DE10136273A1 - Molecular markers in hepatocellular carcinoma - Google Patents

Abstract

The invention relates to molecular markers occurring for hepatocellular carcinoma. The invention more particularly comprises gene sequences or peptides coded thereby which can be regulated upwards or downwards for hepatic cell carcinoma (HCC) in relation to healthy, normal liver cells in the expression thereof. The invention also relates to the use of said sequences in the diagnosis and/or therapy of HCC and for screening purposes in order to identify novel active ingredients for HCC. The invention also relates to an HCC specific cluster as a unique diagnostic agent for HCC.

Description

The present invention relates to molecular markers used in hepatocellular carcinoma occur. It includes in particular Gene sequences or peptides encoded therefrom which are involved in liver cell carcinoma (HCC) compared to healthy, normal liver cells in the expression or are down-regulated, and the use of these sequences for Diagnosis and / or therapy of HCC as well as screening for Identification of new active substances for HCC. The invention further relates to an HCC-specific cluster as a unique diagnostic tool for HCC.

Primary liver cell carcinoma (HCC) (see e.g. M. Sterneck, etiology of the hepatocellular carcinoma, Der Internist, 41 (2000) 185-190; K. Okuda, Heptacellular carcinoma, J. Hepatology 32 (suppl. 1) (2000) 225-237) is in fifth place worldwide among all malignant tumors. 1990 the incidence of this tumor was around 500,000 cases worldwide estimated. The HCC occurrence continues to increase in many countries strong, especially in Japan and in important countries like the USA (H. El- Serag, The New England Journal of Medicine, 340 (10) (1999) 745-750) and in Europe, especially in Southern Europe. The increase is major due to the increasing spread of the main causes for HCC - the viral hepatitis B and C - caused.

So far, the molecular mechanisms of hepatocarcinogenesis are only incompletely understood. Genetic changes are true have been described, but their relevance to date despite numerous Examinations cannot be classified exactly.

Early detection of an HCC is critical to that Survival of affected patients. The 5-year survival rate at this tumor is only 2% worldwide. The forecast of the primary Liver cell carcinoma is directly related to its size and number of tumor nodes Diagnosis dependent. The poor survival rate and the late Diagnoses are mainly based on the relatively unspecific diagnostic possibilities that are currently available and the often attributed to late tumor detection.

The previous diagnosis is mainly based on three methods (see e.g. C. Ziske et al., Diagnosis and therapy of hepatocellular carcinoma, Fernkolleg: Practical hepatology, episode 10; K. P. Maier, hepatitis Consequences of hepatitis, chap. 14.6 "Hepatocellular Carcinoma (HCC)", Thieme Verlag, pp. 339-363; A. Aguayo et al., "Liver Cancer" in Current and Future Treatment Therapies for Liver Disease, Clinics in Liver Disease, Vol. 5 (2), 2001, pp. 479-502).

1. Serology

The serological determination is carried out with alpha-fetoprotein (AFP), one in 30% of all liver cancers were overexpressed and secreted into the blood Protein. However, two thirds of the HCCs do not produce or show AFP no increased AFP values. The AFP marker is therefore an efficient marker for early detection. Newer approaches such as enzyme tests with Des-γ-carboxy Prothrombin (PIVKA II), have brought no decisive advantage and cannot establish themselves.

2. Imaging procedures

Above all, ultrasound and Computed tomography available. However, this diagnosis is relative unspecific and only leads to advanced stages of the tumor visible and thus trend-setting changes.

3. Histology

There is the possibility of invasive diagnostics with the acquisition of a Tissue sample using fine needle aspiration of the liver for histological Analysis. Neoplastic changes in the liver are often not clearly recognizable and sometimes compared to other pathological Changes such as regenerated nodes in cirrhosis or liver cell adenomas very difficult or impossible to define. Furthermore, the assessment of histological specimens depends on the examiner and is therefore subject to a clear variability.

All diagnostic procedures show clear shortcomings and are for that joint recognition of late detection of liver cell carcinoma. So there is from the medical-clinical side, there is a clear need for tumor markers with very high sensitivity and specificity for HCC.

