Combined application technology for assisting in monitoring recurrence risk of solid tumor

Abstract

The invention provides a combination of a PTC platform and a ctDNA-based MRD detection technology, which is used for assisting in monitoring the recurrence risk of solid tumors. The PTC microenvironment contains tumor cells, interstitial cells, fibroblasts and immune cells from epithelium, the cell components are rich, tumor heterogeneity can be reproduced, the culture success rate and clinical consistency can reach 90% or above, and detection can be completed within 10-14 days. The curative effect of the medicine can be predicted by observing the retraction degree of the PTC, and prognosis can be predicted to a certain extent. Based on an MRD detection technology of the ctDNA, a Tumor informed strategy is adopted, customized MRD detection can be completed, the sequencing depth is 100000 X, and the ctDNA of which the concentration is greater than or equal to 0.02% can be stably detected. By combining the two technologies, the prediction accuracy of the recurrence risk can be improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a combined application technology for assisting in monitoring the recurrence risk of solid tumors. Background Art

[0002] The treatment of cancer has always been a difficult problem that the medical community urgently needs to overcome. Although precise individualized treatment is being gradually realized, the key reason for the failure of tumor treatment is the risk of cancer metastasis or recurrence. Most patients will experience a recurrence of cancer after undergoing standardized treatments such as surgery, chemotherapy, and radiotherapy. According to statistics, about 70%-80% of patients die from tumor recurrence and metastasis. After undergoing local treatment + systemic radiotherapy and chemotherapy, there are still some patients who develop subclinical occult metastases, and most patients with clinical recurrence and metastasis have a short survival period. The main reasons for cancer recurrence include: not all cancer cells are removed during surgery, and not all cancer cells are destroyed through treatments such as drugs. At the same time, gene mutations and the cancer cells themselves can also develop drug resistance, affecting the treatment effect and leading to cancer recurrence. Cancer recurrence can be divided into local recurrence, regional recurrence, and distant recurrence. For most types of cancer, local recurrence and regional recurrence can still be treated to reduce the cancerous lesion and slow down the development of cancer, relieve pain and other symptoms, extend the patient's life, and improve the quality of life. Therefore, it is very important to take the right measures to prevent cancer recurrence and detect the symptoms and signs of cancer recurrence and metastasis as early as possible.

[0003] Currently, the traditional technical means used to monitor tumor recurrence mainly include checking the levels of blood tumor markers, imaging examinations such as CT and magnetic resonance, and gene detection technology. For patients who have completed 5 years of follow-up after standardized treatment, still have no abnormal symptoms, and no obvious metastatic lesions are found by imaging examinations such as CT, magnetic resonance imaging, and ultrasound, they can be considered to have achieved "clinical cure". However, imaging examinations also have limitations. Usually, when a lesion can be detected by imaging examination, it often means that billions of cancer cells have aggregated into a mass. In fact, whether in the early stage of tumor occurrence, during treatment, or after treatment, as long as there is damage and death of tumor cells, it will lead to the possible existence of a small amount of tumor cells in the body and the release of ctDNA into the blood. The number of these remaining cells may be very small, so that they do not cause any signs or symptoms and are usually even undetectable by traditional methods. However, these cells are an important hidden danger of cancer recurrence. Therefore, the minimal residual disease (MRD) detection technology based on ctDNA can play a certain role in tumor prediction, treatment effect tracking, and prognosis detection. By detecting blood samples at multiple time points, it can monitor in real time and dynamically reflect the tumor burden and tumor variation in the patient's body, with great clinical application value. According to relevant research reports, MRD detection based on ctDNA can identify disease recurrence earlier than traditional clinical or imaging methods to provide an opportunity for early intervention. In different cancer types, the lead times are as follows: nasopharyngeal carcinoma 6 months, breast cancer 7.9 - 11 months, pancreatic cancer 6.5 months, lung cancer 70 days - 5.2 months, colorectal cancer 5.1 - 10 months. At the same time, there are already relevant expert consensuses in breast cancer and lung cancer.

