: squamous cell carcinoma in situ, papillary type, focal inverted growth pattern with microinvasive areas). In November 2021, the tumor recurred in the left ear and was surgically removed at another hospital; the pathology report was "non-specific type (NOS)". In December 2021, the patient underwent further external auditory canal lesion and lymph node resection. Postoperative pathological examination confirmed an invasive tumor of the left middle ear, exhibiting a dual growth pattern: one was an exophytic papillary component with sparse cytoplasm, deeply stained nuclei, weakened intercellular adhesion, and scattered neutrophils visible in the fibrovascular axis (…). Figure 4 (A and B). The patient refused adjuvant radiotherapy and chemotherapy. Subsequently, the tumor recurred multiple times and metastasized to the larynx, and the patient eventually passed away in January 2025. Using the gene panel and probe composition of Example 1, molecular profiling analysis according to the method of Example 2 detected the DEK-AFF2 gene fusion (specifically, the fusion of DEK exon7 and AFF2 exon4). Figure 5 Based on integrated molecular and histopathological findings, the tumor was reclassified as a DEK-AFF2 squamous cell carcinoma. This tumor exhibits invasive potential and extensive histological mimicry. Accurate diagnosis requires confirmatory AFF2 IHC or molecular testing, and a good response to immune checkpoint inhibitors. This case illustrates the direct clinical value of identifying this rare subtype through comprehensive molecular analysis, potentially unlocking specific and effective treatment options.

[0184] In summary, Example 1 provides a comprehensive, customized gene panel for the diagnosis and targeted therapy of salivary gland tumors, as well as a probe composition for capturing this gene panel. This application provides a panel specifically designed to assist in the diagnosis of salivary gland tumors and guide targeted therapy. This application systematically applies this detection method to a large cohort of diagnostically challenging cases, providing a crucial assessment of how molecular diagnostics can optimize accurate patient triage and guide clinical management. It was applied in a cohort containing 25 different histological subtypes. Clinical cohort studies showed that the gene panel of Example 1 provided a clear classification for 83% of diagnostically challenging cases (Group 2), superior to conventional methods where limited biopsies (21% in this study cohort) and morphological ambiguity often lead to nonspecific diagnoses. It also completely corrected 5% of diagnoses. This salivary gland tumor gene panel and probe composition not only contribute to accurate diagnosis; it assists clinicians in diagnosis by identifying targeted therapy options, revealing missed treatment opportunities, and defining disease subgroups with different prognoses. It contributes to the accurate diagnosis of challenging cases and the optimization of patient treatment strategies.

[0185] The results of the above embodiments clearly demonstrate that this integrated molecular detection approach is a transformative tool in the diagnostic pathway, effectively addressing the significant diagnostic challenges posed by the histological complexity and rarity of salivary gland tumors. More importantly, it bridges the critical gap between diagnostic optimization and direct clinical action, enabling diagnosis to transcend academic classifications and move towards the realm of personalized treatment.

[0186] References:

[0187] [1]Bolger,AM;Lohse,M.;Usadel,B.Trimmomatic:AFlexibleTrimmerforIlluminaSequenceData.Bioinforma.Oxf.Engl.2014,30(15),2114-2120.https: / / doi.org / 10.1093 / bioinformatics / btu170.

[0188] [2]Li,H.;Durbin,R.FastandAccurateShortReadAlignmentwithBurrows-WheelerTransform.Bioinforma.Oxf.Engl.2009,25(14),1754-1760.https: / / doi.org / 10.1093 / bioinformatics / btp324.

[0189] [3]Koboldt, DC; Zhang, Q.; Larson, DE; Shen, D.; McLellan, MD; Lin, L.; Miller, CA; Mardis, ER; Ding, L.; Wilson, RKVarScan2: SomaticMutationan dCopyNumberAlterationDiscoveryinCancerbyExomeSequencing.GenomeRes.2012,22(3),568-576.https: / / doi.org / 10.1101 / gr.129684.111.

[0190] [4]Talevich, E.;Shain,AH;Botton,T.;Bastian,BCCNVkit:Genome-WideCopyNumberDetectionandVisualizationfromTargetedDNASequencing.PLoSComput.Biol.2016,12(4),e1004873.https: / / doi.org / 10.1371 / journal.pcbi.1004873.

[0191] [5]Nicorici,D.;Şatalan,M.;Edgren,H.;Kangaspeska,S.;Murumägi,A.;Kallioniemi,O.;Virtanen,S.;Kilkku,O.FusionCatcher-aToolf orFindingSomaticFusionGenesinPaired-EndRNA-SequencingData.BioinformaticsNovember19,2014.https: / / doi.org / 10.1101 / 011650.

[0192] [6]Audemard,EO;Gendron,P.;Feghaly,A.;Lavallée,V.-P.;Hébert,J.;Sauvageau,G.;Lemieux,S.TargetedVariantDetect ionUsingUnalignedRNA-SeqReads.LifeSci.Alliance2019,2(4),e201900336.https: / / doi.org / 10.26508 / lsa.201900336.

