A probe set for assessing systemic tumor burden of pancreatic cancer and use thereof

Abstract

The application discloses a probe set for evaluating systemic tumor burden of pancreatic cancer and application thereof, wherein the probe set comprises probes for detecting gene markers, the gene markers comprising KRAS, TP53, CDKN2A, SMAD4, RNF43, TGFBR2, PIK3CA, BRAF, GNAS and CTNNB1, covering high-frequency and drug-related sites of pancreatic cancer, and the systemic tumor burden of a pancreatic cancer patient is monitored in real time and dynamically by performing targeted capture on plasma ctDNA alone or in combination with a personalized probe set.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to a probe set for assessing the systemic tumor burden of pancreatic cancer and its application. Background Technology

[0002] Pancreatic cancer is one of the most malignant gastrointestinal tumors, characterized by low early detection rates, high invasiveness, poor prognosis, high recurrence and metastasis rates, and strong drug resistance. Most pancreatic cancer patients are diagnosed at a locally advanced stage or with distant metastasis, thus losing the opportunity for radical surgery. Accurate assessment of the systemic tumor burden in pancreatic cancer patients is crucial for treatment selection, prognostic evaluation, and efficacy monitoring.

[0003] However, existing technologies still have significant limitations in assessing the systemic tumor burden of pancreatic cancer patients. Currently widely used imaging modalities (CT or MRI) lack sensitivity in detecting micrometastases and exhibit delays in detecting tumor shrinkage, failing to reliably and in real-time reflect the actual treatment efficacy. CA19-9 is currently the most commonly used serum tumor marker for pancreatic cancer, but it suffers from insufficient sensitivity and specificity: approximately 20% of pancreatic cancer patients do not have elevated CA19-9 levels, and inflammation or other malignancies can also cause elevated levels. Circulating tumor DNA (ctDNA), as a non-invasive biomarker, has demonstrated important applications in precision oncology. By monitoring the ctDNA status of pancreatic cancer patients before, during, and after treatment, the systemic tumor burden can be accurately assessed, providing guidance for the scientific selection and timely adjustment of treatment plans.

[0004] However, there is currently a lack of probe sets specifically designed for assessing the systemic tumor burden in pancreatic cancer. Existing probe sets either lack versatility or fail to specifically and efficiently cover key gene loci in pancreatic cancer. Summary of the Invention

[0005] To address the above issues, one embodiment of this application provides a probe set specifically designed for assessing the systemic tumor burden of pancreatic cancer. This probe set covers high-frequency and drug-related sites in pancreatic cancer and can be used alone or in combination with a personalized probe set to target and capture plasma ctDNA. This allows for real-time and dynamic monitoring of the systemic tumor burden in pancreatic cancer patients, providing strong support for the planning of personalized treatment plans, efficacy monitoring, and prognosis assessment, and helping to overcome the challenges in the diagnosis and treatment of pancreatic cancer.

[0006] The technical solution of this application includes:

[0007] In a first aspect, this application provides a gene biomarker for assessing the systemic tumor burden of pancreatic cancer, the gene biomarker including KRAS, TP53, CDKN2A, SMAD4, RNF43, TGFBR2, PIK3CA, BRAF, GNAS and CTNNB1.

[0008] In one specific embodiment, the gene marker includes the following gene regions: exons 2 and 3 of the KRAS gene, exons 2-11 of the TP53 gene, exons 1 and 2 of the CDKN2A gene, exons 2, 3, 6, 9-12 of the SMAD4 gene, exons 2 and 4 of the RNF43 gene, exons 4 and 7 of the TGFBR2 gene, exon 10 of the PIK3CA gene, exons 12 and 15 of the BRAF gene, exon 8 of the GNAS gene, and exon 3 of the CTNNB1 gene.

[0009] Secondly, this application provides a probe set including probes for detecting the genetic marker.

[0010] In one specific implementation, the probe set is used to assess the systemic tumor burden of pancreatic cancer.

[0011] In one specific embodiment, the probe set includes nucleotide sequences as shown in SEQ ID NO:1-65, or reverse complementary to them, or nucleotide sequences having at least 85%, 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%, or at least 99% sequence identity with them.