Parallel to the problematic situation in diagnostics are also so far Known therapies of HCC largely ineffective (see e.g. A. Grothey, Systemic therapy of hepatocellular carcinoma, The Oncologist, 6 (2000) 327-335). The classic methods of chemoradiation show no effect on HCC. Invasive procedures like that Chemoembolization, which is ethanol injection or radio frequency ablation complex and rarely lead to healing of this tumor. Surgical interventions such as partial liver resections show in particular better 5-year survival rates for small tumors, however, the only definitive curative therapy is liver transplantation. Not least due to the lack of donor organs, this can be therapeutic Option to be used very rarely for the therapy of tumors.

That is why new and innovative treatments for HCC are urgently needed Approaches necessary. There are now intense ones Research activities in the field of gene therapy, this development but is progressing very slowly and will become clinical in the near future are not available. New molecular approaches can do this Driving research area decisively and thus gene therapy and make tumor immunological approaches clinically feasible.

Cancer is known to be a process in which very many and different genetic changes take place and interact with each other. This shows z. B. in the activation or Over-expression of oncogenes and inactivation or complete loss of tumor suppressor genes in the corresponding tumor cells. numerous Studies have shown changes in oncogenes and anti Oncogenes as well as the loss of heterozygosity of chromosomes in HCC show, but so far is not a coherent pattern of such genetic Changes in HCC have been defined.

An object of the present invention was therefore to create new ones To provide tumor markers for HCC, as well as new opportunities for Show therapy of HCC.

This object is achieved by the use at least one nucleic acid shown (i) in Table 1 or in Table 2 (ii) a sequence from (i) in the context of the degeneration of the genetic Corresponds to codes, (iii) a partial sequence of a sequence from (i) or / and (ii) with a length of at least 25, preferably at least 50 and more preferably has at least 100 nucleotides, (iv) with a sequence (i), (ii) or / and (iii) hybridized under stringent conditions or / and (v) a sequence complementary to a sequence from (i), (ii), (iii) or / and (iv) or a polypeptide encoded by such a nucleic acid as a target for hepatocellular carcinoma (HCC).

In the context of the present invention, a new method of modern genome research used to over- and under-expressed genes in liver carcinomas.

With the help of this recently established DNA chip technology (see e.g. P. Brown et al., Nature Genetics Supplement, 21 (1999) 33-37; K. Cole et al., Nature Genetics Supplement, 21 (1999) 38-41; R. Lipshulz et al., Nature Genetics Supplement, 21 (1999) 20-24) has managed several thousand Identify genes in liver tumors of different patients were significantly over- or under-expressed. By the subsequent Software analysis allowed groups of several hundred genes determine the consistent, d. H. in all (100%) or most (80% or 60%) of the liver carcinomas are over- or underexpressed (see also Tables 1 and 2). All of these with the help of Gene chip analysis will identify genes in the presence of an HCC more and more consistently overexpressed or underexpressed than that currently clinically available and routinely used AFP Marker.

Therefore, those identified within the scope of the present invention Genes are effective targets for HCC. Those are particularly preferred Genes of Table 1A or / and the genes of Table 2A used at which have an increase or decrease in expression in 100% of the examined patients has been identified. For many applications however, it is already sufficient to have at least one gene from Table 1C or / and 2C, i.e. genes in which an increased or reduced expression in at least 60% of the examined patients has been determined. The use of the is somewhat more preferred Genes shown in Tables 1B or / and 2B, where: here genes are involved in at least 80% of the patients examined were up or down regulated.

According to the invention, genes are preferably used which, compared to healthy, non-cancerous liver cells by a factor of at least 1.5 are down-regulated or up-regulated, more preferably by a factor of at least 2, more preferably by a factor of at least 3 and most preferably by a factor of at least 4.

While already by using a single one, in the present Invention identified gene related to HCC improvements can be obtained, further advantageous effects by Use of several of the genes found according to the invention, especially at least 10, more preferably at least 50 still more preferably from at least 100 and most preferably from at least 150 genes can be achieved. Are preferred according to the invention the genes found are grouped together for HCC are specific and clearly distinguish HCC from others Allow cells, also other pathologically modified cells.

