CTC检测

NattaonTansakul, §DongyanGu, § and Jinquan Zhang§*

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China

ABSTRACT:Circulating tumor cells(CTC) are defined as cells that have detached, spontaneously or as a result of clinical operations，from a primary tumor or its metastatic lesions and circulate in the peripheral blood. They are considered as the primary reason for postoperative recurrence and distant metastasis of malignant tumors. In clinical practice, the target treatment of cancer patients needs constant monitoring of the patient's gene mutation, and the traditional method of genetic testing is inconvenient, while CTC is featured with simple sampling, real-time, comprehensive assessment of tumor characteristics, therefore it has provided help for the individualized treatment of the patients with tumor. This article briefly reviews the recent advances in CTC testing and its clinical application.

INTRODUCTION

In tumor metastasis, tumor cells are separated from the primary site and disseminated through the circulatory system and form distant secondary tumors. This is the main cause of death in cancer patients.1In 1869, Ashworth first discovered and proposed the CTC concept.2CTC are defined as tumor cells that travelin the peripheral blood circulation. Tumor cells that have not been cleared by circulation have migrated, adhered and agglutinated to form tiny tumor thrombus, and metastasized under certain conditions.3At present, the latest definition of CTC is the cell that fall off from primary lesion, get into the blood circulation through the blood vessels or lymphatic system, and reflect the tumor tissue. CTC can be used for noninvasive pathology diagnosis, molecular sequencing, disease monitoring, etc. These cells can not only be used to dynamically monitored, but also be used to determine the prognosis. 4

enrich the CTC: Immunomagnetic beads (IMB) and Density gradient centrifugation (DGC).

IMB is to screen tumor cells with tumor-associated antigens ( such as epithelial cellular adhesion molecule，EpCAM）from peripheral blood.The cell surface antigen specific antibody and anti EpCAM antibody beads binding, formation of antigen - antibody - immunomagnetic complexes, the complexes occur in the presence of an external magnetic field the magnetic attraction, temporarily fixed, which contains a target antigen to CTC cells separated from the other, so as to the specific purpose of separation. Because the magnetic beads used are in nanometer size (10~100 nm), the detection process will not cause cell damage. The method has high specificity, complete cell morphology preservation, which can reduce interference factors in subsequent identification, and the CTC antigen have multiple targets and can improve detection rate.6

DGC is used the density of cells, which can be stratified after centrifugation, this method helps to separate CTC from granulocytes and erythrocytes in the blood.7, 8This method has low cost, simple operation, after centrifugation can retain the activity of CTC.But the similarity of mononuclear cell density and CTC in the peripheral blood is difficult toseparatefrom each other completely. Sothe specificity to separate these two cells is not high,if the blood samples collection is improperly, the blood would form clots and can alter tumor cell density, then affect the separation.

In addition to the above two methods,there are also some other ways to enrich the CTC, for example, Qin used the resettable cell trap mechanism to separate cells based on their size and deformability using an adjustable aperture that can be periodically cleared to prevent clogging. 10

Emerging CTC capture strategies typically distinguish these cells based on cultured cancer cells.9 Detection

According to the detection principle, the current methods of CTC number detection can be divided into two categories: cell

CTC CELL TEST TECHNIQUE

There are many methods to test the number of CTC，but the key technology of all can be divided into two parts: the first one is Enrichment of CTC, and the second is the Detection of CTC. In simple terms;because CTCare present in the bloodstream at a low concentration, for example, ranging between 1-10 cells per 10 ml in the majority of patients with PCA (prostate cancer) cancer, we need to use physical or chemical methods to extract and enrich the CTC and other suspicious cells and remove other cells we do not need. The second step, detection, is to further identify whether the cells enriched is CTC based on immunochemistry or PCR. Thus the entire technique is screening the peripheral blood cells with a sieve, then removing the impurity cells from a large number of blood cells, and finally identifying them according to the characteristics of the tumor cells themselves, such as tumor cell surface specific antigens. Enrichment

CTC can be isolated from blood cells based on cell size, density and immunoselection.There are mainly two ways to

5

counting method and nucleic acid detection method. The former mainly includes a variety of immunocytochemistry and flow cytometry; the latter includes polymerase chain reaction, reverse transcription polymerase chain reaction.

Immunocytochemistry(ICC) is based on antigen-antibody binding. Immunoselection is the most commonly used approach for CTC detection, which relies on specific CTC markers that are detected by antibodies.11

Reverse transcription polymerase chain reaction(RT-PCR) is to expand the tumor cell marker gene or mRNA to prove the presence of tumor cells, but the higher false positives and the possible false negative limits its clinical use. 12-14

Flow cytometry(FCM) is a new technology in laser physics, electronics, computer, photoelectric

measurement, cell

fluorescence chemistry and monoclonal antibody technology. Its advantage is the number of tumor cells can be quantitative counting, accurate detection data, but also for multi parameter analysis of the cells.18

Polymerase chain reaction (PCR)is mainly detected by detecting oncogenes, mutations in tumor suppressor genes, or abnormal

DNA.

