WO2000070085A2

WO2000070085A2 - Method for detecting the effect of different chemotherapeutic agents and/or radiation therapy in malignant diseases

Info

Publication number: WO2000070085A2
Application number: PCT/DE2000/001444
Authority: WO
Inventors: Peter Daniel; Timo Hillebrand; Bernd DÖRKEN; Peter Bendzko
Original assignee: Theragen Molekularmedizinische Informationssysteme Ag
Priority date: 1999-05-14
Filing date: 2000-05-10
Publication date: 2000-11-23
Also published as: EP1181394A2; DE19922052A1; WO2000070085A3; AU5672000A

Abstract

The invention relates to a method for detecting the effect of different chemotherapeutic agents and/or radiation therapy in malignant diseases, wherein the expression profile of tumor and/or cell growth and/or apoptosis associated genes and/or individual differences (mutations) in gene sequences are determined. Changes associated with chemotherapeutic agents and/or radiation therapy are identified, represented and diagnostically evaluated. The invention further relates to a method for selecting more effective therapeutic agents for the therapy of malignant diseases. The status of cell cycle genes and/or apoptosis-associated target genes or gene products thereof in body fluids, cells and/or organs is determined and diagnostically evaluated to determine their effect on corresponding therapeutic agents. In a preferred embodiment, Bax and p53 expressions or mutations are investigated and the findings therefrom are used for deciding individual-specific therapy in leukemia and other malignant diseases.

Description

Methods for demonstrating the effect of different chemotherapeutic agents and / or radiation therapy for malignant diseases, and methods for selecting effective therapeutic agents for their therapy

The invention relates to a method for detecting the effect of different chemotherapeutic agents and / or radiation therapy in malignant diseases, the expression profiles of tumor and / or cell growth and / or apoptosis-associated genes and / or the individual differences (mutations) in the Gene sequences can be determined. Changes in connection with chemotherapeutic agents and / or radiation therapy are identified, represented and diagnosed. Furthermore, the invention relates to a method for selecting effective therapeutic agents for the treatment of malignant diseases. The status of cell cycle genes and / or of apoptosis-associated target genes or of their gene products in body fluids, cells and / or organs is determined and their effects on corresponding therapeutic agents are evaluated diagnostically. In a preferred embodiment, Bax and p53 expression or mutations are examined, and information derived therefrom for individual-specific therapy decisions in leukemic diseases and other tumor diseases is provided.

The process of a malignant change in a cell begins very early, often with just a single change in the genetic material. It runs through various stages up to the degenerated cell and is not yet finished even at this stage.

Advances in modern molecular biology, along with a better understanding of the emergence of malignant changes at the molecular level, have provided a wealth of new information about factors involved in the process of carcinogenesis and tumor progression. However, these results also clearly show how diverse, different and complex changes in the molecular network are in order to ultimately present themselves in a malignant phenotype or therapy-resistant tumor. The involvement of various factors in the process of tumor development is on the one hand an expression of the complexity of such a process, but can also be used for a diagnostic application and prognostic risk assessment.

Despite the knowledge reached to date, the stages and the treatment of tumor patients based on them are still based on histopathological or clinical criteria. This conventional classification is therefore still decisive for all therapy decisions (hematology oncology, ed. PC Ostendorf and S. Seeber, Urban and Schwarzenberg 1997; Compendium of Internal Oncology, ed. HJ. Schmoll, K. Höffken, K. Possinger, Springer 1997).

An individual-specific therapy based on the most comprehensive molecular characterization of the tumor has so far not been possible.

The object of the invention was therefore to use knowledge at the molecular level for an individual-specific tumor therapy and to find an effective selection of therapeutic agents for the patients concerned in order to enable effective treatment.

Tumor development, tumor progression and resistance to therapy are determined by cell cycle and apoptosis regulating factors. The basis of the present invention was the surprising finding that by determining their expression profile this tumor or. Use cell growth-associated genes as prognostic markers and correlate the patient's response to different chemotherapy drugs, radiation therapy and clinical parameters. Depending on the existing marker profile, an effective and promising form of therapy for the patient can be derived from this.

