Probe group for judging systemic tumor load of triple-negative breast cancer and application of probe group

Abstract

The invention provides a probe set for judging the whole-body tumor load of triple-negative breast cancer and application of the probe set, the probe set can be used for detecting a gene marker of triple-negative breast cancer and evaluating the whole-body tumor load, good coverage and capture of a target area can be achieved, and the probe set can be used for detecting the whole-body tumor load of triple-negative breast cancer and evaluating the whole-body tumor load of triple-negative breast cancer. The method can be used for acquiring enough reading information on a target area, overcoming the space-time heterogeneity of tumors to a certain extent, improving the capturing efficiency, stabilizing an experiment system and meeting clinical detection requirements.

Description

Technical Field

[0001] The present disclosure belongs to the field of nucleic acid sequencing, and specifically relates to a probe set for determining the whole body tumor load of triple-negative breast cancer and its application. Background Art

[0002] Triple-negative breast cancer (TNBC) is a special type of breast cancer characterized by the fact that tumor cells do not express estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This type of breast cancer accounts for approximately 15%-20% of all breast cancers and is characterized by an early age of onset, high invasiveness, poor prognosis, early local recurrence and distant metastasis. The ctDNA status of TNBC at the time of initial diagnosis, before and after surgery and systemic treatment can effectively distinguish whether the patient's breast cancer is dominated by systemic tumor load or local tumor load, which can be used for individualized recurrence risk assessment and to guide the upgrade or downgrade of clinical systemic treatment plans.

[0003] Personalized ctDNA testing customizes a personalized panel for each patient based on the mutations detected in the tumor tissue, which can be used to monitor the ctDNA status in the blood of TNBC patients. This strategy has been increasingly widely used due to its advantages of high sensitivity, high specificity, high cost performance, rapidity and convenience. However, due to the heterogeneity of tumors, tissue testing may not cover all mutations, and personalized ctDNA testing cannot effectively monitor new mutations that arise from the selective pressure caused by treatment and other factors that lead to the evolution of tumor clones. In addition, the capture efficiency of using a personalized panel alone is low, and the stability of the experimental system needs to be further improved. Therefore, how to overcome the spatial and temporal heterogeneity of tumors and further improve the capture efficiency and stability of the experimental system is a challenge faced by personalized ctDNA testing. Summary of the invention

[0004] According to one aspect of the present disclosure, a gene marker for detecting triple-negative breast cancer (TNBC) is provided, wherein the marker comprises any one or more of the following genes: TP53, PTEN, RB1, PIK3CA, PIK3R1, AKT1, ERBB2 or NOTCH1.

[0005] In some embodiments, the gene marker includes any one or more of the following regions: exons 3-10 of TP53; exons 1 and 3-8 of PTEN; exons 6, 14-15, 20 and 24 of RB1; exons 2, 8, 10 and 21 of PIK3CA; exons 11 and 13 of PIK3R1; exon 3 of AKT1; exon 20 of ERBB2; and, exon 27 of NOTCH1.

[0006] In some embodiments, the gene marker is used to detect systemic tumor burden in triple-negative breast cancer.

[0007] According to one aspect of the present disclosure, a probe set for detecting triple-negative breast cancer is provided, wherein the probe set is used to detect the gene markers described in the present disclosure.

[0008] In some embodiments, the probe set has a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1 to 68, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto.

[0009] In some embodiments, the probe panel is used to detect systemic tumor burden in triple-negative breast cancer.

[0010] In some embodiments, the probe set includes any one or more combinations of the following:

[0011] A probe for detecting the PIK3CA gene: having a nucleotide sequence as shown in SEQ ID NO: 1 to 11, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0012] A probe for detecting the PIK3R1 gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 12 to 16, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0013] A probe for detecting NOTCH1 gene: having a nucleotide sequence as shown in SEQ ID NO: 17 and / or 18, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0014] A probe for detecting the PTEN gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 19 to 34, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0015] A probe for detecting the RB1 gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 35 to 47, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0016] A probe for detecting the AKT1 gene: having a nucleotide sequence as shown in SEQ ID NO: 48 and / or 49 or more, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0017] A probe for detecting the TP53 gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 50 to 66, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or

[0018] A probe for detecting the ERBB2 gene: having a nucleotide sequence as shown in SEQ ID NO: 67 and / or 68 or more, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto;

[0019] Optionally, the probe set further comprises a personalized probe set.

