Cancer detection probes

Details Cancer detection probes Cancer detection probes

1995-12-21

2002-02-26

Whisenant, Ethan (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300

Reexamination Certificate

active

06350576

ABSTRACT:

TECHNICAL FIELD
The field of this invention is the identification of lesions in neoplastic cells.
BACKGROUND
The etiology of neoplasia is an extremely complex one. A large number of genes have been found to be associated with normal cells being transformed into tumor cells. Genes that have been identified include genes which enhance cell proliferation and suppress cell proliferation. The present view is that it takes more than one mutation event to take a cell from the phenotype of the normal state to the phenotype of the tumor state.
It appears today that human tumors are genetically heterogeneous by the time of clinical presentation, even though they are rather monoclonal in origin. As the tumor progresses, there may be changes in the genome, such as deletions, insertions, substitutions, chromosomal arm exchanges, gene amplification, and the like. These changes may be associated with various characteristics of the cancer, which are important to the diagnosis and therapy of the patient. Information about the cancer, whether it is aggressive, metastatic, or responsive to a particular treatment as a result of particular genomic changes, can greatly aid in the choice of therapy of the patient. For example, more intensive treatment may be warranted for more aggressive cancers.
There is, therefore, substantial interest in identifying specific genetic differences which are associated with neoplastic cells. These differences provide the opportunity to dentify groups of patients having analogous lesions, where the course of the cancer ay be mapped. In this way, epidemiological data can be adduced as to the nature of he cancer, its response to different therapies, and probable outcomes.
Relevant Literature
U.S. Pat. No. 5,569,753 describes a method called representational difference analysis RDA) for analyzing differences between complex but related genomes.
Salomon et al. (1991)
Science
254:1153 and Lasko et al. (1991)
Annu. Rev. Genet
. 25:281-314 describe genetic lesions found in tumors. Lisitsyn et al. (a)(1993)
Science
259:946-951 also describe (RDA). See also Lisitsyn et al.(b) (1993)
Nature Genetics
, 6:57-63. Presti et al. (1991)
Cancer Research
51:1544-1552 report loss of at least portions of the Y chromosome in renal cell carcinoma cells. Schwab and Amler (1990)
Genes, Chromosomes and Cancer
1:181-193 report the amplification of N-myc in neuroblastoma cells.
SUMMARY OF THE INVENTION
Nucleic acid probes for detecting cellular lesions associated with tumor cells are provided. The probes are associated with genomic lesions, such as: loss of information, e.g. loss of heterozygosity (“LOH”), hemizygous loss and homozygous loss; gain of information, e.g. gene amplification or acquisition of viral genomes; gene rearrangement; point mutation and the like. By combining the probe with genomic DNA of candidate cells and detecting the lesion, one can evaluate the cancer stage or provide a prognosis. The lesion can be detected by Southern blotting or other hybridization techniques, polymerase chain reaction, and the like.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Compositions and methods are provided for detecting genomic lesions associated with cancer. Specific sequences are provided which may be used to detect the lesion in candidate cells, where cells having a normal phenotype may be evaluated for cancer susceptibility or cancer cells may be evaluated as to prognosis and therapy. The sequences may also be used to walk the genome to identify other sequences at the locus of the specific sequence. (See, for example, Molecular Cloning: A Laboratory Manual, 2nd. ed., eds. Sambrook et al., CSHL Press, Cold Spring Harbor, N.Y., 1989, Sections 3.2, 3.9, 3.23, and 9.3) These other sequences at the locus provide additional probes, identify genes associated with the particular lesion and phenotype, and allow for the detection of specific mutations at the locus.
Specific sequences that can be employed in the subject invention are found in the Sequence Listing, infra, as SEQ ID NOS: 1-35. To obtain additional sequences at the locus of the subject sequences, human genomic fragments may be cloned in various sizes, generally ranging from about 10 kbp to 600 kbp or more. By identifying clones to which the probes base pair, one can then walk the probe to identify the sequences 3′ and 5′ of the probe. See Wahl et al. (1987)
P.N.A.S. U.S.A
. 84:2160; Triglia (1988)
Nucleic Acid Research
16:8186; and Sambrook et al., infra. Depending upon the size of the cloned fragment, one can sequence the entire cloned fragment or further fragment the cloned fragment and sequence a smaller portion. With each extension of the subject sequences, one can then use the additional sequence to identify the next adjacent or contiguous sequence.
The sequences detected by walking the subject sequences and sequences uncovered this way can be screened in an analogous manner to the subject sequences to determine their usefulness as probes. The sequences can be used to screen normal genomes, as well as genomes from tumor cells. Where an unacceptable degree of binding to normal cells is uncovered or after screening a significant number of fresh tumor cells without significant observation of the same lesion, the sequence may be discarded and usually one will not proceed further in walking the DNA. This will be particularly true, where there is a high incidence of the lesion in normal cells. For example, some particular sequences may be found absent in tumors but have a high incidence of being absent in normal human DNA. We call these deletion or insertion polymorphisms. For lesions not associated with gene amplification, generally, the lesion will be absent in 20 randomly selected normal cells, usually absent in 100 randomly selected normal cells, and absent in the normal cells of the source. The lesion is desirably absent in at least a statistically significant proportion of the normal population to provide a cancer diagnostic, but may be present in the normal population, where it is directed to prognosis of an existing tumor, recurrence or remission.
The loci of the subject invention identified by the subject probes will generally be not more than about 300 kbp, usually not more than about 100 kbp and may not exceed about 10 kbp, with the exception that if the lesion comprises an amplified region, the region may be as large as 1 million base pairs in length. The probes employed in this invention as obtained from the specified loci will generally be at least about 18 bp, more usually at least about 30 bp and may be 1kbp or more, usually not exceeding about 40 kbp. While the probes may be either DNA or RNA, as a practical matter the probes will normally entail DNA.
The loci can be present on both autosomal chromosomes or sex chromosomes. For particular types of cancer, there will normally be an association between the type of cancer and the particular lesion. In addition, particular lesions can provide for susceptibility of cells to cancer formation, the aggressiveness of the cancer, particularly as to rate of proliferation and metastatic capability, as well as the response of the cancer cells to particular forms of therapy. The subject loci are associated with carcinomas, as associated with cells from the kidney, colon, esophagus, lung, skin, and brain.
The probes of this invention are further characterized by detecting lesions which are present in neoplastic cells, but not present in normal cells of the same patient, as well as normal cells from other individuals. Frequently, the same lesion may be found in human tumor cell lines and human cells from the same cellular type.
For use in detection of lesions, the probes may be modified by various labels, which will allow for their detection. The labels may be directly detectable, such as fluorescers, enzymes, radioisotopes, particles, and the like. Alternatively, the labels may be indirectly detectable by binding to another molecule which provides for the direct detection. Thus, one may have various ligands which have a complementary pair binding member, where t

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