CN101313221A - Comprehensive diagnostic testing procedures for personalized anticancer chemotherapy(pac) - Google Patents
Comprehensive diagnostic testing procedures for personalized anticancer chemotherapy(pac) Download PDFInfo
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Abstract
The present invention provides methods of assessing and selecting treatment modalities for cancer.
Description
The cross reference of related application
The application requires the right of priority of the 60/718th, No. 724 U.S. Provisional Patent Application of applying on September 21st, 2005 and the 60/778th, No. 901 U.S. Provisional Patent Application of applying on March 6th, 2006.Clearly the content of these two applications is introduced the application as a reference.
The statement of relevant federal government-funded research
The application partly obtains authorizing CCC diagnosis company limited (Diagnostics, the support that the NCI SBIR that is numbered CA081903 LLC) authorizes.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to field of cancer.The present invention be more particularly directed to the cancer of individual patient is carried out the discriminating of effective treatment.
The background of invention
Cancer is the disease of height individuation, the good response rate (response rate) lower (～20%) that adopts single medicine to treat at present.In order to improve responsiveness, select correct medicine particularly important at every patient.Different patients has nothing in common with each other to the response approach of same medicine, and this is probably owing to the individual difference that is caused by heredity, and this point has obtained generally acknowledging.The field of having opened up pharmacogenomics at the clinical observation of the heredity difference of drug effect.In many cases of having reported, the interindividual variation of medicine response owing to the series jump body in the gene of coding drug metabolic enzyme, drug transporter or drug target (for example, referring to Evans, W.E.Johnson, J.A.Annu.Rev.Genomics Hum.Genet.2001; 2:9-39 and McLeod, H.L.Evans, W.E.Annu.Rev.Pharmacol.Toxicol.2001; 41:101-121).
Except the heterogeneity of host to drug metabolism, the variation of tumour also influences the response of tumour to certain drug, for example:
1) if colon cancer patient's thymidylate synthetase, thymidine phosphorylase and dihydropyrimidine dehydrogenase expression are low, most colon cancer patients can have response to the 5-FU chemotherapy.If the expression of one or more enzymes is in high level, then patient's response is just very low;
2) patient of beta tubulin sudden change responds not goodly to the chemotherapy based on taxol (paclitaxel), and about 40% the patient with wild-type beta tubulin then has complete or to the response of small part.In addition, the The median survival time (mediansurvival) of carrying the patient of wild-type beta microtubule protein gene also has been enhanced;
3) can use the HER-2/neu gene magnification of receptor protein or strong cross to express (immunohistochemistry+3) to identify the more likely patient's of the combination results response of pair cell toxicity chemotherapy and Herceptin (trastuzumab) subclass.As at the first-line treatment of suffering from the metastatic breast cancer women, the single medicine Herceptin is activated and tolerated well, and this breast cancer shows HER-2/neu and crosses and express or gene magnification; And
4) high nucleotide excision repairing activity and non-small cell lung cancer cell are closely related to the resistance of cis-platinum, relevant for the report of the relation between the reparation of the dna damage of ERCC-1 expression and cisplatin induction in the Proliferation of Human Ovarian Cell.
Above-mentioned example shows that the expression of one or more biomarker-specific things on the tumour cell is relevant to the response of certain drug with tumour.Because more cancer therapy drugs recently in life process (for example are designed to, repair, mitosis) (for example resist specific cell component in, acceptor, enzyme), therefore may can find such biomarker, promptly on mechanism during relevant cancer drug therapy, this label can tumor cells showed response (referring to for example, Park etc., Clinical CancerResearch 2004; 10:3885-3896 and Vande Woude, G.F. etc., Clinical CancerResearch 2004; 10:3897-3907).
Art-recognizedly the specific tumors of individuality and the most effective therapeutic modality need be complementary, promptly need personalized anticancer chemotherapy (PAC).The present invention satisfies this and other requirement.
Summary of the invention
The present invention has set up the molecular diagnosis detection method that is used for personalized anticancer chemotherapy (PAC) by external imaging technique.PAC has comprised with the tumour cell of medicine response indicator/biomarker (DRI) sign available from individual patient.The appearance of target chemotherapy medicine and make this method be subjected to welcome to illustrating of expressing of tumour cell biomarker, this method can be associated with the resistance of tumour to medicine relevant on the mechanism.Adopted computerized fluorescence microscopy system that new fresh cell or the file cell (archival cell) that is kept in the paraffin mass are carried out the external imaging of tumour.Utilize fluorescent microsphere as a reference measured value to be carried out standardization.
In some specific embodiments, the present invention includes from the patient and obtain one or more tumour cells and tumour cell is characterized.Sign can comprise that particularly monoclonal antibody characterizes tumour by antibody.Typical such antibody can be with the tumour cell composition as target spot, and these cell components are relevant with the mechanism of action of chemotherapeutics.The existence of these compositions, disappearance or quantity have all reflected susceptibility or the resistance of tumour cell to the related drugs of use in the treatment.The cell component that the present invention mentions is relevant with the mechanism of action of specific chemotherapeutics, and it can be considered to the medicine response indicator (DRI) at this chemotherapeutics.
In some specific embodiments, antibody can be labeled, and for example can have different exciting and non-overlapped emission spectrum in the individuality detection that these dyestuffs carry out at the same time with every kind of antibody of different fluorochrome labels.These different combinations can be measured, for example quantitative measurment can be carried out through fluorescently-labeled antibody.In some specific embodiments, fluorescence microscopy system and register system that can using a computer be measured, and described fluorescence microscopy system carries out quantitatively fluorescence intensity with respect to the benchmark that is used for optical standardization.Quantitatively be to measure with cell Chinese traditional medicine response indicator corresponding to the difference of different chemotherapeutics response indicator clearly according to the fluorescence that is target spot through the antibody of dye marker, can contrast from different associated cancer clone above-mentioned quantitatively, and these clones all show the cytotoxicity response to specific anticancer chemotherapeutic agent in various degree.Explain that from the data of the medicine response indicator of different associated cancer clones there is different cytotoxicity susceptibility in system at relevant drug therapy in cancer cell described in the culture from the data of the medicine response indicator of patient tumors by extrapolation.Positive and/or the negative influence of medicine response indicator is estimated the Expected Results of chemotherapeutics by record, and described medicine response indicator is relevant with certain drug, be the disappearance or the existence of some drugs response indicator in the tumour cell, and the high or low meeting of quantity cause tumour that particular medication is not responded (or insensitive).It is invalid that this expection can be described in the antineoplaston that any chemotherapeutics through government (FDA) approval is most likely at individual patient.Therefore, in some specific embodiments,, select the effective chemotherapeutics that is used for individual patient thereby foreclose by the treatment of all being adopted invalid medicine according to display result from the medicine of individual cancer patient tumors cell response indicator.The present invention passes through the statistical analysis to the cell culture data that adopt drug therapy, has confirmed that the quantity of medicine response indicator is directly related with pharmaceutically active.
In some specific embodiments, the tumour cell sample can be obtained by the circulating cancer cells in the blood, and this circulating cancer cells has been represented metastatic carcinoma in the body.In some concrete enforcement case, the tumour cell sample can be obtained by the lymph node of contiguous primary tumor, and the circulating cancer cells in the lymphatic system can obtain by the biopsy method as the bronchoscope biopsy.In some specific embodiments, the tumour cell sample can be obtained by primary tumor, and tumor tissues to be sample by biopsy or excision obtain.In some specific embodiment, when sample is during available from primary tumor, tumor tissues can be fixed in the formalin, and be embedded in paraffin fast in, can be cut into slice, place to be used on the microslide checking.In some specific embodiments, the microslide of being cut into slices by the paraffin mass preparation band of tumor tissues can adopt dimethylbenzene and ethanol cleaning to dewax, and can be through the processing of antigen retrieval process, for example heating/hydrolysis (hydroloysis)/renaturation, then can be with suitable fluorescently-labeled monoclonal antibody dyeing, described monoclonal antibody is employed different cell components in the quantitative measurment of antagonism discrimination process and medicine response indicator.In some specific embodiments, can adopt computerized fluorescence microscopy system to the tissue on the treated microslide check, imaging, analysis and record.In some specific embodiments, can be simultaneously under same field of view to 5 kinds on the microslide of band section or more kinds of fluorescence antibody is measured, imaging, analysis and record.
The present invention can be used to analyze cancer and any methods of treatment in any source.For example, come from the cancer cell system of chest, lung, colon and the cancer in other epithelial cell source, the differential cytotoxicity medicine that they are all ratified government (FDA) shows resistance (or susceptibility) in various degree.Cytotoxic drug can include but not limited to that the relevant medicine of carboplatin, cis-platinum, oxaliplatin, Docetaxel, taxol (paclitaxel), taxol (taxol), Vinorelbine (vinca alkaloids), 5 FU 5 fluorouracil is (as Xeloda, xeloda), gemcitabine and anthracene nucleus class.In some specific embodiments, cancer therapy drug can comprise the human monoclonal antibody, as Herceptin (trastuzumab) (Trastuzumab, herceptin), Cetuximab (cetuximab) (Erbitux, erbitux) and bevacizumab (beracizumab) (A Wasiting, avastin).
In some specific embodiment, medicine response indicator comprises following cell component (antigen), and can be used to estimate the validity through the corresponding cancer therapy drug of FDA approval:
Medicine response indicator medicine
The ERCC1 carboplatin
Cis-platinum
Oxaliplatin
The estrogen receptor Tamoxifen
Aromatase inhibitor
'beta '-tubulin III hypotype Docetaxel
Taxol (paclitaxel)
Taxane (taxane)
Vinorelbine
The medicine that thymidylate synthetase 5-FU is relevant
Xeloda
The ribonucleotide reductase gemcitabine
Topoisomerase II anthracene nucleus class
The HER-2/neu acceptor, PTEN Herceptin (Trastuzumab)
In some specific embodiments, medicine response indicator can comprise that with the mab treatment medicine through suitable mark be the antigen of target spot, as suitable fluorescently-labeled Herceptin (trastuzumab) (Trastuzumab, herceptin), Cetuximab (cetuximab) (Erbitux, erbitux) and bevacizumab (beracizumab) (A Wasiting, avastin).