[0012] In one specific embodiment, the probe group includes the following combination:

[0013] Probes for detecting the TGFBR2 gene: having nucleotide sequences as shown in SEQ ID NO:1-5, or reverse complementary to them, or having nucleotide sequences with at least 85%, 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%, or at least 99% sequence identity with them;

[0014] The probe for detecting the CTNNB1 gene has a nucleotide sequence as shown in SEQ ID NO: 6-8, or is reverse complementary to it, or has a nucleotide sequence that is at least 85%, 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%, or at least 99% sequence identity with it;

[0015] The probe for detecting the PIK3CA gene has a nucleotide sequence as shown in SEQ ID NO:9-10, or is reverse complementary to it, or has a nucleotide sequence that is at least 85%, 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%, or at least 99% sequence identity with it;

[0016] Probes for detecting the BRAF gene: having nucleotide sequences as shown in SEQ ID NO:11-14, or reverse complementary to them, or having nucleotide sequences with at least 85%, 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%, or at least 99% sequence identity with them;

[0017] The probe for detecting the CDKN2A gene has a nucleotide sequence as shown in SEQ ID NO:15-19, or is reverse complementary to it, or has a nucleotide sequence that is at least 85%, 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%, or at least 99% sequence identity with it;

[0018] Probes for detecting the KRAS gene: having nucleotide sequences as shown in SEQ ID NO:20-23, or reverse complementary to them, or having nucleotide sequences with at least 85%, 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%, or at least 99% sequence identity with them;

[0019] The probe for detecting the TP53 gene has a nucleotide sequence as shown in SEQ ID NO:24-42, or is reverse complementary to it, or has a nucleotide sequence that is at least 85%, 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%, or at least 99% sequence identity with it;

[0020] The probe for detecting the RNF43 gene has a nucleotide sequence as shown in SEQ ID NO:43-46, or is reverse complementary to it, or has a nucleotide sequence that is at least 85%, 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%, or at least 99% sequence identity with it;

[0021] Probes for detecting the SMAD4 gene: having nucleotide sequences as shown in SEQ ID NO:47-64, or reverse complementary to them, or having nucleotide sequences with at least 85%, 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%, or at least 99% sequence identity with them;

[0022] The probe for detecting the GNAS gene has a nucleotide sequence as shown in SEQ ID NO:65, or is reverse complementary to it, or has a nucleotide sequence that is at least 85%, 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%, or at least 99% sequence identity with it.

[0023] In one specific embodiment, the probe set further includes a personalized probe set.

[0024] It should be noted that personalized probes are probes specifically designed for different patients. In one embodiment, the personalized probe set includes probes with nucleotide sequences as shown in SEQ ID NO:66-86 or sequences as shown in SEQ ID NO:87-313. It is understood that the personalized probe set can also be other pancreatic cancer personalized probes known to those skilled in the art.

[0025] Thirdly, this application provides a product for assessing the systemic tumor burden of pancreatic cancer, the product comprising the probe set.

[0026] In one specific embodiment, the product further includes DNA extraction reagents (e.g., cfDNA extraction reagents) and / or other reagents required for ctDNA detection, such as library construction reagents described in the sequencing method and hybridization capture reagents required for the sequencing method.

[0027] In one specific embodiment, the product is detected using a high-throughput sequencing method. In one specific embodiment, the probe set or product is suitable for next-generation sequencing (NGS) based on targeted capture. Optionally, the sequencing platform includes, but is not limited to, Geneplus, BGI Genomics, and Illumina.

[0028] In one specific embodiment, the test sample of the kit includes cell, tissue, or body fluid samples. Optionally, the cell sample includes one or more of pancreatic cancer cell suspension samples and circulating tumor cells. Optionally, the body fluid sample includes one or more of whole blood, serum, and plasma. Optionally, the tissue sample includes one or more of pancreatic cancer tissue and paraffin sections.

[0029] Fourthly, this application provides the use of the probe set in the preparation of products for assessing the systemic tumor burden of pancreatic cancer.

