Patent Application
20050014208
The present invention pertains to a procedure and a kit for the diagnosis or monitoring of breast cancer in humans.
The ability to detect a relapsing malignant tumor in a timely manner via the occurrence of metastasizing tumor cells in the blood is relevant in cancer after-care. So-called “tumor markers” are determined quantitatively at the protein level (immunologically or enzymatically) in the blood, or in other bodily fluids, in cancer patients when using current test methods.
These detection procedures are suitable only to a limited extent for tumor diagnosis, or monitoring/after-care, since increased tumor marker values can also be produced by nontumor diseases (e.g. inflammation of the gastrointestinal tract, cirrhosis of the liver, viral infections), heavy smoking, or as a result of pregnancy.
Breast cancer is the most frequent diagnosis when a tumor disease is found in women (26.4% of all new diseases). Despite massive efforts that are being made in regard to early detection, treatment and after-care, this disease still ranks first in cancer-related deaths in women. The number of cases of this disease in the western industrialized countries has been increasing further over past years despite the intensified efforts in regard to early detection. The high rate of metastasis following the initial treatment is problematical, leading to the death of the patient after only 1-3 years in the majority of cases. The main reason for this is the spread of tumor cells in the early stages of tumor development. Thus, in addition to the initial recognition of a mammary carcinoma, the earliest possible detection of metastasizing cells is of especially decisive importance for a successful treatment. Likewise, a definitive negative detection can be helpful in clinical stage I when one must decide whether the patient is to be subjected to the stress of chemotherapy or an operation.
The currently used diagnostic methods are inexact when one is dealing with the evaluation of the malignant potency of residual tumors after chemotherapy has been carried out in the metastasizing stages. Some clinical studies indicate the prognostic importance of disseminated tumor cells. However, numerous methodological aspects are critical and have not been adequately standardized so far. Thus, detection methods for occult-or residual-metastasis have to be found that permit timely classification into the various primarily curative therapeutic options.
The effort to improve the chances of healing is currently accompanied, on the one hand, by the search for and the use of new tumor markers and, on the other hand, by increases in sensitivity in the methods that are used.
The problem of the present invention is to make available a procedure and a kit with which the diagnosis or monitoring of breast cancer is possible in a simple, safe and repeatable manner.
This problem is solved by the procedure in accordance with claim 1 and with the kit in accordance with claim 30 and the microarray in accordance with claim 50. Advantageous further developments of the procedure, of the kit, and of the microarray are given in the pertinent dependent claims.
In accordance with the invention, the presence or absence of mRNA from the tumor marker proteins EGF-R, CEA, CK20, MAGE-3, GA733.2, MUC-1≅GA15.3, Her-2/neu, claudin-7, PDGF-β and/or stanniocalcin are recognized in a human blood sample by means of the kit in accordance with the invention or by means of the procedure in accordance with the invention.
Since the RNAs of the markers that have been described are not normally present in expressed form in the blood of healthy persons, a direct correlation is found between a positive RT-PCR result detection for these tumor markers and circulating tumor cells in the blood that can lead to metastasis.
Since individual markers can be expressed differently in a therapy-dependent manner, and breast cancer exhibits pronounced heterogeneity in the expression pattern of the breast cancer cells, it is expedient to examine a combination of tumor markers in order to recognize all the tumor cells that are circulating in the blood. As a result of this, tumor cells can also be recognized when the expression of a particular marker is relatively slight in a patient or in a stage of the disease, which could otherwise lead to a supposedly negative result. However, the use of additional markers usually encounters limits if mononuclear blood cells exhibit background expression (“illegitimate transcription”) that impedes exact analysis.
Thus the following combination of markers is proposed in accordance with the invention for the recognition of breast cancer cells.
Two of the markers from the following groups
Use can be made of the primers indicated in the following table for the amplification of segments of the markers.
Markers for Mammary Carcinoma
The designations of the markers are explained in the following section:
In the case of using RT-PCR systems for the detection of tumor cells, specificity is a critical point because of the very high amplification rate. The least contamination, e.g. via extraneous RNA or illegitimate transcription, can in this way falsify the result.
An increase in specificity as a result of concentrating the tumor cells relative to the blood cells and, at the same time, an increase in sensitivity in the detection of tumor cells can be achieved (detection rate of 1 tumor cell per 107 mononuclear blood cells) by using immunocytochemistry with monoclonal antibodies that act against tumor cell antigens. In this way the tumor cells are separated by means of specific antibodies or an antibody mixture of mononuclear blood cells. Separation can take place by means of magnetic particles (Dynal) to which the antibodies are bound. This is described in greater detail in the following segment.