In a specific embodiments, the invention provides the method that a kind of selection is used for the treatment of individual cancer patient's treatment for cancer medicine, comprising from the patient, obtaining the tumour cell sample, adopt antibody to determine that the multiple medicine in the sample responds indicator; And selection chemotherapeutics.In some specific embodiments, antibody can be through fluorescently-labeled, for example monoclonal antibody.When antibody during through fluorescence labeling, specificity can be through different fluorochrome labels at the antibody of every kind of medicine response indicator to be measured, described fluorescent dye has difference and excites and non-overlapped emission spectrum, can carry out quantitatively multiple medicine response indicator simultaneously.Typically, medicine response indicator is the cell component relevant with the mechanism of action of chemotherapeutics, and is based on that the existence/disappearance of the medicine response indicator that exists in the cell of sample or quantity selects.When the existence that detects one or more medicine indicator, and wish the medicine response indicator that exists when quantitative, thisly can comprise that quantitatively fluorescence intensity and one or more reference datas with sample Chinese traditional medicine response indicator compare.Typically, method of the present invention can comprise at least 5 kinds of medicine responses of mensuration indicator.In some specific embodiments, determination step comprises multiple with in the sample, for example the quantity of identical medicine response indicator compares in the quantity of 5 kinds or more kinds of medicine response indicator and the cell, and described cell is known to the response of the chemotherapeutics that responds indicator by medicine and play a role.The sample of any same type all can be used for practice of the present invention, and for example sample can be taken from the circulation cancer cell in the blood samples of patients, takes from the lymph node of contiguous patient's primary tumor or takes from patient's primary tumor.Method of the present invention can be used to any suitable known chemotherapeutics of affiliated technical field is selected, relevant medicine, Xeloda, gemcitabine, anthracene nucleus class, the human monoclonal antibody of carboplatin, cis-platinum, oxaliplatin, Docetaxel, taxol (paclitaxel), taxol (taxol), Vinorelbine, vinca alkaloids, 5 FU 5 fluorouracil for example, Herceptin (Trastuzumab), Cetuximab (Erbitux) and bevacizumab (A Wasiting).In some specific embodiments, at least a medicine response indicator is ERCC1, and chemotherapeutics is selected from carboplatin, cis-platinum and oxaliplatin.In some specific embodiments, at least a medicine response indicator is a 'beta '-tubulin III hypotype, and chemotherapeutics is selected from Docetaxel, taxol (paclitaxel), taxane and Vinorelbine.In some specific embodiments, at least a medicine response indicator is a thymidylate synthetase, and chemotherapeutics is selected from the relevant medicine of 5-FU, formyl tetrahydrofolic acid, Pemetrexel and Xeloda.In some specific embodiments, at least a medicine response indicator is a topoisomerase II, and chemotherapeutics is selected from anthracene nucleus class, adriamycin and epirubicin.In some specific embodiments, at least a medicine response indicator is a topoisomerase I, and chemotherapeutics is an Irinotecan.In some specific embodiments, at least a medicine response indicator is the ribonuclease reductase, and chemotherapeutics is a gemcitabine.
Three kinds of diagnosis detecting methods have been set up to implement this method.These diagnosis detecting methods are: (1) medicine response indicator detects (DRIT); (2) be used for HER-2/neu and cross the Trastuzumab-taxane response detection (HER-Tax detection) that is expressed as positive patient with breast cancer; (3) circulation cancer cell detects (CCCT).
DRIT and HER-Tax are the detection methods that is used for the quantitative measurment of DRI, and the expression of DRI biomarker among the quantity of CCCT measurement circulating cancer cells (CCC) and the CCC.These three kinds of detections provide the integrated information of the medicine response of relevant individual patient tumour, can select the most effective chemotherapeutic treatment method by them.
For example, can utilize monoclonal antibody (MAB) to measure breast cancer and lung cancer, the perhaps expression of DRI in tumor tissue section's microslide, and this monoclonal antibody is the fluorochrome label through being used for sample dyeing.Set up a kind of index system (indexing system) with the expression of DRI and cytotoxicity response (IC with various cancers clone of different pharmaceutical resistance
50) associate.This Cytotoxic relevance relates to measurement and is embedded in the DRI of the cancer cell in the paraffin mass, can be that this benchmark is to express about the DRI that the mankind tumor tissue of downcutting on the paraffin mass cuts into slices as the standard cell lines of benchmark thereby set up a kind of.The term of reference of every kind of DRI in the tumor biopsy of measurement can be calculated by the resistance/response probability corresponding to medicine, and the resistance/response probability of medicine is the IC according to cancer cell in the culture
50Draw.Utilize the clinical effectiveness record to carry out retrospective clinical to verify the term of reference of this DRI index.Innovation in this application is based on: the development of (1) external imaging system; The index system of the cytotoxicity response that (2) is associated with the DRI level; (3) probability by the tumour resistance shows, corresponding to the term of reference of the DRI index of tumour response.Behind the term of reference of the DRI index of considering the patient and clinical response, the attending doctor can have the drug prescription according to this patient of ground decision.
Brief description of drawings
Fig. 1 is the typical curve that adopts InSpeck MIcrosope image intensity calibration kit (6 microns fluorescent microspheres) to generate, and this kit is the HER-2/neu quantitative data that is used to contrast from different tumour cell goods.
Fig. 2 is the digital picture that shows the HER-2/neu fluorescence signal of patient with breast cancer's histotomy.This section is by downcutting on the tumor tissues that is embedded in the paraffin mass, its through Herceptin-Alexa 532 dyeing handle, by the fluorescent microscope analysis and through the CCD camera imaging.
Fig. 3 has shown and has been selected to the quantitative target area (AOI) in the cell membrane zone of Herceptin-Alexa 532 dyeing of HER-2/neu.
Detailed Description Of The Invention
Term " biological marker (biomarker) " is defined as " as normal biological process, pathogenic process, perhaps to the indicator of the pharmacology of therapeutic intervention response and by the feature of objective measurement and evaluation ". For the application's purpose, cell Chinese traditional medicine response indicator is a kind of biomarker that the information how cell respond medicine is provided.
The present invention is based on the development of PAC: (1) is carried out quantitative measurment according to fluorescence intensity to the response indicator of the medicine in the target area of every kind of cell (DRI) and is measured simultaneously, carries out standardization with reference data. Fluorescence display through the dyeing quantity of the MAB of the anti-DRI of mark. (2) adopt one group (battery) that different cancer therapy drugs are had the clone of different resistances, and statistically the inhibition of every kind of medicine to cell in-vitro growth associated with the DRI measurement of relevant this medicine. (3) raw material of measuring as DRI from individual cancer patient's tumour cell and tumor tissues.
The low usefulness of chemotherapeutics and actual side effect, and the required expensive and time of development of new cancer therapy drug promoted the development of PAC. It is a kind of very large demand that existing cancer therapy drug more effectively is used for individual patient. The clinical practice of PAC can provide one or more following advantages:
1) index system provides reference about the probability that prescription medicine is developed immunity to drugs for the patient, and this probability that to be DRI by the patient express is represented, index system will instruct the doctor to open the prescription of a kind of medicine or medicine series like this.
2) simultaneously several medicines (3-6 kind) through FDA approval and several quantitative biomarker for these medicines are detected. In this way, can rapidly invalid medicine be deleted from alternative methods for the treatment of, and replace with selectable more effective medicine.
3) detection can be carried out (2-3 days) in real time, but is cheap (being less than 1000 dollars), large capacity (specialized laboratory reaches 100 detections every day), and can in the general hospital, carry out.
4) image technique is that the quantitative assay of tumour DRI value is laid a good foundation, and it comprises the evaluation to Tumor Heterogeneity (heterogeneity). This mensuration to heterogeneity can demonstrate this patient to the duration of response of this medicine.
According to conscientiously the consulting of document, through being widely used in breast cancer, lung cancer and colon cancer and being respectively applied to breast cancer, lung cancer or colon cancer of FDA approval, and on mechanism the catalogue of the medicine relevant with DRI as described in the following table.
| Cancer therapy drug | DRI | The type of cancer | Note |
| Trastuzumab | HER-2/neu acceptor PTEN | Breast cancer breast cancer | Treat the FDA approval. Another kind of definition is to the factor of the response of Trastuzumab. |
| The TAM aromatase inhibitor | The ERs ERs | Breast cancer breast cancer | Generally be accepted and go through. |
| Taxane family Docetaxel taxol (paclitaxel) taxol (taxol) vinca alkaloids | III type 'beta '-tubulin | Breast cancer colon cancer NSC-lung cancer | It is reported relevant with IV type 'beta '-tubulin hypotype with I, II. |
| Gemcitabine | Ribonucleotide reductase (RR) | Lung cancer | Common and platinum, taxane are used in combination. |
| Platinum family | ERCCI | The main medicine that is used for lung cancer. |
| Oxaliplatin cis-platinum and carboplatin | ERCCI | Colon cancer NSC-lung cancer | Often and 5-FU, taxane etc. be used in combination. Oxaliplatin is used for colon cancer, and cis-platinum and carboplatin are used for lung cancer. XPP or ERCCII also have been used as DRI. |
| The 5-FU FUDR of 5-FU family capecitabine (capecitabine) | Thymidylate synthetase (TS) Thymidine phosphorylase (TP) dihydropyrimidine dehydrogenase (DPD) | The breast cancer colon cancer | Frequent and oxaliplatin, Irinotecan are used in combination. |
| Anthracycline Doxorubicin (and adriamycin) | Topoisomerase II | Breast cancer | Through being usually used in drug development. |
Following table provides the medicine response indicator (DRI) of the target anticancer chemotherapeutics of 6 kinds of cancers, and this table also provides other medicine response indicator. Listed chemotherapeutics comes from comprehensive cancer network (National Comprehensive Cancer Network, NCCN) the oncology clinical practice guideline (Clinical Practice Guidelines in Oncology) in 2006 of country. The DRI of relevant chemotherapeutics is from document.
Annotate:
1) to the fresh cancer cell in the biopsy after paraformaldehyde is fixing, and is fixed in the formalin and is embedded in tumor tissue section in the paraffin mass and carry out DRI and measure.
2) the correlation clinical research that detects for DRI will be carried out IV phase patient or metastatic patient. Tumor size or quantity according to this group patient are determined how known clinical response. But this result is applicable to all stages of chemotherapy, comprises the auxiliary phase (adjuvant stage).
3) with regard to lung cancer, these medicines are used for ED-SCLC (representing with asterisk) and non-small cell lung cancer, except Doxorubicin only is used for ED-SCLC.
4) bevacizumab (Arastin) is used for the combination of breast cancer, colon cancer, lung cancer. An independent DRI detects the evaluation of the combination that can obtain relevant Arastin and tumour cell.
As mentioned above, set up index system and need one group of cancerous cell line that different cancer therapy drugs is had different resistances. The tumour that comes from immortal cell line shows powerful propagation usually, and it satisfies the requirement of functional cancer cell model system. Cancerous cell line has been used to predict the response to cancer therapy drug, and has obtained success to a certain degree. In a research, for the sensitiveness of 39 kinds of human carcinoma cell lines to 55 kinds of cytotoxicity cancer drugs, these 39 kinds of human carcinoma cell lines are analyzed. Conclusion is: the database of complete gene expression and chemical-sensitive implementations can be used for the system that exploitation is used for the prediction pharmaceutical efficacy. In another research, the II phase result of the test of 31 kinds of cytotoxic drugs is relevant with the screening of National Cancer Institute human tumor cell line group. Its conclusion is that the body outer cell line model can be predicted non-small cell lung cancer, breast cancer and oophoroma in a different manner. Although research is not aimed at clinical application system, the cytotoxicity sensitive property that they show the human carcinoma cell line is enough in the correlation and prediction to clinical test results.
The effective application of target anticancer treatment has formed personalized anticancer chemotherapy (PAC) with molecular diagnostic techniques. At present, PAC is tool prospect in the treatment of cancer development. HER-2/neu expresses and the Herceptin that is used for the treatment of metastatic breast cancer (MBC) patient is that first is through Rx-Dx associating (coupling) of FDA approval, and as the model system of PAC.
The present invention includes the application of external imaging technique and molecular diagnosis, it is characterized in that being used for the quantitative of medicine response indicator/biomarker available from the tumour cell of individual patient, and use the therapeutic scheme that this information selects to be suitable for the patient. Molecular diagnosis method is comprised of following three steps:
(1) obtains the cancer cell sample from the individual cancer patient; These cancer cells can or be taken from the file cell of serial section available from the circulating cancer cells in freezing tumor biopsy, biopsy raw material, the blood, and described section is downcutting through the fixing tumor tissues of formalin from be embedded in paraffin mass.
The biomarker of (2) expressing in the qualitative assessment tumour cell (medicine response indicator, DRI), because the expression of these biomarkers is relevant to the resistance of specific targeted therapy with tumour cell. Assessment can comprise to be adopted through suitably fluorescently-labeled, the monoclonal antibody take the cell biological label as target spot (Mab), and this cell biological label is tumour cell produces resistance to targeted therapy reason. Can adopt computerized fluorescence microscope system, suitable software and reference data to finish quantitatively.