[0004] Patient-Derived Tumor-like Cell Clusters (PTC, micro-tumor PTC model), an in vitro model based on primary tumor cells from patients, has tissue specificity, stem cell characteristics, epithelial cells, stromal cells, and immune cells, and can simulate the in vivo tumor microenvironment to the greatest extent. Its culture success rate and clinical consistency can both reach over 90%. The effectiveness of drugs can be reflected by the degree of shrinkage of tumor cell clusters in this model. The killing effect of anti-tumor drugs on tumor cell clusters can be observed under a microscope, and the residual situation of PTC microspheres can provide certain indication ability for PFS. At the same time, the detection period of this model is short, and the detection can be completed within 14 days. Moreover, high-throughput drug sensitivity experiments can be carried out to determine the sensitivity of tumor drugs, which can meet the clinical requirements for the detection time window. By jointly applying MRD and PTC technologies, the accuracy of detecting the recurrence risk of solid tumors can be improved, the recurrence probability can be reduced, and timely intervention treatment measures can be taken for patients with high recurrence risk, improving the survival period and prognosis of patients with malignant tumors, better serving clinical practice, and providing important reference value for clinical practice. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a combined application technology of a micro-tumor PTC in vitro model and a ctDNA-based MRD detection method for the deficiencies of the prior art, which can improve accuracy and reduce costs.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is: A combined application technology for assisting in monitoring the recurrence risk of solid tumors, and the combined technologies are a tumor-like cell cluster derived from patients - the micro-tumor PTC in vitro model and a ctDNA-based MRD detection method; the solid tumors include four major solid tumors: colorectal cancer, gastric cancer, breast cancer, and lung cancer.

[0007] For the above micro-tumor PTC in vitro model, the clinical consistency reaches 96.6% in gastrointestinal tumors, 91.4% in breast cancer, and 89% in overall lung cancer.

[0008] For the above micro-tumor PTC in vitro model, the culture success rate reaches over 90%.

[0009] For the above micro-tumor PTC in vitro model, the drug sensitivity is further judged by observing the degree of shrinkage of cell clusters.

[0010] The above-mentioned observation of the degree of shrinkage of tumor-like cell clusters can be counted and statistically analyzed by a self-developed microscope AI intelligent software.

[0011] The above MRD detection is a ctDNA-based detection method.

[0012] The above MRD detection is based on the Tumor informed strategy.

[0013] The sequencing depth of the above MRD detection is 100000X.

[0014] The above MRD detection can stably detect ctDNA ≥ 0.02%.

[0015] The advantages of the present invention compared with the prior art are as follows:

[0016] The present invention first uses the in vitro model of microtumor PTC combined with the MRD detection method based on ctDNA to assist in monitoring the recurrence risk, and this invention can improve the prediction accuracy. Brief Description of the Drawings

[0017] Figure 1 It is the microscopic cell mass change at D7 after adding drugs to the PTC microspheres derived from lung cancer patients in Specific Embodiment 1 of the present invention. Detailed Description of the Invention

[0018] The technical solution of the present invention will be further described below, but the present invention is not limited thereto.

[0019] Example 1

[0020] The in vitro model of microtumor PTC described in this example is a lung cancer cell cluster. The method is to dissociate the lung cancer experimental samples into single tumor cells by physical shearing and enzymatic digestion. After 5 - 7 days, the cells can form PTC microspheres through proliferation and self - assembly. After adding anti - tumor drugs, the lung cancer PTC microspheres can be effectively killed, and their quantity is significantly reduced and their volume is significantly shrunk.

[0021] Place the collected lung tissue samples in a culture dish, cut them into pieces, add collagenase for digestion, blow evenly, time the digestion time, observe with a microscope, and then place them in an incubator and observe the digestion situation regularly.

[0022] After adding a serum - containing digestion termination solution (DMEM medium containing 10% FBS) to terminate the digestion, filter and collect the filtrate with a 100μm cell sieve. After centrifuging at 400×g for 5 min, resuspend with lung cancer PTC medium and place it in a 37°C, 5% CO2 constant temperature incubator for 3 - 7 days. The cells form PTC microspheres through self - assembly and amplification. During the culture process, use a microscope to take pictures to record the culture status.

