DE10337368A1 - Diagnosis of pancreatic cancer by detecting expression of the a disintegrin and metalloprotease domain 9 (ADAM9) protein using specific antibodies, also therapeutic use of these antibodies and of ADAM9-specific nucleic acids - Google Patents

Abstract

Method for diagnosing pancreatic tumors by determining expression of the ADAM9 protein (a disintegrin and metalloprotease domain 9). Isolated cells or tissue sections of pancreas are incubated with ADAM9-specific antibodies (Ab) then examined under a microscope for binding of Ab. Independent claims are also included for the following: (1) Ab, for diagnosis of pancreatic tumors, that are bound to a marker, preferably a radioisotope such as technetium-99, that enables representation by tomographic or scintigraphic methods; (2) Ab, bound to a cell-killing agent, for treating pancreatic tumors; (3) use of nucleic acid (I), or its derivatives, specific for the ADAM9 gene, or its RNA, for treating pancreatic tumors; and (4) nucleic acid sequences (II), or their derivatives, having any of the 26 fully defined sequences of 20-23 nucleotides in length (SEQ ID Nos: 1-26) as given in the specification. An example of the sequences (SEQ ID Nos: 1-3) are as follows: GTTCCTGTGG AGCAAAGAGC CCAGCGTCCA CCAACTTATT GCATTTGTGG GAACAGTGTG ACTIVITY : Cytostatic. No details of tests for cytostatic activity are given. MECHANISM OF ACTION : Gene therapy; Immunotoxin; Radioimmunotherapeutic.

Description

The The invention relates to methods and means for diagnosis and treatment of pancreatic tumors, especially for the differential prognosis of the Ductal adenocarcinoma of the pancreas (PDAC). Field of application of Invention is medicine, especially radiology and oncology. PDAC is the most common Tumor of the pancreas (Pancreas) and the 5th most common Cause for Cancer death in the USA. Patients with this tumor have very poor chances of survival.

The Histological differential diagnosis of pancreatic tumors is often difficult. There are several applied in the pathological routine antibody (eg against cytokeratin 18), but with which not always one Forecast for disease progression possible is. In the investigation of settlements (metastases) of different Carcinomas of the pancreas, biliary tract and gallbladder, such as The Papilla vateri is usually the histological mapping to the primary tumor not possible. An assignment of these metastases to the primary tumor is to choose an adapted important for palliative chemotherapy.

For early detection, the laboratory chemical differential diagnosis of pancreatic tumors to other pancreatic diseases and the follow-up after surgery, there is only one Marker, the CA19-9. This tumor marker to be determined in the blood is but not very specific and sensitive, i. there are patients with chronic pancreatitis (pancreatitis), which is a strong increased Ca19-9 Have value and patients with normal Ca19-9 who have a pancreatic tumor to have.

The Treatment of ductal pancreatic cancer is currently on surgery and chemotherapy limited. The surgical removal of the tumor offers the only option a chance of recovery. Chemotherapy with the most commonly used chemotherapy only offers in a few make a life improvement, but no cure.

task The invention therefore provides means and methods for diagnosis and treatment of pancreatic tumors, especially for the differential prognosis of the Ductal adenocarcinoma of the pancreas (PDAC).

According to the invention, the Task solved by a method of diagnosing tumors of the pancreas, in particular for the diagnosis of ductal adenocarcinoma of the pancreas (PDAC), at in pancreatic tissue or cell samples, ADAM9 protein expression is determined. These are isolated pancreas cells or tissue sections with ADAM9-specific antibodies incubated and examined under a microscope for binding of the ADAM9-specific antibody analyzed.

The Evaluation of the ADAM9 binding under the microscope is preferably carried out with automated image recognition.

ADAM9 represents a membrane protein that plays a role in proteolytic Process membrane-bound precursor molecules and modulate cell-cell and cell-matrix interactions (Nath D et al., J. Cell Sci. 2000 Jun; 113: pp. 2319-2328). The name ADAM9 derives from that ADAM9 contains a disintegrin and a metalloprotease domain ("A DISINTEGRIN AND METALOPROTEINASE DOMAIN 9 "). The protein sequence of ADAM9, according to SEQ ID NO. 27, is known (Sequence entry NP_003807.1, Genbank, NCBI, Bethesda, USA).

