Diagnosis kit, dna chip, and methods for diagnosing or supervising the ...

DIAGNOSIS KIT, DNA CHIP, AND METHODS FOR DIAGNOSING OR SUPERVISING THE TREATMENT OF TESTICULAR CANCER

[0001] The present invention concerns a diagnostic kit, a DNA chip, and a process for diagnosis or treatment control of testicular cancer. Within the framework of preliminary and later cancer care, it is of great importance to be able to detect a malignant testicular tumor or recurrent malignant testicular tumor early by means of the appearance of metastasizing tumor cells in the blood.

[0002] Testicular cancer is responsible for less than 2% of all malignant new formations of tumors in men. However, 20-30% of all cancer diseases in men under 40 years of age involve testicular cancer. The annual number of new diseases in the Federal Republic of Germany, for example, is about 3600, of which about 240 men die of testicular cancer. The highest incidence is found between age 25 and 40. Through advances in oncological therapy, long-term healing is achieved for more than 90% of all affected patients. The high survival rates in this case are based on the pronounced effectiveness of chemotherapy based of cisplatin.

[0003] In this case, it must be decided in clinical stage 1 of the disease whether the patient must be burdened with chemotherapy and/or with an operation in order to achieve a permanent successful cure. A large number of patients are treated with chemotherapy in this case, even though no certain evidence of a metastasis exists. In concepts based on pure monitoring, however, recurrence happens in 25% of cases, some with fatal outcomes.

[0004] In the research methods currently in use with cancer patients, so-called tumor markers at the protein level (immunological or enzymatic) are determined quantitatively in the blood or other body fluids of cancer patients. These detection processes are suitable for tumor diagnosis or treatment control/care only under certain conditions, however, since elevated tumor-marker values can also be caused by non-tumor diseases such as, for example, inflammations of the gastrointestinal tract, cirrhosis of the liver, virus infections, or heavy smoking.

[0005] Molecular genetic processes seem to be helpful here for detecting tumor cells in the peripheral blood, since at the start of the metastatic process, tumor cells can pass into the venous blood.

[0006] EP 0,520,794 B1 discloses a process of this kind, in which metastases can be detected in body tissue or fluids. In this case, nucleic acids are detected, for example by means of copying by a polymerase chain reaction. Now the process is based critically on the fact that the detected nucleic-acid sequence in cells of future tumor tissue is also expressed, i.e., in tumor cells and markers, under certain conditions in healthy cells of the future tissue. Another condition is that this sequence is not expressed only in cells of the tissue being studied. If, therefore, a corresponding sequence is found in the sample being studied, then this must be based on transported, i.e., metastasized cells of a tumor in another place. This process is therefore based in the end on detecting cells that should not appear in blood samples from healthy persons.

[0007] Overall, it can be stated that the currently used diagnostic methods are too imprecise when evaluation of the malignancy of residual tumors after chemotherapy has been

performed in the metastasizing stages is involved. This is also true for finding evidence of hidden or residual metastases, which makes a timely assignment to one of the many primary curative therapeutic options possible.

[0008] The problem of the present invention is therefore to make available a process, a diagnostic kit, and a nucleic-acid microarray with which tumor cells can be detected in the blood in testicular-tumor diseases.

[0009] This problem is solved by the diagnostic kit according to claim 1, the microarray according to claim 20 as well as a process according to claim 22 and its application according to claim 45. Advantageous developments of the diagnostic kit, the microarray, and the process or applications will be given in the various dependent claims.

[0010] The present invention is based essentially not on detecting testicular-tumor markers at the immunological or enzymatic level in the blood of patients, but on the fact that the mRNA (messenger ribonucleic acid) of testicular-tumor markers in a blood sample is detected, whereby the tumor markers represent a gene expression associated with a tumor. As markers for testicular tumors, placenta-specific alkaline phosphatase (PLAP) and germ-cell specific alkaline phosphatase (GCAP) are used according to the invention, which are expressed by testicular tumors, epithelial glycoprotein (GA733.2 or EGP40), the high-mobility group protein isoform I-C (HMGI-C), and the gastrin-releasing peptide receptor (GRPR), are detected. The detection can be made here for only one of the markers or also for any arbitrary number of these four testicular-tumor makers in combination with one another, whereby, however, at least the mRNA of the marker GA733.2 and/or GCAP/PLAP is detected. The kit according to the invention can therefore contain oligonucleotide pairs for only one or for any arbitrary number of the four testicular-tumor markers.

[0011] Not all cases are detected by this means in which, for example because of other diseases, testicular-tumor markers are expressed in the original tissue and enter the blood circulation as proteins. Consequently, only cells are detected that, on the one hand, are themselves found in the blood sample, and on the other, express the particular testicular-tumor makers. This consequently involves tumor cells that derived from their original testicular-tumor tissue and are transported in the blood of the patients. Since the mRNA of the marker being studied is not expressed in the blood of a patient with a testicular tumor, there is a direct correlation between the appearance of the associated mRNA and a metastasis already in an early stage of the metastatic process.

[0012] Advantageously, not only the mRNA of a single testicular-tumor marker is detected, but a combination of markers is studied. In this way, it is possible, for example, to be able to detect all forms of testicular cancer through the blood because of their metastasizing cells in the blood. This means that both seminous and non-seminous forms as well as mixed tumors with portions of a seminoma, and thereby 90-95% of all malignant testicular tumors, specifically all germ-cell tumors, can be detected.

[0013] Since individual markers are expressed in different ways, depending on the therapy, it is advantageous to study