CN102735844B - Protein composition and preparing the application in pulmonary cancer diagnosis kit - Google Patents
Protein composition and preparing the application in pulmonary cancer diagnosis kit Download PDFInfo
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Abstract
The invention belongs to molecular biology, clinical detection technique field, be specifically related to comprise the protein composition being selected from P53, P63, Ki67, MCM7 and EGFR albumen, the ligand combination thing that protein-specific in protein composition is combined, and the application in preparation lung cancer, particularly early stage of lung cancer diagnostic kit of described protein composition or ligand combination thing.The present invention can be used for the diagnosis of lung cancer, especially the diagnosis of the early stage of lung cancer, and its sensitivity, specificity are high, are better than the detection of single protein marker.
Description
Technical field
The invention belongs to molecular biology, clinical detection technique field, be specifically related to the protein composition comprising P53, P63, Ki67, MCM7 and EGFR five albumen, and the application of described protein composition in preparation lung cancer, particularly lung squamous cancer and adenocarcinoma of lung diagnostic kit.
Background technology
Lung cancer is the modal malignant tumour of world, forms great threat to human health and life.U.S.'s statistics display in 2010, the mortality ratio of lung cancer is all positioned at the first place of parts of body tumour at masculinity and femininity, be respectively 29% and 26%.New cases all account for second in masculinity and femininity, are respectively 15% and 14% of each region tumors summation.5 years survival rates of 1999-2005 Nian Jian U.S. lung cancer are 16%, and wherein focal person 5 years survival rates are 53%, have metastases in local lymph node person to be 24%, have blood DISTANT METASTASES IN person to be only 4%.Overall 5 years survival rates of Europe lung cancer are 10.9%, well below 79% and 78% of breast cancer and prostate cancer.
In China, the data display that whole nation treatment and prevention of tumour office of the Ministry of Public Health provides, from between 2000-2005, the number of the infected of lung cancer in China estimates increase by 120,000 people, wherein male lung cancer patient was increased to 330,000 people of 2005 from 260,000 people of 2000, and the same period, female lung cancer patient was increased to 170,000 people from 120,000 people.Current China lung cancer morbidity rate increases by 26.9% every year, and as taked effective control measure not in time, expect 2025, China's patients with lung cancer will reach 1,000,000, becomes the first in the world lung cancer big country.Although in recent years all have new development in diagnostic method, surgical technic and chemotherapeutics, the total 5 years survival rates of patients with lung cancer are still very low.In China, lung cancer about causes 400 every year, 000 routine death.Why so low lung cancer overall 5 years survival rates are, main because be in late period when most patients with lung cancer is gone to a doctor, or occur more for a long time sending out transfer, so the key reducing patients with lung cancer mortality ratio is early warning, early detection, so that early intervention.Research display I phase lung cancer postoperative 10 years survival rates can reach 92%.
Screening lung cancer method conventional at present comprises chest x-ray, low-dose spiral CT (low-dosespiralCT, LDCT), expectorative cytology inspection, brushing piece or get biopsy, BALF cytology inspection etc. under bronchoscope, but all there is limitation in various detection methods in susceptibility, specificity, relevance grade etc.
Except above physical property test mode, also there is application blood serum tumor mark diagnosing at present clinically.Common mark comprises carcinomebryonic antigen (CEA), Squamous intraepithelial lesion (SCC-Ag), cytokeratin 21-1 fragment (CYFRA21-1), CA125 (CA125), CA153 (CA153), CA199 (CA199), neuronspecific enolase (NSE), tissue polypeptide antigen (TPA) etc.But it is all limited that these tumor markerses detect separately its Sensitivity and Specificity, though joint-detection increases, still face the problem that many benign lesions also can cause these tumor markers serological levels to increase as benign tumour, inflammation, degenerative disease etc.The mark of lung cancer marker, particularly early warning that further exploration is efficiently special is of crucial importance.
Lung cancer can be divided into non-small cell lung cancer and small-cell carcinoma of the lung, respectively accounts for 85% and 15% of lung cancer.Wherein non-small cell lung cancer comprises again squama cancer, gland cancer and large cell carcinoma.Because squama cancer and gland cancer are being in the great majority by the patient that diagnoses out, at present the Study on Molecular Mechanism of development is occurred to this two types lung cancer more.
Comprehensively current research, relate to cell cycle regulating, cell proliferation and differentiation, DNA to the relevant gene alteration of lung cancer morbidity mechanism to repair and Apoptosis etc. (as p53, CCND1, ki-67, Telomerase, TTF1 etc.), and the change of the exception of chromosome structure and gene expression dose also can cause tumour to occur.In recent years research proves, some molecular changes occurs in sequence, the development degree (squamous metaplasia-atypical hyperplasia-carcinoma in situ of the frequency that they occur and precancerous lesion; Atypical adenoma hyperplasia-gland cancer) synchronously raise.Therefore, the heterogeneity of tumor biological behavior can be reflected more rightly using the abnormal tumor markers as lung cancer of molecular level, and then provide more valuable guidance for the early warning of lung cancer.
