CN112946271A - Method and kit for diagnosing malignant dysplasia in a subject - Google Patents
Method and kit for diagnosing malignant dysplasia in a subject Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention discloses methods and kits for diagnosing malignant dysplasia in a subject. The method comprises the step of identifying marker cells in an enriched liquid phase obtained by removing red blood cells and white blood cells from peripheral blood by using a reagent, wherein the marker cells are DAPI+/PTPRC‑/PECAM1‑A cell, and the chromosome is heteroploid. The method can effectively distinguish benign tumors from malignant tumors, and has higher diagnostic value compared with the traditional tumor marker-based method.
Description
Technical Field
The present invention relates to the field of diagnostics, in particular to methods and kits for diagnosing malignant dysplasia in a subject.
Background
Various factors stimulate the living body to actively divide cells and increase the number of cells, resulting in various diseases called abnormal hyperplasia or abnormal proliferation including benign tumors and cancer after malignant or malignant abnormal hyperplasia or proliferation. Early diagnosis of malignant dysplasia is therefore crucial for early and timely intervention of the disease. For example, Ovarian Cancer (OC), which is the second most common gynecological cancer, is characterized by a higher number of deaths than any other gynecological malignancy. Due to the lack of an effective early diagnostic method, more than 70% of cases are diagnosed as advanced. Despite optimal cytoreductive surgery and standard platinum chemotherapy, most patients remain incurable and more than 70% relapse within 2-3 years. These factors, taken together, result in a 5-year survival rate of less than 30%. The difficulty of early diagnosis and the ease of metastasis are the major causes of death. The exploration of biomarkers for OC diagnosis and relapse monitoring is of great importance to improve survival in OC patients.
Circulating Tumor Cells (CTCs) are considered as seeds of blood-borne metastases, are a novel tumor biomarker, represent the major fluid biopsy method, and have the advantages of being noninvasive and real-time. CTCs have been approved by the U.S. food and drug administration and have shown diagnostic, prognostic, and predictive value in many types of solid malignancies. In OC, studies on CTCs have been limited because intra-abdominal implantation metastasis has traditionally been considered as the primary pathway of OC metastasis, and blood-borne metastasis has not been considered.
Although recent studies have shown that hematogenous metastases are also an important mode of OC metastasis, including the common peritoneal retinal metastasis (Pradeep S, Kim SW, Wu SY, et al. It is believed that there may be two major transfer mechanisms in the OC: passive diffusion and blood-borne transfer (Tsz-Lun Yeung, Cepilia S.Leung, Kay-Pong Yip, et al. cellular and Molecular Process in OC measurement.A review in the same: Cell and Molecular Process in Cancer measurement.Am J physical Cell physical 2015; 309: C444-C456). The discovery of CTC in OC has also been an important evidence for blood-borne transfer of OC (Van Berckelaer C, Brouwers AJ, Peeters DJ, et al, Current and future role of circulating tumor cells in tissues with intrinsic viral cells Eur J Surg Oncol 2016; 42: 1772-. A number of studies have demonstrated that CTCs have potential clinical utility in the diagnosis, efficacy assessment and prognosis assessment of OCs.
The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.
Disclosure of Invention
In view of the current state of the art, the present inventors have found, after intensive studies, that malignant dysplastic diseases in vivo can be effectively diagnosed by identifying a specific type of cells as marker cells in an enriched liquid phase obtained by removing erythrocytes and leukocytes from peripheral blood. The present invention has been accomplished, at least in part, based on this. Specifically, the present invention includes the following.
In a first aspect of the invention, there is provided a method for diagnosing malignant dysplasia in a subject, comprising the step of identifying marker cells in an enriched liquid phase obtained by removing red blood cells and white blood cells from peripheral blood using an agent, wherein the marker cells are DAPI+/PTPRC-/PECAM1-A cell, and the chromosome is heteroploid.
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention, wherein the presence or absence of the heteroploid in the cell is detected by CEP8 (chromosome 8 centromeric probe).