[0020] In some embodiments, the personalized probe set is a personalized probe set designed for a patient based on the mutation profile of a tissue sample of a patient with triple-negative breast cancer.

[0021] In some embodiments, the personalized probe set includes a nucleotide sequence as shown in any one of SEQ ID NOs: 69 to 277, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto.

[0022] According to yet another aspect of the present disclosure, a kit for detecting triple-negative breast cancer is provided, the kit comprising the probe set.

[0023] In some embodiments, the kit is used to detect systemic tumor burden in triple-negative breast cancer.

[0024] According to another aspect of the present disclosure, a method for detecting triple-negative breast cancer using the probe set and / or the kit of the present disclosure is provided, the method comprising using the probe set and / or the kit to detect the whole body tumor burden of triple-negative breast cancer.

[0025] In some embodiments, the method is used to detect systemic tumor burden in triple-negative breast cancer.

[0026] According to another aspect of the present disclosure, provided is the use of the gene markers and / or the probe set described in the present disclosure in preparing a kit for detecting triple-negative breast cancer.

[0027] In some embodiments, the use comprises detecting whole body tumor burden in triple negative breast cancer.

[0028] In some embodiments, the kit is used for one or more of triple-negative breast early screening, diagnosis, and recurrence monitoring.

[0029] In some embodiments, the kit is used for detecting cell, tissue or body fluid samples.

[0030] In some embodiments, the cell sample includes a triple-negative breast cancer cell suspension sample and / or circulating tumor cells (CTCs).

[0031] In some embodiments, the body fluid sample includes: whole blood, serum, plasma, emulsion, lymph fluid, and processed products thereof.

[0032] In some embodiments, the tissue sample includes triple-negative breast cancer tissue, paraffin sections, and processed materials thereof.

[0033] Beneficial effects:

[0034] Based on a large sample of TNBC clinical cohort, this paper designs a TNBC-specific probe set, which can be used in combination with a personalized probe set for targeted capture, and is used for personalized ctDNA detection of TNBC to assess systemic tumor burden. The TNBC-specific probe set includes TNBC-specific high-frequency mutations, which can overcome the temporal and spatial heterogeneity of tumors to a certain extent during ctDNA detection, while also stabilizing the experimental system and improving capture efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Shown are the capture efficiency test results of the TNBC-specific probe set.

[0036] Figure 2 The results of the TNBC-specific probe set testing for ≥0.2 times the mean depth percentage are shown.

[0037] Figure 3 The results of the TNBC-specific probe set testing for percentage of ≥0.5 times the mean depth are shown.

[0038] Figure 4 The results of the TNBC-specific probe set depth factor test are shown.

[0039] Figure 5 The coverage of TNBC-specific probe sets for TNBC patients is shown.

[0040] Figure 6 Shown is the patient mutation coverage of the TNBC-specific probe set in the clinical cohort.

[0041] Figure 7 The sensitivity and specificity results of the TNBC-specific probe set for detecting ctDNA are shown.

[0042] Figure 8 Shown are the results of survival analysis using a TNBC-specific probe set for detecting ctDNA. DETAILED DESCRIPTION

[0043] The present disclosure designs a ctDNA detection probe set specific for triple-negative breast cancer based on tissue samples from 488 clinical TNBC patients. The present disclosure screens out 8 high-frequency mutation genes specific to TNBC cancer based on clinical sample data, which can be used for ctDNA detection in TNBC patients to assess systemic tumor burden. The probe set provided by the present disclosure can achieve good coverage and capture of the target regions of these 8 high-frequency mutation genes, obtain sufficient read information on the target region, thereby overcoming the spatiotemporal heterogeneity of the tumor to a certain extent, while also stabilizing the experimental system and improving capture efficiency.