Surprised it was found that in 98.3% of ductal pancreatic carcinomas (PDAC) the ADAM9 protein overexpressed becomes. This makes ADAM9 suitable as a target for the diagnosis of PDAC.

there was still surprising found that in different tumors the ADAM9 protein in different cellular Compartments of pancreatic tissue is expressed.

Microscopic may be between an ADAM9 protein expression on the cell membrane and in the cytoplasm. When expressed on the Cell membrane can further express on the apical and the basolateral side of the pancreatic cells are differentiated.

Nearly all PDACs tested show apical ADAM9 protein expression. Surprisingly, it was found that about 50% of the PDACs additionally contain the ADAM9 protein at the basolateral membrane and approx 30% additional expressed in the cytoplasm.

Next surprisingly was found to be patients with either a cytoplasmic or basolateral ADAM9 protein expression in pancreatic tissue much shorter mean survival time as patients have no or exclusively apical Show ADAM9 protein expression in the pancreas.

By the inventive method it will be based on the localization of ADAM9 protein expression in the tumor patients with PDAC in at least two groups (group 1: none or exclusively apical ADAM9 protein expression; Group 2: cytoplasmic or basolateral ADAM9 protein expression). By the method according to the invention it becomes possible for this Groups a different prognosis about the clinical course of the PDAC, an estimate the survival time to create. About that can out for this Groups adapted treatment schemes are made.

In a particularly preferred embodiment the inventive method Therefore, the localization is analyzed in the analysis of ADAM9 protein expression of the ADAM9 protein in the cell is determined and distinguished is between apical, basolateral and cytoplasmic expression of the ADAM9 protein.

Preferably is as ADAM9-specific antibody a monoclonal or a polyclonal antibody selected.

For the purposes of this description, the term antibody includes monoclonal and polyclonal sera, diabodies, antibody fragments such as Fab and (Fab ') ₂ , single-chain (SCFv single-chain) fragments.

Of the ADAM9-specific antibodies recognizes an epitope from the ADAM9 protein sequence according to SEQ ID NO. 27, preferably an extracellular domain of the ADAM9 protein.

Preferably is called ADAM9-specific antibody an antibody selected from the following group: monoclonal Mouse anti-human ADAM9 (AF949, R & D Systems, Wiesbaden or 610750 Becton & Dickinson, Heidelberg), polyclonal Rabbit anti-human ADAM9 (CL2ADAM9, Cedarlane, Ontario, Canada or SA-376, Biomol, Hamburg), polyclonal goat antiHumanADAM9 (PC669, Novabiochem, Laufelfingen, Switzerland) or polyclonal goat anti-mouse ADAM9 (AF939, R & D Systems, Wiesbaden). The polyclonal goat anti-mouse ADAM9 AF939 also recognizes the humane ADAM9.

In a particularly preferred variant of the method according to the invention the detection of the ADAM9-specific antibody is indirect. For this purpose, the tissue sample to be examined first with an unmarked ADAM9-specific antibody, such as B. a human ADAM9 cross-reactive goat anti-mouse ADAM9 antibody (R & D Systems, Wiesbaden, Catalog No. AF949).

The bind this primary antibody is incubated with a species-specific secondary antibody (e.g. A biotinylated anti-goat IgG antibody - available as a ready-to-use reagent system at Vector Laboratories). Because in this second step to a primary Several antibodies secondary antibody bind, the signal is beneficial by incubating with the amplified secondary antibody. A further signal amplification is preferably done in a third step by incubating with tertiary antibodies or streptavidin / avidin.

At the antibody or antibody used in the last incubation step Streptavidin / avidin bound a marker that allows the Localization of antibodies to detect.

In In a preferred variant, this marker is an enzyme, especially preferably a luciferase, a peroxidase, such as. As the peroxidase horseradish (horse radish), or an alkaline phosphatase. peroxidases and phosphatases in a known manner chromogenic or fluorogenic substrates to dyes or to convert fluorescent dyes. These dyes will be thus locally, in the places at the originally the ADAM9-specific antibody have bound, formed and enable a detection with known microscopic methods.

In In an alternative variant, the marker is detectable in the microscope Fluorescent dye, such as. As fluorescein, or even a fluorescent Protein, such as B. the "Green Fluorescent protein "(GFP).

In Another variant of the method according to the invention is the marker directly to the primary ADAM9-specific antibody bound. In this variant, the detection of the ADAM9-specific antibody is carried out directly by the detection of the labeled antibody.