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention, wherein the heteroploids are triploid and tetraploid.
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention, wherein the agent comprises DAPI, an anti-PTPRC antibody and an anti-PECAM 1 antibody.
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention, wherein the abnormal cell proliferation is malignant abnormal proliferation.
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention comprises identifying marker cells by performing immunofluorescent staining simultaneously with chromosomal fluorescent in situ hybridization.
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention comprises the steps of:
(1) adding an antigen retrieval buffer solution and a staining mixed solution into the enriched liquid phase for a binding reaction, centrifuging and removing a supernatant to obtain a cell sap, wherein the staining mixed solution comprises a DAPI (deoxyribose nucleic acid), an anti-PTPRC (protein tyrosine phosphatase receptor) antibody and an anti-PECAM 1 antibody, and each antibody is coupled with different fluorescent groups respectively;
(2) adding a fixing solution into the cell sap, uniformly mixing, coating the cell sap on a glass slide, drying, and adding a chromogenic reagent containing a CEP8 probe for hybridization to obtain a detection sample;
(3) selection of DAPI by color under fluorescent microscope+、PTPRC-、PECAM1-And simultaneously, the cells with chromosome abnormality are used as the marker cells for diagnosis.
In certain embodiments, the method for diagnosing malignant dysplasia in a subject according to the present invention, wherein the enriched liquid phase is obtained by a method comprising the steps of: centrifuging peripheral blood collected from a subject, removing supernatant, adding cleaning solution, mixing, performing density gradient separation to obtain three layers of liquid, mixing the top layer of liquid and the middle layer of liquid, adding leukocyte antibody combined with magnetic beads, and performing magnetic separation to obtain an enriched liquid phase.
In certain embodiments, the method for diagnosing malignant abnormal proliferation in a subject according to the present invention, wherein the subject is diagnosed as having malignant abnormal proliferation or being at high risk of having abnormal proliferation of cells in the subject when 2 or more marker cells are present per 5-7ml of peripheral blood, and the subject is diagnosed as not having malignant abnormal proliferation or being at low risk of having abnormal proliferation of cells in the subject when less than 2 marker cells are present per 5-7ml of peripheral blood.
In a second aspect of the present invention, there is provided a kit for diagnosing abnormal proliferation of cells in an organism, comprising an agent for revealing the presence of PTPRC and PECAM1 on the cell surface or within the cells, DAPI and an agent for revealing chromosomal abnormalities of the cells. Preferably, the kit further comprises a reagent for displaying a tumor marker.
The method of the present invention can effectively diagnose malignant proliferative diseases. Compared with the traditional tumor marker-based method, the method has higher diagnostic value.
Drawings
FIG. 1 is an image of candidate cells identified from ovarian cancer peripheral blood.
FIG. 2 is a graph of the differential distribution of candidate cells between benign and malignant ovarian cancer groups.
FIG. 3 is a graph showing the differential distribution of different ploid subclasses of candidate cells.
FIG. 4 is a graph of the differential distribution of different ploidies of cell lines CAOV-3 and SKOV-3.
FIG. 5 is a distribution of differences in cell size subsets of candidate cells.
FIG. 6 is an aneuploidy analysis of candidate cells of different sizes.
FIG. 7 is a ROC curve for different ploid subclasses.
FIG. 8 is a ROC curve for each subset classified according to ploidy number and cell size.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is a percentage based on the amount.
There were more contaminating cells in the CTC cells identified in the previous studies. The presence of these contaminating cells leads to the presence of more false positives in the diagnosis of the disease, which in turn greatly reduces accuracy. The inventors found that interference of foreign cells can be effectively eliminated by selecting a specific antigen as an index. In addition, given that CTC cells are not uniform in size, but vary, conventional methods of isolating CTC cells from peripheral blood inevitably exclude a fraction of small diameter cells, which are also of significant value for diagnosis. This is extremely disadvantageous for the detection of CTC cells with an inherently small number of cells.