[0030] Fifthly, this application provides the use of the probe set in the preparation of products for prognostic assessment of pancreatic cancer.

[0031] Sixthly, this application provides the use of the probe set in the preparation of products for evaluating the therapeutic effects of pancreatic cancer.

[0032] Seventhly, this application provides the use of the probe set in the preparation of products for monitoring pancreatic cancer recurrence.

[0033] Eighthly, this application provides a method comprising any one of the following:

[0034] (1) A method for assessing or detecting the systemic tumor burden of pancreatic cancer;

[0035] (2) A method for prognostic assessment of pancreatic cancer;

[0036] (3) A method for evaluating the therapeutic effect of pancreatic cancer;

[0037] (4) A method for monitoring pancreatic cancer recurrence;

[0038] (5) A method for detecting ctDNA.

[0039] In one specific embodiment, the method includes detection or evaluation using the probe set. By detecting ctDNA in subjects using the pancreatic cancer-specific probe set of this application, the systemic tumor burden changes in pancreatic cancer patients can be assessed, providing real-time dynamic feedback on treatment efficacy, significantly differentiating patient prognosis (P<0.01), and predicting whether pancreatic cancer progression will occur.

[0040] In one specific implementation, compared to the control, zero ctDNA, decreased ctDNA, and increased ctDNA represent extremely low or completely zero tumor burden, reduced tumor burden, and increased tumor burden, respectively. Patients with zero ctDNA have the longest progression-free survival, followed by those with decreased ctDNA, and those with increased ctDNA have the worst. Patients with zero ctDNA have the lowest risk of progression, followed by those with decreased ctDNA, and those with increased ctDNA have the highest risk of progression.

[0041] In one specific implementation, the control is baseline blood.

[0042] Compared with the prior art, the beneficial effects of this application are as follows:

[0043] The probe set provided in this application covers high-frequency and drug-related mutations and regions in pancreatic cancer. It can be used alone or in combination with personalized probes for ctDNA detection to assess the systemic tumor burden in pancreatic cancer patients. Clinical sample validation has shown that the probe set of this application exhibits excellent capture efficiency and depth coefficient, good probe homogeneity, and meets clinical testing requirements.

[0044] In addition, the pancreatic cancer-specific probe set of this application can monitor tumor evolution and new mutations, and can overcome the spatiotemporal heterogeneity of tumors to a certain extent. When used in combination with personalized probe sets, it can also significantly improve the capture efficiency.

[0045] The probe set in this application has excellent coverage of the pancreatic cancer population, with coverage rates of 95.1% (940 / 988) and 93.9% (713 / 759) for the clinical sample training set and validation set, respectively.

[0046] Clinical cohort validation has shown that the probe set proposed in this application has excellent clinical performance in assessing the systemic tumor burden of pancreatic cancer patients: the pancreatic cancer-specific probe set alone demonstrates excellent sensitivity for baseline blood tumor burden assessment, while the combined use of the pancreatic cancer-specific probe set and the personalized probe set can further improve the detection sensitivity; in addition, by monitoring the changes in the systemic tumor burden of pancreatic cancer patients before and after treatment, the treatment effect can be monitored dynamically in real time, and the trend of changes in tumor burden is significantly correlated with the patient's prognosis. Attached Figure Description

[0047] Figure 1 Results of the capture efficiency test of the pancreatic cancer-specific probe group;

[0048] Figure 2 Results of 0.2 times average depth base coverage test for pancreatic cancer-specific probe group;

[0049] Figure 3 Results of 0.5 times the average depth base coverage test of pancreatic cancer-specific probe group;

[0050] Figure 4 Results of depth coefficient testing for pancreatic cancer-specific probe groups;

[0051] Figure 5 The coverage of pancreatic cancer-specific probe groups in the pancreatic cancer population;

[0052] Figure 6 A pancreatic cancer-specific probe set was used to assess the sensitivity of baseline blood tumor burden.

[0053] Figure 7 The pancreatic cancer-specific probe set was used to assess changes in ctDNA levels in baseline blood and initial efficacy evaluation blood.

