Application of plasma cfDNA marker in parathyroid carcinoma diagnosis

Abstract

The invention relates to application of a plasma cfDNA marker in diagnosing parathyroid carcinoma. Specifically, the disclosure relates to detection of a plurality of free DNA (cfDNA) molecular markers in blood (such as plasma) for early diagnosis or preoperative diagnosis, and establishment of a prediction formula to assess the risk of parathyroid carcinoma of a patient. The method comprises the following steps: collecting a blood (such as plasma) sample of a patient, detecting the content, copy number and mutation condition of a group of cfDNA through (but not limited to) NGS, ddPCR or nucleic acid mass spectrometry detection technology, establishing a formula in combination with clinical information and prognosis of the patient, and calculating the probability that the patient suffers from malignant tumors, thereby taking corresponding treatment measures.

Description

Technical Field

[0001] The present disclosure relates to the fields of medicine, biology and clinical medicine, and in particular to markers for diagnosing parathyroid cancer. Background Art

[0002] Parathyroid cancer is a malignant endocrine tumor with a poor prognosis. Its main clinical feature is the large secretion of parathyroid hormone, which causes patients to have uncontrollable hypercalcemia and ultimately leads to complications such as cardiopulmonary failure and death.

[0003] The best technical approach for parathyroid cancer is to perform radical surgery during the first operation to completely remove the parathyroid tumor, the surrounding thyroid gland, and other soft tissues to avoid tumor rupture, residual tumor, or spread. However, there is currently a lack of effective preoperative diagnostic methods in clinical practice to differentiate parathyroid cancer from benign parathyroid lesions. Therefore, most patients with parathyroid cancer fail to undergo radical tumor resection during the first operation, and only receive a final diagnosis after postoperative pathological diagnosis or tumor recurrence and metastasis. If a diagnosis can be obtained before surgery and radical tumor resection is performed in a timely manner, the patient's long-term survival time can be effectively improved.

[0004] There is currently a lack of effective methods for the preoperative diagnosis of parathyroid cancer. It mainly relies on the clinical manifestations of patients before surgery and makes inferences based on the tumor manifestations during surgery. If the patient has severe clinical symptoms before surgery and serum PTH and blood calcium are significantly increased, parathyroid cancer is suspected. During surgery, exploration revealed severe adhesions between the tumor and surrounding tissues, which is also a manifestation of parathyroid cancer. However, these manifestations are not specific, and some benign parathyroid adenomas (especially those with larger tumors or longer course of disease) may show the above manifestations. Intraoperative rapid frozen pathology examination has a good differential diagnosis effect for most other tumors, but the diagnostic accuracy for parathyroid cancer is only about 15%.

[0005] Existing methods mainly infer the benign or malignant nature of the tumor based on preoperative clinical manifestations and intraoperative exploration results. They lack objective indicators, have poor accuracy and repeatability, and often result in missed or misdiagnosed cases. For patients with missed parathyroid cancer, a second surgery after tumor recurrence significantly increases the risk of tumor metastasis and recurrence, and significantly increases the difficulty of surgery, leading to surgery-related complications such as recurrent laryngeal nerve injury. However, if a benign parathyroid tumor is misdiagnosed as parathyroid cancer, the surgical resection will be too large, resulting in the problem of overtreatment, which will bring risks such as hypothyroidism to patients.

[0006] In view of this, there is still a need in the art to provide a marker, reagent and method that can identify parathyroid cancer at an early stage, as well as a marker, reagent and method that can distinguish benign parathyroid tumors from parathyroid cancer. Summary of the invention

[0007] Free DNA (cfDNA) refers to DNA that is free outside the cell and exists in human plasma, serum, urine, etc. Under normal circumstances, macrophages in the blood can remove free substances after cell damage or death. However, when cells gradually become cancerous and generate tumor cells, cell metabolism increases, the cell death rate is greater than the rate of macrophage clearance, and the cfDNA content will increase. The concentration of cfDNA in the blood of tumor patients is higher than that of normal people, and it has tumor-specific information in fragment distribution, fragmentation information, terminal sequence, etc. Therefore, cfDNA is often used as a target for tumor monitoring.