[0044] In a specific embodiment, the probe set disclosed herein can cover 8 TNBC high-frequency mutation genes: TP53, PTEN, RB1, PIK3CA, PIK3R1, AKT1, ERBB2, NOTCH1. Table 1 shows the regions on the TNBC high-frequency mutation genes covered by the probe set disclosed herein, and Table 2 shows the sequences of the probe set. In a specific embodiment, the 5' end of the probe can be labeled with biotin.

[0045] The TNBC-specific probe set disclosed in the present invention can play a role in monitoring tumor evolution and new mutations. After verification by clinical samples, the probe set disclosed in the present invention has excellent capture efficiency and depth coefficient, good probe uniformity, and meets clinical detection requirements. It also has excellent coverage of the TNBC population, with a coverage rate of more than 80% for clinical samples.

[0046] The probe set provided by the present disclosure can accurately detect mutations related to the whole body tumor burden of triple-negative breast cancer, has good sensitivity and specificity, and has good application prospects in the detection of whole body tumor burden of triple-negative breast cancer.

[0047] Those skilled in the art will appreciate that the probe set disclosed herein can be used alone or in combination with any other conventional probe set.

[0048] In some embodiments, the probe set disclosed herein and the personalized probe set are used in combination to monitor the ctDNA status of TNBC patients and have excellent clinical performance.

[0049] Verified by clinical samples, the probe group disclosed in the present invention and the personalized probe group used in combination have higher sensitivity and specificity, and can predict recurrence earlier than imaging. Patients with positive ctDNA mutations have a higher risk of recurrence than patients with negative mutations. Using only the probe group provided by the present invention can cover at least 80% of ctDNA-positive patients.

[0050] definition

[0051] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0052] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0053] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0054] In this article, the term "triple-negative breast cancer" is a subtype of breast cancer, referring to breast cancer in which the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are all negative, with the characteristics of high invasiveness, high recurrence and metastasis rate and poor patient prognosis.

[0055] Herein, the term "cfDNA" refers to circulating free DNA, including DNA molecules naturally present in an extracellular form in a subject (e.g., in blood, serum, plasma, or other body fluids such as lymph, cerebrospinal fluid, urine, or sputum). Although cfDNA is initially present in one or more cells in a large complex biological organism (e.g., mammal), cfDNA undergoes release from the cells into the fluid present in the organism and can therefore be obtained by obtaining a sample of the fluid without the need for an in vitro cell lysis step.

[0056] In this article, the term "ctDNA" refers to circulating tumor DNA (ctDNA), which is a DNA fragment derived from apoptosis, necrosis or secretion of tumor cells, and contains the same genetic mutations and epigenetic modifications as tumor tissue DNA, such as point mutations, gene rearrangements, fusions, copy number variations, methylation modifications, etc. The detection of ctDNA can be applied to early cancer screening, diagnosis and staging, guidance of targeted drug use, efficacy evaluation, recurrence monitoring, etc. Combining the information on tumor-specific gene mutations and methylation carried by ctDNA can help improve the sensitivity and specificity of detection, detect cancer traces earlier, and is of great significance for early cancer screening.

[0057] As used herein, the term "genetic marker" refers to a molecular indicator having a specific biological property, biochemical characteristic, or aspect that can be used to determine the presence or absence and / or severity of a particular disease or condition.

[0058] In this article, the term "sequence identity" refers to the "percentage of sequence identity" or "percentage of sequence identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences in the comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for the optimal comparison of the two sequences. A matching position is any position in which the same nucleotides are present in both the target sequence and the reference sequence. Since the gap is not a nucleotide, the gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides are counted, and the nucleotides from the reference sequence are not counted, the gap present in the reference sequence is not counted. At least 85% sequence identity includes at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the total length of the sequence having a continuous segment with sequence identity.