[0023] Lay the cultured PTC microspheres in a 96 - well plate, with 30 - 50 PTC microspheres in each well. Add the lung cancer clinical treatment plan, and set 3 replicate wells for each drug plan. Set a control group (3 replicate wells). Observe the tumor regression degree at 24 hours, 48 hours, 72 hours and day 7 respectively, including the quantity and volume changes. Take pictures and count with AI.

[0024] After co-incubating the PTC microspheres with the drug in the 96-well plate, tilt the plate slightly and stand it up. Carefully aspirate 100 μl of the supernatant along the liquid surface. Add 80 μl of the celltiter cell viability test solution. Then transfer the mixed liquid from the transparent plate to the white plate. Vigorously pipette and mix well before transfer. Set the shaker for 20 minutes and shake at medium speed. After transfer, measure the cell viability value with a microplate reader.

[0025] Collect the primary tumor tissues and matched blood samples of the patients. Extract genomic DNA from the tumor tissues and plasma, perform 1021-gene detection, and analyze the somatic mutations of the patients.

[0026] Remove clonal hematopoiesis mutations and design multiplex PCR primers for the detected somatic mutations.

[0027] Collect blood samples within a predetermined time. Collect 20 ml of blood samples in Streck tubes for longitudinal MRD monitoring.

[0028] Perform pretreatment of the blood samples, separate the plasma, extract cfDNA, detect with a customized PCR pool. After multiplex PCR amplification, perform ultra-deep sequencing and analyze the MRD results.

[0029] The results of the present invention show that the MRD result is positive. Although the number and volume of the cell clusters in the microscopic view of the PTC drug sensitivity test results have decreased significantly, there is still residue in the core part of the cell clusters and it is relatively compact. There is a good consistency between MRD and PTC drug sensitivity results, and they can be combined for predicting the recurrence risk.

Claims

1. A combined application technology for assisting in monitoring the recurrence risk of solid tumors, wherein, The combined application technology only includes the PTC platform and the ctDNA-based MRD technology, and is characterized by comprising the following steps: Step 1: Collect available experimental specimens from patients with malignant tumors, construct a microtumor PTC in vitro model, and perform MRD detection based on the Tumorinformed strategy; Step 2: Perform drug sensitivity test based on the microtumor PTC in vitro model and observe the shrinkage degree of tumor cell microclusters under a microscope; Step 3: Perform customized MRD testing at a scheduled time based on tissue-specific variation.

2. The method according to claim 1, wherein The malignant tumors include four solid tumors: gastrointestinal tumors, breast cancer, and lung cancer.

3. The method according to claim 1, characterized in that The experimental specimens in step 1 include surgical tissue, puncture tissue, biopsy tissue or pleural effusion.

4. The method according to claim 1, wherein In the drug sensitivity test in step 2, the drug regimen includes a clinical drug regimen for lung cancer.

5. The PTC construction method according to claim 1, wherein The following steps are involved: Step 1: Using physical shearing or enzyme digestion, the experimental specimens from patients with malignant tumors are dispersed into single cells; Step 2: In a specific culture medium, the digested single cells self-assemble to form tumor cell micelles, which contain epithelial tumor cells, stromal cells, fibroblasts, and immune cells.

6. The ctDNA-based MRD technology according to claim 1, wherein The following steps are involved: Step 1: Obtain tissue and blood samples for tissue 1021 gene testing; Step 2: Remove the influence of clonal hematopoiesis and design specific multiplex PCR primers based on the detected mutations. Step 3: Obtain 20 ml of blood sample, separate plasma, extract cfDNA, and test it with a customized PCR pool. After multiplex PCR amplification, perform ultra-deep sequencing and analyze the MRD results.

7. The screening method according to claim 1, wherein For the first time, the microtumor PTC in vitro model and MRD detection technology were combined to assist in monitoring the risk of recurrence of solid tumors.

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