(3) according to the expression of tumour cell Chinese traditional medicine response indicator and tumour cell to the correlation between the cytotoxicity response of medicine relevant on the mechanism, the therapeutic scheme that selection suits. Can use one group of relevant cancerous cell line that different cancer therapy drugs is had different resistances. These medicines to the effect of the cancerous cell line in culture propagation and in these clones the expression of corresponding DRI together be determined. Because these data are relevant to the resistance of target therapeutic agent with tumour, so these data can illustrate the DRI expression (passing through fluorescence) of tumour cell. To promote (extending) to the correlation between the clinical response of this drug therapy for DRI expression and the particular patient of specific medication, its experiment basis is exactly the relation of external medicine cytotoxicity-DRI. This correlation is generalized in the DRI measurement that is embedded in the cancer cell in the paraffin mass, and it can be used as the reference of expressing from the DRI of the people tumor tissue section that paraffin mass downcuts. Can determine the term of reference of every kind of DRI measured value in the tumor biopsy, it has shown the IC based on cancer cell in the culture50Accordingly to the resistance/response probability of medicine.
According to DRI in the tumour cell actively and/or negative influence, the validity of the targeted therapy that is applied to patient tumors is predicted, namely in the tumour cell disappearance of DRI/existence or quantity low/height can cause tumour that particular medication is not responded. Therefore, the DRI measurement result according to cancer patient's tumour cell adopts the treatment of invalid medicine to exclude all, thereby has finished the selection to the targeted therapy that is effective to the patient.
The tumour of individual patient can also recently estimating by the percentage with drug-fast tumour cell and the percentage that does not have drug-fast tumour cell (heterogeneity of tumour) to the duration of response of specific medication. When the non-resistant part of tumour was attacked by medicine and eliminated, so remaining resisting cell became dominant part, and whole tumour develops immunity to drugs. This reasoning shows that the mensuration to Tumor Heterogeneity will be the sign of resistance, and further shows the life span that must prolong with another kind of active drug the patient as combination and/or successive treatment.
In clinical practice, patient's responsiveness (RR) and/or PD time (TTP) are associated statistically with the DRI measured value of their tumour. Term of reference with resistance (non-response) probability of every individual patient of different DRI indexes will be fabricated. Behind the term of reference of the DRI of reference individual patient index and clinical response, the attending doctor can make with regard to this patient's drug prescription the decision of foundation.
In order to implement the PAC method, three kinds of diagnosis detecting methods have been set up to serve the cancer patient.These diagnostic test methods are: (1) medicine response indicator detects (DRIT); (2) Trastuzumab-taxane response detects (HER-TAX test); And (3) circulating cancer cells detects (CCCT).These detections will further describe hereinafter.
Cancerous cells has two kinds of obvious characteristics:
1) in the host, duplicate uncontrollably, form tumour, and
2) tumour increases, spreads and hinder the vital functions of health, causes death.
When cancer or tumour can not when removing or destroying, so just be needed the anticancer chemotherapy of general by physical force (operation or radiation).
The key of anticancer chemotherapy success is to destroy tumour cell and does not damage Normocellular ability, and normal cell is the cell that accounts for absolute majority in the host.Owing to uncontrollablely duplicate and harm the feature that normal function is a cancer cell, therefore most of anticancer cytotoxic drugs all are designed to pair cell entity (entities) and the related process of reproduction process is attacked.
Modern anti-cancer cytotoxic medicine all belongs to targeted therapy, and its target is the related cell component of life process, for example dna replication dna enzyme, nucleotide (structural unit of nucleic acid) enzyme, DNA repairase, transmit the acceptor of reproducing signals etc.According to these crucial protein parts (moieties), can easily prepare and have scope 10
11-13Mol
-1The antibody, particularly monoclonal antibody (Mab) of high affinity costant.In addition, when Mab and fluorescent dye when chemical bond is connected, can detect the quantity and the position of fluorescence Mab-target spot compound, imaging and record.When different Mab uses the different fluorochrome label with different and non-overlapped excitation/emission spectral position, (ROI) imaging in the target area simultaneously of these different Mab-target spots, but will detect respectively.
A kind of instrument that is effective to detect fluorescent composition is computerized fluorescence microscope system (FMS).Can the intracellular region territory that comprise these compounds be limited, the detected value of fluorescence can be with total fluorescence in finite space zone, the perhaps mean fluorecence of every pixel, and the maximum intensity of every pixel is represented perhaps or even in should the zone.Shown this ability among the embodiment 1.
Except carrying out quantitative measurment, can come standardization is carried out in the FMS measurement by using fluorescent microsphere.After utilizing fluorescent microsphere under different wave length, FMS to be calibrated, can measure from the angle contrast FMS of time, and can the FMS measurement of carrying out on the different FMS be compared.This operation sees embodiment 2.
The tumor tissues of individual patient is taken from three kinds of sources usually: (1) biopsy raw material that sampling obtains from tumour or contiguous tumor lymphatic knot, (2) the operation raw material that obtains from the operation of tumor resection, (3) are represented circulating cancer cells in the blood of metastatic tumo(u)r.We on inspection the tumour cell in all these sources, and all there is its unique requirement in every kind of source.In order to characterize the tumour cell that is obtained by biopsy, tumour cell is distinguished in most important requirement from normal cell.When tumour cell is to derive from epithelium, but cell on every side is not to derive from epithelium (haemocyte or lymphocyte), and according to epithelial characteristic cytokeratin skeleton, differentiation is exactly direct relatively so.Can adopt the Mab of specificity anti-cell skelemin.For circulating tumor cell being distinguished from blood and using a kind of enrichment method, by this method most of normal plasma cells to be got rid of, carcinous epithelial cell is left and is used for characterizing.The most at large, from specimens from pri, collect tumor tissues; These are organized in the formalin fixing, and are embedded in the paraffin mass and store.Through after the process of dewaxing, cleaning and antigen retrieval, make the microslide of band section so that observe then by these pieces.Embodiment 3 has shown such preparation process.Every kind of antigen or even the albumen target spot all may need independent activation/repair process.
In embodiment 4, we will describe detection by quantitative and pass through the middle experimentation of being with the Mab-antigenic compound of fluorescence of viewing area (ROI) of the microslide of FMS recording strip section.This process need deducts the autofluorescence background by computer system.Adopt not by the paraffin mass serial section of the Mab dyeing of band fluorescence, observe similar area by FMS and can obtain this background.In addition, the optical measurement process will be consistent, and with the information stores of relevant background in computing machine so that cut as a setting subsequently.
Embodiment 5 has described and has been embedded in dyeing and the quantitative measurment that responds indicator in the paraffin mass through tumor tissue section's Chinese traditional medicine of formalin fixed.
Embodiment 6 has described the evaluation to the heterogeneity in the zones of different of tumor biopsy.The tumour of individual patient can also be estimated by the ratio (heterogeneity of tumour) of number percent that drug-fast tumour cell is arranged and the number percent that does not have drug-fast tumour cell the duration of response of certain drug.Will be to the mensuration of tumour heterogeneity as the mark of resistance, and further show must be with another kind of active drug as successive treatment to prolong patient's life span.
Described the structure of DRI index among the embodiment 7, this DRI index is expressed DRI relevant on the cytotoxic effect of chemotherapeutics and the mechanism and is associated.This method established the human tumor cells cultivated the responsiveness of chemotherapeutics and DRI are expressed between statistically significant correlation, wherein DRI expresses with the mechanism of drug effect pattern relevant.As described in embodiment 9, carry out statistical analysis these two numerical value are associated, and establish a DRI expression that can be used as demonstration clinical response index.
Described among the embodiment 8 that above-mentioned external index system is generalized among the structure of DRI index system, its basis is the standard cell lines through paraffin-embedded cultivation.This technology has been established a kind of reference standard that reflects the influence of fixation of tissue and processing.
Describe three kinds of diagnostic assays methods that relevant individual cancer disease of patient management information can be provided among the embodiment 10 in detail.In order to implement the PAC scheme, these diagnosis detecting methods have been set up to serve the cancer patient.Diagnostic assays is: (1) medicine response indicator detects (DRIT); (2) Trastuzumab-taxane response detects (HER-TAX detection); And (3) circulating cancer cells detects (CCCT).All describe in detail in an embodiment for every kind.
Describe the necessary step of the PAC that sets up the patient with breast cancer among the embodiment 11 in detail, and provide related data to support this scheme.
The design proposal of the necessary clinical correlation research of confirmation PAC system has been described among the embodiment 12.Thereby at first carrying out retrospective study associates the DRI measured value of the tumour cell of known clinical effectiveness and those same patient.Subsequently, the perspective study meeting of relevant DRI data associates patient's treatment results.This test figure will be as IDE, and causes FDA to become PMA.
The end user uses the mab treatment agent, as when being used for the Herceptin (Trastuzumab) of breast cancer and being used for the Cetuximab (Erbitux) of colon cancer, such scheme can play a role especially effectively.In the method, we after fluorescent dye is connected to therapeutic Mab, use the probe of monoclonal antibody as target spot-acceptor itself.Obviously, if therapeutic Mab can not be connected on the tumour cell, tumour cell can not produce response to therapeutic Mab.It should be noted that its inverse proposition may be incorrect, that is to say that Mab is connected to may not produce the cytotoxicity response on the tumour cell.Therefore, the important information that is responded the method for quantitative measuring acquisition of indicator by this medicine is owing to there is not medicine response indicator, has determined that thus which kind of cell can not show the cytotoxicity response to medicine.Yet favourable medicine response indicator is measured certain information may not can be provided, and promptly these tumour cells can show the cytotoxicity response.In other words, the negative tumour cell that indicating of medicine response indicator can not show the cytotoxicity response, can not determine that tumour cell can show the cytotoxicity response but medicine response indicator is positive, because also have the influence of other key factor.Therefore, we have " invalid medicine indicator " thereby will exclude by invalid medicine (but still showing spinoff) the tumour of individual patient.Understanding to invalid medicine indicator is very useful and reliable, and this is that in cell culture, this conclusion has obtained the support on the statistical significance because under a series of qualificationss.
Instructions has above been summarized the ingredient that comprises in the personalized anticancer treatment (PAC) substantially.At first, target therapeutic agent is by FDA exploitation and approval.Secondly, basic cell target spot process evaluation in laboratory and clinical research.The 3rd, the antibody (specific monoclonal antibody) of anti-this albumen target spot of specificity has been produced and the fluorochrome label through suiting.The 4th, assemble computerized fluorescence microscope system to the quantity and the position of these fluorescence Mab-target spot compound in the tumour cell measure, imaging and record.The 5th, representational tumour cell raw material can obtain by biopsy method, operation sample or blood circulation tumour cell.The 6th, in the external environment of clearly definition, the quantity of completed correlation research proof medicine response flag thing is associated statistically with the cytotoxicity response of tumour cell, selects suitable chemotherapeutics thus.
These six ingredients connect the basis that has formed " personalized anticancer treatment " each other, and this personalized anticancer treatment excludes those according to advisory information to the invalid medicine of particular cancers patient.This scheme is proved in clinical correlation research, and this research relates to the medical treatment contribution of the information of relevant " invalid medicine indicator ".The starting stage of this demonstration provides in the program of FDA approval use Herceptin, and can be described in independent part.
PAC system organization is got up, and we provide to using the method for FMS in available from the quantitative measurment of the biomarker of individual patient tumour cell.To describe in an embodiment at ad hoc approach available from the tumour cell of separate sources.
