CN112067589A - Malignant tumor recurrence and metastasis risk assessment system based on circulating tumor cell detection - Google Patents
Malignant tumor recurrence and metastasis risk assessment system based on circulating tumor cell detection Download PDFInfo
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Abstract
The invention discloses a malignant tumor recurrence and metastasis risk assessment system based on circulating tumor cell detection, which comprises the following steps: the system for evaluating the recurrence and metastasis risks of the malignant tumor realizes the recurrence and metastasis risk evaluation of the malignant tumor based on the detection of the circulating tumor cells by enriching the circulating tumor cells and counting immunofluorescence and combining the independent threshold comparison of the number of the circulating tumor cells in human blood before and after the treatment intervention of the malignant tumor, has the advantages of high sensitivity and strong specificity on the prediction of the recurrence and metastasis risks of the tumor after the treatment intervention, and can provide auxiliary information for clinical decision and treatment in time.
Description
Technical Field
The invention belongs to the technical field of biotechnology and medical detection, and particularly relates to the evaluation of malignant tumor recurrence/metastasis risk through detection of Circulating Tumor Cells (CTC) in human peripheral blood.
Background
Malignant tumors, also known as cancers, still show a rapidly increasing incidence and mortality. The leading cause of death in patients from cancer is relapse and metastasis. The traditional imaging detection technology (CT, MRI, PET, etc.) has certain requirements (at least 1-2mm) on the detection diameter of malignant tumors; meanwhile, the size of the tumor is sometimes not completely consistent with the malignancy degree and infiltration metastatic capacity of the tumor, and the imaging detection technology is not suitable for being used as a real-time monitoring means of the malignancy due to reasons such as radiation and the like. Therefore, the imaging detection technology often cannot find the occurrence, metastasis and recurrence of the malignant tumor in time, and the prognosis of the recurrence and metastasis of the malignant tumor has hysteresis. Serum tumor markers, such as carcinoembryonic antigen (CEA), Alpha Fetoprotein (AFP), and the like, are not screened with sufficient specificity, false positive results are easy to occur, and not all tumors derived from tissues have specific markers which can be used for tumor screening.
Ashworth found epithelial-like cells in the blood of cancer patients that were morphologically similar in size to tumor cells, and first reported the presence of Circulating Tumor Cells (CTCs). The blood transmission is an important way for cancer metastasis, most circulating tumor cells are quickly subjected to apoptosis or phagocytosis in the blood circulation, and few circulating tumor cells can form a micrometastasis focus, so that the death risk of cancer patients is increased. The number of circulating tumor cells represents the ability and extent of the malignancy to undergo blood metastases, and for cancer patients after treatment, if the circulating tumor cells continue to increase, it may be a precursor to or a process of tumor recurrence. The recurrence and metastasis risk of malignant tumor of patients can be improved by finding the trend of micrometastasis focus in early stage and changing the treatment scheme in time.
The number of circulating tumor cells in peripheral blood is rare, and most of them are white blood cells and red blood cells (each 10mL of blood contains about 1 hundred million white blood cells and 500 hundred million red blood cells, while the number of circulating tumor cells may be only a few to several tens). Thus, a prerequisite for an analysis using circulating tumor cells is an enrichment of the circulating tumor cells in the blood sample. Circulating tumor cells may undergo epithelial mesenchymal transition when migrating to a metastatic focus through peripheral blood circulation, so that epithelial specific markers are lost, and meanwhile, the epithelial specific markers are not only expressed in epithelial source tumor cells, but also in non-tumor epithelial cells, so that false negative and false positive of analysis results are increased, and sensitivity and specificity requirements are difficult to meet.
At present, a recurrence/metastasis risk assessment scheme with high sensitivity and strong specificity needs to be established, so that a more scientific and effective approach is provided for cancer recurrence/metastasis risk assessment.