Around preferably To examine a few samples microscopically is in a preferred Variant of the method according to the invention before analysis for ADAM9 protein expression carried out a preliminary analysis in which, in tissue or cell samples of the pancreas, ADAM9 gene expression via ADAM9 mRNA expression (messenger RNA) is analyzed.

To This preliminary analysis will only include the tissue or cell samples in those ADAM9 mRNA expression could be detected as before described for ADAM9 protein expression.

The Analysis of ADAM9 mRNA expression in tissue or cell samples is preferably carried out by means of the common methods of RT-PCR and / or microarray analysis.

For the analysis by RT-PCR (reverse transcriptase polymerase chain reaction) For this purpose, it is included in the RNA present in the tissue or cell samples Non-specific primers in cDNA (complementary DNA) rewritten. there If, in the samples, an ADAM9 mRNA is also present corresponding ADAM9 cDNA was formed.

These ADAM9 cDNA is used in a subsequent PCR (polymerase chain reaction) with ADAM9 specific primers (i.e., oligonucleotides specific hybridize with the ADAM9 cDNA). The proof of this specifically duplicated ADAM9 cDNA is made by using specific probes the PCR (TagMan), or by Southern blot.

For microarray analysis is the present in the tissue or cell samples RNA, or a through a cDNA obtained by RT-PCR, incubated with microarrays, to the oligonucleotides bound specifically to hybridize with the ADAM9 cDNA. The detection of this specifically bound ADAM9 RNA or cDNA takes place by incorporation of fluorescent dyes or radioactive markers into the RNA or cDNA.

Preferably are used to analyze ADAM9 mRNA expression (by RT-PCR or Microarray Analysis Oligonucleotides of SEQ ID NO. 1 to SEQ ID NO. 11 used. These oligonucleotides, preferably DNA oligonucleotides, specifically hybridize with ADAM9 RNA and ADAM9 cDNA and therefore can as a primer for the PCR and / or probes (eg as TagMan probes or Southern blot) or used to make a microarray.

One The invention also relates to the use of ADAM9 specific antibodies for the diagnosis and treatment of pancreatic tumors.

Preferably, humanized or human monoclonal antibodies (mAbs) are used for this purpose. In particular, in a repeated treatment, to avoid an unwanted immune response, antibodies are used whose invariable areas are deleted, for. F (ab ') ₂ fragments or scFv fragments or humanized, humanized mAbs.

The production of these F (ab ') ₂ fragments or scFv fragments or humanized mAbs is carried out by conventional methods (Roland Kontermann, Stefan Dübel, Antibody Engineering (Springer Lab Manuals) Springer Verlag Berlin).

Next the method according to the invention for the diagnosis of pancreatic tumors on isolated cells or tissue of the pancreas, the ADAM9-specific antibodies according to the invention also to Diagnosis of pancreatic tumors, especially PDAC, in vivo, d. H. in the human body uses.

To is preferably a marker on the ADAM9-specific antibody, preferably a radioactive isotope, such as technetium-99 or iodine-131, bound, over the emitted radiation is the representation in tomography method or scintigraphy procedures allowed.

By diagnosing pancreatic tumors, particularly PDAC, with ADAM9-specific antibodies in the body of patients, the size of a pancreatic tumor can be determined. Advantageously, any existing secondary tumors (metastases) can be localized. The determination of The size and presence of secondary tumors of a pancreatic tumor is an important prerequisite for dividing patients into groups with different treatment regimens.

Furthermore the localization of secondary tumors is the prerequisite for these can be surgically removed.

at the diagnosis and treatment with ADAM9-specific antibodies exploited that the antibodies by the ADAM9 expression in PDAC, pancreatic tumor cells also in recognize vivo.

The Use of ADAM9-specific antibody for the treatment of pancreatic tumors preferably carried out alone or in combination with chemotherapy happen (eg during a simultaneous treatment with gemcitabine).

at the use of antibodies specific for ADAM9 for the treatment of pancreatic tumors, is preferably a cell-killing agent directly to the antibody bound.

Prefers becomes the cell-killing Agent from the group of cell-killing Radioisotopes such as iodine 131, rhenium 186, or more preferred Yttrium 90. Alternatively, the cell killing agent is selected from the group chemical, anthracyline like daunorubicin (DNR) or Doxorubicin (DOX) and its derivatives (Rosik LO Bioconjug Chem. 1990 Jul-Aug; 1 (4): 251-6) or from the group of biological toxins, as preferably Pseudomonas exotoxin A or diphtheria toxin selected.

