CN102735844A - Protein composition and application in preparing lung cancer diagnosis reagent kit - Google Patents
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Abstract
The invention, pertaining to the field of molecular biology and clinical testing technology, particularly relates to a protein composition selected from P53, P63, Ki67, MCM7 and EGFR proteins, a ligand composition in combination with protein specificity of the protein composition, and application of the protein composition or the ligand composition in preparing a lung cancer (especially an early lung cancer) diagnosis reagent kit. The protein composition of the invention can be used for the lung cancer diagnosis, especially for the early lung cancer diagnosis, has high sensitivity and high specificity, and is better than testing of a single protein marker.
Description
Technical field
The invention belongs to molecular biology, clinical detection technique field; Be specifically related to comprise P53, P63, Ki67, MCM7, and the protein composition of five albumen of EGFR; And the application of said protein composition in preparation lung cancer, particularly lung squamous cancer and adenocarcinoma of lung diagnostic kit.
Background technology
Lung cancer is the modal malignant tumours in various countries, the world today, and human health and life are constituted greatly threat.U.S.'s statistics in 2010 shows, the mortality ratio of lung cancer all is positioned at the first place of parts of body tumour at masculinity and femininity, is respectively 29% and 26%.New cases all account for second in masculinity and femininity, be respectively 15% and 14% of each position tumour summation.5 of U.S.'s lung cancer years survival rates are 16% in the period of the 1999-2005, and 5 years survival rates of wherein focal person are 53%, and it is 24% that metastases in local lymph node person is arranged, and have the capable DISTANT METASTASES IN person of blood to be merely 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 that whole nation treatment and prevention of tumour office of the Ministry of Public Health provides shows; In the period of 2000-2005; The number of the infected of China's lung cancer is estimated to increase by 120,000 people, and wherein the male lung cancer patient is increased to 330,000 people in 2005 from 260,000 people in 2000, and the same period, the female lung cancer patient was increased to 170,000 people from 120,000 people.At present China's lung cancer morbidity rate is annual increases by 26.9%, takes effective control measure as untimely, expects 2025, and China's patients with lung cancer will reach 1,000,000, become the first in the world lung cancer big country.Although in recent years new development is being arranged all aspect diagnostic method, surgical technic and the chemotherapeutics, the total 5 years survival rates of patients with lung cancer are still very low.In China, lung cancer causes 400,000 routine deaths every year approximately.Why so low overall 5 years survival rates of lung cancer are, has been in late period when mainly going to a doctor because of most patients with lung cancer, or occurred sending out transfer more for a long time, is early warning, early detection so reduce the key of patients with lung cancer mortality ratio, so that early intervention.Research shows that 10 years survival rates of I phase lung cancer postoperative can reach 92%.
Screening lung cancer method commonly used at present comprises chest x-ray, low-dose spiral CT (low-dosespiral CT; LDCT), expectorative cytology inspection, bronchoscope brush sheet or get biopsy, BAL fluid cytolgical examination etc. down, but all there is limitation in various inspection means at aspects such as susceptibility, specificity, relevance grades.
Except above physical property test mode, also there is the blood serum tumor of application to learn the mark diagnosing at present clinically.Common mark comprises carcinomebryonic antigen (CEA), squamous cell cancer associated antigen (SCC-Ag), cytokeratin 21-1 fragment (CYFRA21-1), sugar antigen 125 (CA125), sugar antigen 153 (CA153), sugar antigen 199 (CA199), neuronspecific enolase (NSE), tissue polypeptide antigen (TPA) etc.But these tumor markerses detect its susceptibility separately and specificity is all limited; Though joint-detection increases, still face many benign lesions such as benign tumour, inflammation, DD etc. and also can cause the problem that these tumor markers serology levels increase.The mark of further exploring efficient special lung cancer marker, particularly early warning is of crucial importance.
Lung cancer can be divided into non-small cell lung cancer and ED-SCLC, respectively accounts for 85% and 15% of lung cancer.Wherein non-small cell lung cancer comprises squama cancer, gland cancer and large cell carcinoma again.Because of squama cancer and gland cancer are being in the great majority by among the patient who diagnoses out, at present more to the Study on Molecular Mechanism of these two types of lung cancer incidence and development.