With these identified groups of genes, unique ones can be identified Identifiers for discrimination between different Identify (liver) tumors. For example, an HCC specific pattern for the diagnosis of HCC positive tissue samples against other tissue patterns of the liver.

In a particularly preferred embodiment, 150 genes used with which a particularly good differentiation can be achieved, namely genes 1 to 46 in Table 1A, genes 47 to 75 in Table 1B and genes Nos. 1 to 28 in Table 2A and genes No. 36 to 84 in Table 2B. In this sentence, 75 are regulated and 75 downregulated genes were used, whereby a 100% differentiation between tumor liver tissue and non-malignant Liver tissue is possible. In a further preferred embodiment are all listed in Tables 1A, 1B, 1C and 2A, 2B and 2C Genes used.

Additional gene groups or Gene clusters are put together to provide specific analytical Solve problems. This way, for example, clusters to discriminate against various tumors of the liver, e.g. B. benign-malignant (e.g. liver adenoma-liver carcinoma) or malignant-malignant (liver carcinoma- Colon cancer metastases) or clusters for differentiating Stages of individual tumors are created, whereby the genes from the Tables 1A to C and 2A to C are used, in which a particularly significant or particularly consistent change observed can be. These differentiations are currently with the Available methods are often not or only very difficult accomplish.

The presence of a consistent pattern of genetic changes at the HCC, as has been determined according to the invention in principle the possibility of different stages and causes of the HCC in To be examined for certain genetic patterns. So here z. B. an indication of the likelihood of developing a malignant Tumor can be given from previously damaged tissue. Such one Pattern also offers the possibility of histological Differentiation between tumor cells and non-malignant transformed Clearly differentiate cells. So one can largely diagnosis independent of the investigator can be made (GenCluster analysis - Genome pathology, gene profile pathology). Furthermore, persistent overexpressed genes (or the proteins they encode) as much more specific and - if they are in the early stages of Tumors emerge - more sensitive tumor markers than previously known and applied act. The selected genes can also can be used as a coupling point for potential therapeutic targets.

From the genes shown in the tables, fetal genes are expressed as Tumor markers of particular interest. Are suitable for therapeutic approaches especially genes that code for receptors as well as oncogenes as well genes involved in the cell cycle (cdc) and signal transduction elements.

If the gene products (proteins) or part of them are secreted into the blood, there is enormous potential for a simple detection method for the emergence of an HCC. With the help of this marker or this Detection methods can for example be in the form of a simple one Serum blood tests for patients at risk of developing HCC without any problems, be tested safely and quickly. As a result, a positive Detection test then another specific to the result of this test coordinated diagnostics and / or therapy can be carried out. A Detection of tumor-specific cell surface proteins is also the same possible.

The invention thus encompasses gene sequences as diagnostic Detection methods and as potential therapeutic targets in the Connection with an HCC as well as an HCC-specific cluster z. B. 150 genes (indicator genes - genome pathology) as unique diagnostic instrument.

The economic benefit arises on the one hand z. B. in the use of Expression pattern as a detection method for the molecular Tumor identification (specific serum markers, specific gene clusters, immunohistochemical markers), on the other hand, those genes which are considered suitable for therapeutic approaches for molecular therapies such as Gene therapy and / or tumor vaccination can be viewed accordingly checked and used for therapy development.

The genes identified by gene chip analysis are in Tables 1 and 2 shown. The database access numbers in the tables are Genes and the name of the genes or the code encoded by them Polypeptide or EST number specified.

Table 1A shows genes compared in a human primary HCC with non-cancerous liver cells upregulated, in 100% of the examined patients by at least 1.5 times. Likewise the extent of the up-regulation is indicated.

Table 1B shows genes compared to human primary HCC non cancerous liver cells, in 80% of the examined patients were upregulated and

Table 1C shows genes compared to non in human primary HCC cancerous liver cells, upregulated in 60% of the cells examined were.

Table 2A shows genes that were found in 100% of the patients examined Are downregulated compared to normal liver tissue.

Table 2B shows genes compared to human primary HCC non-cancerous liver cells in at least 80% of patients Are down regulated 1.5 times.

Table 2C shows genes compared to human primary HCC non-cancerous liver cells, down-regulated in 60% of patients.