Disclosure of Invention
The invention aims to provide a system for evaluating the recurrence and metastasis risk of malignant tumors based on circulating tumor cell detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
the malignant tumor recurrence and metastasis risk assessment system comprises a circulating tumor cell enrichment module, an immunofluorescence counting module and a risk judgment module;
the circulating tumor cell enrichment module is used for separating circulating tumor cells in a blood sample from non-circulating tumor cells such as most blood cells (e.g., red blood cells and white blood cells) in the sample, so as to obtain a cell system enriched with the circulating tumor cells, so that the cell system can be used for immunofluorescence experiments and analysis (dyeing, identification and counting);
the immunofluorescence counting module is used for identifying circulating tumor cells in a cell system obtained by separation by means of immunofluorescence staining and determining the number of the circulating tumor cells in a blood sample;
the risk determination module is used for threshold comparison of the numbers of circulating tumor cells in blood samples collected before and after a therapeutic intervention (e.g., surgery) on the malignant tumor, so as to predict the recurrence or metastasis risk of the malignant tumor.
Preferably, the circulating tumor cell enrichment module comprises a device (e.g., ClearCellFX system) that can separate circulating tumor cells from peripheral blood separately collected before and after surgery of a malignant patient using a microfluidic principle based on cell size and inertia.
Preferably, the number of sample volumes of peripheral blood is 7.5-10 mL/person.
Preferably, the peripheral blood is collected from 1 day to 1 month before surgery and from 1 day to 1 month after surgery.
Preferably, the immunofluorescence counting module comprises a fluorescence microscope and a computer platform for analyzing cellular immunofluorescence staining results collected by the fluorescence microscope; wherein, the immunofluorescence staining comprises the following steps: transferring the cell line obtained by separation to a slide glass, and treating with CK and DAPI fluorescent antibodies capable of binding to circulating tumor cells and CD45 fluorescent antibody capable of binding to blood cells; the computer platform can identify and count cells (cells with positive nuclear DAPI staining, positive CK staining and negative CD45 staining) that appear as DAPI +/CK +/CD45 "after immunofluorescent staining on the slide, i.e., the circulating tumor cells have an immunofluorescent staining result of DAPI +/CK +/CD 45-.
Preferably, the standard for judging immunofluorescence staining of the circulating tumor cells is (fluorescence intensity threshold): 1500< GFP < 5000 and CY5< 3800; or 5000< GFP and CY5< 19000.
Preferably, the risk determination module determines that the tumor recurrence or metastasis risk is high for a malignant tumor patient whose number of circulating tumor cells in peripheral blood collected before and after an operation is more than 10, and proposes intervention of the malignant tumor patient for further diagnosis and treatment; conversely, a malignant tumor patient is judged as having a low risk of tumor recurrence or metastasis (e.g., no recurrence or metastasis).
Preferably, the malignant tumor is selected from any one of lung cancer, breast cancer, digestive system tumor (e.g., gastric cancer, colorectal cancer). For these cancers (especially digestive system tumors), distant metastasis of tumor cells is closely related to circulating tumor cells present in blood, and the corresponding tumor recurrence or metastasis risk prediction has higher sensitivity and specificity.
The invention has the beneficial effects that:
according to the invention, by enriching the circulating tumor cells and counting immunofluorescence, and combining with independent threshold comparison of the number of the circulating tumor cells in the blood sample before and after malignant tumor treatment intervention, the evaluation of the recurrence or metastasis risk of malignant tumor based on circulating tumor cell detection is realized, the method has the advantages of high sensitivity and strong specificity on the prediction of the recurrence or metastasis risk of tumor after treatment intervention, can provide auxiliary information for clinical decision and treatment in time, does not need complex experimental equipment, and is low in cost.