Embodiment 1
The mensuration of the fluorescent composition in the fixed cell on the microslide adjusts the telescope to one's eyes.
Our system is the computer assisted LeicaDMRXA fluorescent microscope that is equipped with CCD camera, eight filter rotating discs (eight-filter cube turret) and is used for the Image-Pro Plus software of picture catching and image processing.At present, we can measure the fluorescence of isocellular five kinds of spectral ranges; Two kinds are used for cell and identify that three kinds are used to measure biomarker.For with the dyeing of multiple mouse monoclonal antibody pair cell, directly every kind of antibody of mark.This is to adopt to finish from fluorescein isothiocynate and the Alexa dyestuff succinimide ester derivant of Molecular Probes.
This embodiment demonstrated to the HER-2/neu of breast cancer cell line quantitatively.With Alexa 532 labeled monoclonal antibody Herceptins, and with the anti-wide spectrum cytokeratin of FITC mark (anti-pancytokeratin).(SKBR-3) hatches with above-mentioned antibody with breast cancer cell line, cleans then and places the antidamping medium to redye with DAPI.Adopt filter (filter cubes) to obtain digital picture through the suitable time shutter, this filter can be distinguished DAPI, FITC and Alex 532 signals in the same cell.Utilize cytokeratin fluorescence to delineate out the area of space of every kind of cell.These profiles are preserved, called and cover on Alexa 532 images, be i.e. the HER-2/neu signal.Following table has shown the quantitative data that HER-2/neu expresses in four kinds of cells.The 1st row have provided object number, the 2nd row are represented cell compartment with pixel count, and the 3rd row have provided the average fluorescent strength of every pixel in every kind of cell, and the 4th row have provided the accumulation fluorescence intensity of every kind of cell.HER-2/neu in the data presentation cell expresses and can change.
| The cell numbering | The zone of pixel | The fluorescence intensity of every pixel | Total fluorescence |
| 1 | 4444 | 362.2061 | 1609644 |
| 2 | 3783 | 314.2744 | 1188900 |
| 3 | 3130 | 395.1147 | 1236709 |
| 4 | 3126 | 233.0742 | 728590 |
Embodiment 2
The calibration of microscopic system.
In order to carry out quantitative immunofluorescence research, must be able to obtain digital picture and with in the different time sections with different microscopes on the fluorescence measurement value obtained compare.This is the Leica microscopic system to be calibrated from the simple and effective fluorescence standard of Molecular Probes by employing to realize.These standard items are to be (Inspeck MIcrosope image intensity calibration kit, the Inspeck Microscopy Image Intensity Calibration Kit) that 6 microns fluorescent microsphere constitutes by diameter.The suspending liquid of microballoon is placed on the microslide, and air drying also places antidamping matrix, covered.Obtain image through the different time shutter, the described time shutter is overtime scarcely, in order to avoid the degree that reaches capacity, promptly for the image of 12 bits, every pixel reaches 4096 flat fluorescents.Adopt Image-Pro Plus software processes image, obtain every pixel fluorescence intensity of about 4 or 5 microballoons under each time shutter.Draw the typical curve that adopts every kind of filter to obtain, wherein every pixel average fluorescent strength is corresponding to every millisecond of time shutter.The example of this linear standard curve sees Fig. 1.According to reference data, slope and intercept are used to calculate the generation required time shutter of 2000 unit mean fluorecences.With regard to curve map shown in Figure 1, the numerical value when having obtained 176 milliseconds.Therefore, by using identical fluorescence standard, select the suitable time shutter, thereby each microscope/filter can be calibrated and produces identical fluorescence intensity.
Embodiment 3
Be used to the preparation and the dyeing of the tumour cell preparation differentiating and characterize.
Can obtain lung carcinoma cell by the bronchoscope biopsy.After from the patient, taking out the biopsy raw material, can put it in the neutral physiological saline.Can in PBS, clean cell and cellular incubation to designated volume is used for counting.In the zone that the cell deposition of suitable number can be delineated by PapPen on microslide.After the drying, can with cell fixation in 2% paraformaldehyde, hatch, redye with the identification epithelial cell with DAPI with anti-wide spectrum cytokeratin-FITC.Obtain image and to having the epithelial cell counting of intact cell nuclear.Determine the ratio of epithelial cell in the cell on the microslide/wbc nuclear DNA, with this measured value as heteroploidy (aneuploidy), by the expression of afp receptor in the epithelial cell being carried out quantitatively, like this tumour cell and normal epithelial cell are made a distinction with monoclonal antibody specific dyeing.As described in embodiment 1, can use fluorescently-labeled antibody to carry out quantitatively.
The histotomy that obtains in operation or the biopsy can be fixed in the formalin, and be embedded in the paraffin mass.Can downcut 4 microns serial section from these paraffin masses, and place on the microslide.Can be according to the following steps to the section dewaxing: in the dimethylbenzene 2 times, each 5 minutes; In 95% ethanol 2 times, each 3 minutes; In 70% ethanol 2 times, each 3 minutes; In tap water at least 10 minutes then.For antigen retrieval, can be in 10mM EDTA, pH8 or 10mM citrate, pH6 heating microslide 30 minutes under 95 degree, at room temperature placed identical solution subsequently 20 minutes.Can in PBS, clean microslide and with the fluorescently-labeled antibody staining of anti-wide spectrum cytokeratin-FITC and other selection.
Can separate and identify the circulation cancer cell (CCC) in the blood in accordance with the following methods: adopt the cancer cell in two gradient centrifugation enrichment 15-20ml blood, remove most of haemocytes (negative selection, negative selection) with the immune magnetic pearl subsequently.In the cell deposition zone that PapPen delineates on microslide, and hatch together with mixed antibody (antibody cocktail) (antibody with reactive FITC mark of 9 kinds of cytokeratins of antagonism, and the glycoprotein relevant of expressing on people's cancer) with tumour.Place the antidamping matrix that comprises DAPI to redye.With fluorescent microscope scanning microslide and to having the FITC positive cell counting of intact cell nuclear.
In the CCC of standard detects, reclaimed above 100,000 leucocytes (WBC) with CCC.WBC hinders sometimes with the dyeing (but without HER-2/neu or cytokeratin) of dyestuff-MAB compound to cancer cell.Therefore, developed super enrichment method (SuperEnrichment), this method makes and is less than 3000 WBC and is recovered with cancer cell.In these preparation process, cell dyeing is not influenced by a small amount of WBC can.This method sees for details hereinafter:
The super enrichment method of CCC:
The peripheral blood of 15-20ml being handled through anticoagulation with PBS (6 hours, (spiked) comprises 100 MCF-7 breast cancer cells at most) is diluted to total 30ml;
Carefully with the cell suspending liquid layering of dilution, its gradient is 1.083 and surpasses 15mL in the 50ml conical tube, and under 20 ℃ in swinging bucket rotor (swinging-bucket rotor) with centrifugal 30 minutes of 2000rpm (not braking).
Shift out whole supernatants and collect the upper part at the 1.083G interface (interface) of 6-8ml;
Clean twice through the centrifugal 10min of 1200rpm with Hanks ' solution;
Carefully remove whole supernatants;
Adding 1 times of dilution buffer liquid to final volume is 40ml, and stirs;
Add 5ml MACS cell fixation solution (CellFix Solution), mix and hatch 30 minutes after, add 5ml MACS CellPerm solution, mix and under room temperature (RT), hatched lucky 5 minutes;
Centrifuge cell suspending liquid is 10 minutes under 1200rpm;
In final volume is 1 times of MACS cell dyeing solution (CellStain Solution) of 600 μ l, cell mass is suspended again;
Add 200 μ l FcR closed reagent and mixings;
Add 200 μ l MACS CK microballons and under 20-25 ℃, hatched 45 minutes;
Adding 100 μ l anti-cell keratin-FITC also hatched 10 minutes again;
Add 4ml 1 times of MACS cell dyeing solution (Cell Stain Solution) and centrifugal suspending liquid under 1200rpm;
In the magnetic field of MACS separation vessel, place the positive post of selecting;
The cell that suspends again is added in the post, makes leucocyte (WBC), and clean with 1 times of dilution buffer liquid of 3 * 500 μ l degassing by pillar;
Pillar is removed from separation vessel, placed the 15ml test tube;
Draw 1 times of dilution buffer liquid through the degassing at 1ml pillar top, and the circulating cancer cells that is retained of plunger (plunger) wash-out that uses with pillar of employing;
Cell mass is centrifugal and directly be deposited on the microslide, and described microslide was room temperature (RT) dry 8-24 hour down; And
Add the mounting medium that contains DAPI, and sample is carried out the area of computer aided microscopic analysis.
Embodiment 4
Biomarker to the tumor tissue section of downcutting on the paraffin mass is expressed quantitatively.
The fluorescence intensity measurement value of striding (across) histotomy reflected combine with antigen through fluorescently-labeled one anti-amount, itself so represented the amount of target protein (biomarker) in the cell.Yet, need to consider through the tissue of formalin fixed intrinsic autofluorescence, to obtain repeatably quantitative data.According to said method, but do not use fluorescently-labeled one anti-ly, the serial section of the identical tumour of microslide is in contrast handled, record autofluorescence.
Obtain the digital picture of 3 to 4 zoness of different of every kind of tumour.Adopt C1 (DAPI), C2 (FITC), C3 (Alexa 532), C4 (Alexa 594) and C5 (Alexa 647) filter, according to our reference data, the time shutter of being adopted will produce 2000 flat fluorescents.Contrast microslide and experiment glass slides are through identical processing.In addition, obtain image, do not comprise tissue on the described microslide thereby all the background area on the microslide is filtered at every turn.Adopt Image-Pro Plus software processes image.By the every pixel fluorescence mean value of histogram acquisition, and from suitable experimental image, deduct this numerical value at every kind of image background.Select 3 to 4 target areas (AOI) on each image, each zone comprises about 10 cells, is used for biomarker is carried out quantitatively.Fig. 2 has shown the image of the tumor of breast section of representational HER-2/neu dyeing.Can observe strong completely dyeing.The fluorescence intensity of diaphragm area is carried out quantitatively, and following table has shown 5 different patient with breast cancers' data.Wherein 4 HER-2/neu expresses about 2 to 4 times that are higher than autofluorescence.1 patient's autofluorescence and HER-2/neu signal similar.HER-2/neu across different tumor regions expresses the existence heterogeneity.We have observed the high expressed zone and low the expression between the zone has 3.3 times difference.
By UMM and the exposure of WR-877 microsecond
The Trastuzumab of the breast cancer tissue that obtains-A1532 dyeing
| The patient | Mean fluorecence in the image/every pixel *Picture number 1234 | The number percent picture number 1234 of reference data |
| The unknown autofluorescence of UMM 1,804 111914 A4 HER2 states does not have ABY | 305,285,303 mean values are that 298 231,285,311 mean values are 276 | 15.2 14.2,15.2 mean values are 14.9 11.6,14.2,15.6 mean values are 13.0 |
| UMM 01 505 1295 A9 HER2 state+3 autofluorescences do not have ABY | 473,496,564 mean values are that 511 151,147,135 mean values are 144 | 23.6 24.8,28.2 mean values are 25.5 7.6,6.8,7.4 mean values are 7.2 |
| UMM01 501 2051 C8 HER2 state+3 autofluorescences do not have ABY | 380,522,533,586 mean values are 505 not do | 19.0 26.1,26.7,29.3 mean values are 25.3 |
| WR 505 3597 B2 Exp1 HER2 state+3 autofluorescences do not have ABY | 191,167,153,172 mean values are 171 not do | 9.5 8.3,7.7,8.6 mean values are 8.5 |
| WR 505 3597 B2 HER2 state+3 autofluorescences do not have ABY | 182,170,220 mean values are that 191 115,137,118 mean values are 123 | 9.51 8.5,11.0 mean values are that 9.5 5.8,6.8,5.9 mean values are 6.2 |
| WR 505 2759 A1 HER2 state+3 autofluorescences do not have ABY | 136,135,163,148 mean values are that 146 66,87,79 mean values are 77 | 6.8 6.8,8.2,7.4 mean values are that 7.3 3.3,4.4,4.0 mean values are 3.9 |
*3 to 4 zones to each image are analyzed, demonstration be mean value.ABY represents the monoclonal antibody of the mark that is used to dye.