The term "abnormal cell proliferation" as used herein refers to a general term for a group of diseases caused by excessive cell division in vivo, and in general, abnormal cell proliferation includes tumors and cancers after malignant transformation. In the present invention, the preferred abnormal cell proliferation is malignant abnormal proliferation such as cancer. Examples of cancer are not particularly limited and include, but are not limited to, ovarian cancer, endometrial cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, bladder cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, nasopharyngeal cancer, skin cancer, and the like. Gynecological cancers, particularly ovarian cancers, are preferred in the present invention.
The "marker cell" of the present invention means a cell which can be used as a basis or index for diagnosis or judgment of a disease. The marker cells of the invention belong to a particular class of CTC cells, which are in particular peripheral blood-derived DAPI+/PTPRC-/PECAM1-Cells that were positively stained for DAPI, negative for both PTPRC and PECAM1, and chromosomal, particularly chromosome 8, abnormal. If the chromosome is abnormal, the marker cell of the present invention can be identified. In certain embodiments, the marker cells of the invention not only present chromosomal abnormalities, but also require that the chromosomes, particularly chromosome 8, be polyploid, preferably 3-and/or 4-fold. The size of the labeled cells of the present invention is also not limited, and cells having any diameter may be used as long as they are positively stained by DAPI. In certain embodiments, the marker cells of the invention comprise in particular cells of diameter below 5 microns, and such cells of diameter represent more than 30%, preferably more than 35% of the total amount of marker cells.
The "enriched liquid phase" in the present invention refers to a mixed solution enriched in candidate cells obtained by removing red blood cells and white blood cells, which account for a large part of blood, from peripheral blood. Since only red blood cells and white blood cells as impurities are removed from peripheral blood, the target cells are not lost. The rich liquid phase of the present invention is therefore also referred to as a poor rich liquid phase. The preparation of the enriched liquid phase can be carried out in a known manner. The preparation process is exemplified below.
In the present invention, the numerical value indicating the number or number of cells is generally a natural number, for example, 1, 2, 3, 4, 5, or the like. In the case of number statistics, such as counting multiple times or measuring and counting the average, the number of cells can be expressed as a decimal number, such as 1.5, 1.8, etc.
In a first aspect of the present invention, there is provided a method for diagnosing abnormal proliferation of cells in a body, sometimes referred to simply as "the diagnostic method of the present invention", which comprises the step of identifying marker cells in an enriched liquid phase obtained by removing erythrocytes and leukocytes from peripheral blood using an agent.
In the present invention, the reagent is not particularly limited as long as it can indicate the presence or state of marker cells. The reagent may be one reagent or a combination of reagents. In general, the agents of the present invention include an indicator capable of indicating whether a cell chromosome is abnormal, DAPI, and an agent capable of indicating whether a cell chromosome is abnormal, which are selected from the group consisting of PECAM1 and PTPRC. Preferably, the indicator may comprise a moiety capable of displaying a color and a moiety capable of specifically binding to a specific component within or on the surface of the marker cell. Preferably, the two moieties are bound to each other by means of, for example, a covalent bond. The portion capable of displaying color may be a known colorant, pigment, dye, fluorescent agent, or the like. Moieties capable of specifically binding to a particular moiety within or on the surface of a marker cell include substances capable of binding to nucleic acids, such as DNA probes or other complementary fragments of nucleic acids, and also include antibodies, receptors or ligands, or modifications or derivatives thereof.
The antibody of the present invention includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, nanobodies, humanized antibodies or fully human antibodies. The antibody of the invention may also be a single chain antibody. In the present invention, the modified antibody includes a chemical modified antibody and a conjugate of an antibody and another material. Examples of chemical modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, cross-linking cyclization, disulfide bonding, demethylation, covalent cross-linking, cysteinylation, pyroglutamylation, formylation, gamma-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolysis, phosphorylation of antibodies, and the like. Examples of the conjugate include, but are not limited to, conjugates with a nano polymer material, magnetic beads, and the like.
In certain embodiments, the reagents of the invention comprise an antibody and a detectable group, such as a fluorescent group, coupled thereto. In the present invention, in the case of a plurality of reagents, it is preferable that the detectable group of each reagent is different.