By the binding of the antibody the cell-killing agent with the tumor cells to be killed becomes the tumor cells contacted and killed these off.

One Another object of the invention is the use of nucleic acids or Nucleic acid derivatives, which specifically bind to the ADAM9 gene and / or the ADAM9 mRNA, to Treatment of pancreatic tumors, especially the PDAC. By the Binding to the ADAM9 gene and / or the ADAM9 mRNA may be Substances the ADAM9 expression and thus the growth of pancreatic tumors, especially PDAC, suppress.

in the As used in this description, nucleic acids and nucleic acid derivatives in particular the following molecules to understand: deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and peptide nucleic acids (PNA), as well as all derivable from these basic structures modifications such as As phosphorothioates, phosphoramidates, O-methyl derivatives and locked nucleic acids (LNA).

The in the attached Sequence protocols disclosed nucleic acid sequences SEQ ID NO. 1 to SEQ ID No. 26 also includes the exchange of bases for other bases the analogous hydrogen bonds can be able to such as B. the exchange of t (thymidine) for U (uracil). The revealed Sequences SEQ ID NO. 1 to SEQ ID NO. 26 also include modifications the nucleic acid backbone, and close especially DNA, RNA, PNA, as well as all of these basic structures derivable modifications such. B. phosphothioates, phosphoramidates, O-methyl derivatives and locked nucleic acids (LNA).

Preferably become a suppression ADAM9 Expression in Pancreatic Tumors Nucleic acids or nucleic acid derivatives with the base sequences of SEQ ID NO. 1 to SEQ ID NO. 26 individually or mixed. These sequences and their mixtures are also the subject of the application.

The Use of nucleic acids or nucleic acid derivatives, which specifically bind to the ADAM9 gene and / or the ADAM9 mRNA, for the treatment of pancreatic tumors is preferably carried out alone or in combination with chemotherapy (eg during a simultaneous treatment with gemcitabine).

Based the following embodiments the invention will be closer explains:

Embodiment 1: Immunohistochemistry for the detection of ADAM9 protein expression in pancreatic tumors:

1 shows the results of an immunohistochemical analysis of ADAM9 protein expression in pancreatic tissue sections. For the preparation of these sections, pancreatic tissue is removed in an operative procedure and inserted for fixation in buffered 4% formalin. After fixation, the tissue becomes embedded in paraffin according to a common method (Carlos Thomas, pathology, histopathology, textbook and atlas on general and special pathology, Schattauer Verlag). Sections 4 μm thick are prepared from this paraffin-embedded tissue with a microtome (Leica, Nussloch) and mounted on specially prepared slides (Superfrost, Menzel Gläser, Braunschweig). The sections are then freed from the paraffin by incubation in xylene. The sections are then rehydrated by incubation in a descending alcohol series (100% ethanol to 0% ethanol).

After that will the cuts for Cooked in a pressure cooker for 2 minutes while in a Tris-EDTA citrate Buffers remain and then washed with distilled water. Then the cuts are with a solution of an ADAM9 specific primary antibody added. This antibody, such as B. a human ADAM9 cross-reactive goat anti-mouse ADAM9 antibody (R & D Systems, Wiesbaden, Catalog no. AF949) is added thereto in a concentration of 15 μg / ml in one solution from PBS containing 2% horse serum (available from Vector Laboratories, Alexis, Grünberg) contains solved. After incubating the sections with the antibody solution for 45 min at room temperature, The sections are washed 2 times with PBS for 5 min each.

The binding of the antibody to ADAM9 is detected by the following reaction:
The sections are incubated with a biotinylated anti-goat IgG antibody (available as a finished reagent system from Vector Laboratories) at a concentration of 5 μg / ml. The sections are then incubated with the reagent system avidin-biotin peroxidase (ABC ELITE, Vector Laboratories). The procedure is as described in the reagent systems. Finally, the sections are incubated with a solution of diaminobenzidine (Sigma, Taufkirchen) in 0.05 mol / l Tris-HCl pH 7.0 at room temperature. The sections are then stained and covered with a standard hematoxylin procedure.