We have also carried out quantitatively 'beta '-tubulin III hypotype and ERCC-1 in estrogen receptor expression in the breast tumor tissues and the colon tumor tissue (DNA repairase).Following table has been listed data.At ERCC-1, 'beta '-tubulin III hypotype and estrogen receptor, dyeing organizes the ratio of mean fluorecence and autofluorescence (no antibody) to be respectively 5.7,3.6 and 15.8.
Quantitative measurment by the histotomy Chinese traditional medicine response indicator that downcuts on the paraffin mass
| Tissue markers | Mean fluorecence/pixel | Scope fluorescence/pixel |
| Colon ERCC-1 | The mean value standard deviation | Max min |
| Zone 1 | 1537 395 | 3760 799 |
| Zone 2 | 1420 393 | 3760 738 |
| Zone 3 | 1256 345 | 3760 434 |
| Contrast no Aby | ||
| Zone 1 | 243 28 | 356 146 |
| Zone 2 | 242 26 | 334 120 |
| Zone 3 | 251 39 | 410 42 |
| Colon β-Tub | ||
| Zone 1 | 533 90 | 876 301 |
| Zone 2 | 486 89 | 805 253 |
| Zone 3 | 485 84 | 754 186 |
| Contrast no Aby | ||
| Zone 1 | 136 23 | 245 73 |
| Zone 2 | 129 20 | 215 63 |
| Zone 3 | 150 28 | 358 72 |
| The chest estrogen receptor | ||
| Zone 1 | 1278 307 | 3326 639 |
| Zone 2 | 1489 269 | 3196 840 |
| Zone 3 | 1399 318 | 3218 844 |
| Contrast no Aby | ||
| Zone 1 | 73 20 | 161 18 |
| Zone 2 | 96 25 | 340 30 |
| Zone 3 | 94 21 | 394 47 |
Embodiment 5
With breast cancer is example, and the medicine in the tumor tissue section of formalin fixed that is embedded in the paraffin mass is responded dyeing and the digitized measurement that indicator carries out.
We have set up a kind of repeatably fluorescence microscopy method, be used for to by the HER-2/neu expression of receptor of the breast tumor tissues that downcuts on paraffin mass section quantitatively.The histotomy that is opposite on the microslide according to following steps dewaxes: microslide was placed dimethylbenzene 5 minutes, repeat once; Microslide was placed 95% ethanol 3 minutes, repeat once; Microslide was placed 70% ethanol 3 minutes, repeat once; In distilled water, clean microslide.Then by at 10mM, 95 degree heating 30 minutes down in the citrate of pH 6.0 was carried out antigen retrieval to microslide in 20 minutes and is handled thereby at room temperature cool off subsequently.Simultaneously microslide is hatched with Herceptin-Alexa 532 conjugates and anti-wide spectrum cytokeratin-FITC.According to reference data, the time shutter that employing can produce 2000 flat fluorescents is obtained 3 to 4 digital pictures of every kind of tumor biopsy.Cross on the intact cell film of the cell of expressing HER-2/neu and show very strong fluorescent dye.Check 3 to 4 target areas (AOI) on each digital picture.Can make the histogram (every pixel mean fluorecence, standard deviation, minimum and maximal value and complete fluorescence intensity) of the quantitative fluorescence data that show AOI.Thereby this fluorescence data is used to select the zone of the film that is colored quantitative to the expression of HER-2/neu.The zone that Fig. 3 one of has shown among the above-mentioned AOI, this zone is selected and delineated out and be used for quantitatively.Following table has shown quantitative measurement data (with zone and the every pixel mean fluorecence that pixel quantity is represented, density brightness).
| Target # | Zone (pixel) | Every pixel mean fluorecence |
| 1 | 476 | 203.9727 |
| 6 | 60 | 196.6833 |
| 14 | 215 | 198.4419 |
| 25 | 82 | 194.2927 |
| 30 | 226 | 192.2876 |
| 33 | 1788 | 205.6356 |
| 65 | 51 | 184.9412 |
| 89 | 79 | 202.6456 |
| 96 | 99 | 191.2121 |
| 100 | 52 | 189.9038 |
| 114 | 198 | 208.0404 |
| 116 | 100 | 189.540 |
Embodiment 6
The assessment of the heterogeneity that HER-2/neu in the zones of different of tumor biopsy is expressed.The heterogeneity that HER-2/neu expresses in the patient tumors cell can determine the whole and duration to Herceptin treatment response.We can utilize above-mentioned quantivative approach to come this heterogeneity of assess patient tumor tissue section.Data from four AOI of the digital picture of two patient with breast cancer's tumor tissues are as shown in the table.We observe the difference that has 2.1 times (patient is 1) to 3.3 times (patient is 2) between the minimum and maximum fluorescence intensity of film of HER-2/neu-dyeing.
The heterogeneity that Her-2/Neu in the zones of different of patient tumors expresses is assessed
| The every pixel mean fluorecence of patient #1 mean value standard deviation | The every pixel mean fluorecence of patient #2 mean value standard deviation | |
| Zone 1 | 473(25) ** 40 | 380(22) ** 31 |
| Zone 2 | 493(24) 34 | 522(24) 41 |
| Zone 3 | 559(26) 37 | 533(21) 31 |
| Zone 4 | 586(24) 73 | |
| The autofluorescence that is deducted | ||
| Maximum fluorescence/tumour | 491 | 569 |
| Minimum fluorescence/tumour | 230 | 173 |
| Maximum/minimum | 2.1 | 3.3 |
*Measure the numerical value of film
Embodiment 7
The vitro system that the expression of relevant medicine response indicator associates on a kind of cytotoxic effect with chemotherapeutics and the mechanism.
This embodiment has described the human cancer clone that will cultivate medicine relevant on the mechanism in the cytotoxicity response of cancer therapy drug and the cell has been responded the vitro system that the expression of indicator (DRI) associates.These data are carried out statistical analysis to determine to be used to the DRI level of sub-category ground showed cell toxicity response.
The derivative tumour of immortal cell line shows strong propagation usually and satisfies the requirement of functional cancer cell model system.Cancerous cell line has been successfully used in the prediction to the response of cancer therapy drug to a certain extent, and this shows that the chemical-sensitive implementations can be used to develop the system of prediction pharmaceutical efficacy.
In present embodiment, adopt the monoclonal antibody that links to each other with fluorescent dye the DRI in the clone of multiple breast cancer derivation to be carried out quantitatively as probe.The DRI expression levels is represented with numerical value, the fluorescence standard standardization of this numerical value through obtaining easily, thus can be used between each day and the contrast of the data between each laboratory.All medicines all have potential cell target spot relevant with binding mode on mechanism.
Breast cancer cell ties up to and is fixed on the microslide and dyes, this is by with the Trastuzumab that combines Alexa 532 (Herceptin, anti--HER-2/neu acceptor) or combine anti--ER of Alexa 594 or anti--cytokeratin of combining anti--TUB III of Alexa 647 or combining FITC is hatched realization simultaneously.Under the suitable time shutter (be produced as according to the fluorescence benchmark 2000 numerical value), obtain the digital picture of FITC signal (470nm/497nm/522nm), Alexa 647 signals (630nm/649nm/667nm), Alexa 594 signals (581nm/593nm/617nm) and Alexa 532 signals (546nm/557nm/567nm), and it is analyzed to determine the mean fluorecence of every pixel in every ROI (cancer cell).Determine the area of space of each ROI according to very strong cytokeratin fluorescence.Profile is saved, calls and covers on Alexa 532 images, Alexa 594 images or Alexa 647 images under the identical cell visual field.Software has formed the zone that shows each ROI and the table of every pixel mean fluorecence.
With regard to experiment condition, will comprise 10
4The cell suspension of individual living cells places 96 orifice plates that comprise 100 μ l nutrient culture media, is containing 5%CO
2In the air, made cell attachment 24 hours under 37 ℃.After this hatches process, the regulation analyzed according to disclosed pharmacokinetics, under the medicine of prescribed dose, described dosage is based on peak serum concentration (PPC) with cellular exposure.In the example of Tamoxifen, cell is grown 72 hours until converging.The nutrient culture media that will comprise 0.5%FCS then is added in each hole so that ER expresses maximization.Control wells comprises 100 μ l suitable culture bases, and carries out identical processing with instrument connection.After the processing the 72nd hour carried out WST-8 to orifice plate and analyzed.WST-8 is a kind of tetrazolium salts, and it forms colored first through cell dehydrogenasa biological reducing
Product.First
The amount of product and living cells quantity are directly proportional.Based on first
Analysis be successfully used in the detection of chemosensitivity.Use Biotek microwell plate reader under 460nm, to measure the absorption value in each hole.Calculate mean absorbance ± SE at every kind of drug concentration.The result recently represents with the growth inhibited percentage, compares with drug concentration.Use (log fa/log fu) to determine IC than the half effect figure of log C
50Value, wherein fu is unaffected component, and fa is affected component, and C is a drug concentration.
Different breast cancer cell lines is expressed with relevant DRI the biological response of chemotherapeutics and is met fully.Adopt DRI, HER-2/neu that two kinds of quilts characterize in detail and estrogen receptor (ER) to carry out initial research and come the verification model system.What HER-2/neu oncogene coding and EGF-R ELISA had an extensive homology strides the film tyrosine kinase receptor.HER-2/neu crosses and expresses the susceptibility raising that causes Trastuzumab (Herceptin) treatment.Another kind of is Tamoxifen to the effective targeted therapy of breast cancer, and it combines and hinder the influence of estrogen cell growth with the lip-deep estrogen receptor of cancer cell.ER is known significant predicted value in the process of the susceptibility of determining Tamoxifen is treated.
It is lip-deep when fluorescently-labeled Trastuzumab shows highest level to be combined in clone, shows the peak response of this clone to the Trastuzumab treatment.The clone that these are specific, HCC2218 and SKBR-3 show the propagation relevant with dosage and suppress, and are being low to moderate 0.032 and response arranged during 0.125mg/ml concentration respectively.Relatively, as shown in the table, in the Trastuzumab breast cancer cell line low, perhaps breed undisturbedly in conjunction with level, perhaps only when high drug dose, produce response.
The comparison of biological response and DRI expression in the breast cancer cell of cultivating.-Her-2/neu/ Trastuzumab
| Cell | IC50-1 | IC50-2 | The average combination rate * * of Trastuzumab | SD | Trastuzumab intermediate value combination rate * * | Trastuzumab combination rate scope * * | The cell that # is analyzed |
| HCC 2218 | 0.06 | 0.12 | 53.83 | 16.54 | 51.05 | 31.9-87.3 | 48 |
| SKBR 3 | 0.30 | 0.65 | 21.45 | 6.60 | 20.3 | 1.7-37.1 | 52 |
| MCF-7 | 1.25 | 1.36 | 3.11 | 1.16 | 3.1 | 1.12-5.8 | 57 |
| T47D | 2.0 | 2.0 | 3.55 | 1.46 | 3.45 | 0.85-7.7 | 59 |
| HCC 38 | NR * | NR * | 3.86 | 1.67 | 3.45 | 0.9-8.95 | 37 |
| HCC 202 | NR * | NR * | 2.96 | 0.82 | 2.85 | 1.6-4.75 | 54 |
Annotate: * NR-does not have response to the Trastuzumab treatment up to 2mg/ml
* is with respect to the mean fluorecence/pixel of reference data, and this reference data produces 2000 unit fluorescence after the exposure of experience certain hour.
