DE102006007249A1 - Determining bladder cancer of differentiation grade G2 by measuring the ratio, in urine or urinary sediment, of mRNA encoding CC3 and TC3 proteins - Google Patents

Abstract

Method for determining bladder carcinoma with differentiation grade G2 comprises (a) determining the amount of mRNA (I) encoding CC3 protein in the urine or urinary sediment; (b) determining the amount of mRNA (II) encoding TC3 protein in the same sample; and (c) calculating the ratio (Q) of (I):(II). Bladder cancer of G2 stage is indicated if Q exceeds a predetermined threshold value x.

Description

The The invention relates to a method for the determination of bladder carcinomas the degree of differentiation G2.

In According to cancer registries, Germany suffers a total of 15,000 deaths annually new to bladder cancer, taking men affected about twice as often are like women. Related to all neoplastic diseases that lies Bladder cancer in men 5th (6.2%) and 11th (2.7%) among women. In the majority of the tumors (90%) is a carcinoma that originates from the transitional epithelium of the urinary tract, the urothelium. Only in 5-6% are squamous cell carcinomas and less than 2% are adenocarcinomas before (review article Kirkali Z, Chan T, Manoharan M, Algaba F, Bush C, Cheng L, Kiemeney L, Warmair M, Montironi R, Murphy World Cup, Sesterhenn L4, Tachibana M, Weider J. (2005): Bladder cancer: epidemiology, staging and grading, and diagnosis. Urology 66 (6 Suppl 1): 4-34.).

As a general rule Tumors become through a histo-pathological classification system described in the various parameters such as the spread and Size, infiltration in the surrounding tissue, removal of daughter tumors (metastasis) and the degree of differentiation, the so-called grading. The grading (G1-G4) the histological changes of the tumor with respect to the tissue of origin. The classification G1 describes Tumor tissue, which still has many characteristics of the original tissue has. The degeneration of the tissue increases towards the G4 tumors gradually. The cells of a G4 tumor are highly "degenerate," i. the transitional epithelial character (the tissue of origin) is no longer recognizable. The so-called "moderately differentiated Carcinomas "G2 are by certain histo-pathological parameters of the well-differentiated G1 and poorly differentiated G3 tumors can be differentiated. In spite of defined criteria, this distinction is not always clear since they are counting of cells in the field of view done in the microscope. As a result, that two independent, competent persons with regard to different findings can come to the degree of differentiation. This degree of differentiation has authoritative Impact on the prognosis of patients. The 5-year survival rate for the superficial G1-G2 tumors is after therapy 80-95%. For the superficial G3 Tumors, the so-called locally-limited high-risk carcinomas, is the 5-year survival rate only 64%. Furthermore, the degree of differentiation - besides the spread of the tumor - relevant the form of therapy (Ref. 1). This is at the transition from superficial G2 tumors are more crucial to the local-limited high-risk carcinomas (G3) Meaning, since here in part of the superficial G3 carcinomas a radical cystectomy (surgical removal of the bladder) occurs while at G2 tumors a superficial Removal of the tumor tissue (with preservation of the organ) with subsequent medical treatment is made.

Next the classical histological examination of tumor tissues becomes For some time now the presence of molecular biological Tumor marker proteins included in the tumor diagnosis. With the Development of highly sensitive molecular biology methods is now approaching the detection and quantification of the corresponding tumor marker proteins coding mRNA molecules in the foreground. The analysis of the amount of transcript from peripheral Blood or urine allows it, in some cases on invasive measures like operations or over Endoscopic methods obtained biopsies for diagnostic purposes to renounce.

From the DE 103 26 773 A1 For example, a diagnostic method is known that takes advantage of the finding that overexpression of nucleophosmin / B23 mRNA is a prognostic marker for detecting reoccurring and advanced bladder carcinoma. Although this method already makes it possible to detect advanced tumor stages on the basis of biopsy material or tumor tissue and to allow conclusions about the likelihood of recurrence of the tumor disease after surgical removal of the tumor, it is not suitable for a sensitive and specific diagnosis of the only moderately differentiated (G2 ) To allow bladder carcinoma. Furthermore, the described method relates to the examination of biopsy material or tumor tissue, ie the patient must undergo this procedure psychologically and physically burdensome invasive procedures or operations.