[0054] Figure 8 Survival analysis results of pancreatic cancer-specific probe group used to evaluate the efficacy of treatment in pancreatic cancer patients. Detailed Implementation

[0055] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0056] 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 is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0057] In this document, the term "tumor burden" can refer to the number of cancer cells, the size of a tumor, or the amount of cancerous lesions in the body, whether generalized or localized. A higher tumor burden indicates a greater number of cancerous lesions, whether generalized or localized, and a larger tumor size with a greater number of cancer cells. As used in this disclosure, the term "systemic tumor burden" refers to the total number of cancer cells, the total size of tumors, or the total amount of cancerous lesions in the body of a patient. In some embodiments, systemic tumor burden can be obtained by detecting the type and quantity of mutations in ctDNA released from cancer cells, tumors, or cancerous lesions in a body fluid sample. In some embodiments, the patient includes a patient with pancreatic cancer.

[0058] In this paper, the term "cfDNA" refers to circulating cell-free DNA, including DNA molecules that are naturally present in the subject in an extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). Although cfDNA is originally present in one or more cells of a large, complex biological organism (e.g., a mammal), cfDNA undergoes release from the cell 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.

[0059] In this article, the term "ctDNA" refers to circulating tumor DNA, which is a DNA fragment derived from apoptosis, necrosis, or secretion of tumor cells. It contains the same gene variations and epigenetic modifications as tumor tissue DNA, such as point mutations, gene rearrangements, fusions, copy number variations, and methylation modifications. ctDNA detection can be applied to various aspects of cancer, including early screening, diagnosis and staging, guiding targeted drug therapy, efficacy evaluation, and recurrence monitoring.

[0060] In this article, the term "baseline blood" refers to a blood sample collected before treatment begins.

[0061] This application presents a pancreatic cancer-specific ctDNA detection probe set (Table 1) designed based on a clinical cohort. It covers high-frequency and drug-related mutations and regions in the pancreatic cancer population and can be used in conjunction with a personalized probe set for targeted capture and detection of plasma ctDNA, assessing the systemic tumor burden in pancreatic cancer patients. This probe set achieves good coverage and capture of the target region, obtaining sufficient read information. Because this probe set covers high-frequency and drug-related mutations in the pancreatic cancer population, its combination with the personalized pancreatic cancer probe set for ctDNA detection can, to some extent, overcome the temporal and spatial heterogeneity of tumors. Simultaneously, it stabilizes the experimental system and improves capture efficiency. This probe set covers 10 high-frequency and drug-related genes in pancreatic cancer: KRAS, TP53, CDKN2A, SMAD4, RNF43, TGFBR2, PIK3CA, BRAF, GNAS, and CTNNB1. In some embodiments, each probe is 120 bp in length, and the 5' end of the probe is biotin-labeled.

[0062] Those skilled in the art will understand that the probe set of this application can be used alone or in combination with any other conventional probe set. The pancreatic cancer-specific probe set covers high-frequency and drug-related mutations and regions in pancreatic cancer. In some specific embodiments, it is used in conjunction with a personalized probe set for ctDNA detection, which can overcome the temporal and spatial heterogeneity of tumors to a certain extent and significantly improve the capture efficiency of the personalized probe set. This demonstrates excellent clinical performance for monitoring the ctDNA status of pancreatic cancer patients.

[0063] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, 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.

[0064] 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.

[0065] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0066] Example 1: Target screening and probe sequence for pancreatic cancer-specific probe sets

[0067] Using the test results of 988 clinical pancreatic cancer samples, high-frequency mutated genes and regions of pancreatic cancer were screened according to the following rules.

[0068] The screening rules for high-frequency mutated genes and regions in pancreatic cancer are as follows:

[0069] (1) Obtain the mutation information of each sample from the pancreatic cancer dataset, and annotate the corresponding transcripts and exons based on the mutation information;

[0070] (2) Calculate the mutation density coefficient of the probe covering the target exon:

[0071] Target exon probe mutation density coefficient = (a / m) / length

[0072] a: The total number of mutations in the target exon in the population.