[0008] The inventors unexpectedly discovered that the mutation status of cfDNA (especially the CDC73 gene) in blood samples can be used for the diagnosis of parathyroid cancer, for distinguishing parathyroid cancer (malignant) from parathyroid adenoma (benign), and for predicting whether non-synonymous mutations of the CDC73 gene are present in parathyroid tumor tissue.

[0009] The Gene ID of cell division cycle 73 (CDC73) is 79577; the Genbank accession number is NM_024529.5.

[0010] Determining the mutation status of the CDC73 gene in a blood sample refers to determining the mutation status of the CDC73 gene in the form of cfDNA in the subject's blood sample (whole blood, plasma, serum). This mutation status is a non-synonymous mutation, and the non-synonymous mutation is selected from any one or a combination of the following: the presence of a missense mutation, the presence of a frameshift mutation, the presence of a stop mutation, the presence of a large DNA fragment deletion, and the presence of a gene structure variation. In view of this, the present disclosure provides a reagent for determining the mutation status of the CDC73 gene in a blood sample, which is used to be selected from any one or a combination of the following:

[0011] - Diagnosis of parathyroid cancer in the subject,

[0012] - Differentiate between parathyroid carcinoma and parathyroid adenoma,

[0013] - Predict the mutation status of CDC73 gene in parathyroid tumor tissues.

[0014] The present disclosure provides use of a reagent for determining the mutation status of the CDC73 gene in a blood sample in the preparation of a diagnostic device, wherein the diagnostic device is used for any one or a combination thereof selected from the following:

[0015] - Diagnosis of parathyroid cancer in the subject,

[0016] - Differentiate between parathyroid carcinoma and parathyroid adenoma,

[0017] - Predict the mutation status of CDC73 gene in parathyroid tumor tissues.

[0018] In some embodiments, the blood sample is selected from any one of the following: whole blood, plasma, serum.

[0019] In specific embodiments, the blood sample is plasma.

[0020] In some embodiments, the reagent is used to determine the mutation status of the CDC73 gene in its exon region in a blood sample.

[0021] In some embodiments, the mutation status is selected from any one of the following or a combination thereof: the presence of a missense mutation, the presence of a frameshift mutation, the presence of a stop mutation, the presence of a large DNA deletion, and the presence of a gene structural variation.

[0022] In some embodiments, the reagents for determining the mutation status of the CDC73 gene in a blood sample are in the form of a primer pair or a probe.

[0023] Methods for designing primer pairs and probes are well known in the art.

[0024] Primer refers to a molecule with a specific nucleotide sequence that promotes synthesis at the start of nucleotide polymerization. Primers are usually two artificially synthesized nucleotide sequences, one primer is complementary to one end of the target region (or template, target sequence), and the other primer is complementary to the other end of the target region. Its function is to serve as the starting point of nucleotide polymerization, so that nucleic acid polymerase can start synthesizing a new nucleotide chain along its 3' end.

[0025] The primer can be a DNA primer or an RNA primer. In the specific examples of the present disclosure, a DNA primer is preferred. It should be understood that the RNA primer corresponding to the DNA primer still falls within the scope of the present disclosure. Since primers usually appear in a pair, they are called primer pairs. One primer in the primer pair is specific to the upstream of the target sequence and serves as a forward primer; the other primer is specific to the downstream of the target sequence and serves as a reverse primer.

[0026] When a target sequence is given (such as the CDC73 gene), technicians know the principles of primer amplification of target sequences and probe binding to target sequences, and are also clear about the design principles of primers and probes based on textbooks and the principle of nucleotide sequence complementarity (for example, "Molecular Cloning Laboratory Guide" 2017; P450 "Designing PCR Primers Using Primer3Plus"; Chapter 13 "Preparation of Labeled DNA Probes, RNA Probes and Oligonucleotide Probes"). There are many primer / probe design software in the prior art, such as Primer Premier, Oligo7, BeaconDesigner, etc. When technicians know the target sequence, they can refer to and obtain the sequence information and structure information of specific primers or probes. Therefore, the technical solution disclosed in the present invention is not limited to specific primer pairs or probe sequences. As an example, the length of the primer / probe does not exceed 50 nt, such as but not limited to 1, 2, 3, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 46, 48, 50 nt.