The invention therefore relates to a method for detecting the effect of different chemotherapeutic agents and / or radiation therapy in the case of malignant diseases. According to the invention, the expression profiles of tumor and / or cell growth-associated genes and / or the individual differences (mutations) in the gene sequences are determined and interactions (correlations) with chemotherapeutic agents and / or radiation therapy are identified, represented and diagnostically evaluated.

It has been found that the disruption of apoptosis together with the deregulation of the cell cycle is a crucial mechanism in the development, growth and progression of malignant tumors. The reconstitution of the ability to arrest cells and of genes that promote apoptosis is already an important goal of experimental gene therapy strategies. It has been found that inhibition of apoptosis signal cascades can lead to resistance to cytostatics and radiation therapy, which is of enormous clinical importance, especially in solid tumors. These resistance mechanisms can arise from disorders of the apoptosis and the cell cycle by the fact that pathologically relevant changes are contained in specific genes and their gene products. Important tumor genes that are responsible for resistance mechanisms are, for example, the genes of the bcl-2 Family, vzw. Bax, p53, pl6, caspases, Rb, cyclin, inhibitors of cyclin-dependent kinases (CDKi's), ATM and inhibitors of apoptosis proteins (IAPs).

According to the invention, the expression profiles and / or mutations of the said genes are preferably determined by means of protein or DNA / RNA analysis. The expression profiles and / or mutations of the respective individual genes are evaluated. However, the profiles and / or mutations of different genes can also be combined, whereby the creation of an individual therapy scheme is improved and an individual prognosis and risk assessment is possible. The expression profiles of Bax, p53, pl6, caspase and / or Rb genes or their mutations are preferably used and evaluated for diagnosis. The status of p53 genes and of Bax genes or of their gene products is particularly preferably identified.

The invention relates in particular to a method for selecting effective therapeutic agents for the treatment of malignant diseases. It is characterized by determining the status of cell cycle genes and / or apoptosis-associated target genes or their gene products ex vivo in body fluids, cells and organs, which is evaluated diagnostically in connection with the action of appropriate therapeutic agents.

Therapeutic agents in the sense of the invention are known agents for the therapy of leukemic or lymphoma diseases and other malignant diseases, such as e.g. tumors of the gastrointestinal tract, pancreas, prostate, gynecological tumors (such as ovaries, cervix, breast), sarcomas, brain tumors, tumors of the skin and lungs, and tumors of endocrine organs, such as the thyroid gland

These are known cytostatics, preferably steroid hormones (e.g. prednisone, prednisolone, methylprednisolone, and other glucocorticoids), antimetabolites (cladribine (2-CDA), fludarabine, mercaptopurine, arabinoside C, 5-substituted dideoxynucleosides, such as 5-fluorouracil, azidothymidine) to alkylants (e.g. mafosfamide, chlorambucil, melphalan, cyclophosphamide), taxanes (such as paclitaxel, docetaxel), anthracyclines (e.g. idarubicin, doxorubicin, epirubicin, mitoxanthrons), topos-isomerase, topos-isomerase, Vinca alkaloids, (vincristine, vinblastine, vinorelbine), cis-platinum and other platinum analogues u. v. a. m. as well as radiation therapy.

The invention will be explained in the following using the example of leukaemic diseases. It was found that preferred for the therapy of chronic lymphoblastic leukemia (CLL), evaluating Bax expression or mutations in Bax Loss of expression therapy with alkylating agents, anthracyclines and vinca alkaloids is far less effective. Such therapy should therefore be avoided and other forms of therapy preferred. The data on which the invention is based clearly prove that in patients with CLL with a low Bax expression, a very poor response to cytostatics therapy in vitro compared to the alkylating agents mafosfamide, chlorambucil, melphalan, to the anthracyclines doxorubicin, epirubicin and to that Vinca alkaloid vincristine is present. These in vitro sensitivity data correlate with the response to therapy in vivo.

