A quality control material for detecting minimal residual disease in solid tumors, its preparation method and application

Abstract

This invention discloses a quality control product for detecting minimal residual disease (MRD) in solid tumors, its preparation method, and its applications. This invention simultaneously addresses the limitations of existing quality control products derived from natural tumor cells, such as limited mutation profiles and inability to support multi-site validation, as well as the complex background and inability to provide paired samples in quality control products derived from mixed tumor cell sources. This invention provides simulated tumor tissue samples, paired detection samples, and a series of cell-free DNA samples required for the entire detection process, applicable to all detection methods and platforms, comprehensively meeting the quality control requirements for detecting MRD in solid tumors. All mutations originate naturally within the cellular genome, are randomly distributed, and highly replicate the molecular biological characteristics of real tumors, allowing for further evaluation of mutation clonal analysis capabilities using detection platforms. Furthermore, this invention ensures the comparability and authenticity of the quality control product with clinical samples at the physical level, and based on clonably amplifiable edited cell lines, it exhibits good batch-to-batch consistency and can be stably obtained over a long period.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a quality control product for detecting minimal residual disease in solid tumors, its preparation method, and its application. In particular, it relates to a cell line constructed using gene editing technology that contains a large number of tumor-related mutations, and a quality control product for minimal residual disease in solid tumors prepared from this cell line that more closely resembles real clinical samples. Background Technology

[0002] Minimal residual disease (MRD), also known as measurable residual disease or molecular residual disease, refers to the trace amounts of cancerous lesions that may remain in the body of cancer patients after radical treatments such as surgery and radical radiotherapy / chemotherapy. Because these lesions are difficult to detect using conventional methods such as imaging, they are often the root cause of disease recurrence. In the past decade, circulating tumor DNA (ctDNA), as an emerging biomarker, has received widespread attention in the medical community due to its non-invasive nature and ability to provide dynamic information about tumors, and is therefore considered a potential biomarker for MRD detection in solid tumors. In recent years, MRD assessment guided by next-generation sequencing (NGS) of ctDNA gene mutations has developed rapidly in various solid tumors, including colorectal cancer, non-small cell lung cancer, breast cancer, and invasive bladder cancer. Numerous prospective studies have confirmed the strong prognostic value of ctDNA-MRD detection after surgery for these solid tumors. Furthermore, subgroup analyses from different studies have confirmed that ctDNA-MRD positivity and negativity can serve as biomarkers for escalating or downgrading treatment in terms of predictive value for adjuvant therapy. Therefore, ctDNA-MRD testing has been recommended by numerous clinical guidelines and expert consensus statements both domestically and internationally, and is gradually being integrated into the clinical diagnosis and treatment pathway for solid tumors.

[0003] However, the clinical translation of this technology currently faces a fundamental challenge: the effectiveness and reliability of the detection technology itself lack unified standards. It is well known that the clinical value of any biomarker depends on its ability to be accurately and stably detected at the technical level. Currently, however, there are very few rigorously clinically validated ctDNA-MRD detection platforms. The market is flooded with commercial products with varying technical approaches and panel ranges. These include Tumor-informed analysis products that require sequencing of tumor tissue to identify patient-specific mutation profiles before developing personalized panels for ctDNA mutation detection, and Tumor-agnostic analysis products that use immobilized panels and rely solely on a set of known tumor-associated mutation sites for ctDNA detection. Although many detection platforms claim to perform "ultra-high sensitivity" MRD detection, their claimed performance parameters are mostly based on internal validation processes and lack standardized validation by third-party quality control materials. Therefore, the actual sensitivity and specificity of different platforms may vary significantly, raising questions about the consistency and comparability of test results. This inconsistency will not only affect the reliability of MRD clinical research but may also lead to serious clinical decision-making risks, hindering further development in this field. Therefore, developing ctDNA-MRD detection quality control products suitable for multi-platform comparison, reagent performance verification, internal quality control (IQC) and external quality assurance (EQA) has become key to promoting the application of this technical specification.