[0027] It should be understood that although a specific detection method is used in the specific example, the realization of the technical effect of the present disclosure does not depend on a specific method (such as operating steps, instrument model, instrument setting parameters, captured target sequence, supplier, primer sequence, probe sequence). This is because the core of the technical solution of the present disclosure lies in the discovery of the relationship between the mutation status of the marker in the sample and the disease, so any means that can determine the mutation status are available.

[0028] The aforementioned reagent for determining the mutation status of the CDC73 gene in a blood sample can also be used in combination with other detection reagents, and the other detection reagents are used to determine the mutation status of the following genes in the blood sample: ABCB4, AHCTF1, AKAP9, BRAF, CCND1, CDKN2A, CDKN2C, CLIP1, COL12A1, CTNNB1, DHX38, EPRS, EZH2, FAT3, FH, GNAS, KRAS, LIFR, MAF, MAP1B, MEN1, MTOR, MYH1, NCOR1, NEB, NFKBIE, NTRK1, PIK3CA, PRUNE2, PTEN, QRICH2, RAD54L2, RB1, RET, RNF43, SHH, SMAD4, SMARCA4, SYNE1, TERT, TP53, TPM3, TRRAP, TTK, TTN, VHL, and ZEB1.

[0029] The "differentiation" in "differentiating parathyroid carcinoma from parathyroid adenoma" means: 1) determining that the subject has the indication of interest (or has a higher probability) and does not have other indications (or has a lower probability); and / or 2) distinguishing different subjects (or groups). The technician understands that "differentiation" in the clinical field is a statistical distinction, that is, different groups are distinguished at a statistically specified significant level.

[0030] Therefore, the reagent disclosed in the present invention is used to distinguish between parathyroid cancer and parathyroid adenoma by: determining whether the mutation status of the marker exists in the blood sample; and classifying the subject as parathyroid cancer or parathyroid adenoma according to the mutation status.

[0031] "Diagnosing parathyroid cancer in a subject" means determining whether the subject suffers from parathyroid cancer or the probability of suffering from parathyroid cancer. "Suffering from" should be understood in the broadest sense, including: already suffering from; or the probability of suffering from the disease at a set significance level is statistically significantly higher than that of the control. The context will be able to suggest the specific meaning of "suffering from".

[0032] "Positive" or "presence" means that the detection method used can detect the expected mutation status.

[0033] "Negative" or "absent" should never be understood as the complete absence of the expected mutation state. Rather, it means that the detection method used cannot detect it or cannot be distinguished from the background signal at a specific sensitivity level.

[0034] In some embodiments, the diagnostic device is selected from any one of the following: a kit, a solid phase carrier. In some embodiments, the solid phase carrier is selected from any one of the following: a chip, a particle, a test paper, a test card.

[0035] In some embodiments, when the mutation status of the CDC73 gene is detected in a blood sample, the subject is determined to have parathyroid cancer, or to have a high risk of having parathyroid cancer, or to have a non-synonymous mutation of the CDC73 gene in parathyroid tumor tissue.

[0036] In other embodiments, when the mutation status of the CDC73 gene is not detected in the blood sample, the subject is judged to: not have parathyroid cancer, or have a low risk of having parathyroid cancer, or there is no non-synonymous mutation of the CDC73 gene in the parathyroid tumor tissue.

[0037] In some embodiments, the non-synonymous mutation is selected from any one or a combination of the following: missense mutation, frameshift mutation, stop mutation, large DNA deletion, gene structural variation.

[0038] In some embodiments, the aforementioned reagents for determining the mutation status of the CDC73 gene in a blood sample can also be used in combination with any one of the following: the subject's age, gender, blood calcium level, blood phosphorus level, blood parathyroid hormone level, or a combination thereof; thereby being used to predict the probability that the subject has parathyroid cancer, or to predict the probability of the presence of non-synonymous mutations in the CDC73 gene in parathyroid tumor tissue.

[0039] The present disclosure provides a method and formula for predicting the probability that a subject has parathyroid cancer.

[0040] In some embodiments, the probability P that the subject suffers from parathyroid cancer is predicted according to the following formula (1):

[0041] Logit(P)=4.0+3.914×mutation status of CDC73 gene in blood sample-0.1

[0042] × subject age formula (1).