produced by chromosome

rearrangements.15

However, except for a few solid tumors of chromosome translocation or gene mutations are specific, DNA change in the vast majority of solid tumors have no obvious specificity, and rarely suitable for CTC detection of,19 so the method is of high sensitivity, but prone to false positives, and the application range is limited. In addition, because in the peripheral blood CTC and the half-life of nucleic acid is not stable, the detected free D NA may be a nucleic acid instead of the tumor cells, so the application of PCR technology through the specific detection of CTC marker DNA index is not high, in the clinical practice is still not reach good results CellSearch system (CSS)

It employs ferromagnetic nanoparticles coupled to EpCAM antibodies for CTC capture, and became the first validated CTC assay approved by the Food and Drug Administration (FDA) in 2008.16The commonly used steps of CSS are: 1) Measure 7.5ml peripheral blood from the CellSavc tube into the centrifuge tube, then add 6.5ml buffer, centrifuge it after mixing. The cells in the blood will precipitate at the bottom of the centrifuge tube; 2) After the centrifugation, the supernatant in the tube was aspirated, then the buffer and the nano-immunomagnetic beads were added to the bottom cell layer; 3) The cellular components with the nano-immunomagnetic beads and the buffer were incubated in the magnetic field; 4) After the incubation, the liquid and unbound magnetic particles are aspirated; 5) Remove the magnetic field, and re-suspended the cells enriched by the use of immune magnetic particles in the buffer; 6) Add the fluorescent dye-binding antibody to the enriched cells. Wash

after dyeing; 7) The system captures the cells stained with the fluorescent dye automatically into the sample box with the MagNest and incubates them for more than 20 minutes in the dark; 8) Due to the magnetic field of MagNest, the captured target cells will move to the analytical surface of the sample box to form a single cell layer;9) The system analyzes the results automatically and the final interpretation will be obtained by the operator.

And there are also some limitation in CellSearch System: 1)EpCAM‑negative tumor cells may not be detected. Downregulation of EpCAM may occur during the EMT (epithelial‑mesenchymal transition); 2) Unstable: The morphology, characteristics and cell markers of CTC will change with the changes in the internal environment: for example, kidney cancer cell expresses CK8/18/19, but the tumor cell antigen of CTC mutates after removing into the peripheral blood tumor, such as CK8/18/19 low expression or even not expressed; 3) There is no unified criteria to identify if the sample is positive or not.17

FUNCTION

Diagnosis

For routine testing of tumors, for example, imaging, the tumor is less than a centimeter, and the doctor does not think it is abnormal, even if the PET-CT with the highest resolution can only detect tumor tissues above 5 mm.20 But in many cases, tumor cells have entered the blood circulation at 2-4 mm, and in this sense, it has an understated significance for early diagnosis. Determine the stage

CTC can also guide the staging of tumors and the higher the CTC count, the higher the staging of the disease. Schwarzenbach detected peripheral blood CTC in 69 patients with stage M0 and 12 cases of stage M1 prostate cancer by means of epithelial dot blot, and found that the occurrence of CTC was significantly related to the stage of tumor. In addition, in patients with esophageal cancer and lung cancer, the positive rate of CTC in patients with stage III / IV is higher than that in patients with stage I / II, and the stage is also higher than that in stage I / II.21-23

Monitor the effect of drug treatment

The change of CTC count before and after treatment can reflect the curative effect, and it is a reliable method for treatment and monitoring. It provides an important basis for clinicians to evaluate the curative effect and correct the treatment plan.24

Guide individualized treatment

Targeted therapy has become the main treatment of malignant tumors. The efficacy of molecular targeted drugs is closely related to whether tumor cells have corresponding targets, suggesting that treatment needs to vary from person to

person, that is, individualized treatment. Targeted drugs play an important role in specific target genes, and their efficacy is closely related to the genetic information of cancer patients. In targeted therapy, the patient's gene discovery mutation requires real-time detection of the patient's genetic status, and pathological examination as an invasive test, such as repeated testing in the short term, will lead to patient intolerance. CTC, as a \"liquid biopsy\comprehensive assessment of tumor characteristics, and can monitor gene changes of patients in real time.25 Prognosis assessment

Tien found that the number of CTC in the portal vein could also be an important predictor of liver metastasis. CTC count is associated with prognosis. The higher the count, the worse the prognosis.26

CONCLUSION

The biggest obstacle to the clinical application of CTC is still the test technique.CSS is the relatively more authoritative, accurate, highly automated CTC test technique, has reached satisfactory results in breast cancer, prostate cancer, colorectal cancer and other CTC measurements, but according to the domestic and foreign literature reports and experience of the detection of kidney cancer by CSS, the results in patients is not satisfied, and costly.Tumor markers of various tumor cells need to be further elucidated so that different and more specific antibodies can be used to bind to CTC surface antigens for morphological analysis to improve accuracy.However, due to the advantages of CTC detection compared with the traditional biopsy technique and its potential application in clinical practice, it inevitably becomes a far-reaching research field. More and more test technology is reported, and researchers attach more importance to CTC.

AUTHOR INFORMATION

Corresponding Author

*Email: jinquanzhang@zju.edu.cn

Author Contributions

§

These authors contributed to the manuscript preparation equally.

Notes

The authors declare no competing financial interest.

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