According to similar mode, treatment shows hypersensitivity to the clone of ER high expressed (MCF-7) to Tamoxifen.On the contrary, treatment shows quite low sensitivity response to the clone that the ER conjugation is lower (SKBR-3, MDA-MB 231) to Tamoxifen, and is as shown in the table.
The comparison of biological response and DRI expression in the breast cancer cell of cultivating.-estrogen receptor/Tamoxifen
| Cell | IC50-1 μg/ml | IC50-2 μg/ml | ER mean value * * | SD | ER intermediate value * * | ER/ cell scope * * | The cell that # is analyzed |
| mcf-7 | 9.8 | 13 | 57.0 | 12 | 60.0 | 35-91 | 45 |
| T47D | 28.3 | 21.8 | 49.0 | 16 | 45.0 | 16-80 | 45 |
| SKBR 3 | 41.6 | 35 | 43.0 | 10 | 43.0 | 25-61 | 32 |
| MDA-MB231 | 42.5 | 42.5 | 23.0 | 7 | 22.0 | 9-41 | 61 |
| HCC 202 | NR * | NR ** | 25.0 | 8 | 23.0 | 13-42 | 33 |
| HCC2218 | NR * | NR ** | 29.7 | 12 | 25.7 | 18-88 | 34 |
| HCC 38 | NR * | NR ** | 32.1 | 7 | 31.4 | 22-53 | 30 |
Annotate: * NR-does not have response to the Tamoxifen treatment up to 50 μ g/ml
* is with respect to the mean fluorecence/pixel of reference data, and this reference data produces 2000 unit fluorescence after the exposure of experience certain hour.
These experiments are extended in relevant beta tubulin III (TUB III) expression and the consideration to correlativity between the biological response of anti-cancer medicine paclitaxel (PTX) and Docetaxel (DTX).These medicines combine and bring into play by stabilize microtubules their growth inhibited effect with TUB III.By inference, can change the antitumor action of these medicines by the expression of TUB III in several human cancers that comprise tumor of breast.
The comparison of biological response and DRI expression in the breast cancer cell of cultivating.-taxol (Paclitaxel)/beta tubulin III
| Cell | IC50-1 μg/ml | IC50-2 μg/ml | TUB III mean value ** | SD | TUB III intermediate value * | Cell scope TUB III | The cell that # is analyzed |
| T47D | 3 | 3.6 | 2.22 | 0.52 | 2.18 | 1.4-3.4 | 19 |
| MCF-7 | 6 | 7.1 | 3.58 | 1.03 | 3.42 | 2.1-6.3 | 39 |
| SKBR 3 | 10 | 8.5 | 4.64 | 2.07 | 4.01 | 2.6-14.2 | 19 |
| HCC2218 | NR * | NR * | 31.79 | 9.61 | 28.1 | 20.6-60.4 | 30 |
| HCC 202 | NR * | NR * | 34.49 | 10.5 | 32.53 | 20.7-80.7 | 78 |
| HCC 38 | NR * | NR * | 49.19 | 15.95 | 47.16 | 30.8-98.4 | 56 |
Annotate: * NR-does not have response to taxol (Paclitaxel) treatment up to 50 μ g/ml
* is with respect to the mean fluorecence/pixel of reference data, and this reference data produces 2000 unit fluorescence in experience certain hour exposure back.
The comparison of biological response and DRI expression in the breast cancer cell of cultivating.-Docetaxel/beta tubulin III
| Cell type | IC50-1 μg/ml | IC50-2 μg/ml | TUB III mean value ** | SD | TUB III intermediate value * | Cell scope TUB III | The cell that # is analyzed |
| MCF-7 | 1.0 | 0.8 | 2.2 | 0.5 | 2.2 | 1.4-3.4 | 19 |
| T47D | 5.5 | 4.5 | 3.6 | 1.0 | 3.4 | 2.1-6.3 | 39 |
| SKBR 3 | 6.6 | 12.5 | 4.6 | 2.1 | 4.0 | 2.6-14.2 | 19 |
| HCC2218 | 25.0 | 18.0 | 31.8 | 9.6 | 28.1 | 20.6-60.4 | 30 |
| HCC 202 | 25.5 | 22.0 | 34.5 | 10.5 | 32.5 | 20.7-80.7 | 78 |
| HCC 38 | 58.0 | 59.9 | 49.2 | 16.0 | 47.2 | 30.8-98.4 | 56 |
Annotate: * * is with respect to the mean fluorecence/pixel of reference data, and this reference data produces 2000 unit fluorescence after certain time shutter of experience.
As the example of Trastuzumab and Tamoxifen, different breast cancer cell lines meets fully to the biological response of PTX and DTX and the expression of TUB III.TUB III is in conjunction with the treatment sensitivity of the low clone of level (T47D, MCF-7, SKBR-3) to PTX and DTX.On the contrary, the higher clone (HCC 2218, and HCC 38) of TUB III level only the DTX to higher dosage show response.These clones also show response to PTX, but do not show IC
50Value.Confirm that a kind of clone (HCC 202) all has resistance to the treatment of PTX and DTX.
In another embodiment, the expression of thymidylate synthetase (TS) is associated with the response of breast cancer cell to chemotherapeutics 5 FU 5 fluorouracil (5-FU).TS is the key enzyme in the DNA biosynthesizing, and it has been assumed to be at prediction to playing an important role in the response based on the chemotherapy of 5-FU.Experimentize and check this relation.Experiment condition is similar during to the use Trastuzumab with analytical approach.Dosage range is 5-300 μ g/ml.
The comparison of biological response and DRI expression in the breast cancer cell of cultivating.-5 FU 5 fluorouracil/thymidylate synthetase
| Clone | IC50-1 FU | IC50-2 FU | TS mean value * * | SD** | TS intermediate value * * | Cell scope TS | The cell that # is analyzed |
| T47D | 8.64 | 6.72 | 25.2 | 7.6 | 23.2 | 18.1-56.6 | 77 |
| MCF-7 | 10.0 | 15.2 | 20.4 | 2.1 | 20 | 15.5-23.4 | 14 |
| SKBR-3 | 23.3 | 33.7 | 18.2 | 3.3 | 18.2 | 11.6-25.5 | 46 |
| HCC 202 | 181.3 | 162.5 | 15.1 | 2.2 | 14.6 | 11.0-20.0 | 24 |
| HCC 38 | 215.7 | ND | 11.9 | 2.3 | 12.1 | 9.1-16.5 | 33 |
| HCC2218 | 246.1 | ND | 10.6 | 1.9 | 10.4 | 7.3-15.3 | 49 |
Annotate: * * is with respect to the mean fluorecence/pixel of reference data, and this reference data produces 2000 unit fluorescence after certain time shutter of experience.ND is undetermined.
Different breast cancer cell lines meets fully to the biological response of 5-FU and the expression of TS.TS than the clone (T47D, MCF-7 and SKBR-3) of the 5-FU of low dosage response is expressed than those only to clone (HCC 38, HCC 202, the HCC 2218) height of the 5-FU response of higher dosage.Correlativity was significantly statistically between the grade of 5-FU response expressed with TS, as determined by following visible Pearson ' s related coefficient.
According to Pearson ' s related coefficient, DRI may be relevant statistically with the inhibition of the relevant external cell growth of medicine on the mechanism.This method is measured the linear relationship intensity between two kinds of variablees.Pearson ' s related coefficient is usually by r (rho) expression, can be in-1.0 to 1.0 scope value, wherein-1.0 be that complete negative sense (opposite) is relevant, 0.0 is uncorrelated, 1.0 is that complete forward is correlated with.Can use following formula to calculate Pearson ' s related coefficient:
The analysis of Pearson ' s related coefficient shows IC
50There are strong and significant statistics correlativity in value and DRI between expressing, and be as shown in the table.
The correlativity that biological response and DRI express
| Medicine | DRI | Pearson ' s coefficient | The p value |
| Trastuzumab | Her 2/neu | -0.941 | 0.0005 |
| Tamoxifen | Estrogen receptor | -0.976 | 0.02 |
| Docetaxel | Beta tubulin III | +0.978 | .001 |
| Taxol (Paclitaxel) | Beta tubulin III | +0.990 | .001 |
| 5 FU 5 fluorouracil | Thymidylate synthetase | -0.943 | .002 |
In order further to determine the separation that DRI expresses, thereby determine whether to exist the DRI expression that can be used to show biological response sub-categoryly, will analyze these correlation data by simple cluster analysis and tr pt analysis.
Embodiment 8
DRI index system based on paraffin-embedded standard cell lines through cultivating.
Carry out indexing for the DRI that is deposited in the people's tumor tissues in the paraffin mass is expressed, must set up a kind of reference standards that reflects the influence of fixation of tissueization and processing.At this purpose, many laboratories have used target antigen to show the clone of variable expression.According to the mode identical these clones are fixed, handled and analyze with clinical sample.This technology successfully has been applied to the immunocytochemical assay of HER-2/neu sensitivity tests, estrogen receptor, the standardization that dna ploidy body (ploidy) is analyzed and bred the quality control of label MIB-1.
Collection is used for the human breast cancer cell of same batch through cultivating of the human tumor cells system through cultivating.Then with these cell embedding in agar embolism (plug) and use Treating Cuttings with Paraffin Wax.Adopt 4 μ m section to make microslide, fixing and dye with HER-2-Alexa 532 (546nm/557nm/567nm).Then, as mentioned above, sample is carried out the fluorescent microscope analysis.Result's (following table) is very similar to observed result in the tumour standard cell lines system of cultivating.The Trastuzumab associated value height that HCC 2218 and SKBR-3 show, and MCF-7 and HCC 38 show lower numerical value.The IC that these cellular exposure are produced in Trastuzumab
50The comparison of carrying out, and Trastuzumab be-0.9 in conjunction with the Pearson ' s related coefficient that causes, all once more to the tumour standard cell lines system of cultivation in observed very similar.The paraffin-embedded standard cell lines through cultivating of this models show can be used to indicate the biological response of paraffin-embedded human cancer tissue as mark in living.
The biological response of the human breast cancer cell of cultivating and the comparison of DRI expression.Herceptin/Herceptin-combination
| Cell | IC50 μg/ml | MAB is in conjunction with the fresh paraffin of mean value * * | The fresh paraffin of cell that # is analyzed |
| HCC2218 | 0.06 | 53.8 31.3 | 48 59 |
| SKBR 3 | 0.30 | 21.5 25.1 | 52 62 |
| MCF-7 | 1.25 | 3.1 8.11 | 57 39 |
| T47D | 2.00 | 3.5 ND | 59 ND |
| HCC 38 | NR * | 3.9 5.21 | 37 48 |
| HCC 202 | NR * | 3.0 ND | 54 ND |
ND is that undetermined NR is not for there being response
Embodiment 9
Be the term of reference of determining the DRI index carries out DRI is expressed and statistical analysis to the correlativity of the response of cancer therapy drug.