Furthermore, the invention does not depend on the protein expressed on the cell surface, such as EPCAM protein (epithelial cell adhesion molecule), but selects the circulating tumor cells based on the difference of the cell mechanical properties, the blood sample does not need to be marked, the molecular characteristics and the surface markers of the circulating tumor cells are not influenced, and the circulating tumor cells in the sample can be automatically, rapidly and high-flux enriched only by simple sample processing. And the damage of the cells in the microfluid environment is small, the survival rate of the sorted cells is higher, the cells can be continuously cultured and subjected to subsequent analysis, the sensitivity is higher, and the treatment scheme and the treatment effect can be evaluated to a certain extent through accurate monitoring of the circulating tumor cells before and after treatment.
Furthermore, the invention determines the threshold value of the number of the circulating tumor cells (more than 10 circulating tumor cells exist in a certain volume of blood samples of malignant tumor patients) for risk assessment, improves the prediction capability of the recurrence or metastasis risk of the malignant tumor, and effectively controls false positive and false negative.
Detailed Description
The present invention is further illustrated by the following examples, which are provided only for the purpose of illustration and are not intended to limit the scope of the present invention.
1. Identification of circulating tumor cells in human peripheral blood samples
1.1 efficient enrichment of circulating tumor cells derived from a human peripheral blood sample
Use of
FX1System, based on cell size and inertia, enriches Circulating Tumor Cells (CTCs) from a patient's blood using the microfluidic enrichment principle that does not rely on cell surface expressed proteins such as EPCAM proteins (epithelial cell adhesion molecules). Circulating tumor cells can be separated from non-circulating tumor cells such as white blood cells, red blood cells and the like through enrichment, and the circulating tumor cells obtained through enrichment can be used for staining analysis.
1.2 staining of enriched circulating tumor cells
The enriched cell sample is transferred to a slide, and immunofluorescent staining is performed on the cell sample according to the binding of the circulating tumor cells with CK and DAPI fluorescent antibodies and the binding of the blood cells with CD45 fluorescent antibodies.
1.3 determination of circulating tumor cells meeting the criteria by fluorescence microscopy and software analysis
Circulating tumor cell staining satisfies DAPI +/CD 45-/CK + (fluorescence intensity range for determination: 1500< GFP ≦ 5000 and CY5<3800, or 5000< GFP and CY5< 19000).
Circulating tumor cells are >4 μm × 4 μm in diameter, have a morphology (round, oval and/or clustered cells), and are uniformly expressed with CK and DAPI.
2. Detecting the number of circulating tumor cells in a peripheral blood sample of a human body
1) A standard STREK noninvasive tube containing EDTA anticoagulant and special cell stabilizer is selected to collect 7.5mL of human peripheral blood.
2) The STRECK atraumatic tube was gently inverted several times to ensure a uniform blood sample.
3) The blood sample was aspirated from the STRECK non-invasive tube with a pipette into a new centrifuge tube (i.e., a 50mL centrifuge tube provided in ClearCell FX running reagent).
4) Add 4 volumes of red blood cell lysis buffer to the centrifuge tube containing the blood sample.
5) The centrifuge tube was gently inverted 5 times and incubated at room temperature for 10 minutes.
6) The lysed sample was centrifuged at 500Xg for 10 minutes at 15 deg.C-25 deg.C with the centrifuge containing a centrifuge brake (or highest deceleration).
7) The supernatant was gently removed using a pasteur pipette or seropipette until a supernatant volume of 4-5mL remained. The remaining supernatant was then removed using a micropipette tip with a filter tip, leaving approximately 100. mu.L of supernatant.
8) Using a 1000. mu.L pipette tip with a filter tip, 1.0mL of ClearCell FX resuspension buffer (RSB) was added to the wall of the centrifuge tube to avoid introducing air bubbles into the mixture. Gently blow up and down to resuspend the cell pellet.
9) An additional 3mL of ClearCell FX resuspension buffer was added to the wall of the centrifuge tube (total volume 4mL) to avoid introducing air bubbles into the mixture. Gently blow up and down to mix the cell suspension thoroughly.
10) Samples were processed on ClearCell FX and circulating tumor cells were isolated and enriched.
