WO2002065889A1

WO2002065889A1 - Method for examining cell and tissue samples

Info

Publication number: WO2002065889A1
Application number: PCT/EP2001/001987
Authority: WO
Inventors: Peter Nehls
Original assignee: Rubikon Ag
Priority date: 2001-02-21
Filing date: 2001-02-21
Publication date: 2002-08-29

Abstract

The invention relates to a method for examining cell and tissue samples, in order to evaluate the risk of tumours and to identify existing tumour cells. Said method comprises the following steps: (a) an immunocytological assay is carried out on a cell or tissue sample in order to determine the extent of DNA modifications, and (b) the risk of a tumour is evaluated by comparing the result obtained in (a) with a correlation between the extent of the DNA modifications and the probability of tumour development. A sample which is known to contain, or which is thought to contain dysplastic cells can especially be examined in this way.

Description

METHOD FOR EXAMINING CELL AND TISSUE SAMPLES

The present invention relates to a method for examining cell and tissue samples, in which DNA damage is detected in the samples at the individual cell level by an immunocytological assay in order to enable a tumor risk assessment. In addition to the immunocytological results, morphological data can also be taken into account.

In order to reduce the number of cancers and in particular the number of deaths caused by them, it is very important to detect changes in cells and tissues in good time and, if necessary, to carry out suitable treatment. Therefore, regular checkups are an effective means of early cancer detection. With the help of cancer screening, existing tumor cells, but also benign tissue changes, so-called dysplasias, can be identified. In some cases, these dysplasias develop into tumors. In many cases, however, they recede or remain unchanged. An examination of cell and tissue changes is currently done using cytological methods. This includes making colored smears and microscopic examination. A well-known method is Pap dyeing (Papanicolaou dyeing). After fixation there is a core staining with hematoxylin and then a combined plasma staining with Orange G6 and polychrome dye. In this way, it is possible in principle to recognize changes in cells and to classify abnormal cells.

An example of this procedure is the cervical cytology. Depending on the cell pattern, a smear is assessed on the scale specified by Papanicolaou (division into groups I, II, III, III D [mild to moderate dysplasia], IVa [severe dysplasia, cellular atypia; suspected carcinoma in situ], IVb [Suspected micro carcinoma] and V [highly suspect invasive carcinoma]). Classification of precancerous cells can also be called CIN I [light dysplasia], CIN II [moderate dysplasia] and CIN III [severe dysplasia or carcinoma in situ] (CIN = cervical intraepithelial neoplasia).

A reliable progression assessment of the changes is not possible with such procedures. The result is frequent repetitions of the check-ups and additional tests with the corresponding consequences for the psyche and health of the patients and for the costs of the health system. In many cases, dysplasias are also surgically removed for safety reasons without there being any clear signs of cancer.

Another serious problem is that the known analyzes of cell and tissue samples misclassify 10 to 40% of all cases, i.e. for example tumor cells are not recognized.

Because of the above-described problems in cancer detection, there was a need for an improved method for tumor risk assessment, including the identification of existing tumor cells by examining cell and tissue samples. The rate of misclassification should also be significantly reduced.

The starting point for the development of the method according to the invention was the idea that the formation of mutations in genes related to cancer is a critical step for tumor development. Genes relevant in this context include protoonko genes that are involved in controlling cell division and tumor suppressor genes that inhibit neoplastic cell growth. Mutations, i.e. changes in the sequences of these genes, are associated with the development of tumors.

Genetic changes, as mentioned above, are caused by a number of DNA-reactive agents, such as chemical ones Carcinogens, ultraviolet radiation, ionizing radiation and reactive oxygen species induced. A common feature of these agents is that they cause structural changes in genomic DNA. The DNA reaction products can then be converted into inheritable mutations in dividing cells. In other words, it is assumed that the mutations critical for tumor development are preceded by DNA damage.

Most cells have effective defense mechanisms to protect the DNA from such damage. In particular, certain noxae that can act on the DNA can be intercepted and inactivated. In addition, DNA damage is repaired by repair enzymes. If large amounts of DNA-reactive agents are involved and / or if there are insufficient cellular defense or repair mechanisms, DNA modifications may remain. The persistence of mutagenic DNA modifications in the genome of proliferation-competent cells is a possible starting point for cancer development.

For example, E. Scherer et al. , Cancer Lett., Vol. 46 (No. 1), pp. 21-29 (1989) carried out an immunocytochemical analysis of 0 ⁶ -alkylguanine, which arises on exposure to certain nitroso compounds known to be carcinogenic. The authors conclude that DNA modification is required for the initiation of carcinogenesis by chemical carcinogens and that a low repair capacity for promutagenous lesions promotes this process.

There have therefore been repeated attempts to detect DNA damage that occurs after the targeted exposure to a carcinogen. L. den Egelse et al. Provide an overview of the immunocytochemical analysis of such DNA modifications. , Mut. Res., Vol. 233 (No. 1-2), pp. 265-287 (1990). Some other work in this area is briefly mentioned below. For example, it has previously been known that exposure to certain tumor-causing particles and fibers in the lung tissue of rats causes oxidative DNA modifications, including the formation of 8-oxoguanine (P. Nehls et al., Environ. Health Perspect., Vol. 105 ( Suppl. 5), pp. 1291-1296 (1997)). If the rats are exposed to carcinogenic particles in sufficient quantities, the content of 8-oxoguanine in the DNA increases, among other things. The authors of the cited work suspect that inflammatory processes in connection with increased 8-oxoguanine levels in lung cells and increased cell proliferation are important for the particle-induced development of lung tumors in rats. However, experiments that allow a correlation between the detected amount of 8-oxoguanine and the probability of tumor development are not presented.