Ultimate principle and the method for determining the DRI term of reference of tumour response are based on: (1) external index system; (2) paraffin mass in the external index system.The successful structure of these two kinds of vitro system provides such confidence, i.e. the cytotoxicity of tumour cell response can be associated statistically with the DRI measured value after the paraffin embedding.
The purpose of this index is to calculate under the different DRI expressions of individual patient tumour, and it produces the probability of resistance to medicine.Then, the attending doctor can consult this term of reference, thereby the particular cancers patient with certain level DRI expression is opened the cancer therapy drug prescription.
We use the statistics homing method to simulate the inhibition of every kind of medicine to in-vitro cell growth the DRI expression according to relevant every kind of medicine.The standard tumour cell that this method will be applied to cultivate and the standard cell lines of paraffin-embedded cultivation.Outcome variable is the IC that is determined by the clone dose response curve
50Logarithm, independent variable is the DRI level.Adopt log IC
50Reason be data in our preliminary data and the document.In the research that this is suggested, adopted 13 kinds of clones, we have good reason (80% possibility) to illustrate under 5% the level of signifiance, and real correlativity is at least 60%.We have set alternative hypothesis, and promptly the calculated value of correlativity is 0.90, because observed Pearson ' s related coefficient is higher than 0.90 in above-mentioned EXPERIMENTAL EXAMPLE, this hypothesis is seemingly real.
We will further analyze this correlation data by simple cluster analysis and tr pt analysis.This analysis will determine whether to exist the DRI expression that can be used to show the biological response probability sub-categoryly.
Embodiment 10
For providing three kinds of molecular diagnosis of information, the management of individual cancer disease of patient detects.
Medicine response indicator detects (DRIT)
Explanation
1) 6-8 kind cell biological label is dyeed with fluorescently-labeled monoclonal antibody (fMAb), these biomarkers are all expressed in the primary tumor cell, and relevant with tumour resistance and targeted chemotherapy.Adopt computer assisted fluorescence microscope system quantitative, and use it for targeted chemotherapy the expression of these labels.
2) with the fluorescence standard that is purchased the fluorescence measurement numerical value of these biomarkers is carried out standardization, can carry out the data contrast between each day, between each laboratory like this.
3) set up the external calibration system of being made up of the cancer cell of the cultivation of one type of (as breast cancer or lung cancer etc.) cancerous cell line, this cancer cell has in various degree the resistance to targeted therapy.Resistance level is (by IC
50Measure) and biomarker expression (every pixel fluorescence) between significant correlation determined by Pearson ' s coefficient.DRI expression according to relevant with medicine is used to simulate the inhibition of every kind of medicine to in-vitro cell growth with statistical regression methods.Outcome variable is the IC that is determined by the clone dose response curve
50Logarithm, independent variable is the DRI level.Adopt simple cluster analysis or tr pt analysis to determine whether to exist to be used to the DRI expression of the probability that shows unfavorable biological response.
4), these clones are fixed and are embedded in the paraffin mass according to the mode identical with primary tumor.As mentioned above, these cell sections on the microslide are dyeed and measure.Establish the IC of these cells once more
50And the correlativity between the corresponding biomarker expression.
5) above-mentioned IC
50And the correlation research between the corresponding biomarker expression is used to establish the DRI index that is used for biomarker.This index can be used to the expression of the biomarker of tumour is associated with the resistance level of this tumour to drug therapy relevant on the mechanism.The DRI index can be used to describe the probability (treatment failure) of resistance.
6) verify by the research of retrospective clinical correlation and perspective clinical correlation research and improve these laboratory studyes, so that the term of reference of DRI index and probability thereof is based upon on the basis of clinical data.
7) the paraffin mass section at 4 micron thickness of primary tumor detects (needing 6 microslides to take multiple measurements), 2 days consuming time.Fixing tumour cell not by embedding, also can easily detect.
Use
1) a confidentiality examining report the DRI index will be provided and the tumour of the individual patient that calculates by statistical method to the 5-6 kind through the FDA approval, widely used chemotherapeutics produces the probability of resistance, these medicines include that (National Comprehensive Cancer Network is NCCN) in the guide at the comprehensive cancer network of the country that is obtained by national cancer association (National Cancer Society).The doctor can be according to the decision of the testing result of suitable this patient's therapeutic scheme being made foundation.
HER-tax detects
Explanation
1) HER-tax detects and to be applied to the be positive patient with breast cancer of (IHC 3+ or FISH+ showed expression) of HER-2/neu acceptor.
2) according to the mode similar to DRIT, primary tumor detects in the paraffin mass to being embedded in, except dyeing to HER-2/neu acceptor (using fluorescently-labeled Trastuzumab), PTEN and to 'beta '-tubulin III (at the DRI of taxane).
3) purpose is to filter out HER-2/neu to cross the patient that expression is in low scope (but still being IHC 3+), and the patient's that the PTEN expression values is low, 'beta '-tubulin III expression values is high tumour.This patient group's tumour may all have resistance to Trastuzumab and taxane.The probability of resistance is provided.
4) heterogeneity of cancer cell in the measurement tumour relevant with 'beta '-tubulin III with HER-2/neu.Heterogeneous degree is relevant with the duration that Trastuzumab-taxane therapeutic scheme is produced favourable response.
Use
1) owing to only there is the HER-2/neu positive patient of 40-50% in finite duration (most likely being no more than 1 year), to produce favourable response to Trastuzumab-taxane, therefore this HER-tax detects and will select to have the patient of resistance, and the patient may produce duration of favourable response in the predicted treatment.Trastuzumab is quite expensive medicine and has cardiac toxic.
Patient with breast cancer's circulating cancer cells detects (CCCT)
Explanation
1) gathers 20ml patient's vein blood, thereby normal plasma cell is removed enrichment circulating cancer cells (CCC) by density gradient centrifugation and magnetic cell sorting.Collect these cancer cells by negative back-and-forth method, place on the microslide, then with fluorescent monoclonal antibody dyeing and adopt computerized fluorescence microscope system to count.
2) detect in 24 hours behind the collection blood sample.Sample can send by the express mail that places special pig by the CCCDiagnostics checking.
3) can the medicine response biomarker of expressing among the CCC be characterized.
4) the CCC quantity of finding in the metastatic carcinoma (IV phase) is higher than I, II and III phase cancer.A large amount of CCC is associated statistically with prognosis mala with to the drug therapy difference in response.
5) in the study medication that proposes, during 2-3 time of during detection before treatment and the treatment and treatment back (2-3 month is at interval) detects CCC to be counted, this can reflect therapeutic action.If the quantity of treatment back CCC keeps higher, medicine may be invalid, if the quantity of treatment back CCC becomes quite low, then medicine may be effective.This preliminary discovery will be through clinical examination.Developed the technology of in blood, finding CCC at 7 kinds of cancers (prostate cancer, lung cancer, cancer of the stomach, cancer of pancreas, liver cancer, colon cancer and breast cancer).
Use
1) for III phase or IV primary breast cancer patient, can before treatment, carry out CCCT and estimate prognosis, carry out 2-3 CCCT after the treatment with the explanation response condition.Can carry out image inspection (X ray or CT) the patient and obtain the result before.
2) when tumour may produce drug resistance,, can carry out the situation of change that CCCT measures biomarker among the CCC in order to select another kind of medicine again.These results can be associated with external calibration system, and this system is made up of the cancer cell from the cultivation of the clone of one type of cancer (as breast cancer or lung cancer etc.), and these cells have in various degree resistance to the treatment of targeted therapies.This correlativity be based on that specific DRF expresses and cultured cells to the response of drug therapy.
3) relevant CCC quantity data may not the alternative image inspection, but CCCT is faster and can understand the situation of change of the drug resistance feature of metastatic tumour.Can characterize by the formation method pair tumour resistance relevant with biomarker.
Embodiment 11
PAC needs three big steps.Step 1: simultaneously several target spots in the tumour are carried out quantitative measurement.These target spots are referred to as medicine response indicator (DRI), and the immunofluorescence and the computerized fluorescent microscope of the monoclonal antibody by adopting mark are estimated these target spots.For relatively, can carry out standardization to the numerical value that obtains with the fluorescence reference data.
Step II: establish DRI and express and cytotoxicity response statistically the significant correlation of tumour cell to related drugs.Setting up external index system associates every kind of drug cell toxic action and corresponding D RI measurement.Adopted 7 kinds of breast cancer (BC) clone that different cancer therapy drugs is had different susceptibility.Express at the DRI of every kind of medicine in the effect of Tamoxifen, taxol (paclitaxel), Herceptin and adriamycin and these clones and be associated.DRI is respectively estrogen receptor, 'beta '-tubulin III, HER-2/neu and topoisomerase II.The scope of Pearson classification (rank) related coefficient is 0.77 to 1, and p value scope is 0.005 to 0.02.
Step II I: the technology of obtaining cancer cell from the individual cancer patient.For metastatic tumo(u)r, (p.2787) Cancer 2000:Vol.88, no.12 obtain circulating cancer cells (CCC) from peripheral blood, counting also dyes with the Herceptin of mark, thereby HER-2/neu is expressed quantitative to use negative back-and-forth method.101 BC patients are studied, and extract 402 parts of blood samples; The mediant of every patient's draw samples is 4 (1-7).CCC is relevant with remote transfer; 88% IV phase patient has CCC on some time point between sampling date.The CCC quantitative range is at 1-1283 in each sample.
20 patients have carried out 4 times or HER-2/neu detection of expression more frequently, and have obtained the data of tumor tissues.In 18 patients, (90%) takes place in CCC and primary tumor simultaneously, and wherein 6 HER-2/neu are positive, and 12 HER-2/neu are negative.HER-2/neu is negative in 1 patient's the tumor tissues, and HER-2/neu is positive among the CCC.1 patient is accredited as that HER-2/neu is positive in the tumor tissues, and HER-2/neu is negative among the CCC.
The combination of treatment (Herceptin)-diagnosis (HER-2/neu expression) is first PAC model that is used for BC through the FDA approval.Yet though the patient that HER-2 is negative is obviously less to the response as the Herceptin of single therapy medicine, being lower than the metastatic BC patient that 30% HER-2 is positive has response to the Herceptin as the single therapy medicine.The DRI of BC tumor tissues measures and the improvement of CCC can produce the result of treatment that can be predicted better.The DRI that has also reported the microslide of band section measures, and this section is from BC patient's primary tumor tissue cutting-out.
Embodiment 12
DRI to paraffin-embedded (FFPE) tissue of formalin fixed is quantitative, and this tissue is from breast cancer, colon cancer and patients with lung cancer.
Described among the embodiment 1 and 4 and adopted fluorescently-labeled Monoclonal Antibody to place the FFPE histotomy on the microslide and hatch process.As described in embodiment 2, adopt the time shutter of determining by typical curve, utilize filter to obtain digital picture under the same cell visual field, these filters can be distinguished DAPI, FITC, Alexa 532, Alexa 594 and Alexa 647 fluorescence.Obtain the image and the preservation of 5 zoness of different of every kind of tumor tissues.Utilize the microslide that does not comprise any tissue, under the suitable time shutter, utilize every kind of filter to obtain image background.For the autofluorescence of evaluation of tissue, also to obtain the image of the serial section of every kind of tumour, at this moment only hatch with anti-wide spectrum cytokeratin-FITC, utilize above-mentioned every kind of filter, and complete step process according to specimen.