In certain embodiments, the agents of the invention further comprise an agent for revealing a tumor marker. Such agents may use agents known in the art, such as various tumor-targeted antibodies, examples of which include, but are not limited to, antibodies against CA125, HE4, PDL-1, methothelin, Kallikrein, osteopontin, claudin, TP53, c-myc, Bcl2, Bcl6, IgH, HER-2, MUC1, platelet Receptor, E-cadherin, N-cadherin, EpCAM, cytokeratin, vimentin, CD44, CD133, CD24, CD117, and the like. The present invention may use a combination of one or more of the above-described tumor marker antibodies.
In the present invention, the method for identifying marker cells is not particularly limited, and preferably includes a step of performing immunofluorescent staining and a step of fluorescence in situ hybridization of chromosomes. In an exemplary embodiment, the identification of the present invention comprises the steps of:
(1) adding an antigen retrieval buffer solution and a staining mixed solution into the enriched liquid phase for a binding reaction, centrifuging and removing a supernatant to obtain a cell sap, wherein the staining mixed solution comprises a DAPI (deoxyribose nucleic acid), an anti-PTPRC (protein tyrosine phosphatase receptor) antibody and an anti-PECAM 1 antibody, and each antibody is coupled with different fluorescent groups respectively;
(2) adding a fixing solution into the cell sap, uniformly mixing, coating the cell sap on a glass slide, drying, and adding a chromogenic reagent containing a CEP8 probe for hybridization to obtain a detection sample;
(3) PECAM1 selection by color under fluorescent microscope-、DAPI+、PTPRC-And simultaneously, the cells with chromosome abnormality are used as the marker cells for diagnosis.
Optionally, the present invention further comprises (4) a step of obtaining an enriched liquid phase from peripheral blood.
Enrichment by differential phase enrichment is preferred in the present invention. Differential phase enrichment is completely independent of the expression of tumor cell surface markers (such as EpCAM), and the immune magnetic particles of a special monoclonal antibody combination group coupled with anti-human leukocyte surface antigens and a special centrifugal medium for separating blood-derived cells are used for quickly and efficiently removing red blood cells, white blood cells and plasma proteins in the blood of a tumor patient, so that rare cells in body fluid specimens such as blood and the like are effectively enriched, and the rare cells comprise circulating tumor cells, circulating vascular endothelial cells, stem cells and the like derived from solid tumors of all different tissues. The obtained tumor cells are not wrapped by the magnetic beads and are not combined with any antibody of the anti-tumor cell surface marker, so that the signal transduction path in the cells is prevented from being activated. Compared with other conventional methods, the removal of erythrocytes in the differential phase enrichment method avoids the use of hypotonic lysis, thereby minimizing the damage to the enriched tumor cells and maintaining the acquired target cells in a good natural state and cell morphology. In addition, the use of differential phase enrichment can avoid the disadvantage of missing large numbers of small cells when screening cells based on cell size, e.g., microfluidic technology, and in particular can avoid the loss of effective cells, e.g., less than 5 μm in diameter.
In an exemplary embodiment, the enriched liquid phase preparation of the present invention comprises the steps of: centrifuging peripheral blood collected from a subject, removing supernatant, adding cleaning solution, mixing, performing density gradient separation to obtain three layers of liquid, mixing the top layer of liquid and the middle layer of liquid, adding leukocyte antibody combined with magnetic beads, and performing magnetic separation to obtain an enriched liquid phase.
The diagnostic method of the present invention is within the scope of the present invention as long as it comprises the step of identifying the marker cell of the present invention. In a preferred embodiment, the diagnostic method of the present invention further comprises a judging step in addition to the above-mentioned identifying step.