11) Upon completion of the run on ClearCell FX, a sample containing enriched Circulating Tumor Cells (CTCs) was collected from the sample output port into a 15mL centrifuge tube provided in ClearClell FX running reagent, centrifuged at 500Xg for 10 minutes at room temperature, and centrifuge braked.
12) The supernatant was removed with a pasteur pipette or serum pipette until the liquid in the centrifuge tube showed 1 mL.
13) The supernatant in the centrifuge tube was removed using a 1000. mu.L pipette tip with a filter tip, and 100. mu.L of the supernatant was retained in the centrifuge tube. The remaining 100. mu.L of supernatant will be used as resuspension buffer in step 15.
14) A 100 μ L pipette tip was covered with 0.2% pluronic (tip was blown up and down several times in 0.2% pluronic solution). The 0.2% pluronic solution must be completely drained prior to step 15.
15) The cell pellet was resuspended in the remaining 100 μ L of supernatant by gently pipetting the solution 5-6 times up and down using a 0.2% pluronic-coated pipette tip.
16) The cell suspension (100 μ L) was gently dispensed into silicone-rich isolator wells in a spiral motion using a micropipette tip to evenly distribute the sample over the polymer slide sample area.
17) Slides were allowed to dry in a biosafety cabinet at room temperature for at least 12 hours and vented for a maximum of 14 days.
18) To the sample area 100 μ L of freshly prepared 3.7% formaldehyde was added and coverage of the entire sample area was ensured.
19) Incubate at room temperature for 10 minutes.
20) The slide was tilted and all the formaldehyde solution was gently removed from the bottom corner, taking care not to touch the sample.
21) Washing was performed by adding 200. mu.L of 1 XPBS (2 washes were performed in total, 2 minutes for each soak, and 1 XPBS was removed after each wash) to remove the formaldehyde solution.
22) 200 μ L of permeation barrier buffer was added to the sample area and incubated for 30 minutes at room temperature.
23) Using a 200. mu.L pipette tip, tilt the slide to remove the permeation barrier buffer from the bottom right corner of the sample area, taking care not to touch the sample.
24) Add 100. mu.L of primary antibody gently to the sample area and incubate for 30 min at room temperature. Wherein, the primary antibody mixed solution (100 mu L) comprises the following components: Anti-Cytokeratin (1:500), Anti-CD45(1:200), Anti-pan keratin (1:500), Anti-EpCAM (1:1000) and Pierce immunopotentiator.
25) Add 200 u L1 x PBS washing (total 3 washing, each soaking for 3 minutes, each washing after removing 1 x PBS), to remove the primary antibody.
26) Add gently 100. mu.L of secondary antibody and incubate for 30 min at room temperature, protected from light. From this step, exposure of the slide is minimized. Wash 4 times with 1 × PBS, soak 3 minutes each, remove 1 × PBS after each wash. Wherein, the components of the second antibody mixed solution (100 μ L) are as follows: sheep anti-mouse AlexaFluor 488(1:1000), sheep anti-rabbit AlexaFluor 647(1:500), DAPI working fluid (1:200), and Pierce immunopotentiator.
27) The marker pen was used to mark the four corners of the sample location at the bottom of the slide.
28) The separator was gently removed from the slide with forceps, taking care not to destroy the sample.
29) Excess liquid was removed with a dust-free wipe, taking care not to touch the sample area.
30) A drop of mount solution was added in the middle of the coverslip.
31) The slide was inverted with the sample area facing down and gently placed on the Mounting solution and cover slip. The weight of the slide will cause the mount solution to spread over the entire sample area. If necessary, the edges of the slide were gently pressed to evenly disperse the mount solution.
32) Once the mount solution was evenly distributed over the sample area, nail polish coverslips were applied around the perimeter of the coverslip.
33) After the four corners of the nail polish were dry (2-3 minutes), the entire edge of the coverslip was sealed with nail polish.
34) After the coverslips were completely covered and sealed for 15 minutes, the slides were observed under a Lionheat LX (biotek) fluorescence microscope and counted for circulating tumor cell numbers using the matched analytical software gene 5.