[0004] Currently, some ctDNA-MRD quality control products have been reported to be used. For example, some researchers prepared ctDNA-MRD quality control products by mixing genomic DNA from a pair of paired lung cancer cell lines in different proportions and then fragmenting them using ultrasound. However, due to the limited total number of somatic mutations and the scarcity of homozygous mutations in natural lung cancer cell lines, the number of tumor-related mutation sites that can be effectively detected in this quality control product after high-level serial dilution is extremely low. Therefore, this quality control product is difficult to meet the multi-site performance confirmation requirements of MRD detection platforms (especially detection platforms based on tumor-agnostic analysis). In addition, the fragmentation method of this product is ultrasound fragmentation, and the proportion of fragments smaller than 100bp (after fragment screening during sequencing) in the prepared DNA fragments is much larger than that in real clinical samples. This may lead to situations where the same amount of sample is used but the test results are inconsistent with those of clinical samples, resulting in inaccurate evaluation of the performance of the detection platform or failure of quality control monitoring.

[0005] For example, there are reports of preparing ctDNA-MRD quality control products by enzymatically digesting tumor cells carrying various clinically common MRD mutation sites and then mixing them in a predetermined ratio. However, this type of product also has several significant limitations: First, due to the differences in the initial mutation frequencies of tumor cells from different sources, the actual frequencies of each mutation site in the mixed sample deviate significantly from the expected values; second, different tumor cells carry many heterogeneous endogenous variations, and mixing multiple cells will result in an abnormally complex sample background, making it difficult to accurately determine false positive results in MRD detection; third, this product cannot provide the necessary paired samples, a deficiency that severely limits its widespread application in the market, especially in technical pathways that require paired sample analysis.

[0006] Currently, the most widely used product in the overseas market is Seraseq ctDNA MRD Panel Mix developed by Seracare. This product is prepared by mixing DNA from a pair of paired lung cancer cells with 22 exogenously synthesized gene mutation site fragments. Compared with the two quality control products mentioned above, Seraseq ctDNA MRD Panel Mix contains more tumor-related mutations and can provide the paired normal cells required for detection, meeting the performance validation and quality control requirements of most ctDNA-MRD detection platforms. However, because the mutation site fragments in Seraseq ctDNA MRD Panel Mix are all directly chemically synthesized, the mutation sites are located at fixed positions on the sequence. Therefore, it does not have the characteristic of random distribution of mutation sites on ctDNA fragments, and thus cannot be consistent with clinical samples. In addition, the exogenously introduced mutations in this product are all clinically relevant mutations, and there is a lack of real clonal evolutionary relationships between different mutations. Therefore, it cannot be further used to evaluate the mutation clonal analysis capabilities of tumor-informed detection platforms. Summary of the Invention

[0007] To address some of the technical problems existing in the prior art, this invention develops a quality control product for MRD detection with the following advantages: it provides simulated tumor tissue samples, paired samples with the same genomic background, and a series of cell-free DNA samples required for the entire detection process. It is applicable to all detection methods and platforms, comprehensively meeting the quality control requirements for detecting minimal residual disease in solid tumors. Specifically, the series of cell-free DNA samples can well simulate the characteristics of cell-free DNA in real clinical samples, covering high, medium, low, and very low mutation frequency levels. Each level of sample is rich in clearly defined mutations (containing sufficient gene mutation sites commonly detected in clinical MRD). The specific technical solution of this invention is as follows.

[0008] In a first aspect, the present invention provides a cell line for producing quality control material for detecting minimal residual disease in solid tumors, which was deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 46795.

[0009] A second aspect of the present invention provides a method for preparing a quality control material for detecting minimal residual disease in solid tumors, comprising the steps of culturing and processing the cell line or its passaged cell line described in the first aspect.

[0010] In some embodiments, the method for preparing the quality control material for detecting minimal residual disease in solid tumors according to the present invention includes: (1) The cell line and wild-type cell line were cultured separately to obtain cultures; (2) The cell line culture was embedded in paraffin to obtain a simulated tumor tissue sample; (3) DNA was extracted from the wild-type cell line culture to obtain paired samples; (4) The culture was digested with nuclease to obtain first free DNA from the cell line and second free DNA from the wild-type cell line, respectively; (5) Dilute the first free DNA and the second free DNA in the matrix at a preset ratio to obtain a series of free DNA samples.