In Menke TB, Boettcher K, Kruger S, Kausch I, Boehle A, Sczakiel G, Warnecke JM. (2004): Ki-67 protein concentrations in urothelial bladder carcinomas are related to Ki-67-specific RNA concentrations in urine. Clin Chem. 50: 1461-1463 describes a diagnostic method based on analysis of the expression of the tumor marker Ki-67. The amount of Ki-67 protein present in the tumor tissue is routinely used for the immunohistological analysis of bladder carcinomas and other tumors. It has been found that Ki-67 expression in tumor tissue is substantially consistent with the mRNA content in the patient's urine. However, analysis of the mRNA level of Ki-67 in urine does not allow differentiation between bladder carcinomas of the differential degrees of G2 and G3.

It there is a need for non-invasive diagnostic procedures, the one sensitive and specific diagnosis of the only moderately differentiated (G2) Enable bladder carcinoma.

The object of the present invention is therefore to find a non-invasive method for the sensitive and specific differentiation of the moderately differentiated G2 bladder carcinomas from the other grading types of bladder carcinoma. The object is achieved by a method for the determination of bladder carcinomas of the _degree of differentiation G2, _comprising the steps of determining the mRNA quantity mRNA _{CC3 of} the protein CC3 from the urine or the urine sediment of a human, determining the mRNA amount mRNA _{TC 3 of} the protein TC3 from the same _Sample material and calculating the quotient Q = mRNA _CC3 / mRNA _TC3 , wherein the presence of a bladder carcinoma of the _degree of differentiation G2 is displayed when the quotient Q exceeds a predetermined threshold x.

Of the only subclaim gives an advantageous embodiment of the invention again.

The The invention is based on that of the inventors in the context of a screening method Observation made after bladder carcinoma-associated genes that The relationship the gene products of two genes, CC3 and TC3, in the bladder tumor for G2 tumors across from is different from all other degrees of differentiation and thus one Possibility to Subclassification of bladder tumors. This ratio can besides determining the protein content of CC3 and TC3 in the tumor also by the detection of specific mRNA sequences for TC3 (GenAcess No. AF092095) and CC3 (GenAcessNo.NM_006410) in tumor, urine and urine sediment (i.e., essentially the cellular portion in the urine) of a patient.

The CC3 mRNA encodes a protein (synn. TIP30, Htatip2) which is stable is and acts pro-apoptotic and anti-angiogenic and thus has the function of a tumor suppressor protein (Emma Shtivelman (1997): A link between metastasis and resistance to apoptosis of variant small cell lung carcinoma. Oncogene 14: 2167-2173 and Xiao H, Palhan V, Yang Y, Roeder RG. (2000): TIP30 has an intrinsic kinase Activity required for up-regulation of a subset of apoptotic genes. EMBO J. 19: 956-63).

at TC3 is a splice variant of CC3 RNA (Stephanie Whitman, Xia Wang, Refaat Shalaby, and Emma Shtivelman (2000): Alternatively Spliced Products CC3 and TC3 have Opposing Effects on Apoptosis. Molecular and Cellular Biology 20: 583-593.). The translation product TC3 is very unstable, has anti-apoptotic effects and thus can be considered a tumor promoter (Stepahnie Whitman, Xia Wang, Refaat Shalaby and Emma Shtivelman (2000): Optional Spliced Products CC3 and TC3 Have Opposing Effects on Apoptosis. Molecular and Cellular Biology, 20: 583-593.).

The Thus, the invention does not use a single independent molecular Markers, but of the relationship between two functionally competing mRNA splice variants one and the same gene to make a statement about the presence of a bladder tumor with a certain degree of differentiation (G2). The specialist would now in principle expect that with increasing grading and thus increasing aggressiveness of the tumor, the expression of the tumor suppressor protein CC3 decreases and the relationship from CC3 to TC3 in favor of the tumor promoter TC3. It has However, surprisingly shown during the transition from Grading G1 to G2, the mRNA concentration of CC3 in relation to TC3 rises while she at the transition from G2 to G3 drops again significantly.