Although achieve some progress, up to now, there is no any molecular marker determined can really for the early warning of lung cancer.Therefore, this area, and can effectively detection of lung cancer, the especially method of early detection lung cancer in the urgent need to working out the mark that can be used for lung cancer early diagnosis or mark combination.
Summary of the invention
The object of the present invention is to provide one can the protein composition of effectively detection of lung cancer, the particularly early stage of lung cancer, the described early stage of lung cancer be preferably early stage lung squamous cancer and early stage adenocarcinoma of lung.This combination has following characteristics:
A () be high expressed in patients with lung cancer tissue, cut low expression or do not express in end tissue in operation;
(b) low expression or do not express in other non-cancerous pulmonary lesion tissues such as tuberculosis, inflammation, pulmonary cryptococosis;
C () be strongly expressed in tumor patient bronchial brushing sample, low expression in lung inflammation, tuberculosis and other benign lesion/do not express;
D in () people taking physical examination, this protein combination expression positive generation lung-cancer-risk increases.
In the present invention, inventor passes through unremitting effort, from a large amount of cancer markers albumen, screen the protein marker obtained for pulmonary cancer diagnosis combine, the composition of described protein marker comprises following five kinds of albumen, P53, P63, Ki67, MCM7 and EGFR.
Particularly, the present invention relates to the following aspects:
One aspect of the present invention relates to a kind of protein composition, and it comprises at least four kinds that are selected from P53, P63, Ki67, MCM7 and EGFR albumen.In embodiments of the invention, in described protein composition be four kinds or five kinds.
In embodiments of the invention, it is following combination:
(1) EGFR, MCM7, P63 and P53 albumen;
(2) EGFR, Ki67, MCM7 and P53 albumen;
(3) EGFR, Ki67, P53 and P63 albumen;
(4) Ki67, P53, P63 and MCM7 albumen;
(5) Ki67, P63, MCM7, EGFR albumen;
Or
(6) P53, P63, Ki67, MCM7 and EGFR albumen.
In the present invention, described protein composition is the composition of protein marker, and it can be used as the detection of significant albumen for lung cancer.Therefore the invention also discloses the molecule with marker protein specific binding.
Another aspect of the present invention relates to a kind of ligand combination thing, its composition of part for being combined with the protein-specific described in protein composition of the present invention.
In the present invention, described part is the molecule that the protein-specific in protein composition of the present invention is combined.Part can be nucleic acid, polypeptide or compound.Part of the present invention can be peptide ligand, and in embodiments of the invention, described part is antibody.Antibody can be people's antibody, chimeric antibody, recombinant antibodies, humanized antibody, monoclonal antibody or its Fab or polyclonal antibody.In embodiments of the invention, described antibody is monoclonal antibody.
Another aspect of the present invention relates to a kind of chip, and on it, point has the part described in ligand combination thing of the present invention.Described chip and protein array, can be fixed on protein array by the antibody or other part with protein marker specific binding of the present invention, to detect in sample whether there is protein marker of the present invention.
Another aspect of the present invention relates to a kind of kit, and it comprises ligand combination thing of the present invention.
In the present invention, described kit also comprises damping fluid and developer.
Another aspect of the present invention relates to protein composition of the present invention or ligand combination thing or chip and is preparing the purposes in pulmonary cancer diagnosis kit.In embodiments of the invention, described lung cancer is lung squamous cancer or adenocarcinoma of lung.In one embodiment of the invention, described lung squamous cancer or adenocarcinoma of lung are Advanced Squamous all Carcinoma of Lung or advanced pulmonary adenocarcinoma.In one embodiment of the invention, described lung squamous cancer or adenocarcinoma of lung are early stage lung squamous cancer or early stage adenocarcinoma of lung.
In the present invention, described kit is used for the diagnosis of tissue sample or cell sample.In one embodiment of the invention, described tissue sample is that end tissue section sample is cut in lung cancer or operation.In another embodiment of the invention, described cell sample is the cell sample of bronchial brushing.
In the present invention, the determination methods of described kit is, when at least two kinds of parts in ligand combination thing of the present invention and testing sample react present the positive time, be namely judged as lung cancer sample.
In the present invention, described part is the molecule that the protein-specific in protein composition of the present invention is combined.Part can be nucleic acid, polypeptide or compound.Part of the present invention can be peptide ligand, and in embodiments of the invention, described part is antibody.Antibody can be people's antibody, chimeric antibody, recombinant antibodies, humanized antibody, monoclonal antibody or its Fab or polyclonal antibody.In embodiments of the invention, described antibody is monoclonal antibody.
When part is oligonucleotide fragment, described oligonucleotide fragment can with the gene order specific binding of albumen described in protein composition of the present invention; Described oligonucleotide fragment is such as primer or probe.