W. H. Fischer and W. K. Lutz, Proc. Natl. Acad. Be. USA, Vol. 92 (No. 13), pp. 5900-5904 (1995) deal with the formation of 8-oxoguanine after experimental animals were exposed to a carcinogen. The formation of papillomas in mice after treatment with 7, 12-dimethylbenz [a] anthracene is specifically investigated. 8-Oxoguanine is determined chromatographically after isolation and hydrolysis of the DNA. The authors conclude that a multifactorial analysis is required to predict individual cancer risk after exposure to a carcinogen.

From A. Yarborough et al. , Cancer Res., Vol. 56 (No. 4), pp. 683-688 (1996) was known to detect oxidative damage to the DNA in individual cells by an immunoperoxidase method. The cells were previously exposed to the carcinogen aflatoxin Bl or H ₂ 0 ₂ . In addition, cells from the oral mucosa of smokers were compared with cells from non-smokers. An increased 8-oxoguanine content was found in cells that were exposed to chemicals or smoke. A relationship between the extent of DNA damage and the likelihood of cancer However, development cannot be made from the data provided.

H. Bartsch et al. , Recent Results Cancer Res., Vol. 154, pp. 86-96 (1998) deal with the importance of the formation of adducts of. DNA with polycyclic aromatic hydrocarbons associated with certain genetic markers and suggest that individuals at high risk of cancer may be identified by determining the adduct levels resulting from certain risk genotype combinations in individuals exposed to polycyclic aromatic hydrocarbons.

G. Romano et al. , Cancer Epidemiol. Biomarkers Prev. , Vol. 8 (No. 1), pp. 91-96 (1999) describe the determination of adducts of DNA with polycyclic aromatic hydrocarbons after an immunohistochemical assay in oral cells. Larger amounts of adducts are found in smokers. A correlation with the probability of developing cancer is not possible from the data presented.

J.L. Mumford et al. , Mutat. Res., Vol. 359 (No. 3), pp. 171-177 (1996) investigate the concentration of adducts of DNA with polycyclic aromatic hydrocarbons in various tissues. In many cases, higher concentrations are found in smokers than in non-smokers. A connection between the extent of DNA damage and the probability of tumor development cannot be established from this.

M. Yaono et al. , Cancer Lett. , Vol. 98 (No. 2), pp. 145-149 (1996) describe the immunohistochemical detection of 3,2'-dimethyl-4-aminobiphenyl-DNA adducts after administration of the carcinogen to the test animals and come to the same Result that adduct formation is necessary for tumor development, but is not sufficient. WO 93/12258 relates to a method for the diagnostic and prognostic monitoring of genotoxic changes in tissue which result from oxidative modifications of DNA. Specifically, several modified nucleotides have been detected in fish that have been exposed to carcinogens in contaminated water. For this purpose, DNA was isolated from tissue samples and, after chemical modification, analyzed using GC / MS. In addition, DNA from tissue samples from breast cancer patients was examined in one example using the method mentioned. While increased concentrations of modified nucleotides were found in the DNA from cancer tissue in some cases, this was not the case with DNA from surgical marginal tissue, even if microscopic changes compared to normal tissue were discernible in this tissue. From these results there is no indication of the possibility of a progression assessment; rather, they lead away from the present invention. The publication does not mention the examination of dysplastic tissues.

WO 94/25626, a later application by the same inventor as the WO 93/12258 discussed above, suggests using the cellular redox potential as an indicator of the likelihood of cancer. Using breast cancer tissue samples, it is investigated whether analyzes of combinations of different modified nucleotides or their concentration ratios allow a distinction to be made between cancer tissue and normal tissue. The analysis of the isolated DNA is carried out as in the above publication (that is, not for individual cells). Certain concentration ratios are associated with the cellular redox potential and are considered to be meaningful for the distinction between cancerous tissue and normal tissue. Regardless of the complicated methodology, there is no possibility of progression assessment from this document. Studies of dysplastic tissues are not described. Starting from the prior art described above, it is an object of the present invention to provide a method which allows tumor risk assessment by examining DNA damage at the individual cell level. In particular, the invention is intended to enable the examination of dysplastic cells for tumor risk assessment.

It has now surprisingly been found that progression assessment for cells or tissues is possible by using an immunocytological method to detect DNA damage in a sample and to relate the extent of the DNA damage to data that relate to the probability of tumor development correlate the extent of DNA damage. The quantitative determination of DNA lesions thus serves as a parameter for tumor risk assessment. This also includes the identification of existing tumor cells.

Compared to normal cells, tumor cells have increased amounts of damaged DNA, such as oxidatively damaged DNA and especially DNA with 8-oxoguanine. This can be used to identify tumor cells. According to the invention, it is also possible in particular for cells, such as dysplastic or precancerous cells, to receive a prognosis about the development into tumor cells. This is because the invention allows differentiation between cells, in particular dysplastic or precancerous cells, with high DNA damage and high probability of tumor development and cells with low DNA damage and low probability of tumor development. Conventional cytodiagnostics are unable to do this. Specifically, it was found according to the invention that dysplastic or precancerous cells, even if they are classified cytologically the same, can have different levels of damaged DNA, such as oxidatively damaged DNA and especially DNA with 8-oxoguanine. Some such cells have levels of damaged DNA that are about the same as normal cells, while others have levels that are about that of tumor cells (i.e., the biological endpoint) development). According to the invention, it is now proposed to use the quantitative determination of the extent of the DNA damage, for example the oxidative damage and especially the content of 8-oxoguanine, at the individual cell level in addition to or instead of conventional cytodiagnostic methods in order to carry out a tumor risk assessment for a cell or tissue sample enable.