The increase in the ratio of mRNA concentrations from CC3 to TC3 above a certain threshold x can thus be used as a diagnostic parameter for the presence of a bladder carcinoma of differentiation level G2. On the basis of the available data ( 1 - 3 ) generated in the manner of the embodiment below, a threshold x of 29 can be set. If the quotient Q from the amount of CC3 and TC3 is greater than or equal to 29, the test is positive. On the basis of 35 analyzes evaluated and assuming a correct histopathological classification, the correct diagnosis can be made with a sensitivity of 91.7% (correct-positive rate) and a specificity of 82.6% (true-negative rate) ( 2 . 3 ). For the future, the analysis of larger patient groups is planned. It is likely that the threshold x specified here will change with the increasing sample size, so that the threshold x of 29 given here is currently only a guideline.

Further Features and advantages of the invention will become apparent from the following description. The pictures become closer described

1 : Representation of the quotient Q from CC3 mRNA to TC3 mRNA in the examined samples. The presentation was a grouped box plot. The patient groups are grouped according to histopathological grading (G1-G3). Other groups studied are healthy volunteers (oB) and patients with cystitis gene (inflamm.) shown. The median (the value below and above each of which is 50% of the ordered values) is indicated by the bar, the boxes below mark the 25th percentile (the value below which is 25% of the ordered values) and above is the 75th percentile (the value above which is 25% of the ordered values). The bars mark the 95% confidence interval (the range of 95% chance of being the measurement points of this group). Stars and dots denote readings outside the 95% confidence interval. The ratio of CC3 to TC3 is significantly increased in patients with a bladder tumor of differentiation grade G2 compared to all other patient groups. It is also important in this context that the presence of cystitis has no influence on the CC3 / TC3 ratio. Thus, the test is also feasible and meaningful if the patient to be examined suffers from an acute Bla senentzündung, which is often the case especially in patients with bladder cancer.

2 : The table shows that of the ROC curve ( 3 Sensitivity (indicating how many of the positive samples were positively detected by the test) and specificity (indicating how many of the negative samples were negatively recognized by the test) and also contains the information as to which respective cut-off values (see below) assigned to the corresponding value pairs (leftmost column). Defining a cut-off value of 29 as threshold x gives you a value pair of 91.7% sensitivity and 82.6% specificity (represented as 1-0.174). It is possible to achieve the same sensitivity even with a threshold of 22, but then the specificity decreases because more negative controls are erroneously recognized as positive since in this case they are above the threshold x. a The smallest cut-off value is the smallest observed test value (quotient Q = mRNA _CC3 / mRNA _TC3 ) minus 1, and the largest cut-off value is the largest observed test value plus 1. All other cut-off values are averages of two consecutive ordered observed test values.

3 : Possibility of distinguishing G2 tumors from other tumors and control groups by the procedure. Representation in the form of a Reciever-Operator-Graph (ROC-curve). Plotted is the sensitivity (y-axis) versus 1-specificity (x-axis). Based on the in 2 As shown, the graph shows the relationship between the decrease in specificity and the increase in sensitivity. The steeper the curve, the more specifically a process can detect an event with high sensitivity (Weiss HL, Niwas S, Grizzle WE, Piyathilake C. (2003-2004): Receiver Operating Characteristic (ROC) to determine cut-off points of biomarkers in Mark cancer patients Dis: Markers 19: 273-278.

The Measure of area below the curve indicates the diagnostic usability. For values of> 0.75 is the possibility given a diagnostic use. For the illustrated test system the value is 0.87. The arrow indicates that at the current sample size n = 35 based threshold x (a ratio of CC3 RNA to TC3 RNA from ≥ 29) in which the distinction of G2 tumors to all other studied classes with a sensitivity of 91.7% and a specificity of 82,6% possible is.