The genes given in Tables 1 and 2 are each at least 1.5 times up or down regulated.

All 529 genes shown in Tables 1 and 2 meet more stringent ones Criteria as AFP and IGFII and are clear to the known markers think.

The invention is further illustrated by the following examples and figures.

The gene expression analyzes are a selection of the significantly over- (red tones) and under-expressed (green tones) genes (vertical applied) from the respective tissue samples (shown horizontally). The Colors illustrate the significant differences in the activities of the Genes. Not only tumor tissue of "normal" can be impressively Liver tissue can be distinguished, but also a clear one Differentiation between benign liver changes (adenomas) or Metastases of colon cancer and hepatocellular carcinoma (Tumor = HCC).

Fig. 1 shows a Genclusteranalyse a Leberzelladenoms compared to HCC. Each track in the figure shows a tissue sample, the individual color pixels per track each a gene or a hybridization signal. HCC tumor tissue (T) and adenoma tissue (A) are plotted in the individual tracks. A red color pixel means that the corresponding gene in the sample is overexpressed, a green color pixel that the corresponding gene is underexpressed. A subgroup of genes from the whole collective was used, which allowed the strongest differential differentiation between the two entities "liver cell adenoma - benign neoplasm" and "HCC = malignant neoplasm".

Based on the analysis, 100% differentiation is possible (see right page). The aim of the investigation was to obtain the clearest possible differentiation between the two entities in the simplest possible way. From Fig. 1 it can be clearly seen that a differentiation of the different cell types is possible, even for a layperson or untrained staff.

FIG. 2 shows the subclassification of various masses of the liver by means of two-dimensional cluster analysis. In particular, FIG. 2 shows an illustration of the principle of the methodology of the two-dimensional cluster analysis. The gene clusters of 13 masses were created.

As can be seen, the cluster analysis can differentiate the clusters and the Represent the intensity of the relationships between the clusters.

Fig. 3 shows a Genclusteranalyse of AFP positive and negative AFP HCC tissue samples. In particular, FIG. 3 shows a differentiation between two different forms of the HCC, namely the AFP positive (lane 2, 3 in the upper cluster) and the AFP negative (lane 2, 3 in the lower cluster) HCC. As can be seen, in addition to the AFP, several other genes are co-regulated. The aim of the study was to show that other genes correlate with AFP and which genes they are (e.g. genes that are largely fetal overexpressed).

Fig. 4 shows a differentiation between non-viral-induced, and HCV induced HCC. In particular, FIG. 4 shows a differentiation between two different forms of HCC, namely the virally induced (each lane 2 , 5 ) from the non-virally induced (each lane 1 , 3 , 4 , 6 ) HCC. As can be seen, specific gene clusters are overexpressed in the virally induced HCC. The aim of the study was to show that specific gene groups are overexpressed in virally induced HCC compared to non-virally induced HCC.

Fig. 5 shows a two-dimensional cluster analysis of HCC by way of illustration of the 700 most differentially expressed genes and ESTs which are at least just above or underexpressed.

In particular, Fig. 5 shows the principle of two-dimensional cluster analysis: The aim of the cluster analysis is the fact that it is possible with the methodology to identify a cluster with a certain amount of genes that clearly differentiates it from other entities of the same tissue type (here: Liver tissue) allowed. The exact number of genes recorded is irrelevant, since even slight differences in a large number of genes allow such differentiation, which is not possible with individual analyzes of genes using conventional methods. The 529 genes shown in Table 1-2 are included in the cluster analysis, however the cluster analysis is not limited to this. The principle of the cluster is independent of the expression level of individual genes, but is primarily based on the co-regulation of gene groups.

FIG. 6 shows a gene cluster analysis of colon metastases in comparison to HCC tissue (in the case of colon cancer for adenoma).

FIG. 7 shows a gene cluster analysis of HCV versus HBV-induced HCC (in HBV and HCV idem for the adenoma).

example 1 Determination of genes raised in human primary HCC or are down regulated

A chip with 7000 human genes was used, the Selection by specific hybridization with highly purified Liver cancer tissue (RNA) was done. It was high stringency, high Robustness and high consistency as well as specificity are respected. Through the The digital preparation of the data is primarily a cluster analysis with high Specificity possible. This is the basis of the final selection (by stringent selection criteria) of individual genes based on several thousand genes examined.