The method detecting protein marker relates to by detecting with the interaction of protein-specific antibody.Such as can use as described herein for the antibody of protein marker.Standard technique well known to those skilled in the art can be used to produce antibody, or the antibody of purchase can be used.These antibody can be polyclonal antibodies, or are preferably monoclonal antibody.Such as, the antibody fragment that can use complete antibody or be combined with antigen.
The method that can use immunoassays quantitatively or the existence of qualitative detection protein marker.Described immunoassays generally include hatches biological sample together with antibody, and knows technology for detection binding antibody by multiple.
In embodiments of the invention, Sample Method, immunohistochemical method and positive criterion is prepared as follows:
A () prepares sample:
Organization chip: lung cancer and operation are cut end tissue formalin and fixed, and by paraffin embedding, make paraffin specimen.Section statining, with shrewd cancer pathology type and the required cancer nests of mark and surgical resection margins tissue location.Take out in the relevant position of paraffin specimen and organize core, put into the array module designed in advance, be arranged in organization chip module.Organization chip module cut into slices, mount on slide, be placed in drying box and spend the night, immunohistochemistry to be done is used.
Cell suspension microarray: by centrifugal for the conserving liquid having bronchial brushing sample, leave and take appropriate volume according to cell concentration liquid.Draw appropriate by the array point designed in advance on anticreep slide, fixing, immunocytochemistry to be done is used.
B () immunohistochemistry: organization chip bakes sheet, dewaxing, microwave thermal reparation, adds antibody incubation, washing.Add PV9000 reagent 1 to hatch in 37 DEG C of incubators, add PV9000 reagent 2 and hatch in 37 DEG C of incubators, develop the color under DAB solution mirror, distilled water rinsing, haematoxylin is redyed, and ammoniacal liquor returns indigo plant, ethanol dehydration, and dimethylbenzene is transparent, gummy mounting.
Immunocytochemistry without roasting sheet, dewax two steps, the same immunohistochemistry of remaining step.
(c) standards of grading: different standards of grading are taked to colour developing result according to protein expression position
If EGFR is film expression, 0 is divided into feminine gender, completely without colour developing or the colour developing of < 10% tumor cell membrane.1 is divided into feminine gender, and the tumour cell of > 10% occurs very weak film colour developing and develops the color imperfect.2 are divided into weak to moderate positive, and the tumour cell of > 10% occurs that the weak film to medium tenacity develops the color.3 are divided into strong positive, and stronger film colour developing appears in the tumour cell of > 10%.Namely more than 2 points be judged as the positive.
MCM7, P53, Ki67 and P63 are nuclear expression, and scoring need consider positive cell percentage and colored intensity.The tumour cell of number percent: < 10% occurs that core colour developing is 0 point, the tumour cell of 10%-33% occurs that core colour developing is 1 point, the tumour cell of 33%-67% occurs that core colour developing is 2 points, and the tumour cell of > 67% occurs that core colour developing is 3 points.Colored intensity: seedless colour developing is 0 point, more weak core colour developing is 1 point, and medium core colour developing is 2 points, and stronger core colour developing is 3 points.With the scoring of number percent scoring × colored intensity for final appraisal result.The scoring of number percent scoring × colored intensity is more than 1 point and is namely judged as the positive.
The beneficial effect of the invention
The present invention utilizes EGFR, MCM7, P53, Ki67 and P63 albumen in antibody combined detection sample, can effective detection of lung cancer, and especially early stage lung squamous cancer and adenocarcinoma of lung, its sensitivity and specificity are significantly higher than the testing result using single protein marker.Method of the present invention can improve the recall rate of the early stage of lung cancer particularly early stage lung squamous cancer and adenocarcinoma of lung greatly, is conducive to that patients with lung cancer early finds, early treatment, greatly to improve the survival rate of patients with lung cancer.
Accompanying drawing explanation
End micro-array tissue Immunohistochemical detection is cut in Fig. 1 lung squamous cancer and operation
As can be seen from Figure 1, EGFR, MCM7, P53, Ki67 and P63 five albumen are strongly expressed in lung squamous cell carcinoma cancers late, and does not express in cancer beside organism.
End micro-array tissue Immunohistochemical detection is cut in Fig. 2 adenocarcinoma of lung and operation
As can be seen from Figure 2, EGFR, MCM7, P53, Ki67 and P63 five albumen are strongly expressed in pulmonary adenocarcinoma late, and does not express in cancer beside organism.
Fig. 3 bronchial brushing sample cell suspension microarray Immuncytochemical detection
As can be seen from Figure 3, EGFR, MCM7, P53, Ki67 and P63 five albumen strongly expressed in the FOB sample of tumor cases, and do not express in inflammatory case.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
In the present invention, described protein composition comprises following five kinds of albumen, P53, P63, Ki67, MCM7 and EGFR.Wherein:
P53 is divided into wild and sudden change two kinds of hypotypes, and the sudden change of this gene or disappearance cause many tumorigenic reasons, is also apoptotic regulatory factor simultaneously.Wide expression in most tumour, as lung cancer, breast cancer, gastroenteric tumor and hepatocellular carcinoma etc.