The coloring is then assessed microscopically. A positive stain stands for ADAM9 protein expression (ADAM9 staining).

For tumor diagnosis the following parameters are important: staining of the basolateral side of the cells (s. 1 ) and the staining of the cytoplasm (s. 1 ). Any staining of these two parts of the cells is considered positive ("ADAM9 positive") and staining of the apical side is expected to be detected in the epithelial tissues studied on the apical side and is not considered a positive result ("ADAM9 negative").

Photographs of such prepared tissue sections are in 1 shown:

1A Figure 10 shows a picture of an analysis of ADAM9 protein expression in a well-differentiated pancreatic ductal adenocarcinoma (PDAC) with a typical apical ADAM9 staining 1 ,

1B Figure 10 shows a picture of an analysis of ADAM9 protein expression in a tissue section of a pancreatic ductal adenocarcinoma with cytoplasmic 3 ADAM9 staining.

1C and 1D Photographs show an analysis of ADAM9 protein expression in a tissue section of a poorly differentiated ductal pancreatic adenocarcinoma (PDAC) with basolateral membrane staining 2 and selective staining of the cytoplasm 3 ,

1E Figure 11 shows an image of an analysis of ADAM9 protein expression in a pancreatic tissue section of a patient with chronic pancreatitis. This shows a slight apical staining of the duct epithelium 4 ,

1F Figure 10 shows a picture of an analysis of ADAM9 protein expression in pancreatic islets 5 ,

Embodiment 2: Preliminary analysis ADAM9 mRNA expression (variant 1)

The Preliminary analysis of ADAM9 mRNA expression is performed in isolated RNA fabric pieces of the pancreas.

For this purpose, the RNA is isolated from tissue pieces of the pancreas using standard methods and reagent systems (RNeasy from Qiagen, Hilden, Germany). In doing so, proceed according to the protocol of the reagent system. Thereafter, the RNA is transcribed into cDNA with the enzyme reverse transcriptase. The transcription initiation necessary for this is taken over by "random hexamers" (oligonucleotides consisting of 6 nucleotides each with variable sequence) (Invitrogen, Karlsruhe), to which 100 pg of the "random hexamers" are added. The synthesis is otherwise carried out according to the manufacturer's description. After this reaction, the cDNA is used in a quantitative PCR. For this, 1 μl of the cDNA solution is used. The cDNA is added to a batch of 24 μl from a master mix consisting of buffer, Taq DNA polymerase (eg Sybr Green PCR Mastermix from Applied Biosystems, Weiterstadt) and gene-specific primers of SEQ ID no. 1 and SEQ ID NO. 2 given. In this case, the primers are present in a concentration of 300 nmol / l in the mix. To prepare this mix, for each determination, mix 12.5 μl of the Sybr Green PCR Master Mix with 1 μl each of the primers from a solution with a concentration of 3 μmol / l and 9.5 μl distilled water. Thereafter, a PCR under standard conditions in z. B. the ABI Prism Sequence Detection System performed and the values for the amount of RNA contained is calculated after performing the PCR. The dye Sybr Greens intercalates into the DNA formed during the PCR. The fluorescence of the intercalated dye quantifies the amount of DNA formed.

Embodiment 3: Preliminary Analysis ADAM9 mRNA expression (variant 2)

The Preliminary analysis of ADAM9 mRNA expression is performed in isolated RNA fabric pieces of the pancreas.

To For example, the RNA is prepared using standard procedures and reagent systems from tissue pieces of the Pancreas isolated (RNeasy Fa. Qiagen, Hilden, Germany). In doing so, proceed according to the protocol of the reagent system. After that the RNA is transcribed into cDNA with the enzyme reverse transcriptase. The necessary initiation of transcription is described by "Random Hexamers "(Oligonucleotides consisting of 6 nucleotides each with variable sequence) (Invitrogen, Karlsruhe). The approach 100 pg of the "Random Hexamers "given. The synthesis is otherwise according to the description of the manufacturer. After this reaction will be the cDNA used in a quantitative PCR. For this, 1 μl of the cDNA solution is used. The cDNA is composed of a mixture of 24 μl from a master mix from buffer, Taq DNA polymerase (eg TagMan PCR Mastermix of the Fa. Applied Biosystems, Weiterstadt) and gene-specific primers of SEQ ID NO. 1, SEQ ID NO. 2 and the gene-specific probe SEQ ID NO. 11 given. In this case, the primers are in a concentration in the mix of 300 nmol / l. To make this mix will be for each determination 12.5 μl of the TagMan PCR Master Mix, each 1 μl of the primer from a solution with a concentration of 3 .mu.mol / l and 9.5 μl distilled water mixed. Thereafter, a PCR under standard conditions in e.g. performed and the ABI Prism Sequence Detection System the values for the contained RNA amount is during the implementation the PCR determined analogously to the Tagman method.