Comprehensive present research; Relate to (like p53, CCND1, ki-67, Telomerase, TTF1 etc.) such as cell cycle regulating, cell proliferation and differentiation, DNA reparation and Apoptosis with the relevant gene alteration of lung cancer morbidity mechanism, and the change unusual and gene expression dose of chromosome structure can cause also tumour to take place.In recent years research proves that some molecular changes is that order takes place, the frequency that they occur and the development degree (squamous metaplasia-atypical hyperplasia-carcinoma in situ of precancerous lesion; Atypical adenoma hyperplasia-gland cancer) raise synchronously.Therefore, can reflect the heterogeneity of oncobiology behavior unusually more rightly as the tumor markers of lung cancer, and then more valuable guidance is provided for the early warning of lung cancer with molecular level.
Although obtained some progress, up to now, still there is not the early warning that any definite molecular marker can really be used for lung cancer.Therefore, this area presses for and works out the mark that can be used for the lung cancer early diagnosis or mark combination, and effectively detection of lung cancer, the especially method of early detection lung cancer.
Summary of the invention
The object of the present invention is to provide a kind of effectively protein composition of detection of lung cancer, the particularly early stage of lung cancer, the said early stage of lung cancer is preferably early stage lung squamous cancer and early stage adenocarcinoma of lung.This combination has following characteristic:
(a) high expressed in the patients with lung cancer tissue is cut in operation and lowly in the end tissue to be expressed or not express;
(b) lowly in other non-carcinous pulmonary lesion tissues such as tuberculosis, inflammation, pulmonary cryptococosis express or do not express;
(c) strongly expressed in tumor patient bronchial brushing sample, not low express/expression the in lung inflammation, tuberculosis and other benign lesion;
(d) this protein combination is expressed positive person and the lung cancer risk takes place is increased among the health examination crowd.
In the present invention; The inventor is through unremitting effort; Screening obtains being used for the protein marker combination of pulmonary cancer diagnosis from a large amount of cancer markers albumen, and the composition of said 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 the EGFR albumen.In embodiments of the invention, in the said 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;
Perhaps
(6) P53, P63, Ki67, MCM7 and EGFR albumen.
In the present invention, said protein composition is the composition of protein marker, and it can be used as the detection that significant albumen is used for lung cancer.Therefore the invention also discloses the molecule that combines with the marker protein specificity.
Another aspect of the present invention relates to a kind of ligand combination thing, and it is the composition of the part that combines with the protein-specific described in the protein composition of the present invention.
In the present invention, said part be with protein composition of the present invention in the molecule that combines of protein-specific.Part can be nucleic acid, polypeptide or compound.Part of the present invention can be the peptide part, and in embodiments of the invention, said part is an 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, said antibody is monoclonal antibody.
Another aspect of the present invention relates to a kind of chip, and point has the part described in the ligand combination thing of the present invention on it.Said chip is a protein array, can the antibody or other part that combine with protein marker specificity of the present invention be fixed on the protein array, whether there to be protein marker of the present invention in the test sample.
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, said 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 the chip purposes in preparation pulmonary cancer diagnosis kit.In embodiments of the invention, said lung cancer is lung squamous cancer or adenocarcinoma of lung.In one embodiment of the invention, said lung squamous cancer or adenocarcinoma of lung be late period lung squamous cancer or late period adenocarcinoma of lung.In one embodiment of the invention, said lung squamous cancer or adenocarcinoma of lung are early stage lung squamous cancer or early stage adenocarcinoma of lung.
In the present invention, said kit is used for the diagnosis of tissue sample or cell sample.In one embodiment of the invention, said tissue sample is that end histotomy sample is cut in lung cancer or operation.In another embodiment of the invention, said cell sample is the cell sample of bronchial brushing.
In the present invention, the determination methods of said kit does, when at least two kinds of parts in the ligand combination thing of the present invention and testing sample reaction present the positive, promptly is judged as lung cancer sample.