Accordingly, the invention provides a method for examining cell and tissue samples for tumor risk assessment, including the identification of already existing tumor cells, which comprises the following stages:

(a) Immunocytological assay for a cell or tissue sample to determine the extent of DNA modifications;

(b) Estimation of the tumor risk by comparing the result obtained under (a) with a correlation between the extent of DNA modifications and the probability of tumor development.

To identify existing tumor cells, the probability of tumor development also includes the probability that tumor cells already exist.

The extent of the DNA modification relative to control cells is preferably determined. The process then comprises the following stages:

(i) immunocytological assay for a cell or tissue sample for the detection of DNA modifications;

(ii) immunocytological assay for control cells;

(iii) determining the relative extent of the DNA modification from (i) and (ii); (iv) Estimation of the tumor risk by comparing the result obtained under (iii) with a correlation between the relative extent of the DNA modification and the probability of tumor development.

The control cells can come from the same sample; for example, it can be undamaged cells. The control cells can, however, also be present in a separately prepared control preparation.

According to a further aspect, the present invention relates to a method for examining dysplastic cells, in which the cells are subjected to an immunocytological assay for determining the extent of DNA modifications. For example, dysplastic cells can be identified on the basis of morphological features.

A typical procedure according to the invention is to subject a sample to an immunocytological assay that is known or suspected of containing dysplastic cells. The sample can be a sample obtained during a cancer screening. The sample may also be known or suspected to contain tumor cells. Typically, nothing is known about the previous exposure to a carcinogen for the samples examined according to the invention.

The degree of damage to the DNA, in particular the degree of oxidative damage and especially the content of 8-oxoguanine, is then determined with the immunocytological assay at the individual cell level. With a high degree of damage to a cell in the examined sample, as is typically the case with tumor cells, it is determined according to the invention that the examined cell is a tumor cell or a cell developing into a tumor cell. With a low degree of damage, as is typically the case in a healthy cell is given, it is determined according to the invention that there is no risk of developing into a tumor cell.

According to the invention, it is thus possible to detect cells in a sample for which tumor development is likely.

The extent (degree) of damage to the DNA can be determined as the relative extent of the damage, for example based on healthy cells in the same sample or on cells in a control preparation.

The procedure according to the invention can also be specified as follows:

(a) determining the level of DNA damage for cells identified as tumor cells based on morphological features;

(b) determining the level of DNA damage for cells identified as healthy cells based on morphological features;

(c) determining the level of DNA damage for cells of a sample for which a tumor risk assessment is to be made;

(d) Compare the results obtained under (c) with the results obtained under (a) and (b) to determine whether there are cells in the sample that are tumor cells or cells that develop into tumor cells.

The sample mentioned under (c) and (d) above can be a cell or tissue sample in which there are dysplastic or precancerous cells or which are suspected of containing such cells. Dysplastic cells can be identified based on morphological features, as explained elsewhere. The invention is explained in more detail below. Reference is also made to the figures, wherein

1 shows 8-oxoguanine values (relative fluorescence values) for cells in two different cervical smears with a PAP IVa finding;

2 shows 8-oxoguanine values (relative fluorescence values) for cells in two different cervical smears with a PAP III D finding.

A special feature of the method used according to the invention is that the degree of DNA modification in each individual cell can be determined, that is to say that each cell of the sample, for example a cytological preparation, can be assessed individually. This is made possible by sensitive immuno-analytical techniques for the detection and quantitative determination of defined DNA modifications in the DNA of individual cells, for example in suspension or in tissue samples. These techniques can be used to determine any DNA modification for which a suitable monoclonal or polyclonal antibody is available. This antibody (or a fragment thereof, such as preferably F (ab) ₂ , but the use of a largely complete antibody is preferred here) is brought into contact with a suitably treated sample, so that a bond between the antibody and the DNA modification for which it is specific. If the antibody bears a label, such as a fluorescent label, direct detection is possible. Alternatively, however, the detection is carried out by using a second antibody (or a fragment thereof) which reacts specifically with the first antibody and is also labeled, for example with the aid of a fluorescent label. The marking of the second antibody can then be detected by suitable image evaluation methods. A DNA modification, as it is to be detected according to the invention, is a damage or structural change in the DNA. Such a change can be a strand break or the chemical change of one or more reactive groups of the DNA contained in the base, sugar and phosphate parts. A chemical change is to be understood as a reaction product. Reaction products are, for example, alkylation products that are created by the action of alkylating agents, oxidation products that are created by the action of reactive oxygen species, and special add-on products of carcinogens. Of particular relevance to the present invention are mutagenic DNA damage, ie structural changes which lead to sequence changes in proliferating cells. Of further importance is the persistence of DNA modifications, ie the amount of DNA modifications present in the cells, which results in the long term from the relationship between the formation of new modifications on the one hand and the repair of existing modifications on the other.

In a particularly expedient embodiment of the method according to the invention, DNA modifications are detected which are formed by highly reactive oxygen molecules in the genetic material. Such oxygen species lead to the formation of DNA oxidation products, which include 8-oxoguanine in particular. Its detection can be carried out using a specific antibody, for example against 8-oxoguanine or 8-oxodeoxyriboguanosine.

It is surprising that on the basis of the determination of a special DNA modification, such as is caused by highly reactive oxygen species, a progression assessment for the examined cell material or tissue, including the identification of already existing tumor cells, is possible. It was not recognizable from the prior art that the measurement of a particular damage would allow a prediction of the development of dysplasia, since in contrast to the already experiments, the type, scope and timing of exposure to potentially carcinogenic agents are unknown.

According to the invention, it could be shown that, compared to normal cells, the oxidative DNA damage in tumor cells and in precancerous cells, which develop into tumor cells, is significantly measurably increased and the measurement of the oxidative DNA damage allows tumor cells present in a preparation to be clearly identified identify and predict the conversion of precancerous cells into tumor cells.