On the other hand, it could be shown that in vitro therapy with steroid hormones or fludarabine is promising for the therapy of leukemic diseases, preferably CLL, with low Bax expression. There is no influence on the responsiveness to steroid hormones, e.g. Prednisolone and methylprednisolone or versus fludarabine.

These in vitro data on the chemosensitivity of the CLL cells show a good correlation with the clinical response of the patients to the substances in vivo. They also prove that the diagnostic clarification of the Bax status is essential for a therapy decision. Since the loss of expression of Bax can occur at different stages in the implementation of the genetic information, various detection methods must also be included for such a diagnosis. This affects e.g. the detection of mutative changes at the level of DNA (e.g. point mutations; frameshift mutations) and the detection of hypermethylation patterns within the Bax promoter region as a possible cause for transcriptional silencing and studies of the expression or expression level of the Bax protein (immunohistochemistry or Western blot or Flow cytometry or other quantitative detection methods) as well as the analysis of transcriptional and translational regulators and protein degradation.

Analysis of p53 expression or mutations of the p53 gene has shown that for therapy of leukemic diseases, preferably CLL, if mutations are present within the coding sequence sections of the p53 gene, therapy with DNA-damaging substances, in particular with alkylating agents, anthracyclines and with fludarabine. With regard to a positive correlation between mutations of the tumor suppressor protein p53 and a differential response to cytostatics, it could be demonstrated that there is a significantly poorer response to fludarabine and to DNA-damaging substances such as alkylating agents for patients with mutations within the coding sequence sections of the p53 gene. Comprehensive diagnostic analysis at the DNA and protein level is therefore also required for the candidate gene p53 be performed. In principle, this also applies to the p53 homologous candidate genes p73 and p41.

Ultimately, an individualized form of therapy can be applied via the combined diagnosis of the two candidate genes p53 and Bax by creating individual therapy schemes by combining the genetic status of the p53 and Bax genes or their gene products and / or mutations.

The invention further relates to the use of the status determination of cell cycle genes and / or of apoptosis-associated target genes or of their gene products or mutations by means of protein or DNA / RNA analysis for determining therapy resistance and for the targeted selection of therapeutic agents for cytotoxic therapies. The analysis is preferably carried out using Bax expression or mutations or using p53 expression or mutations.

The status determination of Bax and p53 genes is particularly preferably used for risk-adapted tumor therapy in leukemic diseases, such as CLL and other tumors. In a further embodiment of the invention, the use is made in combination of p53 and Bax with further cell cycle and apoptosis regulators, which can also be used either alone or in combination as a molecular pathway (signal path) diagnosis in malignant tumors or precancerous diseases .

While it is known that e.g. due to the existing clear functionality of the tumor suppressor protein p53 within the cell cycle and apoptosis regulation, a loss of p53 function frequently in tumors e.g. is found by mutation, deletion or promoter changes. However, it was surprising that a functionally damaged p53 protein nevertheless enables a selective response to certain cytostatics in patients with chronic lymphocytic leukemia (CLL). This could be demonstrated for the first time. This also affects a large number of other apoptosis-associated target genes and especially their functional combination.

The present investigations were carried out as examples for the tumor suppressor protein p53 and the proapoptotic gene Bax in patients with chronic lymphoblastic leukemia (CLL). Further data are e.g. B. also for these and other apoptosis and cell cycle regulators before in tumors of the gastrointestinal tracts, such as gastric carcinoma, esophageal carcinoma and sarcomas. With the present invention, the two apoptosis-influencing proteins p53 and Bax were able to provide conclusive evidence that diagnostic characterization of corresponding tumor genes at the molecular level (DNA) and at the expression level (protein) makes it possible to selectively select cytostatics, and thus to achieve improved treatment. The findings on the molecular pathogenesis and resistance to therapy of tumors can, according to the invention, be used as the basis for an individual-specific tumor therapy and in this way achieve a more targeted treatment and ultimately a maximum success for the affected patient.

In this way, patients who have a good prognosis according to molecular standards, to respond to cytotoxic therapies and to show better survival after therapy, can save expensive and costly therapies. At the same time, more aggressive treatments can be carried out in patients with a poor risk profile to improve the therapeutic effect. Furthermore, if resistance is proven, less aggressive treatment can avoid unnecessary burdens on the patient and costs.

