DE102010018736A1 - Thyroid hormone receptor expression for the diagnosis and prognosis of breast cancer and ovarian cancer - Google Patents

Abstract

The invention relates to a method for diagnosing a genetic predisposition to breast cancer (breast cancer) and / or ovarian cancer (ovarian carcinoma) of a human individual, which is characterized in that the concentration of the thyroid hormone receptors alpha and in a sample of the mammary tissue and / or ovarian tissue of the individual beta (THRα and THRβ) determined, wherein an increased concentration of THRα and / or THRβ compared to healthy tissue indicates an increased risk of the individual for breast cancer and / or ovarian cancer and a method for predicting the severity of the course of a primary breast cancer and / or a primary ovarian carcinoma of a human individual, which is characterized in that the concentration of the thyroid hormone receptors alpha and beta ((THRα and THRβ) is determined in a sample of the mammary tissue and / or ovarian tissue of the individual, an increased concentration of THRα and / or THRβ being compared with healthy m tissue shows a higher grade and an unfavorable course of the disease. Furthermore, the invention relates to a kit comprising a) a detectable binding molecule directed against the thyroid hormone receptor alpha / beta, b) reagents and / or buffers for the qualitative and / or quantitative determination of the thyroid hormone receptor alpha / beta binding molecule complex, and c) instructions for Carrying out and evaluating the determination method, for use in diagnosing a genetic predisposition to breast cancer (breast cancer) and / or ovarian cancer (ovarian cancer) in a human individual, as well as a therapeutically effective amount of a compound which is capable of increasing the expression or activity of the thyroid hormone receptor alpha and / or to modulate beta for use in prophylaxis and / or treatment of breast cancer and / or ovarian cancer.

Description

The invention relates to a method for the diagnosis of a genetic predisposition to breast cancer and / or ovarian cancer and to a method for predicting the severity of the progression of a primary breast carcinoma and / or a primary ovarian carcinoma in a human individual. The invention further relates to a corresponding kit for use in these methods. Furthermore, the invention relates to a therapeutic concept derived from these diagnostic and prognostic methods for the treatment of breast cancer and / or ovarian cancer.

At 28%, breast cancer is the most common cancer and the leading cause of death in the Western world, between the ages of 30 and 60 years. The disease can occur accidentally, ie sporadically, but can also be hereditary. Breast cancer is the most common malignant tumor in the world. Static risk factors include familial stress, age at onset, menopausal status, carcinogens and co-carcinogens, constitutional factors, high breast density in mammography, and proliferative and precancerous tissue changes in the breast. In about 50% of hereditary breast cancers, the genetic background is unknown. Nearly half of hereditary breast cancers are heterozygous mutations in the tumor suppressor genes BRCA1 (BReast CAncer), located on chromosome 17q21 and BRCA2 (chromosome 13q13). The detection of a BRCA1 mutation is associated with a significantly increased lifetime risk for breast and / or ovarian carcinoma as well as with a much younger age occurring onset of disease (see 1 ). Tumorbiologically, breast cancers with BRCA mutation of sporadic cases are usually distinguished by a high degree of grading (poor degree of differentiation), a negative hormone receptor status and a higher percentage of invasive ductal or medullary carcinomas. The prognosis is often worse than in sporadic forms due to the poorer degree of differentiation and negative hormone receptor status. The detection of a BRCA mutation is associated with an increased risk of developing breast cancer of more than 80%.

Women with ovarian cancer disease are at a 10% probability BRCA1 / 2 mutation carrier ( Kwon JS, Daniels MS, Sun CC, Lu KH: Preventing future cancers by testing women with ovarian cancer for BRCA mutations; J Clin Oncol 2009 In the case of a BRCA1 / 2 mutation, the risk of ovarian cancer is up to 40%. Histopathology and survival data in BRCA-associated ovarian carcinoma indicate that women with proven BRCA mutation and ovarian carcinoma with exit from the surface epithelium have a better prognosis than women with the same disease with no mutation present ( Cass I, Karlan B: Improved survival in women with BRCA-associated ovarian cancer. Cancer 2003; 97: 2187-2195 ). This is due to the better response to platinum derivatives and the different tumor biology The therapeutic methods for sporadic or hereditary breast cancers or ovarian carcinomas are not yet different ( Garcia-Etienne CA, Barile M, Gentilini OD et al: Breast Conserving Surgery in BRCA1 / 2 Mutation Carriers: Are We Approaching an Answer? Ann Surg Oncol. 2009 ; Graeser MK, Engel Ch, Rhiem K et al: Contralateral breast cancer risk in BRCA1 and BRCA2 mutation carriers. J Clin Oncol 2009; 27: 1-6 ). However, there are preliminary indications that the modification of systemic therapy will in future have advantages for women with genetically determined breast / ovarian carcinoma ( Farmer H, McCabe N, Lord CJ et al .: Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005; 434 (14); 917-921 ). The surgical treatment of BRCA-associated ovarian cancer is similar to that in sporadic cases.

To date, only the BRCA1 / 2 test is available to diagnose a genetic predisposition to breast cancer or ovarian cancer. This genetic test detects the presence of a mutation in the BRCA1 or -2 gene, respectively. The evaluation of this test takes about 3 to 12 months, the costs for which are up to EUR 10,000 depending on the laboratory. The evaluation of the test shows a percentage probability for the risk of developing breast and / or ovarian cancer. However, the test does not allow a therapeutic consequence in the sense of a targeted reduction in the risk of onset of the cancer.