In the present invention, said part be with protein composition of the present invention in the molecule that combines of protein-specific.Part can be nucleic acid, polypeptide or compound.Part of the present invention can be the peptide part, and in embodiments of the invention, said part is an 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, said antibody is monoclonal antibody.
When part was oligonucleotide fragment, said oligonucleotide fragment can combine with the gene order specificity of albumen described in the protein composition of the present invention; Said oligonucleotide fragment for example is primer or probe.
The method that detects protein marker relates to through the interaction with protein-specific antibody and detecting.For example can use as described herein to the antibody of protein marker.Can use standard technique well known to those skilled in the art to produce antibody, perhaps can use the antibody of purchase.These antibody can be polyclonal antibodies, or monoclonal antibody preferably.The antibody fragment that for example, can use complete antibody or combine with antigen.
The method that can use immunoassays quantitatively or the existence of qualitative detection protein marker.Said immunoassays generally include hatches biological sample with antibody, and knows the technology for detection binding antibody through multiple.
In embodiments of the invention, preparation sample method, immunohistochemical method and positive criterion are following:
(a) preparation sample:
Organization chip: lung cancer and operation are cut the end tissue and are used formalin fixed, use FFPE, process paraffin specimen.Section statining is with shrewd tumour histological type and required cancer nests of mark and operation margin tissue position.Organize core in the taking-up of the relevant position of paraffin specimen, put into the array module of design in advance, be arranged in the organization chip module.With the section of organization chip module, mount on slide, place drying box to spend the night, wait to do immunohistochemistry and use.
The cell suspension microarray: the preservation liquid that will have a bronchial brushing sample is centrifugal, leave and take the liquid of appropriate volume according to cell concentration.Draw an amount of array point of pressing design in advance on the anticreep slide, fixing, wait to do immunocytochemistry and use.
(b) immunohistochemistry: organization chip is baked sheet, dewaxing, and the microwave thermal reparation adds antibody incubation, washing.Add PV9000 reagent 1 and in 37 ℃ of incubators, hatch, add PV9000 reagent 2 and in 37 ℃ of incubators, hatch, DAB solution mirror is colour developing down, the distilled water rinsing, and haematoxylin is redyed, and ammoniacal liquor returns indigo plant, ethanol dehydration, xylene is transparent, gummy mounting.
Immunocytochemistry does not have roasting sheet, two steps of dewaxing, the same immunohistochemistry of surplus step.
(c) standards of grading: the colour developing result is taked different standards of grading according to the protein expression position
Like EGFR is film expression, and 0 minute is negative, do not have fully colour developing or＜10% tumor cell membrane colour developing.1 minute negative, and＞10% tumour cell very weak film colour developing occurs and develops the color imperfect.2 are divided into the weak extremely medium positive, and weak film colour developing to medium tenacity appears in＞10% tumour cell.3 are divided into strong positive, and stronger film colour developing appears in＞10% tumour cell.Promptly be judged as the positive more than 2 minutes.
MCM7, P53, Ki67 and P63 are nuclear expression, and scoring need be taken all factors into consideration positive cell percentage and colored intensity.Number percent: it is 0 minute that the nuclear colour developing appears in＜10% tumour cell, and it is 1 minute that the nuclear colour developing appears in the tumour cell of 10%-33%, and it is 2 minutes that the nuclear colour developing appears in the tumour cell of 33%-67%, and it is 3 minutes that the nuclear colour developing appears in＞67% tumour cell.Colored intensity: seedless colour developing is 0 minute, and more weak nuclear colour developing is 1 minute, and medium nuclear colour developing is 2 minutes, and strong nuclear colour developing is 3 minutes.With the scoring of number percent scoring * colored intensity is final appraisal result.The scoring of number percent scoring * colored intensity is promptly to be judged as the positive more than 1 minute.