[0043] The mutation status of the CDC73 gene in the blood sample is determined using the aforementioned method and reagent disclosed herein. The mutation status of the CDC73 gene in the blood sample: when the mutation status exists, it is recorded as 1; when it does not exist, it is recorded as 0; the age unit is year (preferably, one decimal place).

[0044] In some embodiments, when P≧0.726, the subject is determined to have parathyroid cancer, or to have a high risk of having parathyroid cancer.

[0045] In some embodiments, when P<0.726, the subject is determined to not have parathyroid cancer or to have a low risk of having parathyroid cancer.

[0046] The present disclosure provides a method and formula for predicting the probability of non-synonymous mutations in the CDC73 gene in parathyroid tumor tissue.

[0047] In some embodiments, the probability P of the presence of a non-synonymous mutation in the CDC73 gene in parathyroid tumor tissue is predicted according to the following formula (2): Logit (P) = 5.26 + 5.159 × mutation status of the CDC73 gene in the blood sample – 0.002 × blood PTH level + 2.778 × subject gender – 0.170 × subject age formula (2).

[0048] In some embodiments, the mutation status of the CDC73 gene in the blood sample: when the mutation status exists, it is recorded as 1; when it does not exist, it is recorded as 0; the subject's gender: male is recorded as 1; female is recorded as 0; the age unit is year (preferably, one decimal place); the blood PTH level unit is pg / mL.

[0049] In some embodiments, when P≥0.54, it is determined that a non-synonymous mutation exists in the CDC73 gene in the parathyroid tumor tissue.

[0050] In other embodiments, when P<0.54, it is determined that there is no non-synonymous mutation in the CDC73 gene in the parathyroid tumor tissue.

[0051] Detection device

[0052] The present disclosure also provides a detection device for the aforementioned purpose, which can be embodied in any known or future form, such as but not limited to a reagent kit, a test paper, a well plate, or a chip.

[0053] In some embodiments, the detection device comprises at least one container, each of which contains one or more reagents (primer pairs or probes) of the present disclosure.

[0054] When the detection device is in the form of a reagent (or a kit), it contains one or more reagents of the present disclosure, which can be prepared in the form of a liquid or a lyophilized powder.

[0055] When the detection device is in the form of a chip, a well plate, a test paper (such as a strip, a card), one or more reagents of the present disclosure are combined or coated on a solid phase carrier to facilitate subsequent steps (such as washing or separation). Solid phase carriers are such as synthetic resins, nitrocellulose, glass plates, metal plates, glass fibers, microspheres, and microbeads. The synthetic resin can be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon.

[0056] The ordinal numbers "first", "second", "1", "2", etc. in the present disclosure are not intended to limit the order or level, but are only used to distinguish different features, molecules, steps, components, elements, etc.

[0057] The term "comprising" is meant to include, but not be limited to, whatever follows the word "comprising." Thus, the use of the term "comprising" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 :Statistics of free DNA mutation status in plasma of patients with benign / malignant parathyroid tumors (PC vs PA). "1" in the figure means positive detection.

[0059] Figure 2 : ROC curve of using CDC73 mutation status in plasma cfDNA to predict CDC73 mutation status in parathyroid tumor tissue. DETAILED DESCRIPTION

[0060] Abbreviations

[0061] cfRNA cell-free DNA

[0062] PC parathyroid carcinoma

[0063] PA parathyroid adenoma

[0064] PTH parathyroid hormone

[0065] NGS Next-generation Sequencing

[0066] dPCR digital PCR digital PCR

[0067] pHPT primary hyperparathyroidism

[0068] LOH loss of heterozygosity

[0069] Example 1. Screening method for markers

[0070] 1. Methods for DNA extraction from plasma and paired leukocytes

[0071] After obtaining informed consent, plasma and paired leukocyte specimens were collected from patients with parathyroid carcinoma (PC) and parathyroid adenoma (PA) before surgery.

[0072] In plasma samples, cfDNA was extracted using a DNA kit, and a sequencing library was established and amplified using a KAPA kit. Magnetic beads were used for fragment sorting and purification, and Qubit was used for quantification.