Thus, there is a need for a reliable test for diagnosing the genetic predisposition to breast cancer and / or ovarian cancer.

The invention is thus based on the object of providing a method for the diagnosis of a genetic predisposition for breast cancer and / or ovarian cancer as well as a method for the prognosis of the severity of the progression of a breast carcinoma or ovarian carcinoma. The method of the invention is intended to be applicable to all types of breast cancer and / or ovarian cancer and is suitable for both male (in breast cancer) and female (in breast and ovarian cancer) individuals.

The procedure should be fast, inexpensive and reliable and suitable for routine diagnostics. The procedure should be minimally invasive. The method should also allow effective therapeutic prophylaxis for the patient in the sense of a targeted risk reduction of the incidence of cancer.

Surprisingly, it has been found that an increased concentration or overexpression of the thyroid hormone receptors alpha and / or beta (THRα and THRβ) in the breast tissue and / or in the ovarian tissue with an increased risk of breast cancer or ovarian cancer disease and a higher grading and unfavorable course of associated with the disease. It was surprisingly found that the presence of a BRCA1 / 2 mutation correlates with overexpression of THRα / β in breast cancer patients. A corresponding correlation is also to be postulated for male patients.

Carcinomas of the breast and ovary of patients with BRCA1 / 2 mutations behave virtually identical in tumor biology. Furthermore, thyroid receptors α and β are detectable in ovarian carcinoma cell lines and in vivo tumors ( Rae MT, Gubbay O., Kostogiannou A. et al: Thyroid hormone signaling in human ovarian surface epithelial cells; J Clin Endocrinol and Metabol 2006: 92: 322-327 ). Thus, it can be assumed that a comparable overexpression behavior, which correlates with a BRCA1 / 2 mutation, is responsible for both tumor entities.

The evidence of overexpression of THRα / β in breast cancer and ovarian cancer patients allows not only a prognosis of the genetic predisposition and the severity of the course of a disease, but also the development of a prophylactic or therapeutic concept for the prophylaxis or treatment of the disease , It has been found that treatment with compounds capable of modulating the expression or activity of the thyroid hormone receptor alpha and / or beta may reduce the risk for the particular disease or reduce the severity of the course of the disease.

The invention thus relates to a method for the diagnosis of a genetic predisposition to breast cancer (breast cancer) and / or ovarian cancer (ovarian carcinoma) of a human individual, characterized in that in a sample of the mammary tissue and / or ovarian tissue of the individual, the concentration of the thyroid hormone receptors alpha and beta (THRα and THRβ), wherein an increased concentration of THRα and / or THRβ as compared to healthy tissue indicates an increased risk of the individual for breast cancer and / or ovarian cancer. The invention further relates to a method for predicting the severity of the course of a primary breast carcinoma and / or a primary ovarian carcinoma of a human individual, which comprises, in a sample of the mammary tissue and / or ovarian tissue of the individual, the concentration of the thyroid hormone receptors alpha and beta (THRα and THRβ), with an increased concentration of THRα and / or THRβ indicating a higher grade and an unfavorable course of disease compared to healthy tissue.

The invention further relates to a kit comprising a) a detectable binding molecule directed against the thyroid hormone receptor alpha / beta, b) reagents and / or buffers for the qualitative and / or quantitative determination of the thyroid hormone receptor alpha / beta-binding molecule complex, and c) instructions for Conducting and evaluating the method of determination, for use in diagnosing a genetic predisposition to breast cancer of the human individual and / or ovarian cancer of a human individual, and a kit comprising a) a detectable binding molecule directed against the thyroid hormone receptor alpha / beta, b) reagents and / or buffers for the qualitative and / or quantitative determination of the thyroid hormone receptor alpha / beta-binding molecule complex, and c) instructions for carrying out and evaluating the determination method, for use in predicting the severity of the progression of a primary breast carcinoma and / or a primary ovaria carcinoma of a human individual.

Another object of the invention is a therapeutically effective amount of a compound capable of modulating the expression or activity of the thyroid hormone receptor alpha and / or beta for use in the prophylaxis and / or treatment of breast cancer and / or ovarian cancer.

The invention further relates to a method for the prophylaxis and / or treatment of breast cancer and / or ovarian cancer, wherein the patient is given a therapeutically effective amount of a compound capable of expressing or activity of the thyroid hormone receptor to modulate alpha and / or beta.

Preferably, the breast cancer and / or ovarian cancer disease is a disease diagnosed or predicted by a method as above, and more preferably a disease characterized by increased expression of THRα and / or THRβ in the particular tissue.

Thyroid hormones are involved in the regulation of numerous metabolic processes. By means of specific nuclear receptors, the binding and thus importance of participation in various differentiation processes becomes clear.

The T3 receptor chromatin complex consists of an actual receptor protein with a molecular weight of 50,000, an accessory protein and DNA. The cellular erb A oncogene product binds triiodothyronine. It contains the genetic information for the T3 receptor. The genes of the (alpha and beta type) thyroid hormone receptors found in two forms belong to a large family of genes that are structurally related to each other and that encode a range of hormone receptors. The interaction of T3 with its receptor causes a conformational change of the receptor complex, which causes changes in the transcription of different genes ( Biochemistry and Pathobiochemistry by Georg Löffler, Petro E. Petrides and Peter C. Heinrich von Springer, 2006 ).