P63 is expressed in the basal cell of epidermis, the substrate/musculoepithelia cell, mammary gland musculoepithelia cell, prostate basic cell by immunohistochemistry. etc. of cutaneous appendages, be the label of squama cancer, basal-cell carcinoma, urinary tract transitional cell carcinoma, can be used for the diagnosis and differential diagnosis of primary cutaneous tumour and adenocarcinoma metastatic, Benign And Malignant Breast Diseases, prostate cancer.
Ki-67 albumen can be combined with DNA, plays an important role in adjustment cell proliferation.Only have G1, the cellular expression Ki-67 albumen of G2, M and S phase, G0 phase and the early stage cell of G1 are not expressed.Utilize Ki-67 monoclonal antibody can detect increment cell.
MCM7 is the important component of minichromosome maintenance protein complex, plays a crucial role in startup DNA replication dna, synthesis.The index judging cell proliferation is can be used as by the expression and location that detect MCM7 albumen.
The memebrane protein of EGFR to be a kind of molecular weight be 170kDa.Its process LAN indication breast cancer and cancer of the stomach poor prognosis.And can the application of targeted drug Cetuximab of guiding treatment colorectal cancer.
In the present invention, P53 antibody is purchased from SantaCruz company, and article No. is sc-6243; P63 antibody is purchased from SantaCruz company, and article No. is sc-8431; Ki67 antibody is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, and article No. is zm-0166; MCM7 antibody is purchased from SantaCruz company, and article No. is sc-9966; EGFR antibody is purchased from Invitrogen company, and article No. is 28-0005.
In the present invention, Sample Method, immunohistochemical method and standards of grading are prepared as follows:
A () prepares sample:
Organization chip: lung cancer and operation are cut after end tissue fixes 48h with 10% neutral formalin and used paraffin embedding respectively, cut HE dyeing, cancer nests and surgical resection margins tissue location needed for shrewd cancer pathology type and mark.At the relevant position of paraffin specimen mark as to draw materials position, rear diameter is that the perforating needle of 1mm takes out one by one organize core from demarcating position, puts into the array module designed in advance, is arranged in organization chip module.Be cut into 4um slab with microtome after frozen 30min in 4 DEG C of refrigerators to mount on anticreep slide, be placed in 65 DEG C of drying boxes and spend the night, immunohistochemistry to be done is used.
Cell suspension microarray: by centrifugal for the conserving liquid having bronchial brushing sample, leave and take appropriate volume according to cell concentration liquid.Evenly, draw 1uL by the array point designed in advance on anticreep slide, 95% ethanol is fixed, and immunocytochemistry to be done is used in piping and druming.
(b) immunohistochemistry: the roasting sheet 30min of organization chip 65 DEG C, dimethylbenzene dewaxing 10min × 3 time, 100%, 85%, 75% ethanol each 3min, PBS5min × 2 times, H
2o
215min, the reparation of liquor sodii citratis (PH=6.0) microwave thermal (after microwave heating to 95 DEG C-99 DEG C, thin slice is inserted, then with in micro-wave oven-low fire heating 20 minutes, be cooled to room temperature), PBS3min × 3 time, add antibody and to insert in wet box 4 DEG C and spend the night.Within second day, take out wet box and return to room temperature, PBS3min × 3 time, add PV9000 reagent 1 and hatch 20min in 37 DEG C of incubators, PBS3min × 3 time, add PV9000 reagent 2 and in 37 DEG C of incubators, hatch 30min, PBS3min × 3 time, develop the color under DAB solution mirror, distilled water rinsing, haematoxylin is redyed, and ammoniacal liquor returns blue 10min, each 3min of 75%, 85%, 100% ethanol dehydration, transparent 5min × 2 time of dimethylbenzene, gummy mounting.
Note: immunocytochemistry without roasting sheet, dewax two steps, the same immunohistochemistry of remaining step.
(c) standards of grading: different standards of grading are taked to colour developing result according to protein expression position
If EGFR is film expression, 0 is divided into feminine gender, completely without colour developing or the colour developing of < 10% tumor cell membrane.1 is divided into feminine gender, and the tumour cell of > 10% occurs very weak film colour developing and develops the color imperfect.2 are divided into weak to moderate positive, and the tumour cell of > 10% occurs that the weak film to medium tenacity develops the color.3 are divided into strong positive, and stronger film colour developing appears in the tumour cell of > 10%.Namely more than 2 points be judged as the positive.
MCM7, P53, Ki67 and P63 are nuclear expression, and scoring need consider positive cell percentage and colored intensity.The tumour cell of number percent: < 10% occurs that core colour developing is 0 point, the tumour cell of 10%-33% occurs that core colour developing is 1 point, the tumour cell of 33%-67% occurs that core colour developing is 2 points, and the tumour cell of > 67% occurs that core colour developing is 3 points.Colored intensity: seedless colour developing is 0 point, more weak core colour developing is 1 point, and medium core colour developing is 2 points, and stronger core colour developing is 3 points.With the scoring of number percent scoring X colored intensity for final appraisal result.The scoring of number percent scoring × colored intensity is more than 1 point and is namely judged as the positive.