In the present invention, the determining step generally includes classifying the results of the identifying step based on a predetermined threshold and confirming the diagnosis result therefrom. The threshold value of the present invention may be the number of marker cells. Since the marker cells of the present application are specific CTC cells, the content thereof in peripheral blood is very small. Therefore, the number of marker cells detected in 5 to 7ml of peripheral blood, particularly 6ml of peripheral blood is generally used as a standard. In an exemplary embodiment, the threshold value of the present invention is generally above 2 per 6ml of peripheral blood, preferably above 3 per 6ml of peripheral blood. It is to be noted that the marker cells of the present invention may be further subdivided into different subsets or subgroups. When a subset of marker cells is used as a marker, the selection of the threshold value of the present invention will generally vary. For example, when the judgment is made based on only 4-fold marker cells, the threshold value at this time is about 1 per 6ml of peripheral blood.
In a second aspect of the present invention, there is provided a kit for diagnosing abnormal proliferation of cells in an organism, which generally comprises an agent for revealing cell surface or cell content selected from PTPRC and PECAM1, DAPI and an agent for revealing chromosomal abnormality of the cells. Optionally, the kit of the present invention further comprises a reagent for displaying a tumor marker.
Examples
Patient and sample
The subjects recruited were 56 women admitted to the Beijing university Hospital at 5-2020-8 months in 2018. Peripheral blood was collected preoperatively from patients who were highly suspected of being malignant or specifically diagnosed but not receiving treatment. Post-operative pathology confirmed 20 newly diagnosed primary OCs and 36 benign tumors of the ovary. Written informed consent was obtained from all patients according to the declaration of helsinki. The study was approved by the ethical committee of the national hospital, Beijing university, and was conducted according to the principles of the declaration of Helsinki.
Differential phase enrichment of candidate cells
6ml of peripheral blood was collected into tubes containing ACD anticoagulant (Becton Dickinson, Franklin Lakes, NJ, USA), and then centrifuged (200 Xg) at room temperature for 15 minutes to separate plasma. The supernatant above the brownish red precipitate was discarded, and the blood cell precipitate was diluted with 6mL of CRC buffer, then gently added to 3mL of sample density separation medium in a 50mL centrifuge tube, and then centrifuged at 450 Xg for 7 minutes. The solution containing WBCs and tumor cells higher than RBCs was collected into a 50ml tube, and then 300 μ L of magnetic beads coated with anti-leukocyte monoclonal antibodies (anti-PTPRCs, etc.) were added to the sample and shaken at room temperature for 30 minutes. Immune beads bound to WBCs were removed using a 50ml size magnetic separator (Cytelligen), followed by washing of non-blood derived cells (including tumor cells and endothelial cells) and enrichment of the remaining 100 μ L of CRC fluid. The sample is suitable for subsequent analysis.
Third, Immunostaining and Fluorescence In Situ Hybridization (iFISH)
To 100. mu.L of the enriched cell solution was added 2. mu.L of antigen retrieval buffer, gently mixed and shaken well, and then allowed to stand at room temperature for 10 minutes. The cells were then purified by direct interaction with fluorescently-labeled lymphocytes monoclonal antibody (anti-PTPRC-Alexa 594), endothelial cells monoclonal antibody (anti-PECAM 1-Cy5) and OC Tumor Marker (TM) monoclonal antibody (anti-CA 125-Cy7, anti-HE 4-Alexa488) at 1: incubate at 200 dilution for 20 min. The stained cell sample was washed with CRC wash and centrifuged (500 Xg) for 5 minutes, and then the supernatant was discarded, leaving 100. mu.L. An equal volume of tissue fixative was used to fix the cells overnight. Samples were coated on glass slides and hybridized in situ with CEP8 probe (Abbott Molecular, usa). Hybridization conditions were denaturation at 76 ℃ for 10min and hybridization at 37 ℃ for 3 h. The coverslip was slowly removed with the aid of a buffer. The cleaned slides were dried with a blower. After washing, the samples were fixed with fixing medium containing DAPI (blue). Images of the identified tumor cells were collected using a fluorescence microscope.