The beneficial effect of the invention
The present invention utilizes EGFR, MCM7, P53, Ki67 and the P63 albumen in the antibody combined test sample, 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 of the single protein marker of use.Method of the present invention can improve the recall rate of particularly early stage lung squamous cancer of the early stage of lung cancer and adenocarcinoma of lung greatly, helps that patients with lung cancer is early found, early treatment, to improve the survival rate of patients with lung cancer greatly.
Description of drawings
Fig. 1 lung squamous cancer and operation are cut end micro-array tissue immunohistochemistry and are detected
As can beappreciated from fig. 1, EGFR, MCM7, P53, Ki67 and five albumen of P63 are strongly expressed in the lung squamous cell carcinoma cancers late, and does not express in cancer beside organism.
Fig. 2 adenocarcinoma of lung and operation are cut end micro-array tissue immunohistochemistry and are detected
As can beappreciated from fig. 2, EGFR, MCM7, P53, Ki67 and five albumen of P63 are strongly expressed in the pulmonary adenocarcinoma late, and does not express in cancer beside organism.
Fig. 3 bronchial brushing sample cell suspension microarray immunocytochemistry detects
As can beappreciated from fig. 3, EGFR, MCM7, P53, Ki67 and five albumen of P63 strongly expressed in the FOB of tumor cases sample, and do not express in the inflammatory case.
Embodiment
To combine embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to explain the present invention, and should not be regarded as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturer's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.
In the present invention, said protein composition comprises following five kinds of albumen, P53, P63, Ki67, MCM7 and EGFR.Wherein:
P53 is divided into wild and two kinds of hypotypes of sudden change, and the sudden change of this gene or disappearance are to cause many tumorigenic reasons, also are apoptotic regulatory factors simultaneously.Wide expression is in most tumour, like lung cancer, breast cancer, gastroenteric tumor and hepatocellular carcinoma etc.
P63 is expressed in substrate/musculoepithelia cell, mammary gland musculoepithelia cell, prostate basal cell of basal cell, the cutaneous appendages of epidermis etc.; Be the label of squama cancer, basal-cell carcinoma, urinary tract transitional cell carcinoma, can be used for the diagnosis and differential diagnosis of skin primary tumor and adenocarcinoma metastatic, the good malignant change of mammary gland, prostate cancer.
Ki-67 albumen can combine with DNA, is playing an important role aspect the adjusting cell proliferation.Have only G1, G2, the cellular expression Ki-67 albumen of M and S phase, the early stage cell of G0 phase and G1 is not expressed.Utilize the Ki-67 monoclonal antibody can detect the increment cell.
MCM7 is the important component that minute chromosome is kept protein complexes, in starting dna replication dna, synthesizing, plays a crucial role.Expression and location through detecting MCM7 albumen can be used as the index of judging cell proliferation.
EGFR is that a kind of molecular weight is the memebrane protein of 170kDa.It is crossed and expresses indication breast cancer and cancer of the stomach poor prognosis.But and the application of the targeted drug Cetuximab of guiding treatment colorectal cancer.
In the present invention, P53 antibody is available from Santa Cruz company, and article No. is sc-6243; P63 antibody is available from Santa Cruz company, and article No. is sc-8431; Ki67 antibody is available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing, and article No. is zm-0166; MCM7 antibody is available from Santa Cruz company, and article No. is sc-9966; EGFR antibody is available from Invitrogen company, and article No. is 28-0005.
In the present invention, preparation sample method, immunohistochemical method and standards of grading are following:
(a) preparation sample:
Organization chip: lung cancer and operation are cut the end tissue and are fixedly used FFPE respectively behind the 48h with 10% neutral formalin, cut HE dyeing, shrewd tumour histological type and the required cancer nests of mark and operation margin tissue position.Mark is as the position of drawing materials in the relevant position of paraffin specimen, and the back uses diameter to take out one by one and organize core from demarcating the position as the perforating needle of 1mm, puts into the array module that designs in advance, is arranged in the organization chip module.Be cut into the 4um slab with microtome behind the frozen 30min in 4 ℃ of refrigerators and mount on the anticreep slide, place 65 ℃ of drying boxes to spend the night, wait to do immunohistochemistry and use.