Example 2

Specificity of the genes

This example shows the much higher specificity and sensitivity of two genes from Tables 1 and 2 compared to known ones Markers currently used in the clinic for the detection of a hepatocellular carcinoma (HCC). In table A is the known one AFP marker shown, which is high in some HCC tumors other is down-regulated. This corresponds to clinical experience, after which the AFP marker is overexpressed in approximately 1/3 of all HCCs and thus does not have a high sensitivity. The well-known marker IGF II (insulin growth factor II) shows a comparably low sensitivity.

In comparison, the two genes RAIF (M94250; No. 3 in Table 1A) and HMG (L17131; No. 7 in Table 1A) provided according to the invention are clearly overexpressed (at least three times) in each of the HCCs. Therefore, these genes as potential markers are clearly superior to known markers and can also be used as therapeutic targets. Table A

Claims (13)

1. Use at least one nucleic acid which a) is shown in Table 1 or in Table 2, b) corresponds to a sequence from (i) in the context of the degeneration of the genetic code, c) has a partial sequence of a sequence from (i) or / and (ii) with a length of at least 25, preferably at least 50 and more preferably at least 100 nucleotides, d) hybridized with a sequence from (i), (ii) or / and (iii) under stringent conditions or / and e) has a sequence complementary to a sequence from (i), (ii), (iii) or / and (iv), or a polypeptide encoded by such a nucleic acid as a target for hepatocellular carcinoma (HCC). 2. Use according to claim 1 for the diagnosis of HCC. 3. Use according to claim 1 for the therapy of HCC. 4. Use according to claim 1 in a screening process for Identification of new active substances for HCC. 5. Use according to one of the preceding claims, characterized, that at least 10 nucleic acids are used. 6. Use according to one of the preceding claims,
characterized,
that at least 10 targets selected from nucleic acids that a) are shown in Table 1, b) correspond to a sequence from (i) in the context of the degeneration of the genetic code, c) have partial sequences thereof with a length of at least 25, preferably at least 50 and more preferably at least 100 nucleotides, d) hybridize with a sequence from (i), (ii) or / and (iii) under stringent conditions or / and e) have a sequence complementary to a sequence from (i), (ii), (iii) or / and (iv) and thereof encoded polypeptides and at least 10 targets selected from nucleic acids that a) are shown in Table 2, b) correspond to a sequence from (i) in the context of the degeneration of the genetic code, c) have partial sequences thereof with a length of at least 25, preferably at least 50 and more preferably at least 100 nucleotides, d) hybridize with a sequence from (i), (ii) or / and (iii) under stringent conditions or / and e) have a sequence complementary to a sequence from (i), (ii), (iii) or / and (iv) and polypeptides encoded thereof are used. 7. Use according to claim 6, characterized, that an HCC-specific pattern consisting of genes No. 1-46 Table 1A, No. 47-75 of Table 1B, No. 1-28 of the Table 2A and No. 36-84 of Table 2A is used. 8. Procedure for the diagnosis of HCC, characterized, that the amount of at least one in a sample Nucleic acid shown in Table 1 or Table 2 determined. 9. The method according to claim 8, characterized, that you have the amount of at least 10 nucleic acids listed in Table 1 or shown in Table 2. 10. Procedure for the therapy of HCC, characterized, that the amount of at least one nucleic acid contained in Table 1 or shown in Table 2 are affected. 11. The method according to claim 10, characterized, that one influences a nucleic acid selected from genes for Receptors, oncogenes, genes involved in the cell cycle (cdc) as well as signal transduction elements. 12. HCC-specific cluster comprising genes 1-46 of the table 1A, No. 47-75 of Table 1B, No. 1-28 of Table 2A and No. 36-84 of Table 2A. 13. Expression profile, especially for use as a diagnostic Means associated with HCC, characterized, that it is genes 1-46 of Table 1A by at least that Increased 1.5 times and genes 1-28 of Table 2A by at least 1.5 times reduced compared to normal liver cells having.