The antibodies which are used according to the invention for the detection of DNA modifications can be polyclonal or monoclonal antibodies. Methods for producing antibodies are known; such procedures can be used. Fragments of antibodies can also be used, it only being necessary that the fragments have the necessary specific binding properties. Some approaches are described below as examples.

The production of antibodies first requires that a suitable antigen is provided. For this purpose, for example, a model compound is chemically synthesized that is representative of the modified base that is to be detected later. The low molecular weight compound obtained in this way is not or only to a small extent immunogenic. It is therefore coupled (conjugated) to a suitable carrier. Proteins, for example, such as bovine serum albumin (BSA), casein, thyroglobulin or perforated screw hemocyanin, are suitable as carriers. The modified base (as the hapten) and carrier conjugate is then used to immunize animals. Antibodies are then obtained using standard methods. It is possible to provide the antibodies with a label, in particular a fluorescent label. The marking is also done using standard methods. An exemplary method for the production of polyclonal antibodies against 7, 8-dihydro-8-oxo-2 'deoxyguanosine, which can be used for the detection of 8-oxoguanine in DNA, is described in P. Nehls et al. , Environ. Health Perspect. , Vol. 105 (Suppl. 5), pp. 1291-1296 (1997). Monoclonal antibodies against the compound mentioned are described in the previously cited work by A. Yarborough, Cancer Res., Vol. 56 (No. 4), pp. 683-688 (1986) and in YS Lee et al. , Biochem. Biophys. Res. Commun. , Vol. 196 (No. 3), pp. 1545-1551 (1993) and VI Bruskov et al. , Biokhimiia, Vol. 61 (No. 4), pp. 737-744 (1996). Further literature references in connection with antibodies against 7,8-dihydro-8-oxo-2 '-deoxyguanosine can be cited as works relating to immunoaffinity techniques, such as P. Degan et al. , Carcinogenesis, Vol. 12 (No. 5), pp. 865-871 (1991), where polyclonal antibodies are used, and EM Park et al. , Proc. Natl. Acad. Be. USA, Vol. 89 (No. 8), pp. 3375-3379 (1992), where monoclonal antibodies are used. Antibodies against 7, 8-dihydro-8-oxo-2 ^■ -deoxyguanosine are also commercially available.

Antibodies have also been raised against various other carcinogen-DNA adducts. For example, T. Shirai et al. , Toxicol. Lett., Vol. 102/103, pp. 441-446 (1998) polyclonal antibodies against 3,2'-dimethyl-4-aminobiphenyl- and 2-amino-1-methyl-6-phenylimidazo [4, 5- b] pyridine-DNA adducts for immunohistochemical detection of the adducts. Monoclonal antibodies against a specific alkylation product of DNA (O ^s -Ethylguanin) are in connection with an immunocytological assay by F. Seiler et al. , Mutat. Res., Vol. 385 (No. 3), pp. 205-211 (1997). J. al-Atrash et al. , Chem. Res. Toxicol., Vol. 8 (No. 5), pp. 747-752 (1995) describe two monoclonal antisera which recognize 4-aminobiphenyl-DNA adducts and are used for immunohistochemical detection. Various monoclonal antibodies against alkylated DNA are described by JH van Delft et al. in environments. Health Perspect., Vol. 99, pp. 25-32 (1993). A polyclonal serum against a further alkylation product is mentioned by GP van der Schans in Chem. Res. Toxicol., Vol. 7 (No. 3), pp. 408-413 (1994). According to the invention, it is essential that the antibodies used have a sufficiently high specificity. The antibodies must therefore bind with high affinity to the DNA modification that is to be detected. In addition, the reactivity with normal components of RNA and DNA should be as low as possible. Since, when used for testing in cell or tissue samples, there are also numerous other components from the cells which can lead to a cross-reaction with the antibodies, it is important according to the invention to choose antibodies so that they also, if possible, include these other components have low affinity. The person skilled in the art can determine whether this is the case by routine experiments. The antibody material in question is used in an immunocytological assay for suitable preparations, such as, for example, the control preparations described in more detail below. The level of binding of the antibody material is then determined to determine how the binding changes depending on the level of DNA modifications. An antibody material is then preferably selected for the method according to the invention which shows on the one hand a low background binding and on the other hand a high specific binding, ie a high degree of binding in a preparation with a large content of DNA modifications.

In the case of polyclonal antibodies in particular, it can make sense to provide additional purification steps in order to separate unsuitable antibodies. For this purpose, it is expedient to use an affinity column in which the column material carries an antigen with which the antibody which is to be used in the method according to the invention reacts. This can significantly improve the specificity of the material. According to the invention, polyclonal antibodies purified in this way have proven to be particularly advantageous.

The antibodies with a suitable specificity are then used according to the invention to examine cell or tissue samples in the context of an immunocytological assay. The line or tissue Specimens originate in particular from suspected or ascertained dysplasia or suspected tissue. The samples can be histological tissue sections, for example. Suitable material can also be obtained by fine needle biopsy or punch biopsy (puncture cytology), by collecting cells that have been detached or rejected by surfaces (exfoliative cytology), or by cells that are present in spontaneously emptied secretions, body cavity fluids or irrigation fluids, or by direct Smear sampling from mucosal surfaces can be obtained. Other biopsy samples can also be used.

The immunocytological assay is carried out with a prepared cell or tissue sample. For example, a cell or tissue sample is treated with a suitable preservation solution after removal. Exemplary solutions are described in U.S. Patent 5,256,571 (Hurley et al.). A fixation can be done with the help of solvents such as methanol, ethanol, acetone or isopropanol. The cell and core membranes are perforated so that the interior of the cell is accessible to the added antibodies and reagents. The fixed sample is rehydrated with buffer solution before the examination.