The cell suspension microarray: the preservation liquid that will have a bronchial brushing sample is centrifugal, leave and take the liquid of appropriate volume according to cell concentration.Piping and druming is even, and on the anticreep slide, fix by 95% ethanol by the array point that designs in advance for absorption 1uL, waits to do immunocytochemistry and use.
(b) immunohistochemistry: 65 ℃ of roasting sheet 30min of organization chip, xylene dewaxing 10min * 3 time, 100%, 85%, 75% each 3min of ethanol, PBS 5min * 2 time, H
2O
215min, the reparation of liquor sodii citratis (PH=6.0) microwave thermal (after the microwave heating to 95 ℃-99 ℃ thin slice is inserted, again with in the micro-wave oven-low fire heating 20 minutes, be cooled to room temperature), PBS 3min * 3 time add antibody and insert in the wet box 4 ℃ and spend the night.Took out wet box in second day and return to room temperature, PBS 3min * 3 time add PV9000 reagent 1 and in 37 ℃ of incubators, hatch 20min, PBS 3min * 3 time; Add PV9000 reagent 2 and in 37 ℃ of incubators, hatch 30min, PBS 3min * 3 time, DAB solution mirror is colour developing down; The distilled water rinsing, haematoxylin is redyed, and ammoniacal liquor returns blue 10min; 75%, 85%, 100% each 3min of ethanol dehydration, the transparent 5min of xylene * 2 times, gummy mounting.
Annotate: immunocytochemistry does not have roasting sheet, two steps of dewaxing, the same immunohistochemistry of surplus step.
(c) standards of grading: the colour developing result is taked different standards of grading according to the protein expression position
Like EGFR is film expression, and 0 minute is negative, do not have fully colour developing or＜10% tumor cell membrane colour developing.1 minute negative, and＞10% tumour cell very weak film colour developing occurs and develops the color imperfect.2 are divided into the weak extremely medium positive, and weak film colour developing to medium tenacity appears in＞10% tumour cell.3 are divided into strong positive, and stronger film colour developing appears in＞10% tumour cell.Promptly be judged as the positive more than 2 minutes.
MCM7, P53, Ki67 and P63 are nuclear expression, and scoring need be taken all factors into consideration positive cell percentage and colored intensity.Number percent: it is 0 minute that the nuclear colour developing appears in＜10% tumour cell, and it is 1 minute that the nuclear colour developing appears in the tumour cell of 10%-33%, and it is 2 minutes that the nuclear colour developing appears in the tumour cell of 33%-67%, and it is 3 minutes that the nuclear colour developing appears in＞67% tumour cell.Colored intensity: seedless colour developing is 0 minute, and more weak nuclear colour developing is 1 minute, and medium nuclear colour developing is 2 minutes, and strong nuclear colour developing is 3 minutes.With the scoring of number percent scoring X colored intensity is final appraisal result.The scoring of number percent scoring * colored intensity is promptly to be judged as the positive more than 1 minute.
1 five protein antibodies of embodiment are combined in lung squamous cancer and sensitivity and the specificity of pulmonary adenocarcinoma in detecting in late period
The end tissue preparation is cut in 84 example lung squamous cancers in late period (III phase, IV phase), 88 example adenocarcinomas of lung in late period and corresponding operation thereof become organization chip, wherein tumor section is got 3 points, and operation is cut the end tissue and got 2 points.Detect P53, P63, Ki67, MCM7, reach the expression of five albumen of EGFR with immunohistochemical method, and mark, analyze.The result is following:
Table 1: the sensitivity of different protein antibodies combinations in the lung squamous cancer
| Combinatorial property | Sensitivity | Positive routine number | Effective routine number | The protein antibodies combination |
| 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 |
Annotate: as long as 4 select 2 to refer to have 2 antibody positive results promptly to be designated as the positive in 4 antibody; As long as 5 select 2 to refer to have 2 antibody positive results promptly to be designated as the positive in 5 antibody; As long as 5 select 3 to refer to have 3 antibody positive results promptly to be designated as the positive in 5 antibody;
Effectively case load refers to that 4 or 5 antibody have the case load of measured value all;
Below each the table identical with table 1.