T3 receptors are located in the nuclei of healthy mammary tissue. The T3 receptor α is the most expressed isoform of the thyroid receptor in breast cancers ( Conde I, Paniagua R, Zamora J et al .: Influence of thyroid hormone receptors on breast cancer cell proliferation; Annals of Oncology 17 (1): 60-64, 2006 ). Other studies have suggested that a major biological role of the β unit of the T3 receptor in mammary carcinomas ( Silva JM, Dominguez G, Gonzalez-Sancho JM et al .: Expression of thyroid hormone receptor / erbA genes is altered in human breast cancer, Oncogene 21 (27): 4307-4316, 2002 ).

Hereditary and sporadic breast cancers differ in their tumor biology as well as their prognosis. The risk of contralateral breast cancer is significantly increased if the BRCA mutation is proven. For example, 25 years earlier, patients with a primary disease of breast cancer have a cumulative risk of developing contralateral carcinoma of 47.4%, with the age of the primary disease appearing to be crucial. So far, women with the diagnosis of a BRCA mutation have only had the possibility of a highly invasive intervention of a prophylactic total mastectomy with an existing residual risk of up to approx. 3% for a breast cancer disease or the possibilities of early detection (mammography, MRI, mammography). With the diagnostic or prognosis method according to the invention, both the risk for a primary as well as a contralateral breast cancer can be quickly and inexpensively displayed with the renewed possibility of metastasis. This can significantly improve the prognosis.

In the prior art, there are no data on studies on the expression level of thyroid receptors in relation to familial breast cancers. The role of the thyroid hormone receptors as a biological marker for the prognosis or early detection of breast cancer is discussed in the prior art rather controversial and poorly informed. In Silva et al. (2002, loc. Cit.) different expression rates for THRα1 and THRβ1 in normal and carcinogenic breast tissue of 70 patients with sporadic breast cancer are described. However, the results of these studies do not allow for an indication of a generally increased expression of THRα1 or THRβ1 in breast cancer tissue.

In Zhang et al. (Novel pathway for thyroid hormone receptor action by interaction with jun and fos oncogenic activities., Mol Cell Biol., December 1991, 11 (12): 6016-6025) an antagonistic effect of THR on proto-oncogenes is described. Decreased expression of THR is considered to be an increased oncogenic effect and thus a marker of increased breast cancer risk. In US 7,056,663 a correlation between a 3p24.5 loss of heterozygosity (LOH) and a decreased expression of THRβ1 is described. The reduced expression of THRβ1 is considered a predictive marker for the risk of recurrence in breast cancer. In the US 2003/0099974 The expression level of proteins, metabolites or mRNA is described as a marker for breast cancer. However, this reference does not specifically indicate that overexpression of THRα and / or THRβ correlates with an increased risk of familial breast cancer or ovarian cancer, respectively.

In summary, it should be noted that the prior art contains no indications that a change in the expression level of THRα and / or THRβ correlates with an increased risk of breast cancer or ovarian cancer. Furthermore, the prior art contains no indications to the effect that a therapeutic or prophylactic treatment of a breast cancer or ovarian cancer disease could be derived from a corresponding expression pattern.

According to the invention, a total of 247 breast carcinoma sections (85 of patients with mutations and 182 with sporadic breast cancer) and six preparations with healthy mammary tissue were examined. They were incubated with antibodies against thyroid hormone receptor alpha (THRα) and beta (THRβ) and detected by the immunohistochemical ABC method. The evaluation of the respective thyroid hormone receptor expression was semiquantitative with the aid of the IRS score according to Remele. Statistical evaluations were performed using SPSS Windows, 17.0 (p <0.05 significant). Significant differences were found in in vitro studies of tumor tissue from sporadic breast and breast cancer with mutations present in the BRCA1 or -2 gene. This means that the detection of THRα / β significantly correlates with the result of the genetic assay for BRCA1 / 2 mutation. Thus, the present studies show for the first time a connection between the expression of THRα and / or β and a BRCA1 / 2 mutation. It has been found that sporadic and familial breast cancers with BRCA mutation differ in the binding capacity of the alpha and / or beta thyroid receptor, which plays a crucial role in the diagnosis, prognosis and treatment of the disease.

According to the invention, the concentration of the thyroid hormone receptors alpha and / or beta in a sample of the breast cancer or ovarian cancer tissue is determined. For this purpose, a biopsy of the respective tissue is taken minimally invasive. This tissue is then further processed in a conventional manner for a receptor determination.

If the body tissue is removed, this must be fixed immediately, otherwise it comes through the body's own enzymes for autolysis. Fixation arrests the activity of the enzymes.

For fixation is usually used 4% neutral buffered formalin, which is an aqueous solution of formaldehyde. Formalin should always be stored in a dark and cool place, otherwise it oxidizes to formic acid. An example of fixation with formalin and the principle of embedding are described below.

Principle of fixation with formalin:

A cross-linking of the protein molecules occurs by attaching a formaldehyde molecule to a protein molecule. This forms a rigid grid. The proteins are almost not denatured.