In many cases it proves to be expedient to use a sample that has been obtained and processed using a method that is already used in the context of conventional cytological or histological examination methods. Corresponding sample preparation methods and the equipment and chemicals required for this are commercially available, for example as ThinPrep from Cytyc Corporation. Alternatively, a procedure called AutoCyte is offered. Both methods have the advantage that, after cells have been obtained for the examination, components such as blood, mucus or non-diagnostic fragments that are additionally contained in the sample are first removed. This improves the informative value of the immunocytological assay. ® In the ThinPrep method, for example, a gynecological sample, which is obtained in the usual way by a cytological / smear, is rinsed into a preservation medium. Blood, mucus and nondiagnostic debris are then broken up in a dispersion step and the sample is thoroughly mixed. Cellular material is then collected on a filter membrane and finally transferred to a slide.

With AutoCyte's procedure, the swab sample is also transferred to a preservation fluid. A density gradient centrifugation then takes place later for preparation. This removes blood, mucus, material from inflammatory processes and other disruptive components, and you get a cell sample that is enriched with cells to be used for diagnosis. The cell sample can then be resuspended in a suitable medium and transferred to a slide.

Proposals for sample preparation procedures as well as suitable devices and chemicals are also described in various patents. Examples include US Patents 5,139,031 (RA Guirguis), 5,256,571 (AA Hurley et al.), 5,346,831 (CL Carrico et al.) And 5,612,540 (R. Richards-Korturn et al.) called .

The person skilled in the art recognizes that - depending on the source and nature of a sample - different sample preparation methods can be considered, it being particularly advantageous for cytological samples to remove the potentially disruptive components of the diagnosis. The sample preparation therefore preferably comprises filtration steps and / or centrifugation steps (such as gradient or equilibrium centrifugation steps).

In the immunocytological assay, depending on the specificity of the antibodies, RNA present in the cells can interfere. On the one hand, it contains components that are also present in unmodified DNA are contained and, depending on the antibody, are bound with a certain affinity and, on the other hand, like the DNA modifications which may be recognized by the antibody. It is therefore preferred to degrade RNA by treatment with an RNAse. In particular, damaged RNA is also broken down. In order to make the DNA damage to be detected accessible to the antibodies, it is also preferred to cause the DNA to be denatured. A mild alkali treatment is particularly preferred. Ethanol can also be used. This treatment partially makes the DNA single-stranded so that it is more accessible to the antibodies. The denaturation can also be carried out by the action of heat and / or chemicals, for example formamide. However, care must be taken to ensure that there is essentially no destruction of the DNA or removal of the modifications to be detected. It is further preferred to carry out a treatment with a proteolytic enzyme. The background in the later detection reaction can thus be reduced. In order to avoid non-specific binding of antibodies, treatment of the sample with a protein which occupies non-specific adsorption sites is also preferably carried out. For example, the treatment can be carried out with sera or solutions which contain animal proteins, such as IgG or a serum albumin, such as bovine serum albumin (BSA). The sample is then incubated with the antibody specific for DNA damage. After a sufficient waiting period, the sample is washed to remove unbound antibodies. If the first antibody bears a label, the DNA damage can now be made visible. Otherwise the incubation takes place with a second antibody which is specific for the first antibody and bears a label. After washing to remove unbound second antibody, the labeling of the second antibody and thus indirectly the DNA damage is then made visible.

The exact method of visualization and quantitative determination depends on the type of marking. If a fluorescence marker used, which is preferred according to the invention, the evaluation can expediently be carried out with a digital camera. This enables electronic amplification of the immunofluorescence. The images can then be evaluated using a multi-parameter image analysis program. Suitable devices and suitable software are commercially available. Details of a suitable method can be found, for example, in F. Seiler et al., Carcinogenesis, Vol. 14 (No. 9), pp. 1907-1913 (1993).

According to an expedient embodiment, the amount of total DNA contained in the cells can also be measured by fluorescence. For this purpose, an image analysis can be carried out in the same way as for the determination of the DNA modification by means of fluorescence, but using a different dye. The amount of DNA found can additionally be used in the tumor risk assessment intended according to the invention, including the identification of already existing tumor cells. Specifically, the amount of DNA modification can be related to the amount of DNA and this size can be used in place of the absolute amount of DNA modification. This procedure can also detect and take into account artifacts caused by the binding of antibodies to material that differs from the DNA modification to be detected (cross-reaction).

In the method according to the invention, the immunocytological assay, as explained above, is preferably carried out both for the cell or tissue sample to be examined and for one or more control preparations. For example, two control preparations per 20 samples examined can be analyzed. These are standard samples, the nature of which is known. Control preparations can be made, for example, by exposing cells in culture to varying concentrations of a DNA-modifying agent and fixing the cells for a predetermined period after treatment. For the study of DNA modifications by reactive oxygen species, such as 8-oxoguanine, are caused, for example, to have an oxidizing agent, such as H ₂ 0 ₂ , act on the cells. A comparison of the measurement results for the cell or tissue sample and the control preparation then allows the extent of the DNA modification in the cell or tissue sample to be determined. This in turn can then be used to estimate the probability of tumor development, including the probability that tumor cells are already present.

More precisely, the extent of the DNA modification, preferably relative to the total DNA content, can be determined for individual cells of the examined sample. Of course, it is also possible to summarize measurement results for cells of the same type and / or to subject the measurement results contained in the sample for different cells to a statistical evaluation. On the other hand, an immunocytological assay for cells that originate from different stages of tumor development can be used to derive threshold values for the extent of the DNA modification, such as, for example, the content of reaction products of reactive oxygen species, in particular 8-oxoguanine. The progression estimate with respect to the sample is then carried out by comparing the measurement results for the sample with the threshold values.