Eukaryotic cells carry a plurality of different molecules on their cell surface. The combination of expressed surface molecules differs depending on the origin and the function of the individual cell, so that patterns are produced that are cell-type specific. Antibodies are utilized in order to recognize these cell-type specific patterns. Antibodies bind with high specificity to their antigen, namely to selected surface molecules in this case. This property is utilized in order to recognize cells and to differentiate them from one another by means of specific antibody binding on the basis of their cell-type specific patterns.
The expression of special surface proteins differentiates tumor cells from nontransformed cells of this cell type. Since, in the case of tumor cells, this special pattern of surface antigens also differs from the patterns that are typical of blood cells, tumor cells can be differentiated in the blood. In order to identify tumor cells, antibodies that specifically recognize these special surface proteins are utilized as tools. This specific antibody binding becomes usable for various analyses and separation methods.
In addition to recognizing cells via their surface epitopes, it is also possible to separate recognized cells from nonrecognized ones because of the intensive binding of immunoglobulins that are specially selected for this purpose.
1. Separation Principle Based on the Liquid Phase; e.g. Continuous Flow Cytometry:
Antibodies are coupled to fluorescent dyes for the purpose of continuous flow cytometry analysis. Isolated cells are individually led past a light source (laser) in a constant stream of liquid. The fluorescent dyes bound to the antibodies are excited during illumination of the cells, and they irradiate light of a particular wavelength. The irradiated light is detected, and the measured signal is stored in digital form. The light signal can be assigned to individual cells. The antibody-labeled cell is recognized in this way, and can now be separated from other cells. The cells are isolated in extremely small drops for separation purposes. After recognizing the antibody-labeled cell, the drops in question are deflected into a collection container.
2. Separation Principle Based on the Solid Phase; e.g. Magnetic Separation:
Antibodies are coupled to pseudomagnetic particles for the purpose of magnetic separation. After introducing the pseudomagnetic particles to a magnetic field, the particles migrate in this magnetic field. During movement in this magnetic field, the cells to which the coupled antibodies are bound are carried along, and separated from other cells.
Thus, in order to recognize tumor cells by means of magnetic particles, antibodies are covalently coupled to pseudomagnetic particles that possess a defined number of chemically activated sites on their surface. The separation specificity is determined by the specificity of the antibodies. A blood sample that contains tumor cells is mixed with antibody-coupled magnetic particles; the particles and blood then move relative to one another. Those (tumor) cells which are recognized by the antibodies (that are bound to the solid phase) and which are firmly bound to them follow the movement of the particles. As a result, it is possible to withdraw, from the blood, the particles with the cells that are bound to them (e.g. toward the wall of the separation vessel) upon applying a magnetic field. The blood that has been tumor-cell depleted in this way can be exchanged for other solutions, whereby the cells that have been separated via magnetic particles remain behind, and are available for additional applications until the point in time of switching off/removing the magnetic field.
Use can advantageously be made of specific antibody mixtures in order to recognize the tumor cells, whereby these mixtures have either been optimized with respect to tumor cells in a general manner, or they have been specifically optimized with respect to breast cancer cells as well. For example, a combination of MOC-31 antibodies (Novocastra) and Ber-EP-4 antibodies (DAKO) is suitable for recognizing tumor cells in the blood.
Recognition that is specially directed toward breast cancer cells can be achieved via an additionally optimized antibody mixture in accordance with the table below. This is based on the selective expression of certain surface proteins, whereby breast cancer cells are differentiated from other cancer cells.
In comparison to the antibodies when used separately in each case, such antibody mixtures show increased sensitivity in terms of cell recognition and cell separation, independent of the method used.
Some examples, in accordance with the invention, of detection procedures for breast cancer cells in blood samples will be described in the following segment.
The following aspects are shown:
FIGS. 2A-C show tumor marker detection by means of a Light Cycler;
In the first example, the RNA from 1 mL of EDTA/whole blood was processed using the QIAamp RNA Blood Mini Kit (Qiagen, Hilden). Contamination by genomic DNA was avoided via additional DNA digestion in the column using an RNA-free DNase Set (Qiagen, Hilden).