The invention makes it possible to use changed cellular tumor markers as a decision criterion for the selection of different standard chemotherapeutic agents, i.e. also to take advantage of positive correlations between changed tumor markers and the effectiveness or ineffectiveness of chemotherapy drugs. This offers the possibility, for the first time after a characterization of selected cellular tumor markers, to selectively select the form of the chemotherapeutic agent to be used or also a radiation therapy or a combination thereof.

From the following Table 1, based on comparative studies of the expression of Bax protein, which is known to be proapoptotic in CLL, and the Bcl-2 protein, which has the task of blocking cell death, the effect of various cytostatics in one CLL treatment can be removed. It is clearly evident that the response to anthracyclines and alkylating agents (doxorubicin, epirubicin, chlorambucil and mafosfamide) and vincristine show a good correlation to the Bax expression level (Western blot) (p-value significant and <0.05). However, no correlation was found when determining Bcl-2 expression. On the other hand, the effect of steroid hormones (methylprednisolone, prednisolone) or of .Fludarabin administration shows no correlation to the level of Bax expression, from which an increased cytotoxic effect of the agents used can also be concluded in Bax-negative tumor cells. These statements are confirmed by the correlation of Bax shown in Figure 1 and the ex vivo response of CLL patients to a panel of cytostatics. The relative levels of Bax protein expression (densitometric values from Western blot analyzes) and the LC90 dose for the respective cytostatic are given.

Figure 2 shows the correlation between Bax expression and the LC90 dose of doxorubicin in 37 CLL patients.

Figure 3 shows the reduced cytostatics sensitivity of CLL cells in vitro against the alkylating agents chlorambucil and melphalan as well as against fludarabine in p53-mutated CLL patients compared to the p53 wild type. The p53 mutations were determined by means of SSCP-PCR for exons 5 to 8. The bar height corresponds to the dose of the cytostatic in μg / ml.

Table 1 :

Relationship between the level of protein expression of Bax, Bcl-2 and the ratio of Bax and Bcl-2 expression (Bcl-2 / Bax) and the ex vivo cytotoxicity of cytostatics in the treatment of CLL.

Significance level of the Pearson correlation coefficient (p-value)

Cytostatic Bax Bcl-2 Bcl-2 / Bax

Fludarabine phosphate 0.816 0.212 0.829

Cladribine (2-CDA) 0.524 0.351 0.658

Chlorambucil 0.034 0.739 0.157

Mafosfamide 0.017 0.250 0.160

Methylprednisolone 0.836 0.415 0.880

Prednisolone 0.807 0.545 0.898

Vincristine 0.011 0.052 0.127

Doxorubicin 0.001 0.920 0.001

Epirubicin 0.002 0.647 0.001

P values less than 0.05 are statistically significant Legends for the pictures:

Illustration 1:

Association of Bax levels and the ex vivo response of CLL patients to a panel of cytostatics. The relative levels of Bax protein expression are given in each case

(Densitometric values from Western blot analyzes) and the LC90 dose for the respective

Cytostatic.

Figure 2:

Association between Bax expression with the LC90 dose of doxorubicin in 37 CLL patients. This relation with regard to the cytotoxic effect of the cytostatic could only be observed in Bax, but not in Bcl-2 or the quotient from Bcl-2 to Bax.

Figure 3:

Decreased sensitivity to cytostatic agents (bar height corresponds to dose in μg / ml cytostatic agent) in p53-mutated CLL patients compared to the p53 wild type. The p53 mutations were determined by means of SSCP-PCR for exons 5 to 8.

Claims

Claims:

1. A method for detecting the effect of different chemotherapeutic agents and / or radiation therapy for malignant diseases, characterized in that the expression profiles of genes regulating apoptosis and / or cell growth and / or the individual differences (mutations) in the gene sequences are determined and changes in connection with chemotherapeutic agents and / or radiation therapy are identified, presented and diagnostically evaluated.