For quantitative, image is carried out flat field (flat-fielded) first by from the DRI image, deducting suitable image background to the DRI fluorescence intensity.This has just eliminated any variation (variations) in the illumination field (illumination field).Then from every pixel mean fluorecence (F/P) of subtracted image background (available from image histogram) through the image of flat field.Treated DRI image is carried out interactive mode inspection, and delineate out the strongest zone of fluorescence (normally 50 to 100 cells).Duplicate this target area (AOI) and save as clip image.Call the clip image of this preservation and be positioned to adopt in the image under the same cell visual field that the FITC filter obtains, be the epithelial cell of the cytokeratin positive to guarantee all cells among the AOI.Fluorescence signal in every width of cloth shearing DRI image is through the Image-Pro software analysis, thereby the intensity of fluorescent target (cell or cell mass) is isolated in selection from background.Subsequently, Image-Pro can delineate, calculates and demonstrate the quantitative data of the target in the relevant clip image.Data are imported in the Excel program, and calculated F/P mean value, and carry out standardization with reference data at the target in 5 images of every kind of tumour each.Adopt and be used to detect the identical method of microslide, calculate the autofluorescence of continuous microslide under every kind of filter of identical tumour.With the formal description of the number percent of reference data deduct every pixel mean fluorecence (every kind of tumour is 5, amounts to 250 to 500 cells) of each DRI image of organizing autofluorescence.
The DRI result of relevant 6 patient with breast cancers' FFPE tissue sees first following table.Estrogen receptor (ER), 'beta '-tubulin III (TUB), thymidylate synthetase (TS), topoisomerase II (TOP), ribonucleotide reductase (RR), HER-2/neu (HER) and mispairing excision repairase-1 (ERCC-1) are carried out quantitatively.Every kind of DRI to 6 different patients measures, and the mean F/P (through the reference data standardization) of selecteed target shows with the mean value (runic demonstration) of 5 numerical value and the coefficient of variation (CV) of 5 numerical value (showing in the bracket) in 5 clip images of every kind of DRI after measured.The CV value scope of 5 DRI images of 6 patients at 8.9 (39 patients' TS) to 37.4 (39 patients' ER).In 18 measured values, there are 5 not use CV because the DRI value is too low.When the series section of the same patient of duplicating was analyzed, as shown in the table, DRI numerical value was in the identical relevant range.
Breast cancer research
| Patient # | ER | TUB III | TS | topO | RR | HER-2 | ERCC-1 |
| 3 | 23，17，18， 29，19 21 (23.2) | 9，24，16， 18，16 16 (32.3) | |||||
| 20 | 11，13，9， 10，18 12 | 34，33，33，39，4036 | 0.5，1.8， 0.4，0，1.8 0.9 |
| (29.2) | (9.6) | (na) | |||||
| 39 | 7，5，9，13， 6 11 (24) 9，19，9，9， 12 * 12 (37.4) | 48，47，42， 34，34 41 (16，5) 52，55，50， 62，59 56 (8.9) | |||||
| 43 | 0.2，0，0， 0.4，0 0.1 (na) | 31，29，35， 40，44 * 36 (17.4) | 0，0，0，0，0 0 (na) | 72，52，63 84，62 67 (18.5) | |||
| 70 | 30，26，28， 24，33 28 (28.2) | 5，5，7，4， 5 (21.1) 3，3，2，4 3 (27.2) | |||||
| 75 | 12，8，3，2， 2 5(na) | 9，5，0，15， 0 6(na) |
The DRI value recently represents with the percentage of reference data (fluorescent microsphere),
*Use two to resist, goat anti-mouse IgG-Alexa 532, and the numerical value in the bracket is the coefficient of variation at the DRI value of 5 zoness of different mensuration of tumor tissues.
The microslide of FFPE tissue that is loaded with breast cancer, lung cancer and colon cancer patient is available from NCI tissue cooperative network (Cooperative Human Tissue Network, CHTN) central Atlantic portion (Mid-Atlantic Division).Following table shows 3 kinds of dissimilar cancer patients' DRI measured value, and also will use the ERCC-1 expression values of the serial section that two different microscopes obtain from same patient to compare by two different operating persons.DRI measured value from 5 clip images of tumor tissues zones of different is shown with the coefficient of variation.The result shows that these four kinds of DRI of the epithelial cell (cytokeratin dyeing is positive) that can organize the FFPE of three types of cancers measure, and two technician use two different microscopes to carry out the DRI measurement and obtained similar data.It should be noted that every kind of DRI measured value will be associated to the response of relevant cancer therapy drug with the patient, use this test to select the potentiality of suitable medicine as individual patient so that estimate the doctor.Such information will obtain by initial retrospective study and perspective clinical testing subsequently.
The DRI quantitative comparison of chest, colon and lung tissue's section
| DRI-microslide A, B, D-date-microscope-technology | 5 images of chest MAD04-00047T-% reference data | 5 images of lung MAD02-00394T-% reference data | 5 images of colon MAD04-00458T-% reference data |
| TS-A 8/14L6SL | 65，43，66，80，80 65 CV 22.1 | 14，38，40，12，42 29 CV 47.2 | 47，59，77，58，70 62 CV 18.2 |
| TS-D 8/22L5DZ | 52，67，53，79，69 64 CV 17.6 | ||
| ERCC-A 8/14L6 SL | 144，134，225，287， 85 174 CV 45.8 | 76，109，72，87，18 72 CV 45.2 | 75，76，109，77，96 86 CV 17.5 |
| ERCC-D 8/24L5 DZ | 206，139，194，238， 209 197 CV 18.4 | ||
| ERCC-D 8/24L5 SL | 83，76，102，97，103 90 CV 13.0 | ||
| ERCC-D 8/24L6 | 116，119，109，116， |
| DZ | 134 119 CV 7.8 | ||
| ERCC-D 8/24 L6 SL | 89，108，90，82，63 86 CV 18.1 | ||
| ERCC-D 8/22 L5 SL | 112，147，148， 157，132 139 CV 12.5 | ||
| RR-B 8/18 L5 DZ | 11，10，10，20，10 11 CV 7.5 | 7，11，7，7，8 8 CV 15.5 | 10，12，6，10，16 11 CV 11.3 |
| RR-D 9/15 L5 DZ | 7，10，107，9 9 CV 12.5 | ||
| TUB-B 8/18 L5 DZ | 19，19，20，23，10 18 CV 24.7 | 8，13，7，7，11 9 CV 25.1 | 7，9，6，9，14 9 CV 29.9 |
CV is the coefficient of variation of the DRI value measured in 5 zoness of different in the tumor tissues.The DRI value represents recently that with the percentage of reference data mean value shows with boldface letter.
In order to be expressly understood, now the present invention has been described to a certain extent in detail and fully by the mode of diagram and embodiment, it is evident that for the person of an ordinary skill in the technical field, in the scope of wide in range and suitable condition, prescription and other parameter, invention made amendment or change equally and can implement the present invention, and do not influence scope of the present invention and any specific specific embodiments thereof, this modification or variation will be included in the scope of appending claims.Those skilled in the art's involved in the present invention technical merit has all been represented in all publications, patent and the patented claim of mentioning in this instructions, the present invention introduces them as a reference, and its degree is equivalent to point out expressly and individually that every piece of publication, patent or patented claim all are introduced into as a reference.
Claims (20)
1. one kind for the individual cancer patient selects to treat the method for the chemotherapeutics of cancer, comprising
Select one group of chemotherapeutics that is used for the treatment of types of cancer that this patient suffers from approval of authority;
Obtain the tumour cell sample from the patient;
In at least one cell of tumour cell sample, use antibody to determine to respond the expression of indicator corresponding to one group of medicine of chemotherapeutics group; And
Chemotherapeutics is selected in expression according to medicine response indicator.
2. method according to claim 1, wherein said antibody is through fluorescence labeling.
3. method according to claim 1, wherein said medicine response indicator is the cell component relevant with the mechanism of action of chemotherapeutics.
4. method according to claim 1, wherein said antibody excites with the different fluorescent dyes of non-overlapped emission spectrum and carries out fluorescence labeling with having difference.
5. method according to claim 1, wherein said determining step comprise carries out quantitatively medicine response indicator.
6. method according to claim 5, wherein said quantitative step comprise fluorescence intensity and reference data are compared.
7. during method according to claim 5, wherein said determining step are included in and detect for 1 time at least 5 kinds of medicine response indicator are carried out quantitatively.
8. method according to claim 5, wherein said determining step comprises that the amount with medicine response indicator identical in the expression of multiple medicine response indicator in the sample and the cell associates the chemotherapeutics generation known response of described cell to playing a role by medicine response indicator.
9. method according to claim 8, wherein the expression at least 5 kinds of medicine response indicator in detecting compares.
10. method according to claim 1, wherein said tumour cell sample be selected from circulating cancer cells, take from the sample of the lymph node of contiguous primary tumor, from the sample of primary tumor and from sample fixing and that be embedded in the primary tumor in the paraffin mass.
11. method according to claim 1, wherein said chemotherapeutics are selected from carboplatin, cis-platinum, oxaliplatin, Docetaxel, taxol (paclitaxel), taxol (taxol), vinorelbine, vinca alkaloids, 5 FU 5 fluorouracil relevant medicine, Xeloda, gemcitabine, anthracene nucleus class, Irinotecan.
12. method according to claim 1, wherein said chemotherapeutics are selected from human monoclonal antibody, Herceptin (Trastuzumab), Cetuximab (liking necessary) and bevacizumab (A Wasiting).
13. method according to claim 1, wherein at least a described medicine response indicator is ERCCl, and described chemotherapeutics is selected from carboplatin, cis-platinum and oxaliplatin.
14. method according to claim 1, wherein at least a described medicine response indicator is a 'beta '-tubulin III hypotype, and described chemotherapeutics is selected from Docetaxel, taxol (paclitaxel), taxane and vinorelbine.
15. method according to claim 1, wherein at least a described medicine response indicator is a thymidylate synthetase, and described chemotherapeutics is selected from 5 FU 5 fluorouracil relevant medicine, formyl tetrahydrofolic acid, Pemetrexel and Xeloda.
16. method according to claim 1, wherein at least a described medicine response indicator is a topoisomerase II, and described chemotherapeutics is selected from anthracene nucleus class, adriamycin and epirubicin.
17. method according to claim 1, wherein at least a described medicine response indicator is a topoisomerase I, and described chemotherapeutics is an Irinotecan.
18. method according to claim 1, wherein at least a described medicine response indicator is the ribonuclease reductase, and described chemotherapeutics is a gemcitabine.
19. method according to claim 1, wherein said determining step comprises that carrying out medicine response indicator at one or more cancers that is selected from breast cancer, lung cancer, colon cancer, cancer of the stomach, cancer of pancreas and cancer of the esophagus detects, and wherein said medicine response indicator detects tumour resistance/sensitive data that all chemotherapeutics that list, that be used for cancer types after diagnosing in national comprehensive cancer network patient treatment (the National Comprehensive Cancer Network Treatment for Patients) guide are provided.
20. method according to claim 19, wherein said tumour resistance/sensitive data can be according to the statistical analysis to the correlativity clinical research, and the number percent probability of being failed by the medicine response that is tried individual patient illustrates.
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| CN104919315A (en) * | 2012-12-11 | 2015-09-16 | 社会福祉法人三星生命公益财团 | Personalized anti-cancer agent screening system |
| CN108717862A (en) * | 2018-04-10 | 2018-10-30 | 四川骏逸富顿科技有限公司 | A kind of careful square evolution model of the intelligence based on machine learning |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104919315A (en) * | 2012-12-11 | 2015-09-16 | 社会福祉法人三星生命公益财团 | Personalized anti-cancer agent screening system |
| CN108717862A (en) * | 2018-04-10 | 2018-10-30 | 四川骏逸富顿科技有限公司 | A kind of careful square evolution model of the intelligence based on machine learning |
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