This is important to subsequently detect antigenic structures.

The duration of fixation depends on the size of the tissue. Small pieces of tissue (eg Abradate) may be well fixed within 12 hours, large pieces of tissue (eg complete uterus) are first cut to size (relevant tissue samples should be taken from the organ.) These should not be cut thicker than 0.5 cm. The tissue samples are then encapsulated and fixed for 24 hours.

After fixation, it is important that the fixative is washed out well with tap water. Now follows the process of embedding.

Principle of embedding:

The fabric is soaked in a liquid that turns into a solid, solid mass (eg paraffin). The embedding agent deposits wherever there is water or can be removed.

For dewatering, an organic solvent having a high affinity for water is used. The fabric goes through a series of 70% and several absolute ethanols. As an intermediate (= liquid that mixes with both ethanol and paraffin) xylene acts. This washes the last remnants of the alcohol out of the tissue. Now the heated to 60 ° C and thus liquid paraffin penetrate. Then we cast the tissue with liquid paraffin in the form of a block.

After cooling the paraffin, the block is removed from the mold and cooled in the freezer compartment.

With the aid of the slide microtome 2-3 μm thick tissue sections are cut off the block and mounted on specially coated slides for immunohistochemistry ("Superfrost Plus").

The thyroid hormone receptors can be detected by any method known in the art. Preferably and simply they are detected with antibodies, preferably monoclonal antibodies. These antibodies are appropriately labeled to be detected. Such antibodies are well known in the art.

In addition to the immunohistochemical method for the determination of THR alpha and beta, the RT (real time) PCR or SSCP (single stranded conformational polymorphisms) examination can be used to determine the m-RNA ( Silva JM et al ). Furthermore, the Western blot analysis can be used.

Overexpression of the thyroid hormone receptors alpha and beta in the respective tissues can also be detected by means of a blood sample. In this case, an analysis of the corresponding mRNA or the methylation state of the respective genes is specifically detected.

Overexpression of thyroid hormone receptors and related receptors in BRCA1 / 2 mutant patients can also be achieved by mRNA analysis.

The tissue sample can also be taken from any other tissue in the body (eg skin).

The concentration of thyroid hormone receptors alpha and beta in breast or ovarian tissue is compared to a basal value known from healthy tissue. On the basis of the obtained values the following evaluation of the genetic predisposition results.

The hormone receptor detection is determined immunohistochemically according to Remmele and Stegner ( Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue, pathologist. 1987 May; 8 (3): 138-40 ) carried out. The immunohistochemical detection [ER-ICA, Pgr-ICA (Estrogen / Progesterone Receptor Immuno Cytochemical Assay)] allows the direct, visually visible and free and occupied receptors-including detection of the nuclear receptor protein by means of monoclonal antibodies. Independently of the chromogen used, positively reacting nuclei show a brown to dark brown staining, which can be characterized by a different color intensity as well as by a homogeneous and heterogeneous expression. Receptor-negative carcinomas show only shadowy pale nuclei. The cytoplasm of the tumor cells and the stroma surrounding the cells show no staining reaction. Depending on the color intensity and percentage of positive cell nuclei, the following immunoreactive scoring pattern is obtained: Immunoreactive Score (IRS): SI x PP

Interpretation:

• • 0-2: negative IRS
• • ≥ 3: positive IRS

• (Source: www.pathologie-vechta.de )

The score obtained for a patient is compared with a basic value known from the literature or a base value (based on healthy tissue) known from an earlier examination of the patient. Generally, any increase in score indicates an increased risk of the patient for breast cancer and / or ovarian cancer. Preferably, increasing the concentration of thyroid hormone receptors by 10%, more preferably 20%, most preferably 30%, indicates a correspondingly increased risk of breast or ovarian cancer. In particular, an IRS> 3 indicates an increased risk of breast cancer and / or ovarian cancer.

The method according to the invention for the diagnosis of a genetic predisposition is suitable both for male and for female subjects with regard to breast cancer, with regard to ovarian cancer only for female subjects. According to the invention, this method can predict the genetic predisposition of any human individuals. A preferred group of human individuals to use the method of genetic predisposition diagnosis of the present invention are individuals with a positive family analysis for breast cancer and / or individuals with contralateral breast cancer and / or individuals with carcinophobia.

The method according to the invention is also suitable for predicting the severity of the course of a primary breast carcinoma and / or primary ovarian carcinoma. In this case, sampling and determination of the concentration of the thyroid hormone receptors alpha and beta in the respective tissue take place as described above. Increased expression of THRα and / or THRβ compared to a basal value known for healthy tissue indicates a higher grade and an unfavorable course of the disease. The evaluation is carried out as described above.

Healthy tissue has an IRS score of zero.

Thus, the method according to the invention is not only suitable for predicting a genetic predisposition to the respective disease, but also allows prediction of the severity of the course with the possibility of a targeted therapeutic intervention even if the disease has occurred.

In general, the term increased risk for breast cancer or ovarian cancer means that the patient has a higher chance of developing breast cancer or ovarian cancer than the average population, which is comparable to the validity of the BRCA test. The term "breast cancer" or "ovarian cancer" refers to all histological subtypes of the particular cancer and includes, for example, carcinomas in situ, primary invasive cancer and metastatic cancer.