3. Human malignancy recurrence/metastasis risk assessment
83 gastrointestinal malignant tumor patients subjected to surgery treatment in a third-level hospital of Changsha city in Hunan province from 2017 in 1 month to 2017 in 6 months are selected, and all the patients are confirmed to be diagnosed through imaging and pathological examination, wherein 35 patients with gastric cancer and 48 patients with colon cancer are treated through radical surgery. Taking 7.5mL of venous blood before and after one month before and after all patients to count CTCs, and comparing the number of CTCs before and after the operation of all patients through a threshold value, thereby verifying the efficacy of predicting postoperative recurrence/metastasis risk of malignant tumor patients based on CTC count.
3.1 Experimental example
TABLE 1 Experimental examples relapse/metastasis and CTC number statistics
| Recurrent and metastatic conditions | N | The number of CTC before and after operation is more than or equal to 10 (example) | Number of CTC before or after operation < 10 (example) |
| Follow-up 3 years relapse/metastasis | 37 | 29 | 8 |
| Non-recurrence/metastasis | 46 | 4 | 42 |
Referring to table 1, 37 relapsing/metastatic patients and 46 non-relapsing/metastatic patients appeared in the three-year follow-up of 83 patients; among 37 patients with relapse/metastasis, 29 patients have the number of CTCs before or after operation more than or equal to 10, and 8 patients have the number of CTCs before or after operation less than 10; among 46 patients without recurrence/metastasis, 4 cases had > 10 pre-operative and post-operative CTCs, and 42 cases had < 10 pre-operative and post-operative CTCs.
The application of the pre-operation and post-operation CTC number more than or equal to 10 for predicting the sensitivity of the high risk of relapse/metastasis: the number of CTCs before and after the operation is more than or equal to 10, and the number of relapse or metastasis/total relapse metastasis is 78.3 percent; i.e., there were 21.7% false negatives.
Applying specificity that the number of CTCs before and after operation is more than or equal to 10 for predicting high risk of relapse/metastasis: specificity ═ number of pre-or post-operative CTCs < 10 and no recurrence or metastases/total non-recurrence metastases ═ 91.3%; i.e., there were 8.7% false positives.
3.2 comparative example
TABLE 2 statistics of relapse/metastasis and CTC number in control cases
| Recurrent and metastatic conditions | N | The number of CTC before and after operation is more than or equal to 5 (example) | The number of CTC after operation is more than or equal to 10 (example) | The number of CTC after operation is more than or equal to 5 (example) |
| Follow-up 3 years relapse/metastasis | 37 | 30 | 29 | 32 |
| Non-recurrence/metastasis | 46 | 10 | 11 | 16 |
According to the statistical results in table 2, the sensitivity and specificity of predicting high risk of recurrence/metastasis in the three control cases are calculated.
1) Application of pre-operation and post-operation CTC number not less than 5 for predicting recurrence/metastasis risk
The application of the pre-operation and post-operation CTC number more than or equal to 5 for predicting the sensitivity of the high risk of relapse/metastasis: the susceptibility is equal to or more than 5 CTC numbers before and after operation and 81.0% recurrences or metastases/total recurrences metastases.
Applying specificity that the number of CTC before and after operation is more than or equal to 5 for predicting high risk of relapse/metastasis: specificity ═ number of pre-or post-operative CTCs < 5 and no recurrence or metastases/total non-recurrence metastases ═ 78.2%.
2) Application of postoperative CTC number more than or equal to 10 for predicting recurrence/metastasis risk
The application of the postoperative CTC number is more than or equal to 10 to predict the sensitivity of high risk of relapse/metastasis: the number of postoperative CTCs was ≥ 10 and 78.3% relapse or metastasis/total relapse metastasis.
Applying specificity that the number of CTCs after operation is more than or equal to 10 for predicting high risk of relapse/metastasis: specificity ═ number of postoperative CTCs < 10 and no recurrence or metastases/total non-relapsed metastases ═ 76.1%.