The processing of the RNA from 1 mL of EDTA/whole blood was verified photometrically via the 260:280 nm ratios. For the purposes of quality and quantity determinations in this connection, 1 μL of the mixture can be analyzed via electrophoretic separation on an RNA 6000 chip using the Agilent Bioanalyzer 2100.
The isolated RNA was denatured in an appropriate volume together with oligo(dT) 15 primers (Promega, Mannheim) for 5 min at 65° C., and then incubated directly on ice. cDNA synthesis took place by means of the Sensiscript™ Reverse Transcriptase Kit (Qiagen, Hilden) in 20 μL of reaction mixture in accordance with Table 1 for 1 h at 37° C. with subsequent reverse transcriptase inactivation for 5 min at 95° C. that was then followed by cooling on ice.
Using the cDNA that was produced in this way, a multiplex PCR was carried out for each of the selected tumor markers stanniocalcin, EGF-R, and CEA and also for β-actin as an internal control. The PCR mixture is illustrated in Table 2 that follows.
(*contains 15 mM MgCl2;
**HotStarTaq ™ DNA polymerase; Qiagen, Hilden
***DMSO azddition in the case of stanniocalcin)
A primer pair, which is seen in Table 3 below, was used for each tumor marker in this regard.
The primer combinations and quantities that were used for the individual tumor marker detections are listed in Table 4 that follows.
The PCR was carried out using the conditions indicated in Table 5 together with the marker-specific melting temperatures and numbers of cycles indicated in Table 6.
1 μL of the PCR product produced in this way was separated in an Agilent Bioanalyzer 2100 on a DNA chip (500), and the result of the separation was documented electronically. The results are shown in
Lanes 2, 3, 6, 7, 10, 11 do not contain any biological material, so that no corresponding PCR products arise there. In
As an alternative to block PCR, this tumor marker detection procedure can also be done by means of a Light Cycler (Roche, Basel).
Reverse transcription of the mRNA was done as described above. The PCR was then carried out with the Light Cycler DNA Master Sybr Green I′ Kit (Roche, Basel) in accordance with data from the manufacturer under conditions that had been optimized for each tumor marker. The oligonucleotides that are indicated in Table 3 were used as primers in this case. Table 7 and Table 8, respectively, show the mixture for the PCR and the PCR conditions using the Light Cycler.
The result of this PCR and the evaluation using Light Cycler technology are illustrated in
In this analysis, the melting curve for the PCR products is analyzed by means of the Sybr Green I detection method. The pertinent graph in
As an alternative to the methods that are illustrated here, use can, of course, be made of conventional methods of analysis as well, such as agarose gel electrophoresis in which, for example, 25 μL of the PCR product synthesized above are separated over a 2.5% agarose gel, and the DNA bands are then stained with ethidium bromide and rendered visible. Documentation can be carried out with the help of e.g. the DUO Store System from Intas.
In addition, fragment analysis by means of the ABI Prism 310 Genetic Analyzer (Applied Biosystems, Weiterstadt) can also be used for the evaluation. In order to do this, a PCR is carried out with fluorescence-labeled primers and then, for example, 1 μL in each case of each PCR product is used at a dilution of 1:50.
Detection by means of sequence-specific fluorescence-labeled hybridization samples is possible as an additional detection procedure, whereby these samples allow the evolution of products to be monitored after each PCR cycle. A conclusion can then be drawn on the basis of special standards in regard to the quantity of starting RNA.
The enrichment of the cell fraction which is used for this purpose and which arises from the blood sample used is central for the quality of the RNA, which is isolated as the basis of the detection procedure, and the cDNA that is synthesized therefrom. Four different methods are available for this as follows.
a) Enrichment by Means of Repeated Centrifugation Following Erythrocyte Analysis:
1 mL of EDTA/blood is lysed for 20 min on ice following the addition of 5 volumes of erythrocyte lysis buffer (“QIAmp Blood Kit,” Qiagen; Hilden). The plasmallysate is removed from the pelletized cells and resuspended, and then renewed centrifugation takes place for 20 min at 3000×g. After removing the supernatant liquor, the pelletized leukocyte fraction is available for RNA preparation.
b) Enrichment by Means of Density Gradient Centrifugation:
Cells of different mean volume-based density can be separated from one another via a density gradient that is produced by means of centrifugation. Mononuclear blood cells are separated by means of a Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden), and then washed twice with PBS/1% FCS.
c) Enrichment of Tumor Cells by Means of FACS Continuous Flow Cytometry:
The mononuclear cells from the fraction enriched under b) are incubated with fluorescence-labeled mononuclear antibodies that act against tumor-specific surface proteins. The labeled cells are washed twice with PBS, and then 107 cells are resuspended in 1 mL of PBS. A FACS Vantage SE continuous flow cytometer (Becton Dickinson) is used in order to isolate the tumor cells. Data recording, instrument control, and data evaluation are done via the CellQuest program. The sorted cells are transferred to a 1.5-mL reaction vessel (filled with 1 mL of PBS). The RNA can then be isolated as described above.