[0073] 2. This example uses exon capture primers to specifically target the following detection genes: ABCB4, AHCTF1, AKAP9, BRAF, CCND1, CDC73, CDKN2A, CDKN2C, CLIP1, COL12A1, CTNNB1, DHX38, EPRS, EZH2, FAT3, FH, GNAS, KRAS, LIFR, MAF, MAP1B, MEN1, MTOR, MYH1, NCOR1, NEB, NFKBIE, NTRK1, PIK3CA, PRUNE2, PTEN, QRICH2, RAD54L2, RB1, RET, RNF43, SHH, SMAD4, SMARCA4, SYNE1, TERT, TP53, TPM3, TRRAP, TTK, TTN, VHL, and ZEB1.

[0074] The Genbank information of the above genes is well known, and technicians can design primers based on their sequences (especially exon regions). The technical effect of the present application is not limited by the specific sequence of the primers, and specific amplification of the target gene (especially exon regions) can be achieved, because the purpose of amplification is to identify the mutation status of the target gene.

[0075] 3. Sequencing analysis: The library was deeply sequenced using the Illumina Hiseq3000 sequencing platform, with cfDNA sequencing depth >5000× and leukocyte DNA sequencing depth >200×. After removing duplicate reads from cfDNA sequencing data, if the effective sequencing depth was less than 1000×, additional testing was performed.

[0076] 4. Sequencing data analysis: After obtaining the raw sequencing data, output it as a fastq file, remove low-quality and repeated reads, obtain net reads, calculate the effective sequencing depth, and count the sequencing quality. Using hg19 (GRCh37) as the reference genome, use BWA+Picard / novosort (Novocraft)+GATK software for mapping, use VarDict+FreeBayes+ANNOVAR software to call SNV and indel, and annotate, use CNVkit software to calculate copy number variation CNV, and use Lichee, Pyclone, and Sciclone software for mutation evolution analysis. SPSS software was used for data statistical analysis, and p<0.05 was used as the standard for statistical difference.

[0077] 5. Criteria for determining effective plasma free DNA mutations:

[0078] 1) After removing duplicate reads, the mutation frequency of a gene in cfDNA (AF, number of mutation reads / total number of reads after deduplication) is > 0.4%,

[0079] 2) The average sequencing quality (QUAL) of mutation-supporting reads is greater than 55, and is supported by both positive and negative strand reads.

[0080] 3) The mutation site is located in the exon region,

[0081] 4) The mutation is annotated as a non-synonymous mutation (selected from any one or a combination of the following: missense mutation, frameshift mutation, stop mutation, large DNA deletion, gene structural variation), which leads to the inactivation of the target.

[0082] 5) The mutation frequency of this site in the 1000 Genomes and Exome Aggregation Consortium Oriental databases is <0.001.

[0083] IGV software was used to compare and verify the sequencing information of cfDNA, leukocytes and tumor tissues ( Figure 1 ).

[0084] Example 2. The CDC73 gene mutation status in cfDNA predicts the CDC73 status in tumor tissue

[0085] 1. Inclusion / Exclusion Criteria

[0086] This example includes plasma specimens and paired leukocyte specimens from 31 patients with parathyroid tumors, including 18 patients with parathyroid carcinoma and 13 patients with parathyroid adenoma. All patients received surgical treatment and obtained a clear pathological diagnosis. All patients had corresponding postoperative follow-up information.

[0087] 2. Clinical information

[0088] Among the 31 patients, there were 4 males and 27 females with an average age of 48.6±2.2 years. After postoperative pathology, 18 patients were diagnosed with parathyroid carcinoma (PC) and 13 were diagnosed with parathyroid adenoma (PA). There was no difference in age, serum PTH, serum calcium, serum ALP and serum creatinine between the two groups. The serum phosphorus level of the cancer group was lower than that of the adenoma group (P=0.043). Thirteen of the 18 patients with parathyroid carcinoma had metastasis or recurrence during follow-up.

[0089] 3. CDC73 mutation status in patient tumor tissues

[0090] Gene sequencing and immunohistochemical staining showed non-synonymous mutations in the CDC73 gene or abnormal expression of parafibromin (protein encoded by the CDC73 gene) in tumor tissues of 10 of the 31 patients with parathyroid cancer.

[0091] 4. CDC73 mutation status in patient plasma

[0092] (1) cfDNA concentration:

[0093] The plasma free DNA concentration in patients with parathyroid carcinoma (PC) was higher than that in patients with benign tumors (PA), but the difference was not statistically significant (1.57±1.92μg / ml in PC group vs 0.81±0.54μg / ml in PA group, P=0.128).