[0059] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of the sequence and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, a part of the EMBOSS suite of Needle, Stretcher, Water or Matcher, bioinformatics programs, and can also be obtained at www.ebi.ac.uk / Tools / psa.

[0060] As used herein, the term "sequencing" refers to the process used to determine the sequence (eg, the identity and order of monomeric units) of a biological molecule, eg, a nucleic acid such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturation temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, DNA nanoball sequencing (DNBSEQ), composite probe anchor polymerase sequencing (cPAS), and combinations thereof. In some embodiments, sequencing can be performed by a genetic analyzer, such as a genetic analyzer commercially available from Illumina, Inc., Pacific Biosciences, Inc., Applied Biosystems / Thermo Fisher Scientific, or Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd., etc. For example, BGI DNBseq sequencing platforms such as BGISEQ-500, BGISEQ-50, MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-T20X2, or Illumina's HiSeq2000, HiSeq2500, HiSeq4000, HiSeqX10, NovaSeq6000, etc.

[0061] In this article, the term "HR value (Hazard Ratio)" is an indicator used to compare the difference in the risk of certain events between two groups of people. An HR value greater than 1 means that one group has a higher risk of occurrence, and a value less than 1 means that the risk is lower. An HR value equal to 1 means that the risk of an event in the two groups is equal.

[0062] As used herein, the term "sensitivity" refers to the proportion of samples that yield true positive results using an assay compared to known positive samples. Sensitivity is typically calculated as the number of true positives identified by the assay divided by the sum of the number of true positives and false negatives determined by the assay on known positive samples.

[0063] Similarly, the term "specificity" refers to the ratio of the number of true negatives determined by the assay divided by the sum of the number of true negatives and false positives determined by the assay performed on known negative samples.

[0064] In this article, the term "sequencing data" generally refers to the data of short sequences obtained after sequencing. For example, sequencing data includes the base sequence of the sequencing short sequence (sequencing read length), the number of sequencing read lengths, etc.

[0065] In this article, the term "sequencing depth" or "depth" generally refers to the number of times a specific region (e.g., a specific gene, a specific interval, a specific base) is detected. Sequencing depth can refer to a sequence of bases detected by sequencing. For example, by aligning the sequencing depth to the human reference genome and optionally removing duplicates, the number of sequencing reads at a specific gene, a specific interval, or a specific base position can be determined and counted as the sequencing depth.

[0066] In this article, the term "tumor burden" can refer to the number of overall or local cancer cells, the size of the tumor, or the amount of cancer lesions in the body. The larger the tumor burden, the more overall or local cancer lesions, the larger the size of the tumor, and the more cancer cells. The term "systemic tumor burden" used in the present disclosure refers to the total number of cancer cells in the patient's body, the sum of the size of the tumor, or the total amount of cancer lesions in the body. In some embodiments, the systemic tumor burden can be obtained by detecting the type and number of mutations of ctDNA released by the patient's cancer cells, tumors, or cancer lesions in the body fluid sample. In some embodiments, the patient includes a patient with triple-negative breast cancer or a patient who has suffered from triple-negative breast cancer. In some embodiments, the body fluid sample is such as whole blood, serum, plasma, lymph fluid, and their processed materials.

[0067] In this article, the term "uniformity" refers to a measure of the uniformity of the sequencing depth of the sequenced reads in the target region corresponding to the probe set. In some embodiments, the ratio of bases with ≥20% (0.2 times the average depth) of the average depth to the total number of bases in the target region is used to characterize the uniformity. The depth of all bases in the target region of the captured sequencing data conforms to the Poisson distribution (approximately normal distribution). For data with good uniformity, the depth of most regions is close to the average sequencing depth, and there will be very few bases with an average depth of <20%, that is, the proportion of bases with an average depth of ≥20% (coverage) will be relatively high; while for data with poor uniformity, the base depth distribution will be more discrete, and a very wide peak will be presented. There are more bases with an average depth of <20%, that is, the proportion of bases with an average depth of ≥20% (coverage) will be low. Therefore, the proportion of bases with an average depth of ≥20% is used to evaluate the uniformity of the probe group capture data. In some embodiments, the ratio of bases with ≥30% (0.3 times the average depth), ≥40% (0.4 times the average depth), or ≥50% (0.5 times the average depth) of the average depth to the total number of bases in the target region is also used to characterize uniformity.