According to a further preferred embodiment of the invention, a cell or tissue sample which has been examined by the immunocytological assay is additionally subjected to histological or cytological staining. This staining then enables the immunocytological data and thus the extent of the DNA modification to be assigned to individual dysplastic cells which, as usual, are classified by histological or cytological staining. A useful coloring is the Pap coloring. A working specification for this can be found, for example, in the book "Gynecological Cytodiagnostics" by Soost and Baur. The 8-oxoguanine content in the DNA of tumor cells, for example cervical tumor cells, is always higher than in the DNA of normal cells, for example normal squamous cells.

Furthermore, the fluorescence intensities for dysplastic cells were determined and the values obtained were compared with those for normal and for tumor cells. It was shown that there are moderate and severe dysplasias in which the precancerous cells (CIN I, CIN II and CIN III) have a higher oxidative DNA damage than the normal epithelial cells. However, there are also dysplasias of the same severity in which the precancerous cells have the same low oxidative DNA damage as the normal cells. Based on the fact that there is a close molecular connection between the persistence of oxidative damage in the DNA of replicating cells and the gradual conversion of these cells into tumor cells, it can be seen that the 8-oxoguanine content in dysplastic cells can be used prognostically for the development of cancer according to the invention.

An exemplary procedure for the present invention with cell-specific determination of oxidative DNA damage using example 8-oxoguanine essentially comprises four steps, namely (1) sample preparation; (2) performing the immunocytological method and quantitative determination of the 8-oxoguanine content in each individual cell of the preparation; (3) cytological staining of the preparation; (4) Combination of the 8-oxoguanine values and the optical characteristics determined after staining for the individual cells.

As already explained elsewhere, optimal and reproducible sample preparation involves removing unwanted accompanying material, such as mucus, bacteria and cell fragments, and separating the cells before applying them to a slide. The cells attached to the slide are then fixed and treated in order to make the cellular DNA accessible to antibody molecules, as also described above. was explained. The preparation is then treated with a monospecific antibody that can recognize and specifically bind to the 8-oxoguanine molecules present in the DNA. To visualize the bound antibody molecules, a second, fluorescence-labeled antibody is applied to the preparation, which binds to the fc part of the first antibody. The preparation can then be treated with a DNA-binding fluorescent dye, which allows the DNA content of each cell to be quantified. The evaluation is carried out with a fluorescence microscope, which is equipped with a digital camera for measuring the fluorescence signals. The preparation is scanned step by step and each cell is registered on a data carrier with regard to its position, its 8-0xoguanin content and, if appropriate, its DNA content. In the next step, the preparation is stained according to Papanicolaou for the cytological examination of the cells it contains, including any dysplasia or tumor cells it contains. The colored specimen is placed under the microscope as before, but this time it is scanned step-by-step in transmitted light. The position of the cells and their specific optical characteristics are then also recorded.

After the fluorescence staining and the cytological staining, the following information is available about each cell of the preparation:

8-oxoguanine content,

• possibly DNA content and

• Optical identification features.

As long as it is not yet known for the type of cell material examined, how the 8-oxoguanine content is quantitatively related to the division into normal, precancerous and cancerous cells or the degree of dysplasia, it is now possible to do so Establish correlation by identifying the type of a cell on the basis of optical distinguishing features, as are known in the field of cytodiagnostics, and assigning 8-oxoguanine content and possibly DNA content. Levels or ranges for levels of 8-oxoguanine and, if desired, total DNA and thus also the relative extent of the damage (8-oxoguanine / total DNA), which typically apply to a specific type of cells, can be determined. However, it should be pointed out that the method according to the invention does not depend on a conventional classification of the sample. Rather, the method according to the invention provides additional information that cannot be obtained by conventional cytodiagnostics, for example by Papanicolaou staining. Thereafter, dysplastic cells can often be recognized; a forecast of their further development is not possible. ^■

On the other hand, according to the invention, optical identification features, preferably those that are accessible for automatic detection / detection, can be used in addition to the precise identification of the cells, for example as tumor cells, or for prognosis (tumor risk assessment) if there is a correlation between 8-0xoguanin content and cell type. For example, cells that show a certain degree of dysplasia can be assessed.

Functional (immunocytologically determined DNA damage) and morphological data are linked here. It is particularly preferred here to use the nucleus morphology, especially the nucleus size, the nucleus shape and the coloring of the chromatin and in particular the ratio of nucleus size to cell size. In addition to increasing reliability, the method can also be accelerated by using suitable features for preselection.

The method according to the invention can in principle be used for all types of cancer. The method is particularly useful for examining dysplastic cells of the cervix. If dysplasias are found in the context of cervical cancer screening cytology, an objective progression assessment can be made with the method according to the invention. More special Areas of application are dysplasia of the female breast or colon. In addition, the method according to the invention is particularly applicable to examinations of the lungs / bronchi (especially examination of expectoration, lung secretions), bladder (especially examination of cells from urine samples), thyroid and malignant effusions. In these cases, especially in the case of bladder and effusions, the identification of existing tumor cells can already be improved considerably in comparison with conventional methods.

An immunocytological assay for determining DNA modifications, as explained above, can also be used to determine the individual radiation sensitivity. This is of great importance insofar as in the case of radiation therapy, for example of cancerous diseases, in about 30% of the patients inpatient treatment is required because of damage to healthy radiation-sensitive tissues adjacent to the tumor, with about 2% even causing permanent damage. On the other hand, for optimal tumor control, it would occasionally be desirable to use higher than usual radiation doses. In order to determine the radiation sensitivity according to the invention, a sample of a healthy tissue that is exposed to radiation during the therapy or of a suitable surrogate tissue is irradiated in vitro after the removal. Then the kinetics of the repair of DNA damage is examined. For this purpose, irradiated tissue or cell material is fixed at various times after the irradiation and then subjected to an immunocytological assay, as has already been described. Radiation sensitivity can now be determined by measuring the temporal decrease in the amount of DNA modifications, in particular the amount of modifications caused by reactive oxygen species, and especially the amount of 8-oxoguanine, and correlating them with standard data. In general, a greater decrease in the amount of modifications up to a certain point in time means better repair capacity and less radiation sensitivity. It is possible to make a comparison with statistical averages. On in this way it is possible to individually determine the total radiation dose and the distribution of the individual doses over time in the case of fractional radiation.