[0094] (2) The total number of sequencing reads and effective sequencing depth in the PC group were comparable to those in the PA group (P = 0.064, P = 0.850).

[0095] (3) Mutation status of cfDNA in plasma:

[0096] Among the 31 plasma samples, each sample had an average of 43.6 mutations in 14.2 genes. The number of mutated genes in the plasma of PC patients was more than that of PA patients (P=0.026).

[0097] 5. Plasma cfDNA predicts CDC73 gene mutation in tumor tissue

[0098] CDC73 mutations were found in tumor tissues of 10 PC patients, including 2 cases of germline mutations. Deep sequencing of plasma cfDNA detected CDC73 mutations in the plasma of 8 of these patients.

[0099] Four CDC73 mutations were detected in the blood of 20 patients without CDC73 mutations in their tumor tissue (i.e., false positives), with a sensitivity of 80% and a specificity of 80%.

[0100] Using logistic regression analysis, it was found that the mutation status of CDC73 in cfDNA (i.e., the presence of non-synonymous mutations) can predict the status of CDC73 in tumor tissue (whether there are non-synonymous mutations). After establishing a prediction formula, a ROC curve was drawn, and the area under the curve (AUC) reached 0.8 (95% CI: 0.622-0.978).

[0101] 6. Predicting CDC73 mutation status in tumor tissue using CDC73 gene mutation status in plasma cfDNA and clinical factors

[0102] After analyzing clinical factors, logistic regression found that plasma CDC73 mutation, patient gender, age and serum PTH level were related to whether the tumor carried CDC73 mutation. After establishing the prediction formula, the ROC curve was drawn, and its AUC reached 0.935 (95% CI: 0.841-1.000).

[0103] The probability of non-synonymous mutation of CDC73 gene in patient tumor tissue is P:

[0104] Logit (P) = 5.26 + 5.159 × CDC73 mutation status in blood sample – 0.002 × blood PTH level + 2.778 × subject sex – 0.170 × subject age;

[0105] The mutation status of CDC73 in blood samples: the presence of nonsynonymous mutations was recorded as 1; when they were absent, they were recorded as 0;

[0106] Gender: male is recorded as 1; female is recorded as 0;

[0107] Age is in years (one decimal place);

[0108] The unit of blood PTH level is pg / mL.

[0109] When P ≥ 0.54, CDC73 mutation was considered to be present in the tumor tissue.

[0110] When P<0.54, it was considered that there was no CDC73 mutation in the tumor tissue.

[0111] Example 3. Markers for distinguishing parathyroid carcinoma (PC) from parathyroid adenoma (PA)

[0112] 1. The results of plasma cfDNA sequencing were used to predict the benign or malignant nature of parathyroid tumors. Using logistic regression analysis, the AUC value of CDC73 mutation status in plasma for differential diagnosis of benign and malignant parathyroid tumors was 0.795 (95% CI: 0.632-0.958).

[0113] 2. Combining clinical characteristic parameters with logistic regression analysis revealed that the CDC73 gene mutation status in cfDNA and the patient's age were correlated with the benign or malignant nature of the tumor.

[0114] 3. After the prediction formula was established, the ROC curve was drawn, and the area under the curve (AUC) reached 0.889 (95% CI: 0.764-1.000) ( Figure 2 ).

[0115] The probability of a patient developing a parathyroid malignancy is P:

[0116] Logit(P) = 4.0 + 3.914 × CDC73 mutation – 0.1 × patient age;

[0117] CDC73 mutation status: the presence of a nonsynonymous mutation was recorded as 1; the absence of a nonsynonymous mutation was recorded as 0;

[0118] Age unit: year.

[0119] 4. Conclusion: The prediction formula established using the mutation status of CDC73 in cfDNA, patient gender, age and blood phosphorus level can predict the benign or malignant nature of the tumor (distinguishing between PC and PA).

[0120] When the probability was P ≥ 0.726, it was considered a malignant tumor;

[0121] When P < 0.726, it was considered a benign tumor.

[0122] The aforementioned embodiments were verified by double-blind tests, demonstrating that the uses, reagents and methods of the present disclosure are repeatable.