[0068] In this article, the term "chromosome structural variation (SV)" refers to large-fragment insertion (INS), deletion (DEL), inversion (INV), translocation (Translocation), duplication (DUP) and other types of variation that occur in the genome. In some embodiments, chromosomal variation can cause gene fusion, which is the process in which all or part of the sequences of two or more genes are fused to form a new gene.

[0069] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail in conjunction with the following examples. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. The actual scope of protection of the present disclosure is set forth in the claims. In the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following examples are not mentioned in terms of their sources, and are all commercially available on the market or obtained by conventional methods known to those skilled in the art.

[0070] Example

[0071] Example 1: TNBC-specific probe set target screening and probe sequences

[0072] Based on the test results of 488 clinical TNBC samples, the inventors screened out the high-frequency mutation genes and regions of TNBC as shown in Table 1.

[0073] Table 1 Combination of cancer markers for TNBC detection

[0074] Gene area TP53 Exons 3-10 PTEN Exons 1,3-8 RB1 Exons 6, 14-15, 20, 24 PIK3CA Exons 2, 8, 10, 21 PIK3R1 Exons 11,13 AKT1 Exon 3 ERBB2 Exon 20 NOTCH1 Exon 27

[0075] A TNBC-specific probe set was designed based on the above TNBC high-frequency mutation genes and regions. The probe sequences are shown in Table 2.

[0076] Table 2. TNBC-specific probe sets

[0077]

[0078]

[0079]

[0080] Example 2: Evaluation of capture efficiency and uniformity of TNBC-specific probe sets for clinical samples

[0081] The performance of the TNBC-specific probe set was tested using cfDNA samples from clinical TNBC patients to evaluate the capture efficiency and uniformity of the probes.

[0082] 1. Test process:

[0083] ①cfDNA extraction: Whole blood samples were centrifuged at 1,600g and 16,000g to separate plasma and remove cell debris in plasma. TM The plasma cfDNA was extracted using the magnetic bead method using the Cell-free DNA Isolation Kit (Thermo Fisher).

[0084] ②Library construction: The cfDNA was end-repaired and "A" was added using a customized detection kit for human molecular residual disease (MRD) (Gene Plus), and then the pre-capture intermediate library was obtained through the process of adapter connection, purification, pre-capture PCR (Non-C-PCR) and purification. The samples with qualified concentration quality control of the intermediate library were subsequently subjected to hybridization elution.

[0085] ③ Hybridization capture: The library that passed the concentration quality control was pooled, evaporated, probe hybridized, eluted, PCR of the eluted product, and purified using the customized detection kit for human molecular residual disease (MRD) (Gene Plus) to obtain the common library after hybridization. The common library was sequenced after passing the concentration and fragment distribution quality control.

[0086] ④ Sequencing and FASTQ data output: Double-end (PE100) sequencing was performed using the Gene+Seq2000 sequencer, and the data was split into fastq files after being downloaded.

[0087] ⑤Data alignment and bam file generation: Before data alignment, first use Realseq2 software (version: 1.1.6):

[0088] (1) Remove the UMI at the end of the read and save it in the read name;

[0089] (2) Filter low-quality reads. The obtained fastq was aligned to the human reference genome (version: hs37d5) using BWA (version: 0.7.15-r1140) software to generate a bam file of the initial alignment result. Then, Realseq2 software (version: 201808) was used to cluster and correct the PCR duplicate reads in the initial alignment result file with the help of UMI. The indel regions within 50 bp of both ends of the detection chip were re-aligned based on the common indel mutations in the 1000-person database and dbSNP (version: 138) database. The base quality values ​​within 50 bp of both ends of the detection chip were re-corrected based on the information in the 1000-person database, dbSNP (version: 138) database, and COSMIC database.