The judgment standard of the marker cell of the invention is as follows: DAPI+/PTPRC-/PECAM1-/CEP8+(1 mer/3 mer/4 mer/. gtoreq.5 mer). Specifically, PTPRC: staining was negative, the most obvious difference from leukocytes was a bright red halo of light that did not surround the nucleus. CEP 8: 1, 3, 4 or more and 5 or less. The signal was clear on either the red or orange channel. PECAM 1: green signal is negative, because PECAM1 binds to platelets, there is a clustered dot staining under green light, and the background level varies from person to person. DAPI: blue, circular or elliptical.
Fourth, data processing
All statistical analyses used SPSS version 24.0 (version 24.0; IBM corporation, N.Y., USA) and GraphPad Prism 7(GraphPad Software, La Holland, Calif., USA). The differences between the two groups were statistically analyzed using one-way analysis of variance (ANOVA) and unpaired student t-test, as were the differences between the three independent groups of data. The Fisher exact test was used to examine the correlation of CTC positivity with clinical pathology. Thresholds for CTC and CTEC were determined by nonparametric receiver operating characteristic curve (ROC) analysis and the maximum ewing index (sensitivity + specificity-1) was used to determine the cutoff. The classification data is expressed in percentiles. Correlation was determined using human correlation analysis. P values <0.05 were considered statistically significant.
Five results
Both CA125 and HE4 have proven to be of significant value in the clinical diagnosis of OC as Tumor Markers (TM). Either CA125 positive or HE4 positive or double positive is considered TM positive. CTCs of the following criteria, i.e., nucleated cells characterized as having TM positivity and/or aneuploidy but not having PTPRC and PECAM1 expression, were selected as candidate cells in the present invention. More specifically, CTC is defined as: DAPI +/PTPRC-/PECAM1-/TMs + and/or DAPI +/PTPRC-/PECAM1-/CEP8 ≠ 2 (FIG. 1). For the purpose of convenience of description, CTCs are sometimes also referred to simply as CTCs hereinafter and in the drawings, but it is specifically stated that CTCs hereinafter and in the drawings belong to a specific class of cells within the scope of CTCs as generally understood in the art.
1. Distribution of CTC counts in OC and control groups
The counts of CTCs from 20 OC patients were compared to 36 diagnosed ovarian benign tumors. FIG. 2 shows that 210 CTCs (10.5/6 ml on average) were detected in 19/20 (95.0%) OC patients. CTCs were also detectable in 34/36 (94.4%) patients with benign tumors of the ovary, totaling 221 (mean 6.14/6 ml).
2. CA125 and HE4 expression characteristics and Chr8 heteroploidy of CTC
Expression of CA125 and HE4 on the cell membrane and cytoplasm of CTCs was identified by immunofluorescence staining. The ploidy of Chr8 in CTCs was detected by fluorescence in situ hybridization. The results are shown in table 1. CA125 positive CTCs were detected in 4 OC patients (4/20, 20.0%) accounting for only a relatively small proportion of total CTCs (10/210, 4.8%). HE4 positive CTCs were detected in 5 OC patients (5/20, 25.0%) accounting for only 4.3% of total CTCs (9/210).
TABLE 120 CA125 and HE4 expression profiles and Chr8 isotopy in CTC in ovarian cancer patients
Regardless of the positive detection rate of OC patients, the overall count ratio of CA125+ or HE4+ CTCs was low. However, CTCs with Chr8 aneuploidy were found in 19/20 (95.0%) OC patients. Furthermore, of the 210 heterogeneous CTCs, only one was diploid CA125 positive cells, and the other 209 CTCs (99.5%) were aneuploidies of chr 8. Taken together, the number of TMs positive CTCs was insufficient and the main feature of CTCs in our study was chr8 aneuploidy.
Analysis of aneuploidy subtype in CTC
In our study, most CTCs exhibit different subsets of Chr8 aneuploidy, i.e., haploid, triploid, tetraploid and above pentaploid. Fig. 3A shows an image of an exemplary CTC.