[0011] In some embodiments, according to the method for preparing the quality control material for detecting minimal residual disease in solid tumors according to the present invention, the nuclease is MNase enzyme.

[0012] In some embodiments, according to the method for preparing the quality control material for detecting minimal residual disease in solid tumors according to the present invention, the main peak of the free DNA is located at 100-200 bp.

[0013] In a third aspect, the present invention provides a quality control product for detecting minimal residual disease in solid tumors, which is obtained according to the preparation method described in the second aspect.

[0014] A fourth aspect of the present invention provides the application of the above-described quality control material in quality control.

[0015] In some embodiments, according to the application described in the present invention, the quality control includes at least one of the following: performance confirmation / verification of the product for detecting minimal residual disease in solid tumors, internal quality control of the testing laboratory, and inter-laboratory quality assessment.

[0016] The beneficial technical effects of this invention include: (1) This invention simultaneously addresses the problems of limited mutation spectrum and inability to support multi-site verification in existing quality control products derived from natural tumor cells, as well as the complex background and inability to provide paired samples in quality control products derived from mixed tumor cell sources. The present invention integrates thousands of individual cell mutations into a gene-edited cell line (MPPP), including a certain number of homozygous mutations. Even under extremely low frequency conditions, it can still provide rich and stable mutation signals, fully meeting the verification requirements of different MRD detection panels for multiple sites and broad coverage. Furthermore, the present invention is based on mixing a single edited cell line with its wild-type background cells, resulting in a clearly defined mutation spectrum. Simultaneously, its wild-type background cells can also serve as paired samples, meeting the filtering requirements for germline mutations and clonal hematopoietic mutations in MRD detection.

[0017] (2) This invention solves the problem of fixed mutation locations in the quality control samples from synthetic fragment sources and the lack of true clonal evolutionary relationships between different mutations. All mutations in this invention originate naturally within the cellular genome, are randomly distributed, and conform to the molecular biological characteristics of real tumors. Therefore, they can be further used to evaluate the ability of the Tumor-informed detection platform to analyze mutation clonality.

[0018] (3) The problem of inconsistent fragment distribution between quality control products and real clinical samples has been solved. This invention uses MNase enzyme digestion to prepare DNA fragments, and the main peak of the product is located in the range of 140-170 bp, which is highly consistent with the fragment characteristics of human endogenous cfDNA, thereby ensuring the comparability and authenticity of quality control products and clinical samples at the physical level.

[0019] (4) Quality control materials are readily available over a long period. This invention is based on a cloneable and amplifiable edited cell line, which exhibits good batch-to-batch consistency and can be obtained stably and continuously over a long period. It can be used as a third-party quality control material to meet the needs of reagent performance confirmation and laboratory internal quality control, while also supporting cross-platform and cross-laboratory performance comparison and quality evaluation. Attached Figure Description

[0020] Figure 1 The distribution of DNA fragments in the free DNA sample of the MRD quality control material is shown. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, the numerical ranges in this invention should be understood to specifically disclose the upper and lower limits of the range and every intermediate value between them. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, 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 invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0024] cell lines In one aspect, the present invention provides a cell line for producing quality control materials for detecting minimal residual disease in solid tumors. The present invention successfully constructed a stable cell line carrying a specific mutation through gene editing combined with screening technology. The cell line was deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 46795.

[0025] The cell lines of the present invention are MPPP cell lines containing at least the following mutations: FAT1:c.9301G>A, FAT1:c.2776A>G, ​​POLE:c.840A>T, TENM3:c.3037G>A, SORBS2:c.939del, MSH2:c.1046C>G, POLE:c.857C>G.

[0026] Preparation method of quality control products In one aspect, the present invention provides a method for preparing a quality control sample for detecting minimal residual disease in solid tumors, comprising the steps of using the above-mentioned cell line or its passaged cell line to generate a simulated tumor tissue sample, a paired sample, and a series of cell-free DNA samples.