Sensitivity during be combined in embodiment 1 five protein antibodies Advanced Squamous all Carcinoma of Lung and pulmonary adenocarcinoma detect and specificity
End tissue preparation is cut in 84 example late period (III phase, IV phase) lung squamous cancers, 88 routine advanced pulmonary adenocarcinomas and corresponding operation thereof and becomes organization chip, wherein tumor section gets 3 points, and operation is cut end tissue and got 2 points.Detect the expression of P53, P63, Ki67, MCM7 and EGFR five albumen by immunohistochemical method, and carry out marking, analyzing.Result is as follows:
Table 1: the sensitivity of different protein antibodies combination in lung squamous cancer
| Combinatorial property | Sensitivity | Positive number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.9375 | 75 | 80 | EGFR、MCM7、P63、P53 |
| 4 select 2 | 0.9125 | 73 | 80 | EGFR、Ki67、MCM7、P63 |
| 4 select 2 | 0.9091 | 70 | 77 | Ki67、P63、P53、MCM7 |
| 5 select 2 | 0.9620 | 76 | 79 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.8250 | 66 | 80 | EGFR、Ki67、MCM7、P53、P63 |
Note: as long as 4 select in 2 finger, 4 antibody and have 2 antibody positive results to be namely designated as the positive; As long as 5 select in 2 finger, 5 antibody and have 2 antibody positive results to be namely designated as the positive; As long as 5 select in 3 finger, 5 antibody and have 3 antibody positive results to be namely designated as the positive;
Effective case load refers to that 4 or 5 antibody all have the case load of measured value;
Following table is identical with table 1.
Table 2: the sensitivity using single protein marker in lung squamous cancer
| Protein name | Positive number of cases | Effective number of cases | Sensitivity |
| P53 | 53 | 80 | 0.6625 |
| P63 | 69 | 82 | 0.8415 |
| Ki67 | 54 | 78 | 0.6923 |
| MCM7 | 68 | 83 | 0.8193 |
| EGFR | 60 | 81 | 0.7407 |
Table 3: the specificity of different protein antibodies combination in lung squamous cancer
| Combinatorial property | Specificity | Negative number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.9383 | 76 | 81 | EGFR、MCM7、P63、P53 |
| 4 select 2 | 0.9634 | 79 | 82 | EGFR、Ki67、MCM7、P63 |
| 4 select 2 | 0.9625 | 77 | 80 | Ki67、P63、P53、MCM7 |
| 5 select 2 | 0.9375 | 75 | 80 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 1.0000 | 83 | 83 | EGFR、Ki67、MCM7、P53、P63 |
Note: specificity refers to ratio negative in Carcinoma side normal tissue.Wherein 4 select 2,5 to select 2 and 5 to select the meaning of 3 identical with table 1, when it does not meet positive criterion, be namely judged as feminine gender.Following table is identical with table 3.
Table 4: the specificity using single protein marker in lung squamous cancer
| Protein name | Negative number of cases | Effective number of cases | Specificity |
| P53 | 76 | 81 | 0.9383 |
| P63 | 77 | 81 | 0.9506 |
| Ki67 | 74 | 75 | 0.9867 |
| MCM7 | 77 | 82 | 0.9390 |
| EGFR | 75 | 84 | 0.8929 |
Table 5: the sensitivity of different protein antibodies combination in adenocarcinoma of lung
| Combinatorial property | Sensitivity | Positive number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.8442 | 65 | 77 | EGFR、Ki67、MCM7、P53 |
| 4 select 2 | 0.8375 | 67 | 80 | EGFR、MCM7、P53、P63 |
| 4 select 2 | 0.8000 | 64 | 80 | Ki67、MCM7、P53、P63 |
| 5 select 2 | 0.8750 | 70 | 80 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.6707 | 55 | 82 | EGFR、Ki67、MCM7、P53、P63 |
Table 6: the sensitivity using single protein marker in adenocarcinoma of lung
| Protein name | Positive number of cases | Effective number of cases | Sensitivity |
| P53 | 51 | 78 | 0.6538 |
| P63 | 44 | 86 | 0.5116 |
| Ki67 | 38 | 86 | 0.4419 |
| MCM7 | 57 | 81 | 0.7037 |
| EGFR | 55 | 85 | 0.6471 |
Table 7: the specificity of different protein antibodies combination in adenocarcinoma of lung
| Combinatorial property | Specificity | Negative number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.9146 | 75 | 82 | EGFR、Ki67、MCM7、P53 |
| 4 select 2 | 0.9405 | 79 | 84 | EGFR、MCM7、P53、P63 |
| 4 select 2 | 0.9639 | 80 | 83 | Ki67、MCM7、P53、P63 |
| 5 select 2 | 0.9136 | 74 | 81 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.9655 | 84 | 87 | EGFR、Ki67、MCM7、P53、P63 |
Table 8: the specificity using single protein marker in adenocarcinoma of lung
| Protein name | Negative number of cases | Effective number of cases | Specificity |
| P53 | 79 | 86 | 0.9186 |
| P63 | 79 | 87 | 0.9080 |
| Ki67 | 78 | 84 | 0.9286 |
| MCM7 | 78 | 82 | 0.9512 |
| EGFR | 72 | 81 | 0.8889 |
Above result shows, EGFR, MCM7, P53, Ki67 and P63 five albumen are strongly expressed (see Fig. 1 and Fig. 2) in lung squamous cancer and pulmonary adenocarcinoma late; Select four or five protein antibodies joint-detection Advanced Squamous all Carcinoma of Lungs, when wherein namely two antibody positives are judged as the positive, its sensitivity can reach more than 90%, specificity can reach more than 93%, joint-detection advanced pulmonary adenocarcinoma, when wherein namely two antibody positives are judged as the positive, its sensitivity can reach more than 80%, specificity can reach more than 91%, all higher than the testing result of single albumen.When selecting five protein antibodies joint-detection, can further improve the sensitivity of detection.