2. The method according to claim 1, characterized in that the expression profiles of the genes of the bcl-2 family, vzw. Bax, p53, pl6, caspases, Rb, cyclins, inhibitors of cyclin-dependent kinases (CDKi's), ATM and inhibitors of apoptosis proteins (IAPs) and / or mutations in the genes can be determined by means of protein or DNA / RNA analysis and evaluated individually or in different combinations.

3. The method according to claim 1 or 2, characterized in that individual differences in the sequence of apoptosis and / or cell growth-regulating genes and / or the expression of their gene products that occur in malignant diseases, in relation to an individually different responsiveness to drugs are set and evaluated, especially with regard to their relevance for therapy responses.

4. A method for the selection of effective therapeutic agents for the treatment of malignant diseases, characterized in that the status of cell cycle genes and / or of apoptosis-associated target genes or of their gene products in body fluids, cells and / or organs is determined ex vivo and for this status effective therapeutic agents are selected.

5. The method according to claim 4, characterized in that agents for the therapy of leukemic diseases and other haematological malignancies and solid tumors, such as e.g. Tumors of the gastrointestinal tract, pancreas, prostate, gynecological tumors, sarcomas, brain tumors, tumors of the skin and lungs as well as tumors of endocrine organs are assessed.

6. The method according to claim 4 or 5, characterized in that therapeutic agents known cytostatics, preferably steroid hormones, alkylating agents, anthracyclines, antimetabolites, taxanes, topo-isomerase inhibitors, vinca alkaloids, cis-platinum and other platinum derivatives and much more are.

7. The method according to claim 1 to 6, characterized in that for the therapy of solid tumors or leukaemic and other haematological, malignant diseases, preferably chronic lymphocytic leukemia, the Bax expression or mutations are evaluated and therapy is given at low Bax expression avoided with alkylating agents, anthracyclines and vinca alkaloids and another form of therapy is chosen.

8. The method according to claim 1 to 6, characterized in that for the therapy of leukemic diseases, preferably chronic lymphoblastic leukemia, the Bax expression or mutations is evaluated and, with low Bax expression, therapy with steroid hormones or fludarabine phosphate / 2- CDA takes place.

9. The method according to claim 1 to 6, characterized in that for the therapy of leukemic diseases, preferably chronic lymphoblastic leukemia, the p53 expression or mutations are evaluated and, if mutations are present within the coding sequence sections of the p53 gene, therapy with DNA - Avoid harmful substances, especially those with alkylating agents, anthracyclines and fludarabine, and avoid another form of therapy.

10. The method according to any one of claims 1 to 9, characterized in that by combining the determination of the status of various apoptosis and / or cell growth-associated genes, preferably of p53 and Bax or of their gene products and / or mutations and / or their Homologous individual therapy schemes can be created.

11. Use of status determination by means of protein or DNA / RNA analysis of genes associated with apoptosis and / or cell growth or of their gene products for determining resistance to radiation therapy and to therapeutic agents and for the targeted selection of therapeutic agents for cytotoxic therapies.

12. Use according to claim 11, characterized in that the status, namely expression profile and / or mutations, of genes of the bcl-2 family, vzw. Bax, p53, pl6, Caspasen, Rb. Cyclins, inhibitors of cyclin-dependent kinases (CDKi's), ATM and inhibitors of apoptosis proteins (IAPs) of individual genes or in various combinations is determined.

13. Use according to claim 12, characterized in that the status of Bax expression or mutations of the Bax gene or Bax promoter and Bax regulators is used.

14. Use according to claim 12, characterized in that the status of p53 expression or mutations of the p53 gene is used.

15. Use according to claim 11 to 14, characterized in that the status of Bax and p53 is used for risk-adapted tumor therapies in malignant haematological diseases, such as the CLL, and other tumor diseases.

16. Use according to claims 11 to 15 of a combination of p53 and Bax and optionally further cell cycle and apoptosis regulators either alone or in their combination in the sense of a molecular pathway diagnosis in malignant tumors or precancerous diseases.