If there is an increased risk for the particular cancer, the method according to the invention is carried out once.

In one embodiment of the invention, further biomarkers are determined in addition to THRα and THRβ. Examples of biomarkers whose determination increases the significance of the THRα / β determination are vitamin D receptor, retinoid acid receptors, retinoid X receptors, peroxisome proliferation-activated receptors, liver X receptors and farnesoid receptors.

The additional biomarkers are preferably selected from vitamin A receptors, vitamin D receptors, prostaglandin-specific receptors and other THR receptors. At least one of these biomarkers can be determined in addition to THRα and THRβ.

These are all factors of the nuclear receptor family: vitamin D receptor, renin acid receptor, retinoid X receptor, peroxisome proliferation-activated receptor, liver X receptor and farnesoid receptor. These receptor families can be determined individually or in combination.

The determination of these biomarkers is known per se and is carried out according to methods known in the art. Examples of biological samples in which these biomarkers can be determined are breast or ovarian tissue and blood.

The determination of these biomarkers in other body tissues are also possible.

The invention also relates to a kit comprising a detectable binding molecule directed against the thyroid hormone receptor alpha / beta, reagents and / or buffers for the qualitative and / or quantitative determination of the alpha / beta-binding molecule complex and instructions for carrying out and evaluating the determination method for use in the diagnosis of a genetic predisposition to breast cancer (breast cancer) and / or ovarian cancer (ovarian carcinoma) of a human subject and to use for predicting the severity of the course of a primary breast carcinoma and / or a primary ovarian carcinoma of a human subject. Appropriate kits are known in the art. In a further embodiment, the kit additionally contains reagents and / or buffers for the determination of further biomarkers. These further biomarkers are preferably vitamin A receptors, vitamin D receptors, prostaglandin-specific receptors and other THR receptors.

The finding according to the invention that a genetic predisposition to breast cancer and / or ovarian cancer is accompanied by an increased expression and thus concentration of the thyroid hormone receptors alpha and beta in the respective tissue permits, in contrast to a genetic test such as the BRCA test, the development of a targeted therapeutic concept for the prophylactic and, if appropriate, therapeutic treatment of the particular cancer.

In the presence of an elevated concentration of the thyroid hormone receptors alpha and / or beta, the administration of a therapeutically effective amount of a compound capable of modulating the expression or activity of the thyroid hormone receptor alpha and / or beta may increase the risk of breast cancer and / or ovarian cancer be lowered or the severity of the course of the disease can be lowered.

One therapy option is the administration of T3 (triiodothyronine), which is already established in the treatment of thyroid dysfunction. Here cell experiments are carried out in order to initiate a Phase 1 study with expected positive result. Duration and dosage will be adjusted here. In need of control are patients with previously normal thyroid function. This will be closely monitored blood. To the effect and NW continue:
Inhibitors of the THR receptors are also therapeutically suitable, for. B. THR antagonists such. B: 1-850, 1- (hexyphenyl) -2-propanes-1-ones.

In this regard, therapeutically active compounds are compounds which downregulate the expression of the thyroid hormone receptor alpha and / or beta at the genetic level or which directly reduce the activity of these receptors in the tissue.

The corresponding compound preferably acts specifically on the expression or activity of the thyroid hormone receptor alpha and / or beta in the mammary gland and / or in the ovarian tissue.

Since other organs have thyroid receptors for THR, effects on other organs should also be considered. Therefore, blood tests for TSH, T3 should be performed at regular intervals (eg every 3 months initially).

These compounds are administered in a therapeutically effective amount. By therapeutically effective amount is meant here that the compounds downregulate the expression or concentration of the thyroid hormone receptor alpha and / or beta in a physiologically acceptable range. The physiologically desirable expression of these receptors in other tissues should not be impaired.

T3 and T4 are dosed in the amounts usually to be administered in thyroid disorders. THR inhibitors are dosed depending on the nature of the drug and the severity of the disease. A usual dosage is in the range of 0.1-10 nmol / ml.

Since evidence has been provided that both receptors are significantly increased, the goal is to downregulate both receptors with the therapy. The main objective is to down-regulate the proliferation of mutant cells.

The above compounds are formulated in a manner known per se for administration according to the invention and depending on the route of administration. Exemplary administration routes are oral, topical, parenteral, cutaneous, subcutaneous, intravenous, intraarterial, intraperitoneal, rectal or inhalation.

Preferred route of administration is oral administration.

The invention will be explained in more detail with reference to the accompanying figures. Show it

1 : Risk distribution for hereditary mammary and ovarian cancer compared to sporadic forms

2 : Representation of a hereditary breast cancer with BRCA mutation (expression of THRα - clear staining of the nuclei)

3 : Representation of a sporadic breast carcinoma without BRCA mutation (no expression of THRα - no staining of the nuclei)

4 : Representation of a normal glandular mammary tissue with BRCA mutation (expression of THRα - clear staining of the nuclei)

5 : Representation of a normal glandular mammary tissue without BRCA mutation (no expression of THRα - no staining of the nuclei)

6 : Representation of a hereditary breast cancer with BRCA mutation (expression of THRβ - clear staining of the nuclei)

7 : Representation of a sporadic breast cancer without BRCA mutation (no expression of THRβ - no staining of the nuclei)

8th : Representation of a normal glandular mammary tissue with BRCA mutation (expression of THRβ - clear staining of the nuclei)

9 : Representation of a normal glandular mammary tissue without BRCA mutation (no expression of THRβ - no staining of the nuclei)

The following examples illustrate the subject matter of the invention in more detail.