Sensitivity during be combined in embodiment 2 five protein antibodies early stage lung squamous cancer and pulmonary adenocarcinoma detect and specificity
Retrospective study: end tissue preparation is cut in early stage (I phase, the II phase) lung squamous cancer of 50 example, the early stage adenocarcinoma of lung of 50 example and corresponding operation thereof and becomes organization chip, wherein tumor section gets 3 points, and operation is cut end tissue and got 2 points.Detect the expression of P53, P63, Ki67, MCM7 and EGFR five albumen by immunohistochemical method, and mark.Result is as follows:
Table 9: the sensitivity of different protein antibodies combination in lung squamous cancer
| Combinatorial property | Sensitivity | Positive number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.8980 | 44 | 49 | Ki67、P53、P63、MCM7 |
| 4 select 2 | 0.8776 | 43 | 49 | EGFR、Ki67、MCM7、P63 |
| 4 select 2 | 0.8367 | 41 | 49 | EGFR、Ki67、MCM7、P53 |
| 5 select 2 | 0.8980 | 44 | 49 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.7347 | 36 | 49 | EGFR、Ki67、MCM7、P53、P63 |
Table 10: the sensitivity using single protein marker in lung squamous cancer
| Protein name | Positive number of cases | Effective number of cases | Sensitivity |
| P53 | 30 | 49 | 0.6122 |
| P63 | 36 | 49 | 0.7347 |
| Ki67 | 40 | 49 | 0.8163 |
| MCM7 | 35 | 49 | 0.7143 |
| EGFR | 5 | 49 | 0.1020 |
Table 11: the specificity of different protein antibodies combination in lung squamous cancer
| Combinatorial property | Specificity | Negative number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.9800 | 49 | 50 | Ki67、P53、P63、MCM7 |
| 4 select 2 | 0.9800 | 49 | 50 | EGFR、Ki67、MCM7、P63 |
| 4 select 2 | 0.9800 | 49 | 50 | EGFR、Ki67、MCM7、P53 |
| 5 select 2 | 0.9800 | 49 | 50 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 1.0000 | 50 | 50 | EGFR、Ki67、MCM7、P53、P63 |
Table 12: the specificity using single protein marker in lung squamous cancer
| Protein name | Negative number of cases | Effective number of cases | Specificity |
| P53 | 49 | 50 | 0.9800 |
| P63 | 42 | 50 | 0.8400 |
| Ki67 | 49 | 50 | 0.9800 |
| MCM7 | 49 | 50 | 0.9800 |
| EGFR | 50 | 50 | 1.0000 |
Table 13: the sensitivity of different protein antibodies combination in adenocarcinoma of lung
| Combinatorial property | Sensitivity | Positive number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.8000 | 40 | 50 | Ki67、MCM7、P53、P63 |
| 4 select 2 | 0.7800 | 39 | 50 | EGFR、Ki67、MCM7、P63 |
| 4 select 2 | 0.7200 | 36 | 50 | EGFR、Ki67、P53、P63 |
| 5 select 2 | 0.8200 | 41 | 50 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.6200 | 31 | 50 | EGFR、Ki67、MCM7、P53、P63 |
Table 14: the sensitivity using single protein marker in adenocarcinoma of lung
| Protein name | Positive number of cases | Effective number of cases | Sensitivity |
| P53 | 25 | 50 | 0.5000 |
| P63 | 36 | 50 | 0.7200 |
| Ki67 | 37 | 50 | 0.7400 |
| MCM7 | 32 | 50 | 0.6400 |
| EGFR | 17 | 50 | 0.3400 |
Table 15: the specificity of different protein antibodies combination in adenocarcinoma of lung
| Combinatorial property | Specificity | Negative number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.9592 | 47 | 49 | Ki67、MCM7、P53、P63 |
| 4 select 2 | 0.9592 | 47 | 49 | EGFR、Ki67、MCM7、P63 |
| 4 select 2 | 0.9592 | 47 | 49 | EGFR、Ki67、P53、P63 |
| 5 select 2 | 0.9592 | 47 | 49 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.9592 | 47 | 49 | EGFR、Ki67、MCM7、P53、P63 |
Table 16: the specificity using single protein marker in adenocarcinoma of lung
| Protein name | Negative number of cases | Effective number of cases | Specificity |
| P53 | 48 | 49 | 0.9796 |
| P63 | 35 | 49 | 0.7143 |
| Ki67 | 47 | 49 | 0.9592 |
| MCM7 | 49 | 50 | 0.9800 |
| EGFR | 49 | 49 | 1.0000 |
Above result shows, EGFR, MCM7, P53, Ki67 and P63 five albumen are strongly expressed in lung squamous cancer and pulmonary adenocarcinoma in early days; Select four or five early stage lung squamous cancers of albumen joint-detection, when wherein namely two antibody positives are judged as the positive, its sensitivity can reach more than 83%, specificity can reach 98%, the early stage adenocarcinoma of lung of joint-detection, when wherein namely two antibody positives are judged as the positive, its sensitivity can reach more than 72%, specificity can reach more than 95%, all higher than the testing result of single albumen.When selecting five protein antibodies joint-detection, can further improve the sensitivity of detection.