[0073] m: The total number of mutations in the population.

[0074] length: The length of the probe covering the target exon, estimated based on the length of the exon.

[0075] Exon length ≤ 40 bp, probe length: 120 bp, exon extension length ≥ 40 bp.

[0076] Exon > 40 bp, probe length: exon length + 80 bp, with each end of the exon extending outward by 40 bp;

[0077] (3) Calculation of the incidence rate of exons carrying mutations:

[0078] Prevalence of exons carrying mutations = b / n

[0079] b: Number of patients carrying the mutation in the target exon

[0080] n: Total number of patients;

[0081] (4) First, sort the target exon probe mutation density coefficient in descending order, and then sort them in descending order according to the population incidence of the exon carrying the mutation. Genes / regions ranked earlier have higher priority.

[0082] (5) Select the high-priority high-frequency mutations and regions according to the sorting, and supplement the uncovered drug-related sites on this basis. The expected probe group region is about 5 kb.

[0083] The high-frequency and drug-related mutated genes and regions identified in the screening for pancreatic cancer are shown in Table 1:

[0084] Table 1

[0085] Based on the above genes and regions, a pancreatic cancer-specific probe set was designed, and the probe sequences are shown in Table 2:

[0086] Table 2

[0087]

[0088]

[0089]

[0090] Example 2: Evaluation of the capture efficiency and homogeneity of pancreatic cancer-specific probe sets on clinical samples

[0091] The performance of the pancreatic cancer-specific probe set was tested using clinical cfDNA samples to evaluate the probe capture efficiency and uniformity.

[0092] (1) Experimental procedure:

[0093] ① cfDNA extraction: After the whole blood sample was centrifuged at 1,600 g and 16,000 g to separate plasma and remove cell debris from the plasma, plasma cfDNA was extracted using the MagMAX™ Cell-Free DNA Separation Kit (Thermo Fisher) using magnetic beads.

[0094] ② Library Construction: The cfDNA was end-repaired and an "A" was added using a customized human molecular residual lesion (MRD) detection kit (GenePlus). Then, the DNA underwent adapter ligation, purification, pre-capture PCR (Non-C-PCR), and further purification to obtain a pre-capture intermediate library. Samples with acceptable intermediate library concentration were then subjected to subsequent hybridization and elution.

[0095] ③ Hybridization Capture: Using a customized human molecular residual lesion (MRD) detection kit (GenePlus), libraries that passed concentration quality control were subjected to pooling, evaporation, hybridization with mixed probes, elution, PCR of the elution products, and purification to obtain a hybridized general library. The general library was then sequenced after passing concentration and fragment distribution quality control.

[0096] ④ Sequencing and FASTQ data output: Paired-end (PE100) sequencing was performed using the Gene+Seq2000 sequencer, and the data was split into fastq files after being processed.

[0097] ⑤ Data alignment and BAM file generation: Before data alignment, Realseq2 software (version: 3.0.6) was used to: (1) remove UMIs from the ends of reads and save them in the read name; (2) filter low-quality reads. The obtained fastq was aligned to the human reference genome (version: hs37d5) using BWA software (version: 2.2.1a) to generate the initial alignment result BAM file. Then, Realseq2 software was used to perform clustering and error correction on the PCR repeat reads in the initial alignment result file with the help of UMIs. The indel regions within a 50bp range extending from both ends of the detection chip were re-aligned using common indel mutations from the 1000-person database and the dbSNP (version: 147) database. The base quality values ​​within a 50bp range extending from both ends of the detection chip were re-corrected using information from the 1000-person database, the dbSNP database, and the COSMIC database (version: v80).

[0098] ⑥ Sample quality control: (1) Sample pairing error: The bioinformatics workflow judges the sample pairing status by calculating the consistency between homozygous sites in the control sample within a 50bp range extending from both ends of the chip interval and those in the tumor sample. (2) Sample contamination: The cross-contamination status of the samples is assessed by combining the BAM file information of the control and tumor samples with the Calculate Contamination module in the GATK (version: 4.1.4) software, and by reading and statistically analyzing the reads information of supporting reference bases in the homozygous sites in the test samples.