Embodiment 4: Localization ADAM9 protein expression and tumor diagnosis:

In tissue samples of the pancreas as in Ausführüngsbeispiel 1 and 1 describe the ADAM9 protein expression analyzed. Here staining of the basolateral side of the cells and the staining of the cytoplasm were rated as "ADAM9 positive" and exclusively basolateral protein expression as "ADAM9 negative".

Table 1: ADAM9 expression in various tumors of the pancreas

It were pancreatic tissue samples from 59 patients with PDAC, 24 patients with azinärem Cell Carcinoma (ACC) and 11 patients with an endocrine pancreatic tumor (ET) examined. In the case of the PDAC, 58/59 ADAM9 cases are positive, the others Pancreatic tumors (ACC and ET) are almost all ADAM9 negative. Only in In the case of the ACC, 2 out of 24 tissue samples tested were positive for ADAM9.

In summary, the table shows that the analysis of the invention ADAM9 protein expression differential discrimination of PDAC from other pancreatic tumors (such as ACC and ET) is possible.

Embodiment 5: Localization ADAM9 Protein Expression and Tumor Prognosis:

In tissue samples of the pancreas was as in Example 1 and to 1 describe the ADAM9 protein expression analyzed. In a statistical analysis, the Kaplan-Meier test investigated the correlation between ADAM9 protein expression and the clinical pathological course of the PDAC (using the SPSS version 11 computer program (SPSS Inc., Chicago, USA), Kaplan, EL, & Meier, P (1958) Nonparametric estimation from incomplete observations (Journal of the American Statistical Association, 53, 457-481).

• (a): 59 Patienten ohne operationellen Eingriff (univariate Analyse) und
• (b): 42 Patienten denen der Pankreastumor in einem operationellen Eingriff entnommen wurde (multivariate Analyse; Cox Regression (SPSS11, Cox, D. R., & Oakes, D. (1984). Analysis of survival data. New York: Chapman & Hall.).

The following table shows the results of 103 PDAC patients. There are two groups of patients:

• (a): 59 patients without surgery (univariate analysis) and
• (b): 42 patients undergoing pancreatic tumor surgery (multivariate analysis, Cox regression (SPSS11, Cox, DR, & Oakes, D. (1984), Analysis of survival data, New York: Chapman & Hall.) ,

In univariate analysis becomes only the influence of a parameter, here the cytoplasmic or basolateral expression, on the variable to be tested, here survival, examined. In the multivariate analysis, on the other hand, the influence becomes of several parameters, here the cytoplasmic and basolateral Expression as well as tumor grade, on the variable to be tested, here survival, examined. The results of the univariate analysis serve to assess to what extent the examined parameters enter into a multivariate analysis should.

The following parameters are used to predict the clinical pathological course of PDAC:
N is the number of patients (total) and number of deaths (death) in the patient group.

Under HR is the relative risk (HR) for a worse disease course, so an earlier death date as a result of multivariate analysis.

Of the Confidence interval (CI) indicates the range of HR.

Of the P value indicates the significance with which a parameter contributes to the HR.

Table 2 Results of univariate and multivariate analysis of ADAM9 expression in patients with PDAC.

These Data show that cytoplasmic ADAM9 expression is related to the assessment the risk for a worse disease course can be used. This is limited with the determination of the tumor grade possible because the determination of tumor grade in pancreatic tumors is not easy to perform: The cytoplasmic ADAM9 staining Consequently, it offers itself as a new method for assessing HR and as a substitute the determination of the tumor grade, since the values are in the HR and in the equal p value. Based on cytoplasmic ADAM9 expression a stratification of patients of at least the same quality as with the tumor grade possible.