Claims

1. Use of a reagent for determining the mutation status of the CDC73 gene in a blood sample in the preparation of a diagnostic device, wherein the diagnostic device is used for any one or a combination thereof selected from the following: - Diagnosis of parathyroid cancer in the subject, - Differentiate between parathyroid carcinoma and parathyroid adenoma, - Predict the mutation status of CDC73 gene in parathyroid tumor tissues; The blood sample is selected from any one of the following: whole blood, plasma, serum, preferably plasma; The mutation is located in the exon region of the CDC73 gene; The mutation status is selected from any one of the following or a combination thereof: the presence of a missense mutation, the presence of a frameshift mutation, the presence of a stop mutation, the presence of a large DNA fragment deletion, and the presence of a gene structural variation; The reagent is selected from any one of the following: a primer pair, a probe.

2. The use according to claim 1, wherein: The CDC73 gene in the blood sample is cfDNA.

3. The use according to claim 1 or 2, wherein the gene further comprises any one or a combination thereof selected from the group consisting of ABCB4, AHCTF1, AKAP9, BRAF, CCND1, CDKN2A, CDKN2C, CLIP1, COL12A1, CTNNB1, DHX38, EPRS, EZH2, FAT3, FH, GNAS, KRAS, LIFR, MAF, MAP1B, MEN1, MTOR, MYH1, NCOR1, NEB, NFKBIE, NTRK1, PIK3CA, PRUNE2, PTEN, QRICH2, RAD54L2, RB1, RET, RNF43, SHH, SMAD4, SMARCA4, SYNE1, TERT, TP53, TPM3, TRRAP, TTK, TTN, VHL, and ZEB1.

4. The use according to claim 1, wherein: The diagnostic device is selected from any one of the following: a kit, a solid phase carrier; Preferably, the solid phase carrier is selected from any one of the following: a chip, a particle, a test paper, a test card.

5. The use according to claim 1, wherein: When the mutation status of the CDC73 gene is detected in the blood sample, the subject is judged to have: parathyroid cancer, or a high risk of parathyroid cancer, or a non-synonymous mutation of the CDC73 gene is present in the parathyroid tumor tissue; When the mutation status of the CDC73 gene is not detected in the blood sample, the subject is judged to: not have parathyroid cancer, or have a low risk of having parathyroid cancer, or there is no non-synonymous mutation of the CDC73 gene in the parathyroid tumor tissue; The non-synonymous mutation is selected from any one of the following or a combination thereof: missense mutation, frameshift mutation, termination mutation, large DNA fragment deletion, and gene structural variation.

6. The use according to claim 1, wherein the reagent for determining the mutation status of the CDC73 gene in a blood sample is used in combination with any one selected from the following: The subject's age, sex, blood calcium level, blood phosphorus level, blood parathyroid hormone level, or a combination thereof; Preferably, the subject's age, gender and blood calcium level.

7. The use according to claim 6, wherein the probability P of the subject suffering from parathyroid cancer is predicted according to the following formula: Logit(P)=4.0+3.914×mutation status of CDC73 gene in blood samples -0.1×subject age; in: The mutation status of the CDC73 gene in the blood sample: when the mutation status exists, it is recorded as 1; when it does not exist, it is recorded as 0; Age is expressed in years.

8. The use according to claim 7, wherein: When P≥0.726, the subject is determined to have: parathyroid cancer or a high risk of having parathyroid cancer; When P<0.726, the subject is determined to have no parathyroid cancer or to have a low risk of having parathyroid cancer.

9. The use according to claim 6, predicting the probability P of the presence of non-synonymous mutations in the CDC73 gene in parathyroid tumor tissue according to the following formula: Logit (P) = 5.26 + 5.159 × mutation status of CDC73 gene in blood sample – 0.002 × blood PTH level + 2.778 × subject sex – 0.170 × subject age; in: The mutation status of the CDC73 gene in the blood sample: when the mutation status exists, it is recorded as 1; when it does not exist, it is recorded as 0; Gender of the subject: male is recorded as 1; female is recorded as 0; Age is in years; The unit of blood PTH level is pg / mL; When P ≥ 0.54, it was determined that there was a nonsynonymous mutation in the CDC73 gene in parathyroid tumor tissue; When P<0.54, it was determined that there was no nonsynonymous mutation in the CDC73 gene in parathyroid tumor tissue.

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