[0027] In a preferred embodiment, the method of the present invention includes: (1) The cell line and wild-type cell line were cultured separately to obtain cultures; (2) The cell line culture was embedded in paraffin to obtain a simulated tumor tissue sample; (3) DNA was extracted from the wild-type cell line culture to obtain paired samples; (4) The culture was digested with nuclease to obtain first free DNA from the cell line and second free DNA from the wild-type cell line, respectively; (5) Dilute the first free DNA and the second free DNA in the matrix at a preset ratio to obtain a series of free DNA samples.

[0028] In step (1), the wild-type cell line includes HEK293T / 17 cells. The specific steps of the expansion culture, such as basal expansion after cell resuscitation, routine passage expansion, large-scale expansion from adherent to suspension or multilayer flasks, and the culture media used in between, are not particularly limited and can be adjusted according to actual needs.

[0029] In step (4), the nuclease is preferably MNase, and the amount used is not particularly limited, as long as it can simulate the fragmentation characteristics of real free DNA produced by apoptosis in vivo.

[0030] In step (5), the first cell-free DNA and the second cell-free DNA are mixed in a preset ratio to obtain a gradient detection quality control. For example, when the tumor DNA content is in the range of 0 to 0.05%, the first cell-free DNA is 0 to 0.5 parts, and the second cell-free DNA is 100 to 99.5 parts. The matrix used is a plasma matrix that has been denucleated.

[0031] Quality control products In one aspect, the present invention provides a quality control material for detecting minimal residual disease in solid tumors, which is obtained by the above-described preparation method.

[0032] In a preferred embodiment, the main peak of free DNA in the quality control sample is located in the range of 100-200 bp, for example, 140-170 bp, thereby ensuring a high degree of consistency with the fragment characteristics of endogenous free DNA in the human body.

[0033] Example 1. Select wild-type background cells for gene editing (1) By comprehensively analyzing the gene mutation sites covered by the current MRD detection product Panel and the driver mutations that can induce cell mutation accumulation reported in the literature, partial mutations in the MSH2 and POLE genes were selected as gene editing target sites. Specific sgRNAs were designed and synthesized, and corresponding expression vectors were constructed. At the same time, ssODNs were designed and synthesized as gene editing repair templates.

[0034] (2) Using homologous recombination editing, the sgRNA expression vector and ssODNs required for gene editing were co-electrotransfected into HEK293T / 17.

[0035] (3) Single-cell cloning was performed 48 hours after transfection.

[0036] (4) Once the monoclonal cells have grown to a sufficient number, a portion of the cells are taken for Sanger sequencing to confirm whether the gene editing was successful.

[0037] 2. Detect and screen the edited cells. Genomic DNA was extracted from the successfully edited MSH2 and / or POLE gene mutant cell lines (MC, MP, MPPP, MPPV) and the wild-type baseline cell line (HEK293T / 17). Whole exome sequencing (WES) was then performed using an MGI DNBSEQ-7RS sequencer to verify the newly added genomic mutations in each edited cell line. Based on the sequencing results, a cell line containing a large number of tumor-related mutations and a significant number of homozygous mutations, capable of realistically mimicking tumor molecular biology characteristics (MPPP, accession number CGMCC No. 46795) was selected. MC and MP were obtained by editing different gene loci in MSH2, while MPPP and MPPV were obtained by editing different loci in MSH2 and POLE genes, respectively. The newly added homozygous mutations in the successfully edited cell lines are shown in Table 1 below.

[0038] Table 1. New homozygous mutations in successfully edited cell lines. 3. Perform mutation truth set calibration on the selected cell lines. To verify the reliability of the mutation profile, genomic DNA was extracted from the preferred cell line MPPP and its homologous wild-type baseline cell line HEK293T / 17, followed by repeated WES analysis using the MGI DNBSEQ-T7 and Illumina Novaseq 6000 dual sequencing platforms. Somatic mutations appearing simultaneously in all filtered MPPP WES data were selected as reliable mutations to establish the MPPP mutation truth set. Validation showed that the MPPP truth set included 7 reliable mutations with a 100% frequency (i.e., homozygous mutations), 100 mutations with a 50% frequency, and 3402 other reliable mutations, of which 100 were known tumor-related mutations indexed in the COSMIC database. This step ensured the accuracy, purity, and traceability of the core mutation information in the quality control materials.