Table 2: use the sensitivity of single protein marker in the lung squamous cancer
| Protein name | Positive routine number | Effective routine number | 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 combinations in the lung squamous cancer
| Combinatorial property | Specificity | Negative routine number | Effective routine number | The protein antibodies combination |
| 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 |
Annotate: specificity refers to negative ratio in the other normal tissues of cancer.Wherein 4 select 2,5 to select 2 and 5 to select 3 meaning identical, when it does not satisfy positive criterion, promptly be judged as feminine gender with table 1.Below each the table identical with table 3.
Table 4: the specificity of using single protein marker in the lung squamous cancer
| Protein name | Negative routine number | Effective routine number | 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 combinations in the adenocarcinoma of lung
| Combinatorial property | Sensitivity | Positive routine number | Effective routine number | The protein antibodies combination |
| 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: use the sensitivity of single protein marker in the adenocarcinoma of lung
| Protein name | Positive routine number | Effective routine number | 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 combinations in the adenocarcinoma of lung
| Combinatorial property | Specificity | Negative routine number | Effective routine number | The protein antibodies combination |
| 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 of using single protein marker in the adenocarcinoma of lung
| Protein name | Negative routine number | Effective routine number | 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 five albumen of P63 are strongly expressed (referring to Fig. 1 and Fig. 2) in lung squamous cancer and the pulmonary adenocarcinoma late; Select four or five protein antibodies joint-detection lung squamous cancers in late period for use, when wherein two antibody positives promptly were judged as the positive, its sensitivity can reach more than 90%; Specificity can reach more than 93%; Joint-detection adenocarcinoma of lung in late period, when wherein two antibody positives promptly were judged as the positive, its sensitivity can reach more than 80%; Specificity can reach more than 91%, all is higher than the testing result of single albumen.When selecting five protein antibodies joint-detection for use, can further improve the sensitivity of detection.
2 five protein antibodies of embodiment are combined in sensitivity and the specificity in early stage lung squamous cancer and the pulmonary adenocarcinoma detection
Retrospective study: the end tissue preparation is cut in early stage (I phase, the II phase) lung squamous cancer of 50 examples, the early stage adenocarcinoma of lung of 50 examples and corresponding operation thereof become organization chip, wherein tumor section is got 3 points, and operation is cut the end tissue and got 2 points.Detect P53, P63, Ki67, MCM7, reach the expression of five albumen of EGFR with immunohistochemical method, and mark.The result is following:
Table 9: the sensitivity of different protein antibodies combinations in the lung squamous cancer
| Combinatorial property | Sensitivity | Positive routine number | Effective routine number | The protein antibodies combination |
| 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: use the sensitivity of single protein marker in the lung squamous cancer
| Protein name | Positive routine number | Effective routine number | 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 combinations in the lung squamous cancer
| Combinatorial property | Specificity | Negative routine number | Effective routine number | The protein antibodies combination |
| 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 of using single protein marker in the lung squamous cancer
| Protein name | Negative routine number | Effective routine number | 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 combinations in the adenocarcinoma of lung
| Combinatorial property | Sensitivity | Positive routine number | Effective routine number | The protein antibodies combination |
| 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: use the sensitivity of single protein marker in the adenocarcinoma of lung
| Protein name | Positive routine number | Effective routine number | 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 combinations in the adenocarcinoma of lung
| Combinatorial property | Specificity | Negative routine number | Effective routine number | The protein antibodies combination |
| 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 of using single protein marker in the adenocarcinoma of lung
| Protein name | Negative routine number | Effective routine number | 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 five albumen of P63 are strongly expressed in lung squamous cancer and the pulmonary adenocarcinoma in early days; Select four or five early stage lung squamous cancers of albumen joint-detection for use, when wherein two antibody positives promptly were 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 two antibody positives promptly were judged as the positive, its sensitivity can reach more than 72%; Specificity can reach more than 95%, all is higher than the testing result of single albumen.When selecting five protein antibodies joint-detection for use, can further improve the sensitivity of detection.