Since few haploid CTCs were detected in our study, no data was analyzed. Figure 3B shows that the differential distribution of triploid and tetraploid CTCs was more pronounced in the two groups compared to CTC counts (0.0068 and 0.0003, respectively). There were no statistical differences in the distribution of CTCs above pentaploid (P-0.7578). Interestingly, although more than pentaploid CTCs were predominantly detected in the benign or cancer group, the difference between triploid and tetraploid CTCs was more pronounced in the OC group compared to the other subtypes.
To explain the existence of a large number of more than pentaploid CTCs in the benign group, we investigated the Chr8 polyploidy in the ovarian cancer cell lines CAOV-3 and SKOV-3. Fig. 4A shows the status of Chr 8-fold cells detected by FISH. The percentage of cells with polyploid Chr8 in both cell lines is shown in fig. 4B. Interestingly, in cell line samples, cells with aneuploid Chr8 were found to be predominantly triploid or tetraploid, which are also common in CTCs of OCs. However, there are few cells with more than Chr8 pentaploid, and these cells account for the majority of CTCs in the OC or benign group.
Distribution of four, different size CTCs
The distribution of small (. ltoreq.5 μm) and large (>5 μm) cells in CTC is shown in FIG. 5A. FIG. 5B shows the distribution of small and large CTCs in the OC and benign groups, with statistically significant differences in the distribution of small CTCs alone (P0.0013). Fig. 5C shows that the proportion of small and large CTCs in OC patients was 38.57% (81/210) and 61.43% (129/210), respectively. For the benign group, small cell and large cell CTCs were 17.65% (39/221) and 82.35% (182/221), respectively. Large cells predominate in the cancer and benign groups, while the proportion of small CTCs in the OC group was higher than in the benign group.
Fifthly, aneuploidy analysis of CTC with different sizes
To further understand the importance of the various subtypes of CTCs, taking into account cell size, Chr8 ploidy, and disease, fig. 6A-C show the distribution of 12 subtypes of CTCs in the benign or OC group. Fig. 6A shows that only triploid and tetraploid cells in small cell CTCs differed significantly between benign and cancer groups (P ═ 0.0003 and 0.0002, respectively). Above pentaploid in small cell CTCs there is no way to distinguish cancer patients from benign patients. For large cell CTCs, only tetraploid cells showed statistically significant distribution differences between the two groups (fig. 6B).
Figure 6C shows the proportion of aneuploid cells of different sizes in benign or cancer groups. Triploid cells are the highest subset in small cell CTCs, and cells above pentaploid are the highest subset in large cell CTCs, whether benign or cancer patients.
Sixth, clinical value of CTC enumeration
To distinguish OC patients from non-malignant tumors, the total number of CTCs detected according to the invention, or the number of aneuploidies or cell size subtypes thereof, was plotted against a ROC curve to determine the sensitivity and specificity of the detection.
The optimal cut-off value was chosen based on the john index, with a cut-off value of 4.5/6ml for total CTC diagnosis of ovarian cancer of 75.00% sensitivity and 58.3% specificity.
In order to find a better index, the threshold and sensitivity and specificity of the CTC subclasses were next determined in the same way. The results are shown in table 2 below. The ROC curves for the subclasses are shown in fig. 7.
TABLE 2 threshold values and sensitivity and specificity of each ploidy subclass
As shown in Table 2 and FIG. 7, aneuploidy CTCs with triploids or tetraploids were of good diagnostic value for the AUC of ROC, 0.792 (cut-off 2.5/6ml, sensitivity 50.00%, specificity 94.44%) and 0.821 (cut-off 0.5/6ml, sensitivity 75.00%, specificity 80.51%), respectively. Based on this result, we tried to combine triploid CTCs and tetraploid CTCs as indicators in order to be able to get better clinical value. The AUC after combination was analyzed to be 0.853 (cutoff value of 2.5/6ml, sensitivity of 70.00%, specificity of 91.67%), higher than for the triploid or tetraploid alone.