The The invention is explained in more detail below with reference to an exemplary embodiment:

1) Isolation of RNA from the urine of tumor patients / suspected persons

First, the urine sample or urinary sediment pellet is mixed with a guanidinium salt solution to a final concentration of 3 M guanidinium. This solution is applied to a commercially available column of activated silica matrix (here Qiagen Midi Kit) wherein the nucleic acids are bound to the matrix. Subsequently, the washing of the silica-bound nucleic acids with high salt buffer for the separation of proteins and soluble substances with a buffer of the manufacturer. To avoid DNA contamination in the following step, an enzymatic degradation of any existing DNA is performed on the silica matrix. In this example, the enzyme DNase I dissolved in 140 μL DNase I buffer was used. This is followed by washing the silica-bound RNA with high-salt buffer of the manufacturer for the separation of DNA fragments, proteins and soluble substances. The elution of the RNA from the silica matrix is achieved by means of the smallest possible volume of RNase-free water. Here 2 × 160 μL H ₂ O were used. To increase the RNA concentration in the sample, the sample volume should be concentrated to 20 μL by vacuum centrifugation. Alternatively, ethanol precipitation is also possible here.

2) Enzymatic synthesis the counterstrand DNA from isolated RNA

For the synthesis of the counterpart DNA (cDNA), 10 .mu.l of the RNA-containing isolate in a reaction volume of 20 .mu.L by means of a commercially available reverse transcriptase system (here Superscript III / Invitrogen) rewritten. The beginning of the reverse transcription reaction on the RNA is achieved by commercially available random primer of 6 nucleotides in length (Invitrogen) in a concentration of 15 ng per μL reaction volume. After the end of the given reaction time (here 50 ° C for 60 min), the enzyme is inactivated by a high-temperature step for 15 minutes at 70 ° C.

3) amplification and quantification the counterstrand DNA by quantitative polymerase chain reaction (QPCR)

The determination of the amounts of CC3 cDNA and TC3 cDNA in the sample is carried out by means of a quantitative polymerase chain reaction. For this purpose, a dilution of the cDNA solution is amplified with suitable buffers and a temperature-stable DNA polymerase. For this purpose, the commercially available "core kit" from Eurogentec was used by way of example in order to set conditions of 1 × PCR buffer, 0.25 U Taq polymerase, 200 μM dNTPs and 3 mM MgCl ₂ in a reaction volume of 10 μl. The sequence-specific, cyclic amplification of the counterstrand DNA is determined by addition of oligonucleotides having the nucleic acid sequences indicated under SEQ ID NO: 1 and SEQ ID NO: 2 (see Sequence Listing) for CC 3 and in a second batch of the oligonucleotides having the nucleic acid sequences indicated under SEQ ID NO: 3 and SEQ ID NO: 4 (see Sequence Listing) for TC3 to a final concentration of 200 nM The quantification is carried out by a further oligonucleotide which also specifically binds to the sequence to be detected and a fluorescence S proportional to the amplification emitted signal. The quantification is carried out here by way of example by a fluorescence-labeled oligonucleotide probe of the TaqMan system with the nucleic acid sequence for CC3 given under SEQ ID NO: 5 and the nucleic acid sequence for TC3 given under SEQ ID NO: 6 (see Sequence Listing), in each case in a final concentration of 125 nM. Sequence-specific amplification is followed by a single denaturation step for 10 minutes at 92 ° C, in 50 temperature cycles of 15 seconds at 95 ° C followed by 1 minute at 60 ° C. Absolute quantitation can be achieved by parallel runs of serial dilutions of the sequences of TC3 and CC3 in known concentrations under identical conditions.

4) Determination of the ratio from CC3 RNA to TC3 RNA in the urine sample

The Determination of the quotient Q from the content of the CC3 RNA and the content The TC3 RNA in the urine sample is calculated by dividing the absolute amount of CC3 cDNA in the PCR sample by the absolute Amount of TC3 cDNA in the PCR sample.

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (2)

Method for the determination of bladder carcinomas of differentiation _grade G2, characterized by the steps of determining the mRNA quantity mRNA _{CC3 of} the protein CC3 from the urine or the urine sediment of a human, determining the mRNA quantity mRNA _{TC3 of} the protein TC3 from the same sample material, and calculating the quotient Q = mRNA _CC3 / mRNA _TC3 , wherein the presence of a bladder carcinoma of the _degree of differentiation G2 is indicated when the quotient Q exceeds a predetermined threshold x. Method according to claim 1, characterized in that that the determination of mRNA levels via a quantitative polymerase chain reaction takes place, and the oligonucleotides used in this case are selected from the group of nucleic acid molecules of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 sequences shown.