As an alternative, the isolated fraction of mononuclear blood cells, which were isolated in accordance with one of the above procedures, was lysed in trizole reagent (Gibco BRL, New York, USA), and homogenized by means of a pipette. Following chloroform extraction, the RNA-containing aqueous phase is precipitated in isopropanol at −80° C. After washing twice in 80% ethanol, the pellet is dried in air, and then resuspended in RNase-free water.
Reverse transcription and mRNA detection as described above then follow on from this isolation of the RNA.
d) Enrichment of Tumor Cells by Means of Immunomagnetic Separation:
The expression of special surface proteins differentiates tumor cells from nontransformed cells of this cell type. Since, in the case of tumor cells, this special pattern of the surface antigens is also different from the patterns that are typical of blood cells, one can differentiate between tumor cells in the blood. In order to identify tumor cells, antibodies which specifically recognize these special surface proteins are utilized as tools. Specific antibody binding is made usable for the procedure in accordance with the invention. Antibodies are covalently coupled to pseudomagnetic particles that possess a defined number of chemically activated sites on their surface. The separation specificity is determined by the specificity of the antibodies. A blood sample that contains tumor cells is mixed with antibody-coupled magnetic particles; two different mixtures of antibodies are used as antibodies in the various examples; the particles and blood then move relative to one another, e.g. by means of “over-end rotators” in samples that are located in a closed container or by means of alternating magnetic fields. Those (tumor) cells which are recognized by the antibodies (that are bound to the solid phase) and which are firmly bound to them follow the movement of the particles. As a result, it is possible to withdraw, from the blood, the particles with the cells that are bound to them (e.g. toward the wall of the separation vessel) upon applying a magnetic field. The blood that has been tumor cell depleted in this way can be exchanged for other solutions, whereby the cells that have been separated via magnetic particles remain behind, and are available for additional applications until the point in time of switching off/removing the magnetic field.
However, tumor cells were recognized with high specificity quite generally by means of the antibody mixture in Table 9. This is based on the selective expression of certain surface proteins that differentiate cancer cells from other cells.
In comparison to the separately used antibodies, an increased sensitivity during cell separation was demonstrated quite invariably, and independently of the method used, as a result of the use of the antibody mixture. This is shown in
A total of four measurements, in which, respectively, 1, 10, 100, or 1000 carcinoma cells in 10 mL of blood were inoculated, were carried out for each of the antibodies or antibody mixtures. Lanes 1a through 4a, 1b through 4b, and 1c through 4c then show the detection of RNA following RNA preparation and RT-PCR with tumor marker-specific primers, as described above, for samples with a volume of 1 μL in each case.
When using magnetic particles labeled with merely one antibody as in
In this example, experimental results have been shown that do not represent the maximum possible sensitivity but, by way of example, they demonstrate the increase in sensitivity that is achievable with the procedure in accordance with the invention.
Cycles: 35
Cycles: 35
Cycles: 35
Cycles: 35
Cycles: 35
Cycles: 40
The diagnosis kit in accordance with the invention and the procedure in accordance with the invention also make it possible to subsequently use the sorted and separated cells further as desired. For example, these can be inserted into a suitable cell culture medium where they can be cultivated in situ.
Since the cells are intact following separation, the properties of the cell membrane and of the cell nucleus are also conserved. This opens up the possibility of microscopically investigating the expression of additional surface markers, and of carrying out chromosome analyses as well. The sorted cells are applied to microscope slides for this purpose. The detection of additional surface markers can take place cytochemically or via fluorescence microscopy. Likewise, genetic analyses can be carried out such as, for example, chromosome analyses by means of FISH (fluorescence in situ hybridization), or via karyogram compilation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 101 43 776.5 | Sep 2001 | DE | national |
| PCT EP02 05489 | May 2002 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP02/09999 | 9/6/2002 | WO |