Embodiment 3 five protein antibodies are combined in the sensitivity in lung squamous cancer and adenocarcinoma of lung bronchial brushing Samples detection
Get cytology with pathology (operation/biopsy) result and the two consistent bronchial brushing (FOB) sample, squama cancer 73 example, the method of gland cancer 17 example immunocytochemistry detects the expression of P53, P63, Ki67, MCM7 and EGFR five albumen, and marks.Result following (see Fig. 3):
Table 17: the sensitivity of different protein antibodies combination in lung squamous cancer
| Combinatorial property | Sensitivity | Positive number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.8889 | 56 | 63 | Ki67、P63、MCM7、EGFR |
| 4 select 2 | 0.8788 | 58 | 66 | Ki67、P53、P63、MCM7 |
| 4 select 2 | 0.8636 | 57 | 66 | Ki67、P53、P63、EGFR |
| 5 select 2 | 0.9077 | 59 | 65 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.7937 | 50 | 63 | EGFR、Ki67、MCM7、P53、P63 |
Table 18: the sensitivity using single protein marker in lung squamous cancer
| Protein name | Positive number of cases | Effective number of cases | Sensitivity |
| P53 | 34 | 64 | 0.5313 |
| P63 | 59 | 68 | 0.8676 |
| Ki67 | 55 | 68 | 0.8088 |
| MCM7 | 49 | 66 | 0.7424 |
| EGFR | 19 | 63 | 0.3016 |
Table 19: the sensitivity of different protein antibodies combination in adenocarcinoma of lung
| Combinatorial property | Sensitivity | Positive number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.7857 | 11 | 14 | Ki67、P53、EGFR、MCM7 |
| 4 select 2 | 0.7692 | 10 | 13 | Ki67、P53、P63、MCM7 |
| 4 select 2 | 0.7143 | 10 | 14 | Ki67、P53、P63、EGFR |
| 5 select 2 | 0.7857 | 11 | 14 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.6154 | 8 | 13 | EGFR、Ki67、MCM7、P53、P63 |
Table 20: the sensitivity using single protein marker in adenocarcinoma of lung
| Protein name | Positive number of cases | Effective number of cases | Sensitivity |
| P53 | 11 | 14 | 0.7857 |
| P63 | 4 | 16 | 0.2500 |
| Ki67 | 9 | 13 | 0.6923 |
| MCM7 | 10 | 15 | 0.6667 |
| EGFR | 4 | 14 | 0.2857 |
Above result shows, EGFR, MCM7, P53, Ki67 and P63 five albumen are strongly expressed (see Fig. 3) in the bronchial brushing sample of lung squamous cancer and adenocarcinoma of lung; And select four or five albumen joint-detection lung squamous cancers, when wherein namely two antibody positives are judged as the positive, its sensitivity can reach more than 86%, joint-detection adenocarcinoma of lung, when wherein namely two antibody positives are judged as the positive, its sensitivity can reach more than 71%, all higher than the testing result of single albumen.When selecting five protein antibodies joint-detection, can further improve the sensitivity of detection.