3 five protein antibodies of embodiment are combined in the sensitivity in lung squamous cancer and the detection of adenocarcinoma of lung bronchial brushing sample
Get cytology and pathology (operation/biopsy) result and bronchial brushing (FOB) sample that the two is consistent; Squama cancer 73 examples; Gland cancer 17 examples detect P53, P63, Ki67, MCM7, reach the expression of five albumen of EGFR with the method for immunocytochemistry, and mark.The result is (referring to Fig. 3) as follows:
Table 17: the sensitivity of different protein antibodies combinations in the lung squamous cancer
| Combinatorial property | Sensitivity | Positive routine number | Effective routine number | The protein antibodies combination |
| 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: use the sensitivity of single protein marker in the lung squamous cancer
| Protein name | Positive routine number | Effective routine number | 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 combinations in the adenocarcinoma of lung
| Combinatorial property | Sensitivity | Positive routine number | Effective routine number | The protein antibodies combination |
| 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: use the sensitivity of single protein marker in the adenocarcinoma of lung
| Protein name | Positive routine number | Effective routine number | 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 five albumen of P63 strongly expressed (referring to 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 for use; When wherein two antibody positives promptly are judged as the positive; Its sensitivity can reach more than 86%, and the joint-detection adenocarcinoma of lung is when wherein two antibody positives promptly are judged as the positive; Its sensitivity can reach more than 71%, all is higher than the testing result of single albumen.When selecting five protein antibodies joint-detection for use, can further improve the sensitivity of detection.
4 five protein antibodies of embodiment are combined in the specificity in the detection of non-carninomatosis human bronchial brush inspection sample
Getting pathology (operation/biopsy) result is not bronchial brushing (FOB) sample 62 examples of lung cancer; Comprising 37 routine inflammation, 11 routine tuberculosis, 1 routine atypical cell, 3 routine hyperplasia, 1 routine optimum cystic lesion and 9 routine negative samples; Method with immunocytochemistry detects P53, P63, Ki67, MCM7, reaches the expression of five albumen of EGFR, and marks.The result is following:
Table 21: have higher specific protein antibodies combination in the non-cancer sample
| Combinatorial property | Specificity | Negative routine number | Effective routine number | The protein antibodies combination |
| 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 five albumen of P63 are expressed (referring to Fig. 3) hardly in the bronchial brushing sample of non-carninomatosis example such as lung inflammation, pulmonary tuberculosis, lung's atypical cell, lung's hyperplasia; When wherein two antibody positives were judged as the positive, its detection specificity can reach more than 77%.
Although 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 modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by accompanying claims and any equivalent thereof.
Claims (10)
1. protein composition, it comprises at least four kinds that are selected from P53, P63, Ki67, MCM7 and the EGFR albumen, for example is four kinds or five kinds.
2. the protein composition of claim 1, 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; Perhaps
(6) P53, P63, Ki67, MCM7 and EGFR albumen.
3. ligand combination thing, it is the composition of the part that combines of the protein-specific described in the protein composition with claim 1 or 2; Wherein said part is preferably antibody; Said antibody is preferably monoclonal antibody.
4. chip, the some part described in the ligand combination thing of requirement 3 of having the right on it.
5. kit, it comprises the ligand combination thing of claim 3.
6. the kit of claim 5, it also comprises damping fluid and developer.
7. the purposes of the chip of the ligand combination thing of claim 1 or 2 protein composition or claim 3 or claim 4 in preparation pulmonary cancer diagnosis kit.
8. the purposes of claim 7, wherein said lung cancer is lung squamous cancer or adenocarcinoma of lung.
9. each purposes of claim 7-8, wherein said kit is used for the diagnosis of tissue sample or cell sample.
10. each purposes of claim 7-9, the determination methods of wherein said kit do, when right requires at least two kinds of parts and testing sample in the chip of 3 ligand combination thing or claim 4 to react to present the positive, promptly is judged as lung cancer sample.
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