[0090] ⑥Sample quality control:

[0091] (1) Sample pairing error: The bioinformatics process determines the sample pairing by calculating the consistency between the homozygous sites in the control sample and the tumor sample within a range of 50 bp extending from both ends of the chip interval.

[0092] (2) Sample contamination: The Calculate Contamination module in the GATK (version: 4.1.4) software was used to combine the bam file information of the control and tumor samples. The reads information supporting the reference bases in the homozygous sites in the test samples were read and counted to evaluate the cross-contamination of the samples.

[0093] ⑦ Mutation calling: This product detects single nucleotide mutations (SNVs) and insertion / deletion mutations (Indels), as well as SVs, within a range of 50 bp extending from both ends of the chip capture interval. The mutations obtained in the above detection process are annotated through the following databases:

[0094] (1) Gene Annotation Database (version: NCBI release 104);

[0095] (2) dbSNP database (version: 147);

[0096] (3) tgp database (version: phase3);

[0097] (4)COSMIC database (version: v80);

[0098] (5) ExAC database (version: 0.3.1);

[0099] (6)clinvar database (version: 20200701).

[0100] The mutations obtained in the above steps are filtered and credible mutations are retained.

[0101] In this example, cfDNA samples from 10 clinical TNBC patients were used to test the probe capture efficiency, uniformity and probe depth coefficient. The probe used in step ③ hybridization capture is a personalized probe set for detecting TNBC (as shown in Table 3, 10 patients are numbered T1 to T10, hereinafter referred to as TNBC personalized probe set) used alone, or a TNBC specific probe set shown in Table 2 (hereinafter referred to as TNBC specific probe set) used alone, or both used in combination.

[0102] Table 3. Personalized probe set sequences for TNBC in 10 patients

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] 2. Probe capture efficiency

[0109] The box plot of the probe capture efficiency test results of clinical samples is shown in Figure 1As shown, where capture efficiency = sequencing data volume of probe target region / total sequencing data volume × 100%. The results showed that the capture efficiency of using only TNBC personalized probe set ranged from 19.1% to 37.4%, with a median capture efficiency of 28.9%, the capture efficiency of TNBC specific probe set ranged from 41.9% to 55.0%, with a median capture efficiency of 51.3%, and the capture efficiency of superposition of TNBC personalized probe set and TNBC specific probe set ranged from 43.1% to 57.7%, with a median capture efficiency of 52.8%. The above results show that compared with TNBC personalized probe set, the capture efficiency of TNBC specific probe set is significantly increased, and the superposition of TNBC personalized probe set and TNBC specific probe set can significantly improve the capture efficiency.

[0110] 3. Probe uniformity

[0111] The results of the probe uniformity test using the TNBC-specific probe set alone are as follows Figure 2 and Figure 3 As shown in the figure, the base ratio of 10 cfDNA samples with ≥0.2 times the average depth is 100%, and the base ratio of 10 cfDNA samples with ≥0.5 times the average depth is 95.8% at the minimum, 99.5% at the maximum, and 97.8% at the median. The above results show that the TNBC-specific probe set has excellent uniformity and meets the needs of clinical testing. Among them, the base ratio of ≥0.2 times the average depth = (the number of bases covered by the probe at ≥0.2 times the average depth / the total number of bases covered by the probe) × 100%, and the base ratio of ≥0.5 times the average depth = (the number of bases covered by the probe at ≥0.5 times the average depth / the total number of bases covered by the probe) × 100%.

[0112] 4. Probe Depth Factor

[0113] The box plot of the depth coefficient results of the TNBC-specific probe set is shown in Figure 4 The test results show that in 10 samples, the median value of the probe depth coefficient of each TNBC-specific probe set covering the region or exon is between 1.02-1.12, indicating that the TNBC-specific probe set has a relatively consistent depth and the depth coefficient is stable in different samples. The probe depth coefficient = the average depth of bases covered by a probe / the average depth of bases covered by the probe set.