Table 3 and fig. 8 show the diagnostic value of subgroups classified by aneuploidy and cell size. The difference in the distribution of these indices between benign and cancer groups was statistically significant. As shown in table 3 and figure 8, all CTC subsets had good clinical value, with AUC over 0.7, with the triploid and tetraploid small cell CTC groups exhibiting the best clinical diagnostic value (cut-off 1.5/6ml, sensitivity 70.00%, specificity 83.3%, AUC 0.809).
TABLE 3 threshold values and sensitivity and specificity for each ploidy, cell size subclass
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Claims (10)
1. A method for diagnosing malignant dysplasia in a subject comprising identifying marker cells from an enriched liquid phase obtained by removing red blood cells and white blood cells from peripheral blood using an agentWherein the marker cell is DAPI+/PTPRC-/PECAM1-A cell, and the chromosome is heteroploid.
2. The method of claim 1, wherein the presence of heteroploids in the cells is detected by CEP 8.
3. The method for diagnosing malignant dysplasia in a subject according to claim 1, wherein said heteroploids are triploids and/or tetraploids.
4. The method for diagnosing dysplasia in a subject according to claim 1, wherein said agent comprises DAPI, an anti-PTPRC antibody and an anti-PECAM 1 antibody.
5. The method of claim 1, wherein the dysplasia is ovarian cancer.
6. The method of claim 1, comprising identifying marker cells by simultaneous immunofluorescent staining and chromosomal fluorescent in situ hybridization.
7. The method for diagnosing dysplasia in a subject according to claim 6, comprising the steps of:
(1) adding an antigen retrieval buffer solution and a staining mixed solution into the enriched liquid phase for a binding reaction, centrifuging and removing a supernatant to obtain a cell sap, wherein the staining mixed solution comprises an anti-DAPI antibody, an anti-PTPRC antibody and an anti-PECAM 1 antibody, and each antibody is coupled with different fluorescent groups respectively;
(2) adding a fixing solution into the cell sap, uniformly mixing, coating the cell sap on a glass slide, drying, and adding a chromogenic reagent containing a CEP8 probe for hybridization to obtain a detection sample;
(3) selection of DAPI by color under fluorescent microscope+、PTPRC-、PECAM1-And simultaneously, the cells with chromosome abnormality are used as the marker cells for diagnosis.
8. The method for diagnosing dysplasia in a subject according to claim 7, wherein said enriched liquid phase is obtained by a method comprising the steps of:
centrifuging peripheral blood collected from a subject, removing supernatant, adding cleaning solution, mixing, performing density gradient separation to obtain three layers of liquid, mixing the top layer of liquid and the middle layer of liquid, adding leukocyte antibody combined with magnetic beads, and performing magnetic separation to obtain an enriched liquid phase.
9. The method of claim 7, wherein the subject is diagnosed as having or at high risk of having dysplastic disease or malignant dysplasia in the subject when more than 2 marker cells are present per 5-7ml of peripheral blood, and the subject is diagnosed as not having or at low risk of having dysplastic disease or malignant dysplasia in the subject when less than 2 marker cells are present per 5-7ml of peripheral blood.
10. A kit for diagnosing malignant dysplasia in a subject, comprising an agent for revealing the presence of PTPRC and PECAM1 on or in a cell, a DAPI agent and an agent for revealing chromosomal abnormalities in a cell.
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Non-Patent Citations (4)
| Title |
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| ZHENLONG YE ET.AL: "Detecting and phenotyping of aneuploid circulating tumor cells in patients with various malignancies", 《CANCER BIOLOGY & THERAPY》 * |
| ZHENLONG YE ET.AL: "Detecting and phenotyping of aneuploid circulating tumor cells in patients with various malignancies", 《CANCER BIOLOGY & THERAPY》, vol. 20, no. 4, 31 December 2019 (2019-12-31), pages 546 - 551 * |
| 赛特: "人外周血循环肿瘤细胞差相富集试剂盒说明书", 《人外周血循环肿瘤细胞差相富集试剂盒说明书》, 30 September 2017 (2017-09-30), pages 1 - 5 * |
| 邵志敏等主编: "《肿瘤医学 下》", 复旦大学出版社 , pages: 1555 * |
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