die kumulierte Gesamtüberlebenswahrscheinlichkeit. In dieser Gleichung ist, S(t) die geschätzte Überlebensfunktion, n die Gesamtanzahl der Fälle und Π beschreibt die geometrische Summe über alle Fälle welche kleiner gleich t sind; ^δ(j) ist eine Konstante die entweder gleich 1 ist, im Falle das von allen Fällen die Überlebenszeit bekannt ist oder 0 wenn das nicht der Fall ist. Dazu wird anhand der vorhandenen Fällen bei Patienten mit PDAC das Überleben geschätzt ("cumulated survival", 1 = Überleben, 0 = Tod). Die Überlebenswahrscheinlichkeit wird dabei in der linken Graphik bei positiver („pos.") oder („neg.") cytoplasmatischer („ADAM9 cyto.") und in der rechten Graphik bei basolateraler („lat.") ADAM9-Expression angegeben. 2 shows the results of a Kaplan-Meier statistical analysis (using the SPSS version 11 computer program (SPSS Inc., Chicago, USA); Kaplan, EL, & Meier, P. (1958).) Nonparametric estimation from incomplete observations Statistical Association, 53, 457-481). The Kaplan-Meier analysis calculates after

the cumulative overall survival probability. In this equation, S (t) is the estimated survival function, n is the total number of cases, and Π describes the geometric sum over all cases which are less than or equal to t; ^δ (j) is a constant that is either equal to 1, in case the survival time of all cases is known, or 0 if it is not. Survival is estimated based on existing cases in patients with PDAC (cumulated survival, 1 = survival, 0 = death). The probability of survival is indicated in the left-hand graph in the case of positive ("pos.") Or ("neg.") Cytoplasmic ("ADAM9 cyto.") And in the right-hand graph in the case of basolateral ("lat.") ADAM9 expression.

there show the X-axes: time after the operation in months and the Y Axis: Cumulative Overall Survival Probability ("cumulated survival").

In the 1 and 2 summarized results show that patients with either one cytoplasmic or basolateral ADAM9 protein expression significantly shorter mean survival time than patients showing no or exclusively apical ADAM9 protein expression.

These Patients should therefore be treated as patients in whom such expression has not been detected can be. Advantageously, by the inventive method the treatment scheme in dependence be adapted by the ADAm9 expression. The different ones ADAM9 expression itself can serve as a starting point for therapy itself, z. As an immune therapy with anti-ADAM9 antibodies are selected.

By the inventive method it becomes possible a forecast about to create the clinical course of the PDAC and the patients with Divide PDAC into at least two groups and for these groups an estimate the survival time to give.

Embodiment 6: Production of ADAM9-specific antibodies:

monoclonal antibody against human mouse ADAM9 are generated by standard techniques preserved and humanized (Roland Kontermann, Stefan Dübel, Antibody Engineering (Springer Lab Manuals) Springer Verlag Berlin). alternative become human antibodies against human mouse ADAM9 (Nina D. Russel, Jose R. F. Corvalan, Michael L. Gallo, C. Geoffrey Davis, Liise-Anne Pirofski. Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoblobulin Loci. Infection and Immunity April 2000, p. 1820-1826).

Embodiment 7: Diagnosis PDAC with ADAM9-specific antibodies in vivo:

The humanized or human antibody are labeled with iodine-131 (Simmel et al., J. Biol. Chem., Vol. 275, 30445-30450, September 29, 2000) and in patients in one Concentration of 2 MBq / kg to 50 MBq / kg body weight in an isotonic solution infused. The binding of antibodies to human ADAM9 is measured with a scintigraph. From the distribution of the recorded Radiation lets the presence, localization and size of tumor tissue in the body determine.

Embodiment 8: Treatment of the PDAC with ADAM9 specific antibodies:

The Humanized (unlabeled) antibodies are in concentration from 1 mg / kg to 20 mg / kg body weight in an isotonic solution Infused on several days.

Embodiment 9: Production and Treatment with Humanized Yttrium-90 Labeled Antibodies:

The humanized or human antibody are labeled with yttrium-90 (Simmel et al., J. Biol. Chem., Vol. 275, 30445-30450, September 29, 2000) and in patients in one Concentration of 5-20MBq / kg body weight in an isotonic solution Infused on several days. By the specific binding of the Radionuclide-labeled antibody targeted tumor cells are destroyed.

Embodiment 10: Treatment of the PDAC with siRNA:

A siRNA according to Seq ID No: 12 is used in a chemical nucleotide synthesis synthesized by standard methods. These are Phosphothioatnukleotide used for the synthesis to prevent the RNA from digesting too quickly Protect ribonucleases. This siRNA is in an isotonic concentration of 2-20 mg / kg solution Infused on several days.