[0114] Example 3: Coverage of TNBC-specific probe sets for TNBC patients

[0115] The tissue sample data of 488 clinical TNBC patients in Example 1 and the tissue sample data of 121 TNBC patients with mutations (Koboldt, Daniel C., et al. Nature 490, 61-70 (2012)) were used to evaluate the coverage of TNBC patient tissues by the TNBC-specific probe set. The coverage was calculated as follows: the number of samples with ≥1 mutation covered by the TNBC-specific probe set / the total number of samples. The evaluation results are shown in FIG. Figure 5 As shown, the coverage of the TNBC-specific probe set for the above 488 clinical TNBC patients' tissue samples and the above 121 TNBC patients' tissue samples was 96.3% (470 / 488) and 88.4% (107 / 121), respectively. Therefore, the TNBC-specific probe set has excellent coverage for the TNBC population.

[0116] Example 4: Clinical performance of TNBC-specific probe set for assessing systemic tumor burden in TNBC patients

[0117] Another 34 clinical TNBC patients were included to evaluate the TNBC-specific probe set to detect ctDNA status and evaluate the clinical performance of systemic tumor burden. First, a comprehensive genomic analysis was performed on the formalin fixed paraffin embedded (FFPE) tissue samples of the 34 patients to identify the mutation profile of each patient. Then, 2-20 mutations were selected based on the patient's mutation profile to design a patient-specific personalized probe set, as shown in Table 4. The personalized probe was superimposed with the TNBC-specific probe set for ctDNA detection. The patient was followed up every 3-6 months after surgery, and 20 mL of peripheral blood was collected for ctDNA detection. A total of 62 cfDNA samples from 34 clinical TNBC patients were tested.

[0118] Table 4. Mutations detected in 34 clinical TNBC patient tissues and patient-specific personalized probes

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] In Table 4, "c." indicates a mutation in the coding DNA sequence, "sv" indicates gene fusion caused by chromosome structural variation (Structure Variation), "del" indicates deletion, ins indicates insertion (insertion), "_" indicates the range in between, fs indicates a frameshift mutation (frameshift mutation), * indicates a stop codon, "p." indicates a mutation at the protein level, and "p.0?" indicates that this mutation may cause a change in the translation start site of the protein, which may affect the normal synthesis of the protein, but the specific impact is unclear.

[0131] No personalized probes were designed for the mutations covered by the TNBC-specific probe set in Table 4. The three mutations detected in the P1 patient tissue were all covered by the TNBC-specific probe set, so no personalized probes were designed.

[0132] First, we evaluated the coverage of TNBC-specific probe sets for mutations detected in 34 patient tissues. Figure 6 As shown, 94% (32 / 34) of the patients could be covered by at least one mutation by the TNBC-specific probe set, indicating that the TNBC-specific probe set had good coverage for the TNBC population.

[0133] Then, the accuracy of the TNBC-specific probe set in detecting the ctDNA status of TNBC patients was evaluated. Figure 7 and Figure 8 As shown, among the 14 patients who relapsed, 13 were identified as ctDNA positive (reference CN202310889084.8 for the judgment method), with a sensitivity of 92.9% (13 / 14), and all 20 patients who did not relapse were identified as ctDNA negative, with a specificity of 100% (20 / 20) ( Figure 7 ). Patients with ctDNA positive had a higher risk of recurrence than those with negative ctDNA (HR=42.1, P<0.0001) ( Figure 8). As shown in Table 5, among the 13 patients with ctDNA positive, 11 patients could predict recurrence earlier than imaging. According to statistics, ctDNA detection could predict recurrence 3.7 months earlier than imaging. The above results show that the TNBC-specific probe set provided by the present disclosure can be used for TNBC ctDNA monitoring, has high sensitivity and specificity, can identify patients with a higher risk of recurrence, and can predict clinical recurrence earlier than imaging.