Example 1:

There were a total of 247 breast carcinoma sections (65 of patients with mutation and 182 with sporadic breast cancer) and 6 preparations with healthy breast cancer

Experimental procedure:

Tumor blocks or sections already prepared by external pathologies on slides of patients with histologically proven breast cancer from several pathological institutes from the whole of Germany were requested and processed. The tumor blocks were examined by patients with sporadic as well as by patients with proven hereditary breast cancer. They were all blocks of tumor from patients who consented to this examination. Placental tissue was used as a positive control.

The patient data was pseudonymized into an SPSS file containing the existing breast cancer clinical data - TNM status, grading, hormone receptor status (estrogen and progesterone receptor status), Her-2 / neu status, and metastasis.

To examine the thyroid receptor status of the paraffin embedded 300 tumor blocks in the gynecological clinic or in the Institute of Pathology using the slide microtome several 2-3 m thick tissue sections were prepared and mounted on specially coated for immunohistochemistry slides ("Superfrost Plus"). The sections were dried overnight at about 56-58 ° C in the incubator. In order to be able to start the immunohistochemical staining, the paraffin was extracted from the sections with the aid of xylene. Then the xylene of a descending alcohol series (ethanol) was removed.

The primary antibodies used were the Rabbit Thyroid Receptor alpha 1 and 2 from Zytomed (Article No. 520-4074, LOT: 70918) and the detection system of Zytochem. Plus HRP polymer kit. The immunohistochemical staining was carried out at room temperature in a humid chamber according to the ABC method (avidin-biotin complex). This is based on the affinity of avidin to biotin. Since it is partly too unspecific Reactions in the use of avidin, was obtained by genetic engineering, a purer product. This product is called streptavidin and is isolated from the bacterium "Stretomyces avidinii".

For the ABC method, a bridge antibody that is biotinylated, that is labeled with biotin, was used. Biotin is a water-soluble vitamin that binds well to a bridging antibody and thus connects to the ABC complex. A multi-link antibody was used. It is a solution composed of bridging antibodies from various animal species (eg mouse, rabbit, etc.). The biotinylated bridge antibody binds to the ABC complex. An enzyme is linked to the complex - in this case the peroxidase. A color reaction occurs in which the enzyme peroxidase forms a colored end product with the substrate buffer H2O2 as catalyst and the respective chromogen (DAB).

Alternatively, the polymer method was used for the immunohistochemical reaction in some antibodies. This was followed by the rinsing with 96% and 70% alcohol and distilled Aqua, the unmasking by heat pretreatment with sodium citrate buffer at pH 6. The preparations were washed 2 times 2 minutes in PBS. The "blocking solution" (reagent-1) was dropped on the slides. After 5 minutes, the rinse was repeated in 2 times 2 minutes PBS. After tilting the solution, the primary antibody in the dilution 1: 200 was pipetted and incubated for 60 minutes at room temperature. After rinsing again, the "Post Block" (Reagent 2) followed for 20 minutes, and after rinsing in PBS again, the HRP-polymer binding (Reagent 3) for 30 minutes. This step was followed by substrate staining with DAB as chromogen (Dako, Order No. 3468). After renewed washing in H2O, the counterstaining with acid hemalaun according to Mayer was carried out and the preparations blued in tap water for 5 minutes. Thereafter, the slides were dehydrated in the ascending alcohol series, placed in xylene and covered by Eukitt.

• 1. 0–10%
• 2. 11–50%
• 3. 51–80%
• 4. > 80%

The preparations were evaluated according to color intensity and number of stained cells (percentage frequency). The intensity of "1 + (weak) over 2 + (strong) and 3 + (very strong colored)" was given. The percentage frequency was ranked 1-4:

• 1. 0-10%
• 2. 11-50%
• 3. 51-80%
• 4.> 80%

The IRS (immunohistochemical score) was determined from the multiplication of color intensity and percentage frequency of the stained cells. As a result, an immunoreactive Score value ( Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue, pathologist. 1987 May; 8 (3): 138-40 ) from 0-12.

Evaluation:

Breast carcinoma tumor blocks from a total of 247 patients were available for evaluation, and 6 women had undergone prophylactic mastectomy without previous breast cancer. These were preparations with normal breast tissue. This group included women with proven mutation and women with no genetic test for an empty family history, who decided to undergo this invasive procedure because of carcinophobia.

In 26% (65/247) of the tumors examined, there was a proven BRCA1 or -2 mutation; in 74% (182/247) of the patients no known family history was known, which is why no genetic test was offered in the latter. Therefore, these carcinomas were classified as sporadic cases.

The median age distribution of patients with sporadic breast cancer was 69 years, that of patients with an existing mutation a median of 48 years, which is self-explanatory due to the significantly younger age at onset of mutation.