Embodiment 4 five protein antibodies are combined in the specificity in non-cancer patient bronchial brushing Samples detection
Get bronchial brushing (FOB) sample 62 example that pathology (operation/biopsy) result is not lung cancer, comprising 37 routine inflammation, 11 routine tuberculosis, 1 routine atypical cell, 3 routine hyperplasia, 1 routine benign cystic pathology and 9 routine ' negative ' specimens, detect the expression of P53, P63, Ki67, MCM7 and EGFR five albumen by the method for immunocytochemistry, and mark.Result is as follows:
Table 21: have in non-cancer sample and combine compared with the protein antibodies of high specific
| Combinatorial property | Specificity | Negative number of cases | Effective number of cases | Protein antibodies combines |
| 4 select 2 | 0.8235 | 42 | 51 | EGFR、MCM7、P63、P53 |
| 4 select 2 | 0.8542 | 41 | 48 | EGFR、Ki67、MCM7、P53 |
| 4 select 2 | 0.8600 | 43 | 50 | EGFR、Ki67、P53、P63 |
| 5 select 2 | 0.7755 | 38 | 49 | EGFR、Ki67、MCM7、P53、P63 |
| 5 select 3 | 0.9000 | 45 | 50 | EGFR、Ki67、MCM7、P53、P63 |
Above result shows, EGFR, MCM7, P53, Ki67 and P63 five albumen express (see Fig. 3) hardly in the bronchial brushing sample of the non-cancer cases such as lung inflammation, pulmonary tuberculosis, lung's atypical cell, lung's hyperplasia, when wherein two antibody positives are judged as the positive, its detection specificity can reach more than 77%.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (10)
1. protein composition, it is selected from least four kinds in P53, P63, Ki67, MCM7 and EGFR albumen, and it is following combination:
(1) EGFR, MCM7, P63 and P53 albumen;
(2) EGFR, Ki67, MCM7 and P53 albumen;
(3) EGFR, Ki67, P53 and P63 albumen;
(4) Ki67, P53, P63 and MCM7 albumen;
(5) Ki67, P63, MCM7, EGFR albumen; Or
(6) P53, P63, Ki67, MCM7 and EGFR albumen.
2. ligand combination thing, its composition of part for being combined with the protein-specific described in the protein composition of claim 1.
3. the ligand combination thing of claim 2, wherein said part is antibody.
4. the ligand combination thing of claim 3, wherein said antibody is monoclonal antibody.
5. chip, on it, point has part, and described part is made up of the part described in the ligand combination thing of any one of claim 2-4.
6. kit, it comprises ligand combination thing, and described ligand combination thing is made up of the ligand combination thing of any one of claim 2-4.
7. the kit of claim 6, it also comprises damping fluid and developer.
8. the chip of the protein composition of claim 1 or the ligand combination thing of any one of claim 2-4 or claim 5 is preparing the purposes in pulmonary cancer diagnosis kit, and wherein said lung cancer is lung squamous cancer or adenocarcinoma of lung.
9. the purposes of claim 8, wherein said kit is used for the diagnosis of tissue sample or cell sample.
10. the purposes of claim 8 or 9, the determination methods of wherein said kit is, when at least two kinds of parts in the ligand combination thing of any one of claim 2-4 or the chip of claim 5 and testing sample react present the positive time, be namely judged as lung cancer sample.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101603966A (en) * | 2008-06-12 | 2009-12-16 | 上海裕隆生物科技有限公司 | A kind of male multi-tumor marker detection protein chip and kit thereof |
| CN101711284A (en) * | 2007-01-25 | 2010-05-19 | 达娜-法勃肿瘤研究所 | The purposes of anti-egfr antibodies in the mutant mediated disease of treatment EGFR |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1871517A (en) * | 2002-02-19 | 2006-11-29 | 免疫公司 | Methods and reagents for the rapid and efficient isolation of circulating cancer cells |
| KR100987961B1 (en) * | 2007-11-08 | 2010-10-18 | 남해현 | Chair including headrest |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101711284A (en) * | 2007-01-25 | 2010-05-19 | 达娜-法勃肿瘤研究所 | The purposes of anti-egfr antibodies in the mutant mediated disease of treatment EGFR |
| CN101603966A (en) * | 2008-06-12 | 2009-12-16 | 上海裕隆生物科技有限公司 | A kind of male multi-tumor marker detection protein chip and kit thereof |
Non-Patent Citations (6)
| Title |
|---|
| Comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma;Babett Bartling et al;《Lung Cancer》;20050831;第49卷;摘要，表1 * |
| HPV16、18E6和MCM7蛋白在肺鳞癌中的表达及其临床意义;郑建伟;《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》;20100815;摘要，正文第9页"方法"部分 * |
| Ki67、 p53和P糖蛋白在肺鳞癌、腺癌中的表达关系及意义;刘树立 等;《中国医科大学学报》;20081231;第37卷(第6期);摘要，第798页第1.1-1.2节 * |
| MCM7、P27在肺腺癌中的表达及其意义;张明明;《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》;20081015;摘要 * |
| P63和TTF-1在肺癌组织中的表达及意义;刘建 等;《中国组织化学与细胞化学杂志》;20080630;摘要，第290页的"材料和方法"部分 * |
| 相近分化的肺鳞癌和腺癌中 PCNA及 EGFR表达的研究;林凤武 等;《白求恩医科大学学报》;20000730;第26卷(第4期);摘要，第358页第1.2-1.3节 * |
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