An immunocytological assay can also be used to determine whether a certain degree of damage (limit value), for example a certain amount of oxidative DNA modifications and especially a certain amount of 8-oxoguanine, is undershot in a sample at a certain time after irradiation. This information can, for example, be used to determine the time and / or dose for further irradiation.

According to a special embodiment, it is also provided to combine the data as determined above, which reflect the repair capacity of the cells with regard to DNA damage, with data which describe the proliferation of the cells in order to arrive at an even more precise determination of the radiation dose ,

Alternatively or additionally, the radiation sensitivity of tumor tissue can also be determined in the manner described above. In this case, the decrease in the amount of DNA modifications in a tumor tissue sample after radiation is found. This makes it possible to estimate the radiation dose required for treatment.

The present invention also includes the provision of a test kit. Such a kit comprises one or more components which are required for carrying out the method for examining cell and tissue samples or for examining the sensitivity to radiation. These components have already been discussed above in the explanation of the methods. As a rule, the test kit comprises an antibody which is specifically directed against a DNA modification, for example against a DNA modification (oxidation product) formed by reactive oxygen species and especially against 8-oxoguanine. It can also is an antibody fragment. This antibody can carry a label, preferably a fluorescent label. If the antibody is not labeled, the test kit preferably contains a second antibody (or a fragment) which has a label, in particular a fluorescent label, and specifically names the first antibody. The test kit may further include one or more of the following components that can be used in the immunocytological assay, as discussed above: RNAse; DNA denaturing agents; protolytic enzyme; Protein for binding unspecific adsorption sites. The test kit also preferably contains buffers and / or solvents used to carry out the method. A cell preservative and / or a fixative may also be included. A test kit for carrying out a preferred embodiment of the method according to the invention, in which the total amount of DNA is determined, further comprises a dye for staining the DNA, as has also already been discussed.

Additional components that were mentioned above in connection with the explanation of the methods or those that result from the following production examples and examples may additionally or alternatively be contained in the test kit.

The test kit according to the invention is typically associated with instructions relating to the execution of the method according to the invention.

The invention is further illustrated below by examples.

Production example: Production of antibodies against 7.8

Dihydro-8-oxo-2 ^■ deoxyguanosine

7, 8-Dihydro-8-oxo-2'-deoxyguanosine and 7,8-dihydro-8-oxoguanosine were synthesized as described by H. Kasai and S. Nishimura, Nucleic Acids Res., Vol. 12, p 2137-2145 (1984). Hapten-protein conjugates were prepared by coupling 7, 8-dihydro-δ-oxoriboguanosine to thyroglobulin as described by BF Erlanger and SM Beiser, Proc. Natl. Acad. Be. USA, Vol. 52, pp. 68-74 (1964). The conjugates used for the immunization contained 400 to 1000 molecules of 8-oxoguanine or the corresponding ribonucleoside per molecule of thyroglobulin.

Rabbits were injected subcutaneously and intramuscularly with emulsions containing 600 μg conjugate and ABM-ZK adjuvant (Linaris GmbH, Bettingen, Germany). The injections were repeated 4, 10 and 22 weeks after the initial immunization. Serum was collected 12 days after the last injection. Antibodies were precipitated with solid ammonium sulfate (50% saturation), dialyzed and stored at -80 ° C. Antibodies with a specificity against 7, 8-dihydro-8-oxo-2 '-deoxyguanosine were obtained with both haptens.

Example 1: Immunocytological Assay

An immunocytological assay was carried out on frozen tissue sections. The tissue sections were fixed in methanol for 15 minutes and rehydrated in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate, pH 7.2). After treatment with RNAse A (200 μg / ml 2xSSC) and RNAse TI (50 U / ml 2xSSC) for 1 hour at 37 ° C., the sections were washed in 0.14 M NaCl. Cellular DNA was denatured by incubation in a solution containing 70 mM NaOH, 40% EtOH and 0.14 M NaCl for 5 minutes at 4 ° C. This was followed by incubation with Proteinase K (2 μg / ml) for 10 minutes at 37 ° C. The sections were then incubated with phosphate-buffered saline (PBS) containing 20% bovine serum albumin (BSA) for 20 minutes at room temperature. The sections were then incubated with rabbit anti-7, 8-dihydro-8-oxo-2 'deoxyguanosine antibody (1 μg antibody / ml PBS containing 1% BSA) for 16 hours at 4 ° C. Unbound antibodies were carefully ch washed with PBS containing 0.1% BSA removed. Goat anti-rabbit IgG F (ab) ₂ fragments (2 μg / ml PBS containing 1% BSA; Dianova, Hamburg, Germany) conjugated to rhodamine isothiocyanate were added for 45 minutes at 37 ° C.

After washing with PBS, the DNA was additionally stained with 0.3 μM 4, 6-diamidino-2-phenylindole in PBS. To reduce fading, the samples were placed in PBS at pH 8.2 containing 20% glycerin, 10% elvanol and 0.03 M dithiothreitol.