[0099] ⑦ Mutation calling: This product detects single nucleotide variants (SNVs) and insertion / deletion mutations (Indels) within a 50 bp range extending from both ends of the chip capture region, as well as SVs. The mutations obtained in the above detection process are annotated using the following databases: (1) Gene Annotation Database (version: NCBI release 104); (2) dbSNP database (version: 147); (3) tgp database (version: phase3); (4) COSMIC database (version: v80); (5) ExAC database (version: 0.3.1); (6) clinvar database (version: 20240312). The mutations obtained in the above steps are filtered, and reliable mutations are retained.

[0100] In this embodiment, cfDNA samples from three clinical patients were used to test probe capture efficiency, uniformity, and probe depth coefficient. The probes used in step ③, hybridization capture, were either a personalized probe set for detecting pancreatic cancer (hereinafter referred to as the pancreatic cancer personalized probe set) used alone, or a pancreatic cancer-specific probe set provided in this application (hereinafter referred to as the pancreatic cancer-specific probe set) used alone, or a combination of both.

[0101] The personalized probe sequences of the three pancreatic cancer cases used in the test are shown in Table 3:

[0102] Table 3

[0103]

[0104] (2) Probe capture efficiency

[0105] Probe capture efficiency test results of clinical samples Figure 1 As shown in the results, the capture efficiency (capture efficiency = number of sequencing bases in the target region of the probe / total number of sequencing bases × 100%) using only the pancreatic cancer personalized probe set ranged from 22.6% to 33.2%, with significant fluctuations and a median capture efficiency of 30.6%. The capture efficiency of the pancreatic cancer specific probe set ranged from 57.4% to 58.7%, with a median capture efficiency of 58.5%. The capture efficiency of using both the pancreatic cancer personalized probe set and the pancreatic cancer specific probe set combined ranged from 57.4% to 59.0%, with a median capture efficiency of 58.6%. These results indicate that compared to the pancreatic cancer personalized probe set, the pancreatic cancer specific probe set significantly increased the capture efficiency with less fluctuation. Furthermore, the combined use of the pancreatic cancer personalized probe set and the pancreatic cancer specific probe set significantly improved the capture efficiency and increased its stability.

[0106] (3) Probe uniformity

[0107] Results of probe homogeneity test for pancreatic cancer probe group Figures 2-3 As shown, the 0.2-fold average depth base coverage (number of bases at probe coverage depth reaching 0.2 times the average sequencing depth / total number of bases covered by the probe) and the 0.5-fold average depth base coverage (number of bases at probe coverage depth reaching 0.5 times the average sequencing depth / total number of bases covered by the probe) of the three cfDNA samples were both 100%, indicating that the pancreatic cancer-specific probe set has excellent uniformity and meets the needs of clinical testing.

[0108] (4) Probe depth coefficient

[0109] Depth coefficient of pancreatic cancer-specific probe set (average depth of bases covered by a single probe / average depth of bases covered by the probe set) results Figure 4 As shown in the figure. The test results show that the median values ​​of the probe depth coefficients of the three samples were 1.02, 1.04 and 1.00, respectively, indicating that the pancreatic cancer-specific probe group has a relatively consistent depth and the depth coefficients are stable in different samples.

[0110] Example 3: Coverage of pancreatic cancer-specific probe group for pancreatic cancer patients

[0111] The 988 clinical tissue samples used in the pancreatic cancer-specific probe group target screening (Example 1) were used as the training set, and an additional 759 pancreatic cancer clinical tissue samples were used as the validation set. The coverage of the pancreatic cancer-specific probe group for pancreatic cancer patients was evaluated (number of samples covered by the pancreatic cancer-specific probe group with ≥1 mutation / total number of samples). The evaluation results are as follows: Figure 5 The results showed that the pancreatic cancer-specific probe group achieved coverage of 95.1% (940 / 988) and 93.9% (713 / 759) of the training and validation sets, respectively. Therefore, the pancreatic cancer-specific probe group demonstrated excellent coverage of the pancreatic cancer population.