[0134] Table 5. Statistical results of ctDNA detection in predicting recurrence compared with imaging

[0135]

[0136]

[0137] In addition, among the 13 patients with relapse and ctDNA positive, the mutations detected in 15 plasma samples of 11 patients were covered by the TNBC-specific probe set. The detected mutations are shown in Table 6. The coverage rate of ctDNA-positive patients using only the TNBC-specific probe set was 84.6% (11 / 13), indicating that the TNBC-specific probe set has a high coverage rate for TNBC ctDNA-positive patients. In this study cohort, if only the TNBC probe set was used for detection, 11 relapsed patients could be identified as ctDNA-positive, with a sensitivity of 78.6% (11 / 14), and all 20 non-relapsed patients were identified as ctDNA-negative, with a specificity of 100% (20 / 20), indicating that the use of only the TNBC probe set to predict the recurrence of TNBC patients is still highly accurate.

[0138] Table 6. Coverage results of TNBC-specific probe sets for ctDNA-positive patients

[0139]

[0140] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A gene marker for detecting triple-negative breast cancer, the marker comprising any one or more of the following genes: TP53, PTEN, RB1, PIK3CA, PIK3R1, AKT1, ERBB2 or NOTCH1.

2. The gene marker according to claim 1, characterized in that The gene markers include any one or more of the following regions: exons 3-10 of TP53; exons 1 and 3-8 of PTEN; exons 6, 14-15, 20 and 24 of RB1; exons 2, 8, 10 and 21 of PIK3CA; exons 11 and 13 of PIK3R1; exon 3 of AKT1; exon 20 of ERBB2; and exon 27 of NOTCH1; Preferably, the gene marker is used to detect systemic tumor burden of triple-negative breast cancer.

3. A probe set for detecting triple-negative breast cancer, wherein the probe set is used to detect the gene marker according to claim 1 or 2.

4. The probe set according to claim 3, characterized in that: The probe set has a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1 to 68, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; Preferably, the probe set is used to detect systemic tumor burden in triple-negative breast cancer.

5. The probe set according to claim 3, characterized in that: The probe set includes any one or more combinations of the following: A probe for detecting the PIK3CA gene: having a nucleotide sequence as shown in SEQ ID NO: 1 to 11, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting the PIK3R1 gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 12 to 16, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting NOTCH1 gene: having a nucleotide sequence as shown in SEQ ID NO: 17 and / or 18, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting the PTEN gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 19 to 34, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting the RB1 gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 35 to 47, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting the AKT1 gene: having a nucleotide sequence as shown in SEQ ID NO: 48 and / or 49 or more, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting the TP53 gene: having a nucleotide sequence as shown in any one or more of SEQ ID NOs: 50 to 66, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; and / or A probe for detecting the ERBB2 gene: having a nucleotide sequence as shown in SEQ ID NO: 67 and / or 68 or more, or a nucleotide sequence that is reverse complementary thereto or has at least 85% sequence identity thereto; Optionally, the probe set further comprises a personalized probe set.

6. A kit for detecting triple-negative breast cancer, comprising the probe set according to any one of claims 3 to 5.

7. A method for detecting triple-negative breast cancer using the probe set according to any one of claims 3 to 5 and / or the kit according to claim 6, the method comprising using the probe set and / or the kit to detect triple-negative breast cancer.

8. Use of the gene marker according to claim 1 or 2 and the probe group according to claims 3 to 5 in preparing a kit for detecting triple-negative breast cancer.

9. The use according to claim 8, characterized in that: The kit is used for one or more of triple-negative breast early screening, diagnosis and recurrence monitoring. Preferably, the gene marker is used to detect systemic tumor burden of triple-negative breast cancer.

10. The use according to claim 8, characterized in that: The kit is used for detecting cell, tissue or body fluid samples; Preferably, the cell sample comprises a triple-negative breast cancer cell suspension sample and / or circulating tumor cells; and / or The body fluid sample includes: whole blood, serum, plasma, lymph fluid and processed products thereof; and / or The tissue samples include triple-negative breast cancer tissues, paraffin sections and processed materials thereof.

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