The tumor stages (TNM classification) were similarly distributed in the 3 breast cancer groups, although there were differences in grading. In the group of sporadic cases, grades 2 and 3 were equally distributed with 46% each. In contrast, grade 3 was the most prevalent in the group of mutated carcinomas with almost 62%.

Differences in the number of stained specimens are due to the fact that in some cases only slides were supplied by the external pathologies, which in some cases were sufficient only for one staining reaction, or the preparations for the evaluation were of too poor quality, which is why the introduction of these preparations was omitted ,

For the evaluation of the THRα, 57 preparations of BRCA mutant patients and 143 preparations of patients with sporadic breast cancer were used. Breast carcinomas with evidence of a BRCA mutation showed a strong positive (brown) staining of the nuclei (s. 2 ) in contrast to carcinoma tissue of sporadic breast carcinomas (see p. 3 ). The IRS score averaged 2.0 (± 2.97 standard deviation) in the presence of a mutation, and in the sporadic cases the median was significantly lower (1.0 ± 2.13 standard deviation) (p = 0.001).

Even more significant significant differences (p <0.0001) were found in the comparison of mutant and sporadic cases in the case of THR β ( - ) to be shown. The median for the preparations with BRCA mutation (n = 56) was 6.0 (± 3.17 standard deviation) in contrast to the sporadic cases (n = 87) with median 1.0 (± 2.45 standard deviation). In normal glandular mammary tissue from women with a proven BRCA mutation, a strong positive staining of the cores of the epithelial cells of the milk ducts was also detected ( 4 ) in contrast to healthy mammary tissue of women who had undergone mastectomy for carcinophobia ( 5 ).

Rating:

In the study, significant differences in in vitro studies on the tumor tissue of sporadic breast and breast cancers with mutations in the BRCA1 or -2 gene were detected, d. H. there is a significant correlation of detection of THR α / β with the result of genetic testing for a BRCA1 / 2 mutation.

The number of prophylactically operated patients in this collective can be compared with that of all-German women, who are undergoing a prophylactic mastectomy due to a proven BRCA mutation or who undergo such a serious operation due to carcinophobia. On average, it amounts to 10% (most patients are already suffering contralaterally) ( Metcalfe KA, Narod SA, et al .: International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers. Int J Cancer. 122 (9): 2017-22, 2008 ). The patients studied were exclusively women who were able to undergo prophylactic surgery without having previously contracted contralaterally, so that the figure given in this collective is 2.4% lower than that reported in the literature. The age group of women with prophylactic surgery showed an as described in the literature ( Metcalfe KA, Narod SA, et al .: International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers. Int J Cancer. 122 (9): 2017-22, 2008 ; Metcalfe KA, Narod SA, et al .: Family history as a predictor of uptake of cancer preventive procedures by women with a BRCA1 and BRCA2 mutation. Clin Genet., May 2008, 73 (5): 474-9 ) typical shift to lower ages.

The individual breast cancer groups show typical distribution patterns of TNM stages and grading, which makes valid statements about the collective possible.

It turns out that the test according to the invention makes it possible to obtain at least just as reliable a statement as to the genetic predisposition to breast cancer and / or ovarian cancer and thus to the risk of one of these diseases, as with the BRCA1 / 2 test. In addition, the inventive method has the advantage that it is faster, easier and inexpensive to perform and provides results that can be directly translated into a prophylactic or therapeutic concept.

The test according to the invention is simpler and thus more widely applicable since it can be carried out in many / all pathologies and not only in specialized laboratories.

QUOTES INCLUDE IN THE DESCRIPTION

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Cited patent literature

• US 7056663 [0021]
• US 2003/0099974 [0021]

Cited non-patent literature

• Kwon JS, Daniels MS, Sun CC, Lu KH: Preventing future cancers by testing women with ovarian cancer for BRCA mutations; J Clin Oncol 2009 [0003]
• Cass I, Karlan B: Improved survival in women with BRCA-associated ovarian cancer. Cancer 2003; 97: 2187-2195 [0003]
• Garcia-Etienne CA, Barile M, Gentilini OD et al: Breast Conserving Surgery in BRCA1 / 2 Mutation Carriers: Are We Approaching an Answer? Ann Surg Oncol. 2009 [0003]
• Graeser MK, Engel Ch, Rhiem K et al: Contralateral breast cancer risk in BRCA1 and BRCA2 mutation carriers. J Clin Oncol 2009; 27: 1-6 [0003]
• Farmer H, McCabe N, Lord CJ et al .: Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 2005; 434 (14); 917-921 [0003]
• Rae MT, Gubbay O., Kostogiannou A. et al: Thyroid hormone signaling in human ovarian surface epithelial cells; J Clin Endocrinol and Metabol 2006: 92: 322-327 [0009]
• Biochemistry and Pathobiochemistry by Georg Löffler, Petro E. Petrides and Peter C. Heinrich von Springer, 2006 [0017]
• Conde I, Paniagua R, Zamora J et al .: Influence of thyroid hormone receptors on breast cancer cell proliferation; Annals of Oncology 17 (1): 60-64, 2006 [0018]
• Silva JM, Dominguez G, Gonzalez-Sancho JM et al .: Expression of thyroid hormone receptor / erbA genes is altered in human breast cancer, Oncogene 21 (27): 4307-4316, 2002 [0018]
• Silva et al. (2002, supra). [0020]
• Zhang et al. (Novel pathway for thyroid hormone receptor action by interaction with jun and fos oncogenic activities., Mol Cell Biol., December 1991, 11 (12): 6016-6025) [0021]
• Silva JM et al [0036]
• Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue, pathologist. 1987 May; 8 (3): 138-40 [0041]
• www.pathology-vechta.de [0041]
• Remmele W, Stegner HE. [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue, pathologist. 1987 May; 8 (3): 138-40 [0085]
• Metcalfe KA, Narod SA, et al .: International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers. Int J Cancer. 122 (9): 2017-22, 2008 [0094]
• Metcalfe KA, Narod SA, et al .: International variation in rates of uptake of preventive options in BRCA1 and BRCA2 mutation carriers. Int J Cancer. 122 (9): 2017-22, 2008 [0094]
• Metcalfe KA, Narod SA, et al .: Family history as a predictor of uptake of cancer preventive procedures by women with a BRCA1 and BRCA2 mutation. Clin Genet., May 2008, 73 (5): 474-9 [0094]