[0112] Then, the coverage of a probe set comprising 223 genes (hereinafter referred to as the 223-gene probe set, from patent CN112951325B, including 10 genes from the pancreatic cancer-specific probe set of this application) in the validation set of pancreatic cancer patients was evaluated. This probe set was designed for early detection of pancreatic cancer and monitoring of ctDNA MRD. The evaluation results are as follows: Figure 5 The results show that the 223 gene probe group achieved a coverage rate of 96.7% (734 / 759) for pancreatic cancer patients. These results indicate that although the pancreatic cancer-specific probe group of this application covers fewer genes, it can achieve similar patient coverage as the 223 gene probe group, demonstrating unexpected technical effectiveness.

[0113] Example 4: Clinical efficacy of pancreatic cancer-specific probe kits for assessing systemic tumor burden

[0114] This study included 37 patients with advanced pancreatic cancer and 20 healthy individuals to validate the clinical efficacy of a pancreatic cancer-specific probe set for assessing systemic tumor burden. Comprehensive genomic analysis of tumor tissues from the 37 patients identified patient-specific tumor mutation profiles. For each patient, 2-20 mutations were selected to create a customized probe set (the personalized site selection algorithm is described in patent ZL 2023 1 0890251.0), and the personalized probe sequences are shown in Table 4. The personalized probe set was then used in conjunction with the pancreatic cancer-specific probe set to perform targeted capture and ultra-high-depth sequencing of baseline blood ctDNA before treatment in the 37 patients to evaluate the sensitivity of the pancreatic cancer-specific probe set in assessing systemic tumor burden. Additionally, 20 probe sets (T4-T23) were selected from the personalized probe sets of the 37 pancreatic cancer patients and used in conjunction with the pancreatic cancer-specific probe set to detect cfDNA in the 20 healthy individuals to evaluate the specificity of the pancreatic cancer-specific probe set for detecting systemic tumor burden.

[0115] Table 4 Personalized probe groups for 37 patients

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] Of the 37 baseline blood samples, 29 were identified as ctDNA positive (ctDNA positivity criteria refer to patent ZL2023 1 0889084.8). The sensitivity of the pancreatic cancer-specific probe group and the personalized probe group used together for baseline blood detection was 78.4% (29 / 37). In the 29 ctDNA-positive samples, the mutations detected in 27 samples could be covered by the pancreatic cancer-specific probe group. The sensitivity of using the pancreatic cancer-specific probe group alone for baseline blood tumor burden assessment was 73.0% (27 / 37). See [link to relevant documentation]. Figure 6 The above results demonstrate that using the pancreatic cancer-specific probe set alone for baseline blood tumor burden assessment exhibits excellent sensitivity, while the combined use of the pancreatic cancer-specific probe set and the personalized probe set further improves detection sensitivity. Furthermore, in the 20 healthy individual samples, regardless of whether the pancreatic cancer-specific probe set and the personalized probe set were used together, or whether the pancreatic cancer-specific probe set was used alone, all 20 samples were ctDNA negative, indicating that the specificity of the pancreatic cancer-specific probe set for assessing tumor burden is 100%.

[0128] Then, the 223-gene probe set was used to perform targeted capture detection and high-depth sequencing on baseline serum cfDNA before treatment in another 20 pancreatic cancer patients (without requiring tumor tissue detection). Results are as follows: Figure 6 As shown, 15 baseline blood samples were identified as ctDNA positive (≥1 mutation positive), with a detection sensitivity of 75.0% (15 / 20). This indicates that although the pancreatic cancer-specific probe group covers fewer genes, it can achieve a detection sensitivity similar to the 223-gene probe group, demonstrating an unexpected technical effect.

[0129] The above results demonstrate that the pancreatic cancer-specific probe set has not only high sensitivity but also excellent specificity for assessing tumor burden, proving that the probe set of this invention has excellent clinical performance for assessing pancreatic cancer tumor burden in real clinical samples.