Example 2: Image analysis

A standard fluorescence microscope (Neofluar 40 / 0.75; Zeiss, Oberkochen, Germany) was prepared for the epifluorescence and equipped with a suitable light source (HBO 100 W mercury lamp). Standard filter number 02 (Zeiss) was used for the excitation (maximum: 350 nm) and emission (maximum: 475 nm) of the 4,6-diamidino-2-phenylindole fluorescence spectrum in combination with filter number 14 (Zeiss) for the excitation (maximum : 550 nm) and emission (maximum: 580 nm) of rhodamine isothiocyanate fluorescence. Fluorescence signals were amplified with a single-stage image intensifier (Proxifier BV2532 EG35; Proxitronic, Weiterstadt, Germany) and a CCD video camera (Hamamatsu, Japan), the connection being made via fiber optics. The amplified images were fed to a multi-parameter image analysis system (Cytometry Analysis System ACAS; Ahrens, Bargteheide, Germany). This system allowed the integration of images with low signal-to-noise ratios and the quantitative determination of both the fluorescence specific for the labeled antibody and the fluorescence specific for the DNA from the same cell nucleus. Automatic detection of cell nuclei was made possible by setting a threshold value to distinguish between background (electronic noise, autofluorescence) and DNA staining signal. Using filter number 14 (Zeiss) the rhodamine isothiocyanate fluorescence recorded in the same positions where nuclei were detected. The fluorescence intensities of all pixels that originated from the nuclei (both DNA-specific and DNA adduct-specific signals) were integrated. In this way, data for the extent of DNA modification and the amount of DNA were obtained at the individual cell level.

Example 3: 8-Oxoguani levels in normal and cancerous human cervical cells

This example shows that tumor cells have a higher 8-oxoguanine content in genomic DNA than normal cells from the same patient. For this purpose, smears were examined from 9 women who had been diagnosed with cervical carcinoma. The swabs were prepared as explained above and then subjected to an immunocytological assay essentially in accordance with the procedure also described above. An antibody against 8-oxoguanine was used.

Comparing the fluorescence intensities of the tumor cells with those of the normal epithelial cells in the same smear shows that the DNA of the tumor cells always has a measurably higher 8-oxoguanine content than that of the normal cells. This is clear from the ratio of the value pairs for the tumor cells and the normal cells (CA / Normal), which is between 1.5 and 2.7 for the individual samples. The mean for normal cells is 23 + 6 relative fluorescence units and for tumor cells 44 + 10 relative fluorescence units. On average, the relative fluorescence intensities of the tumor cells are about twice as high as those of the normal cells (mean CA / normal = 1.9). Example 4: 8-oxoguanine levels in dysplastic cells

Using a procedure similar to that in Example 3, cervical smears were subjected to an immunocytological test. The content of 8-oxoguanine in each cell of the respective preparation was in the form of fluorescence signals quantitatively determined ^• (image analysis).

Following the execution of the immunocytological assay, the samples were cytologically stained on the basis of the Papanicolaou staining protocol, with an extension of the incubation time in Harris hematoxylin solution and a shortening of the subsequent washing steps being chosen to improve the staining.

The evaluation was now carried out in such a way that the fluorescence and color images were compared for each sample. If dysplastic or cancerous cells were identified in the color image on the basis of their optical distinguishing features, a corresponding cytological classification was carried out. In addition, the intensity was determined from the fluorescence image as a measure of the 8-oxoguanine content. Normal epithelial cells served as controls.

While the fluorescence values were consistently higher for all tumor cells than for normal cells, this was not the case for the CIN III cells in all smears with a PAP IVa finding. This can be seen from Fig. 1, in which two smears (PAP IVa, smear 2 and smear 6) are shown with different characteristics. In smear 2, the fluorescence values for all dysplastic cells (CIN I, CIN II and CIN III) are significantly higher than in the normal cells; in smear 6, the fluorescence values for the precancerous cells (CIN II cells were not present in this smear) are not or only slightly higher than those for the normal cells. A similar picture emerges for the precancerous cells in smears with moderate dysplasia (PAP III D). 2 shows an example of a smear (PAP III D 11) with high values and for a smear (PAP III D 13) with slightly higher fluorescence values for the CIN II cells contained therein.

Claims

claims

1. A method for the examination of cell and tissue samples for tumor risk assessment, including the identification of already existing tumor cells, which comprises the following stages:

2. The method of claim 1, wherein the DNA modification is a reaction product of DNA with a reactive oxygen species.

3. The method of claim 2, wherein the DNA modification is 8-oxoguanine.

4. The method according to any one of the preceding claims, wherein a sample is examined, of which is known or suspected of containing dysplastic cells.

5. The method according to any one of the preceding claims, in which additionally the total DNA content of the cells is determined and used for tumor risk assessment including the identification of already existing tumor cells.

6. The method according to any one of the preceding claims, in which a histological or cytological staining of the sample is carried out after the immunocytological assay, which allows the detection of dysplastic cells and / or the determination of the severity of dysplastic cells.

7. The method according to any one of the preceding claims, wherein the cell or tissue sample originates from the cervix, the lungs, the bronchi, the bladder, the thyroid, a malignant effusion, the female breast or the colon.

8. A method for determining radiation sensitivity to cells or tissues, comprising the following stages:

(a) irradiation of a line or tissue sample with one or more defined radiation doses;

(b) immunocytological assay for the irradiated sample to determine the extent of DNA modifications at at least a predetermined time after the irradiation;

(c) Correlation of the data obtained under (b) with standard data.

9. A test kit for carrying out the method according to one of the preceding claims, comprising:

(a) an antibody or antibody fragment that is specifically directed against a DNA modification to be detected and can carry a label;

(b) if the antibody / the antibody fragment mentioned under (a) has no label, a secondary antibody or a corresponding fragment which / which recognizes the primary antibody / the primary antibody fragment according to (a) and carries a label.