[0193] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0194] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A probe composition for detecting the salivary gland tumor gene panel, characterized in that, The probe composition comprises probes that target genes in the salivary gland tumor gene panel, which includes the following genes: AKT1, APC, AR, ARID1A, ATM, AXIN1, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, CDH1, CDK11B, CDKN2A, CDKN2B, CREBBP, CTNNB1, CYLD, EGFR, EP300, FBXW7, FGFR1, FLT3, FOXA1, HER2, HER4, HMGA2, HRAS, IDH1, IDH2, IGF1R, IGF2, KDM6A, KDR, KMT2C, KRAS, LRFN1, MDM2, MET, MLH1, MSANTD3, MSH2, MUTYH, MYB, MYBL1, MYC, NF1, NOTCH1, NOT CH2, NOTCH3, NR4A3, NRAS, PDGFRA, PIK3CA, PIK3R1, PLAG1, PRKD1, PRKD2, PRKD3, PRSS1, PTEN, RAD51B, RB1, SETD2, SF3B1, SMARCA2, SMARCA4, SMARCB1, SMO, SPEN, S TK11, TERT, TP53, TPTE2P1, VHL, ABL1, ALK, BCL6, CACNA1B, DEK, ENOX1, ETV5, ETV6, EWSR1, IRF2BP2, KMT2A, MAML2, NTRK1, NTRK2, NTRK3, NUTM1, RAPGEF6, RET, and SS18.

2. The probe composition for detecting salivary gland tumor gene panels according to claim 1, characterized in that, The probe targets the genomic DNA encoding the following genes: AKT1, APC, AR, ARID1A, ATM, AXIN1, BAP1, BARD1, BCOR, BRAF, BRCA1, BRCA2, CDH1, CDK11B, CDKN2A, CDKN2B, CREBBP, CTNNB1, CYLD, EGFR, EP300, FBXW7, FGFR1, FLT3, FOXA1, HER2, HER4, HMGA2, HRAS, IDH1, IDH2, IGF1R, IGF2, KDM6A, KDR, KMT2C, KRAS, LR FN1, MDM2, MET, MLH1, MSANTD3, MSH2, MUTYH, MYB, MYBL1, MYC, NF1, NOTCH1, NOTCH2, NOTCH3, NR4A3, NRAS, PDGFRA, PIK3CA, PIK3R1, PLAG1 , PRKD1, PRKD2, PRKD3, PRSS1, PTEN, RAD51B, RB1, SETD2, SF3B1, SMARCA2, SMARCA4, SMARCB1, SMO, SPEN, STK11, TERT, TP53, TPTE2P1 and VHL; Furthermore, the probe targets mRNAs encoding the following genes: ABL1, ALK, BCL6, CACNA1B, CDH1, DEK, EGFR, ENOX1, ETV5, ETV6, EWSR1, HMGA2, IRF2BP2, KMT2A, MAML2, MSANTD3, MYB, MYBL1, NOTCH1, NR4A3, NTRK1, NTRK2, NTRK3, NUTM1, PLAG1, PRKD1, PRKD2, PRKD3, RAPGEF6, RET, and SS18.

3. The probe composition for detecting salivary gland tumor gene panels according to claim 1, characterized in that, The probe targets the coding region of the target gene, wherein the coding region does not include the coding region of the TERT gene; and the probe targets the promoter region of the TERT gene. Optionally, in the probe composition, each probe binds to the target molecule at a length of 100-150 nt independently.

4. The probe composition for detecting the salivary gland tumor gene panel according to any one of claims 1 to 3, characterized in that, The nucleotide sequences of the probe-target molecule binding regions are shown in SEQ ID NO.1 to SEQ ID NO.4473, or in complementary sequences of SEQ ID NO.1 to SEQ ID NO.4473, respectively.

5. A method for constructing a library of salivary gland tumor genes using a panel, characterized in that, include: The probe composition for detecting salivary gland tumor genes Panel as described in any one of claims 1 to 4 is used to capture nucleic acid molecules in a sample.

6. The method for constructing a library of salivary gland tumor gene panels according to claim 5, characterized in that, The method includes first constructing a genomic DNA library and a cDNA library of the subject, and then using the probe composition to capture the target nucleic acid molecule from the genomic DNA library and the cDNA library; Optionally, the library construction method includes using probes in the probe composition that target and encode genomic DNA to capture target nucleic acid molecules in the genomic DNA library; Optionally, the library construction method includes using a probe in the probe composition that targets mRNA encoding a gene to capture target nucleic acid molecules in the cDNA library.

7. A sequencing method for a salivary gland tumor gene panel, characterized in that, This includes preparing a library using the library construction method described in claim 5 or 6, and then sequencing the obtained library; Optionally, the sequencing method further includes variation analysis of the gene sequence in the salivary gland tumor gene panel obtained by sequencing; Optionally, variation analysis includes, but is not limited to, analyzing at least one of the following variations: single nucleotide variants, insertion / deletion variants, copy number variants, and RNA fusions.

8. A reagent kit, characterized in that, Includes: (a), or (a) and (b): (a) The probe composition for detecting the salivary gland tumor gene panel according to any one of claims 1 to 4; and, (b) At least one of the reagents used for library construction and the reagents used for sequencing.

9. The probe composition for detecting salivary gland tumor gene panels according to any one of claims 1 to 4, or the kit according to claim 8, in the preparation of products for detecting salivary gland tumors; Optionally, the salivary gland tumor detection product is used to classify salivary gland tumors.

10. A device for detecting salivary gland tumors, characterized in that, Includes sequencing and analysis modules; The sequencing module is used to implement the sequencing method of the salivary gland tumor gene panel as described in claim 7; The analysis module is used to perform bioinformatics analysis on the data obtained by the sequencing module to obtain data information of the gene panel as defined in claim 1 of the sample; Optionally, the device for detecting salivary gland tumors further includes at least one of a nucleic acid extraction module and a library construction module.

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