[0130] Example 5: Pancreatic cancer-specific probe kits for monitoring the efficacy of treatment in pancreatic cancer patients.

[0131] In Example 4, all 37 patients received first-line treatment, and 28 of them underwent ctDNA tumor burden assessment via blood samples at the initial efficacy evaluation. The results are as follows... Figure 7The results showed that, compared with baseline blood levels, at the initial efficacy evaluation, 13 patients had zero ctDNA levels (ctDNA level (MTM / ml) = (ctDNA level × cfDNA input (ng) × 1000 pg / ng) / (3.3 pg × plasma volume (ml)), the ctDNA level calculation method is based on patent ZL 2023 1 1540699.6), 8 patients had decreased ctDNA levels, and 7 patients had increased ctDNA levels. By the end of follow-up, 3 patients in the zero ctDNA group, 4 patients in the decreased ctDNA group, and 5 patients in the increased ctDNA group experienced disease progression, with progression rates of 23.1% (3 / 13), 50.0% (4 / 8), and 71.4% (5 / 7) respectively. Survival analysis showed that the ctDNA zeroing group had the longest progression-free survival, followed by the ctDNA decreasing group, and the ctDNA increasing group had the worst prognosis. The median progression-free survival times for the three groups were not reached, 6 months, and 2.8 months, respectively, indicating that the ctDNA zeroing group had the best prognosis, followed by the ctDNA decreasing group, and the ctDNA increasing group had the worst prognosis (see...). Figure 8 Notably, the risk of progression in the elevated ctDNA group was 5.7 times that in the ctDNA-zero group (hazard ratio 95% confidence interval 1.1–29.8, P = 0.0055). These results demonstrate that the pancreatic cancer-specific probe kit of this application can effectively assess the tumor burden in patients. By monitoring changes in tumor burden before and after treatment in pancreatic cancer patients, the treatment effect can be dynamically reflected in real time, and the trend of tumor burden change is significantly correlated with patient prognosis.

[0132] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

[0133] Although this application has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this application are all within the scope of protection claimed in this application.

Claims

1. A probe set for assessing the systemic tumor burden of pancreatic cancer, characterized in that, The probe set includes probes for detecting gene markers, including KRAS, TP53, CDKN2A, SMAD4, RNF43, TGFBR2, PIK3CA, BRAF, GNAS, and CTNNB1, and the probe set includes probes with nucleotide sequences as shown in SEQ ID NO:1-65. The genetic markers include the following gene regions: exons 2 and 3 of the KRAS gene, exons 2-11 of the TP53 gene, exons 1 and 2 of the CDKN2A gene, exons 2, 3, 6, 9-12 of the SMAD4 gene, exons 2 and 4 of the RNF43 gene, exons 4 and 7 of the TGFBR2 gene, exon 10 of the PIK3CA gene, exons 12 and 15 of the BRAF gene, exon 8 of the GNAS gene, and exon 3 of the CTNNB1 gene.

2. The probe assembly as described in claim 1, characterized in that, The probe set also includes personalized probe sets.

3. A product for assessing the systemic tumor burden of pancreatic cancer, characterized in that, The product includes the probe set as described in claim 1.

4. The product as described in claim 3, characterized in that, The product also includes one or more of the following: DNA extraction reagents, library construction reagents required for sequencing methods, and hybridization capture reagents required for sequencing methods.

5. The product as described in claim 3 or 4, characterized in that, The test samples for the product include cell, tissue, or body fluid samples.

6. The product as described in claim 5, characterized in that, The cell samples include one or more types of pancreatic cancer cell suspensions and circulating tumor cells.

7. The product as described in claim 5, characterized in that, The body fluid samples include one or more of whole blood, serum, and plasma.

8. The product as described in claim 5, characterized in that, The tissue samples include one or more of pancreatic cancer tissue and paraffin sections.

9. The product as described in claim 3 or 4, characterized in that, The products include testing reagents, testing kits, testing chips, or testing devices.

10. The use of the probe set of claim 1 in the preparation of a product for assessing the systemic tumor burden of pancreatic cancer.

Images