3) Application of postoperative CTC number more than or equal to 5 for predicting recurrence/metastasis risk
The application of the postoperative CTC number is more than or equal to 5 to predict the sensitivity of high risk of relapse/metastasis: the number of postoperative CTCs was ≥ 5 and the number of relapses or metastases/total number of relapsed metastases was 86.4%.
Applying specificity that the number of CTCs after operation is more than or equal to 5 for predicting high risk of relapse/metastasis: specificity ═ number of postoperative CTCs < 5 and no recurrence or metastases/total non-relapsed metastases ═ 65.2%.
In conclusion, the invention can more conveniently and timely reflect the tumor recurrence/metastasis risk of patients compared with the traditional imaging examination and tissue sample biological index identification based on the detection and evaluation of the CTC number of peripheral blood samples. Meanwhile, the kit has the advantages of convenience in sample collection, no side effect, high sensitivity, strong specificity, capability of real-time detection, easiness in acceptance by patients and the like.
Claims (7)
1. A system for assessing risk of recurrence and metastasis of malignant tumor based on circulating tumor cell detection is characterized in that: the malignant tumor recurrence and metastasis risk assessment system comprises a circulating tumor cell enrichment module, an immunofluorescence counting module and a risk judgment module;
the circulating tumor cell enrichment module is used for separating circulating tumor cells in a sample;
the immunofluorescence counting module is used for detecting the number of the circulating tumor cells obtained through separation;
the risk judgment module is used for predicting the recurrence or metastasis risk of the malignant tumor by respectively comparing the numbers of circulating tumor cells in samples collected before and after the malignant tumor treatment intervention with threshold values.
2. The system of claim 1, wherein the risk of recurrence and metastasis of malignant tumor is assessed by using circulating tumor cells as the basis: the circulating tumor cell enrichment module comprises equipment for separating circulating tumor cells from peripheral blood respectively collected before and after operation of a malignant tumor patient by utilizing a microfluid principle based on cell size and inertia.
3. The system of claim 2, wherein the risk of recurrence and metastasis of malignant tumor is assessed by using circulating tumor cells as the basis: the sampling volume number of the peripheral blood is 7.5-10 mL/person.
4. The system of claim 2, wherein the risk of recurrence and metastasis of malignant tumor is assessed by using circulating tumor cells as the basis: the peripheral blood is collected 1 day to 1 month before the operation and 1 day to 1 month after the operation.
5. The system of claim 1, wherein the risk of recurrence and metastasis of malignant tumor is assessed by using circulating tumor cells as the basis: the immunofluorescence counting module comprises a fluorescence microscope and a computer platform for analyzing the cellular immunofluorescence staining result collected by the fluorescence microscope; the immunofluorescence staining result of the circulating tumor cells is DAPI +/CK +/CD 45-.
6. The system of claim 1, wherein the risk of recurrence and metastasis of malignant tumor is assessed by using circulating tumor cells as the basis: the prediction result of the risk judgment module comprises the following steps: and judging the malignant tumor patients with more than 10 circulating tumor cells in the samples collected before and after the therapeutic intervention as high tumor recurrence or metastasis risk.
7. The system of claim 1, wherein the risk of recurrence and metastasis of malignant tumor is assessed by using circulating tumor cells as the basis: the malignant tumor is selected from any one of lung cancer, breast cancer and digestive system tumor.
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| CN113161000A (en) * | 2021-05-06 | 2021-07-23 | 复旦大学附属中山医院 | Mixed cell type liver cancer prognosis scoring model and construction method thereof |
| CN113552375A (en) * | 2021-06-02 | 2021-10-26 | 宁夏金域医学检验所（有限公司） | Automatic discard cap and divide blood device |
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| CN114354596A (en) * | 2021-11-12 | 2022-04-15 | 万贝医疗健康科技(上海)有限公司 | Equipment for evaluating activity state of liver and gall bladder cancer cells after chemoradiotherapy |
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