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AssigneeRoche Diagnostics GmbH
InventorsJosef Ruschoff, Wolfgang Dietmaier, Richard Fishel
Estimating the term…
The invention concerns a method for the determination of the genomic instability at 5 selected microsatellite loci. The analysis of these selected loci is suitable for making prognostic tumour diagnoses, for analysing hereditary tumor predisposition as well as for early tumor detection. This method is of particular importance for the diagnosis of tumors of the gastrointestinal tract such as colorectal tumors.
A method for determining the sizes of at least 5 different microsatellite loci comprising the following steps:
A method for determining the sizes of 5 microsatellite loci comprising the following steps:
A method for determining familial tumor predisposition in a human, said method comprises the following steps:
A method for detecting a tumor of the gastrointestinal tract or endometrium in a human, said method comprises the following steps:
A method for early detection of a tumor of the gastrointestinal tract or endometrium in a human, said method comprising the following steps:
A kit consisting essentially of 5 different primer pairs wherein at least 2 of said 5 different primer pairs are suitable for DNA amplification of mononucleotide repeat loci and the remaining primer pairs of said 5 different primer pairs are suitable for the DNA amplification of
A method for detecting a colorectal tumor in a human, comprising
The method of 56, wherein said 5 different microsatellite loci are BAT25, BAT26, APC, Mfd15 and D2S123.
A method for determining the prognosis or the familial predisposition of a colorectal tumor in a human, comprising
Dependencies are inferred from the claim text.
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Involving nucleic acids: nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
For diseases caused by alterations of genetic material: for cancer
Oligonucleotides characterized by their use: disease subtyping, staging or classification
Oligonucleotides characterized by their use: primer sets for multiplex assays
Term ended 25 Mar 2018 (estimated)
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The invention concerns a method and a kit for the prognostic diagnosis, predisposition diagnosis and early recognition of tumours of the gastrointestinal tract preferably colorectal tumours. The basis for this is the detection of the genomic instability of so-called microsatellites with the aid of PCR.
Microsatellites (MIS) are short tandem repeats which are distributed over the entire human genome. Microsatellites occur statistically about once every 100,000 base pairs. Up to now 5 classes of MIS have been described which differ from one another in the length of their smallest repetitive unit as a mono-, di-, tri-, tetra- or pentanucleotide repeat. As a rule these repetitive units occur repeatedly 10 to 40 times in a tandem arrangement. Microsatellite instability (MIN) in the form of small deletions or insertions can be detected in many tumour patients if one compares DNA from tumour material with normal DNA of the same individual (Thibodeau et al., (1993), Science, 260, 816-819) (WO 94/19492). This is achieved by amplifying the DNA with the aid of PCR and subsequently separating the amplification products by gel electrophoresis. A permanent replication defect of the tumour cells is regarded to be the cause of MIN (Parsons et al., (1993), Cell, 75, 1227-1236; Shibata et al., (1994) Nat. Genet. 6, 273-281). Such tumours are classified as "replication error-positive" (RER+). An RER+ phenotype is characteristic for colorectal tumours in families with HNPCC (hereditary non-polyposis colon cancer) (Aaltonen et al., (1993), Science, 260, 812-816).
The analysis of microsatellites is an extremely attractive method for diagnostic applications as well as for the examination of the tumourigenesis of RER+ tumours. Because it is simple to carry out the determination of MIN before sequencing the mismatch repair gene of HNPCC families is a suitable aid in identifying potential RER+ patients. MIN analysis is also of major importance for the prognostic diagnosis of sporadic colorectal carcinoma because the occurrence of MIN correlates with a better prognosis (Lothe et al. (1993) Cancer Res., 53, 5849-5852; Thibodeau et al. (1993), Science, 260, 816-819; Bubb et al. (1996) Oncogene, 12, 2641--2649).
FIG. 1 shows a table of the most important features of the analysed MIS loci (locus symbol, marker name, chromosomal location, repeat type) and the parameters for the respective PCR amplification (PCR-Tm: hybridization temperature).
FIG. 2a shows the gel electrophoretic analysis of 25 examined microsatellite loci. The various alleles of the loci BAT26, BAT40, APC, MfD15, D2S123 and TP53Alu to be analysed according to the invention can be clearly distinguished from one another. FIG. 2b shows an example of a duplex analysis of the loci BAT40 and D3S1283 in one reaction mixture.
FIG. 3 shows the result of the study carried out in an overview. 27 Patients with colorectal tumours were examined at 25 different alleles for MIN. The result shows that (i) different MIS are affected by polymorphic changes at different frequencies and (ii) in different patients different classes of microsatellites are affected at different frequencies of genomic instability.
The invention is further elucidated by the following examples:
Selection of 2 Mononucleotide Repeat Loci, 1 Dinucleotide Repeat Locus of the Class 2a, 1 Dinucleotide Locus of the Class 2b and 1 Pentanucleotide Repeat Locus
As the study showed, there were no false positive results when using mononucleotide repeat loci to determine the RER+ phenotype so that an analysis of these loci appears to be particularly suitable. On the other hand it was not possible to detect all RER+ tumours by analysing mononucleotide repeat loci. Thus for example tumour No. 5 was unambiguously RER+ but was not unstable with regard to the loci BAT25, BAT26 and BAT40 although it was possible to detect MIN at 9 loci with other repeat types. It was therefore necessary to select a combination of various repeat types for the most exact possible determination of the RER phenotype with a limited number of 5 analysed loci. This ensures that, despite the low number of analysed loci, on the one hand all RER+ carriers can be identified with the highest possible degree of probability and on the other hand the number of lowMIN+ cases that cannot be unambiguously classified is minimized as much as possible.