In The description uses the following abbreviations:

ACC:

Azine cell carcinoma

ADAM9:

A DISINTEGRIN AND METALOPROTEINASE DOMAIN 9

cDNA:

complementary DNA

CI:

confidence interval

Cyto:

cytoplasmically

DNA:

deoxyribonucleic acid

ET:

endocrine pancreatic tumor

MR:

relative risk

IgG:

Immunoglobulin gamma

Lat:

basolateral

MAb:

monoclonal antibody

MBq:

Mega Becquerel

N:

number

Neg:

negative for ADAM9 - none ADAM9 staining

PBS

Dulbecco's phosphate buffered Saline

PCR:

Polymerase chain reaction

PNA:

"Peptide nucleid acids" - peptide nucleic acids

PDAC:

Ductal adenocarcinoma of the pancreas

Pos:

positive for ADAM9 - ADAM9 staining visible

RNA:

ribonucleic acid

siRNA:

"Small Interfering RNA" - short interfering RNA

RT-PCR:

Reverse transcriptase PCR

Taq:

Thermophilus aquaticus

Tris-HCl:

Tris (hydroxymethyl) aminomethane hydrochloride

In the figures use the following reference numerals:

1

apical ADAM9 membrane staining

2

cytoplasmic ADAM9 staining.

3

basolateral ADAM9 membrane staining

4

apical ADAM9 staining of the duct epithelium.

5

ADAM9- staining in islet cells of the pancreas

Claims (16)

Method for the diagnosis of tumors of the pancreas in tissue or cell samples of the pancreas, ADAM9 protein expression is determined using isolated pancreatic cells or tissue sections with ADAM9-specific antibodies and incubated under a microscope on binders of the ADAM9 specific antibody to be analyzed. Method according to claim 1, characterized in that that in analyzing the ADAM9 protein expression localization of the ADAM9 protein in the cell is determined and distinguished is between apical, basolateral and cytoplasmic expression of the ADAM9 protein. Method according to claim 1 or 2, characterized that the ADAM9-specific antibody monoclonal or polyclonal antibody. Method according to claim 3, characterized that the ADAM9-specific antibody selected from the following group AF949, AF939, 610750, CL2ADAM9, PC669, SA-376. Method according to one of claims 1 to 4, characterized that the detection of binding of the ADAM9-specific antibody by Fluorescence or by means of a color reaction. Method according to one of claims 1 to 5, characterized that prior to analysis for ADAM9 protein expression, a pre-analysis carried out in tissue or cell samples, the pancreas is ADAM9 gene expression or ADAM9 mRNA expression is determined. Method according to Claim 6, characterized that after pre-analysis for ADAM9 gene expression or ADAM9 mRNA expression the tissue or cell samples in which ADAM9 gene expression or ADAM9 mRNA expression could be detected, incubated with ADAM9-specific antibodies and analyzed under a microscope for ADAM9 protein expression. Method according to claim 6 or 7, characterized that analysis of ADAM9 mRNA expression in the tissue or cell samples by RT-PCR and / or microarray analysis. Method according to one of claims 6 to 8, characterized in that at least one oligonucleotide is used to analyze ADAM9 mRNA expression SEQ ID NO. 1 to SEQ ID NO. 11 is used. Use of ADAM9 specific antibodies to Diagnosis and / or treatment of pancreatic tumors. ADAM9-specific antibody for the diagnosis of pancreatic tumors, being the antibody a marker, preferably a radioactive isotope such as technetium-99, bound to the representation in tomography or scintigraphy allowed. ADAM9-specific antibodies for the treatment of pancreatic tumors, being the antibody a cell-killing Agent is bound. ADAM9-specific antibody according to claim 12, characterized characterized in that the cell killing agent a radionucleotide, preferably yttrium 90, or a toxin. Use of nucleic acids and / or nucleic acid derivatives, which specifically bind to the ADAM9 gene and / or the ADAM9 mRNA, for the treatment of pancreatic tumors. nucleic acids or nucleic acid derivatives with a base sequence from the group of SEQ ID NO. 1 to SEQ ID NO. 26th Use of nucleic acids or nucleic acid derivatives according to claim 15 for the diagnosis or treatment of pancreatic tumors.