[0039] 4. Preparation of simulated tumor tissue samples The selected preferred cell line MPPP was embedded in paraffin and sectioned to prepare simulated tumor tissue samples required for detection by the Tumor-informed method.

[0040] 5. Paired Sample Preparation DNA was extracted from the wild-type baseline cell line HEK293T / 17 to prepare paired samples for testing.

[0041] 6. Preparation of a series of gradient cell-free DNA samples The selected superior cell line MPPP and the wild-type baseline cell line HEK293T / 17 were expanded and cultured, followed by digestion with MNase enzyme to simulate the fragmentation characteristics of real cell-free DNA generated by in vivo apoptosis. The digestion products of the two were diluted and mixed according to the preset ratio shown in Table 2, and then added to a nucleic acid-free plasma matrix to finally prepare a series of cell-free DNA samples for MRD detection.

[0042] Table 2. Mixing ratio of digestion products 7. Capillary electrophoresis verification Cell-free DNA was extracted from a series of MRD-detected cell-free DNA samples prepared using the MagMax cell-free DNA isolation kit. The distribution of extracted cell-free DNA fragments was then analyzed using an Aglient 2100 capillary electrophoresis system to verify whether it conformed to the distribution of cell-free DNA in real clinical samples. The results showed that the main peak of cell-free DNA in each sample was located in the 140-170 bp range, which was highly consistent with the fragment characteristics of endogenous human cfDNA. Figure 1 ).

[0043] 8. NGS Verification To comprehensively evaluate the applicability and platform compatibility of the quality control products of this invention, NGS validation of the series of quality control products was performed using the Gene+1021+MRD detection product, which combines the features of tumor-informed personalized analysis and tumor-agnostic fixed panel analysis. Given that the detection limit of this product is 0.1% (mutation level), high-, medium-concentration, and negative samples S1, S2, and S6 were selected for this validation. Sequencing data quality assessment showed that the average sequencing depth of each sample was >80000×, the Q30 ratio of base quality was >80%, and the genome alignment rate was >90%, meeting the basic requirements for data quality in MRD detection. The detection details of the seven homozygous mutations in each sample are shown in Table 3. The results further confirm the detection effectiveness and reliability of the mutation signals of the quality control products at the set concentrations.

[0044] Table 3 NGS Validation Results for Each Sample Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell line for generating quality control samples for detecting minimal residual disease in solid tumors, characterized in that, It was deposited on March 27, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, with accession number CGMCC No. 46795.

2. A method for preparing a quality control material for detecting minimal residual disease in solid tumors, characterized in that, The steps include culturing and processing the cell line or its passaged cell line as described in claim 1.

3. The preparation method according to claim 2, characterized in that, The method includes: (1) The cell line is expanded to obtain a culture; (2) The culture was embedded in paraffin to obtain a simulated tumor tissue sample derived from the cell line; (3) The culture was digested with a nuclease to obtain the first free DNA fragment derived from the cell line.

4. The preparation method according to claim 3, characterized in that, The method further includes: (1) The wild-type cell line was expanded to obtain a culture; (2) Extract DNA from the culture to obtain paired samples derived from the cell line; (3) The culture was digested with a nuclease to obtain a second free DNA fragment derived from the wild-type cell line; (4) Dilute the first free DNA fragment and the second free DNA fragment in the matrix at a preset ratio to obtain a series of free DNA samples.

5. The preparation method according to claim 3 or 4, characterized in that, The nuclease is MNase.

6. The preparation method according to claim 2, characterized in that, The cell line was obtained by gene editing of a wild-type cell line.

7. The preparation method according to claim 3 or 4, characterized in that, The main peak of the free DNA is located at 100-200bp.

8. A quality control product for detecting minimal residual disease in solid tumors, characterized in that, The preparation method according to any one of claims 2-7 is obtained.

9. The application of the quality control material according to claim 8 in quality control.

10. The application according to claim 9, characterized in that, The quality control includes at least one of the following: performance confirmation / verification of products for detecting minimal residual disease in solid tumors, internal quality control of testing laboratories, and inter-laboratory quality evaluation.

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