Claims (17)

Method for the diagnosis of a genetic predisposition to breast cancer (breast cancer) and / or ovarian cancer (ovarian carcinoma) of a human individual, characterized in that the concentration of the thyroid hormone receptors alpha and beta (THRα and THRβ) is determined in a sample of the mammary tissue and / or ovarian tissue of the individual determined, wherein an increased concentration of THRα and / or THRβ compared to healthy tissue indicating an increased risk of the individual for breast cancer and / or ovarian cancer. Method for predicting the severity of the progression of a primary breast carcinoma and / or a primary ovarian carcinoma of a human individual, characterized in that the concentration of the thyroid hormone receptors alpha and beta ((THRα and THRβ) is determined in a sample of the mammary tissue and / or ovarian tissue of the individual , wherein an increased concentration of THRα and / or THRβ as compared to healthy tissue indicates a higher grading and an unfavorable course of the disease. A method according to claim 1 or 2, characterized in that the human individual is selected from the group of individuals with a positive family history for breast cancer or ovarian cancer and / or individuals with a contralateral breast cancer / ovarian cancer and / or individuals with carcinophobia. Method according to one of claims 1 to 3, characterized in that one determines the concentration of the thyroid hormone receptors alpha and beta with anti-thyroid hormone receptor antibodies. Method according to one of claims 1, 3 or 4, characterized in that a positive immunoreactive score (preferably IRS greater than 3 to RS) of THRα and / or THRβ is associated with an increased risk of breast cancer and / or ovarian cancer. Method according to one of claims 2 to 4, characterized in that a positive immunoreactive score (preferably IRS greater than 3 to RS) of THRα and / or THRβ is associated with a higher grading and an unfavorable course of the disease. Method according to one of claims 1 to 6, characterized in that in addition at least one biomarker selected from vitamin A receptors, vitamin D receptors, prostaglandin-specific receptors and other THR receptors, determined in a biological sample of the individual , A detectable binding molecule directed against the thyroid hormone receptor alpha / beta for use in diagnosing a genetic predisposition to breast cancer (breast cancer) and / or ovarian cancer (ovarian carcinoma) of a human subject. A detectable binding molecule directed against the thyroid hormone receptor alpha / beta for use in predicting the severity of the progression of a primary breast carcinoma and / or a primary ovarian carcinoma of a human subject. Kit, comprising a) a detectable binding molecule directed against the thyroid hormone receptor alpha / beta, b) reagents and / or buffers for the qualitative and / or quantitative determination of the thyroid hormone receptor alpha / beta-binding molecule complex, and c) instructions for carrying out and evaluating the determination procedure, for use in diagnosing a genetic predisposition to breast cancer (mammary carcinoma) and / or ovarian cancer (ovarian carcinoma) of a human subject. Kit, comprising a) a detectable binding molecule directed against the thyroid hormone receptor alpha / beta, b) reagents and / or buffers for the qualitative and / or quantitative determination of the thyroid hormone receptor alpha / beta-binding molecule complex, and c) instructions for carrying out and evaluating the determination procedure, for use in predicting the severity of the course of a primary breast carcinoma and / or a primary ovarian carcinoma of a human subject. Kit for use according to claim 10 or 11, characterized in that it further comprises detectable binding molecules / reagents and / or buffers for the determination of further biomarkers. A therapeutically effective amount of a compound capable of modulating the expression or activity of the thyroid hormone receptor alpha and / or beta for use in the prophylaxis and / or treatment of breast cancer and / or ovarian cancer. Therapeutically effective amount of a compound for use according to claim 13, characterized in that the compound is capable of specifically modulating the expression or activity of the thyroid hormone receptor alpha and / or beta in breast tissue and / or in ovarian tissue. Therapeutically effective amount of a compound for use according to claim 13 or 14, characterized in that the compound is selected from thyroid hormones and THR inhibitors. Therapeutically effective amount of a compound for use according to any one of claims 13 to 15, characterized in that the breast cancer and / or ovarian cancer has been diagnosed or predicted by a method according to any one of claims 1 to 7. Therapeutically effective amount of a compound for use according to any one of claims 13 to 16, characterized in that the breast cancer and / or ovarian cancer is characterized by an increased concentration of THRα and / or THRβ in the respective tissue.

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