Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-12
2001-12-04
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06326148
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains to the field of cancer genetics and cytogenetics. In particular, this invention pertains to the identification of an association between amplification of regions on chromosome 20 and colorectal cancer.
BACKGROUND OF THE INVENTION
Chromosome abnormalities are often associated with genetic disorders, degenerative diseases, and cancer. The deletion or multiplication of copies of whole chromosomes and the deletion or amplifications of chromosomal segments or specific regions are common occurrences in cancer (Smith (1991)
Breast Cancer Res. Treat
. 18: Suppl. 1:5-14; van de Vijer (1991)
Biochim. Biophys. Acta
. 1072:33-50). In fact, amplifications and deletions of DNA sequences can be the cause of a cancer. For example, proto-oncogenes and tumor-suppressor genes, respectively, are frequently characteristic of tumorigenesis (Dutrillaux (1990)
Cancer Genet. Cytogenet
. 49: 203-217). Clearly, the identification and cloning of specific genomic regions associated with cancer is crucial both to the study of tumorigenesis and in developing better means of diagnosis and prognosis.
One of the amplified regions found in studies of breast cancer cells is on chromosome 20, specifically, 20q13.2 (see, e.g. WO98/02539). Amplification of 20q13.2 was subsequently found to occur in a variety of tumor types and to be associated with aggressive tumor behavior. Increased 20q13.2 copy number has been found in 40% of breast cancer cell lines and 18% of primary breast tumors (Kalliioniemi (1994)
Proc. Natl. Acad. Sci. USA
91: 2156-2160). Copy number gains at 20q13.2 have also been reported in greater than 25% of cancers of the ovary (Iwabuchi (1995)
Cancer Res
. 55:6172-6180), colon (Schlegel (1995)
Cancer Res
. 55: 6002-6005), head-and-neck (Bockmuhl (1996)
Laryngor
. 75: 408-414), brain (Mohapatra (1995)
Genes Chromosomes Cancer
13: 86-93), and pancreas (Solinas-Toldo (1996)
Genes Chromosomes Cancer
20:399-407).
A number of studies have elucidated genetic alterations that occur during the development of colorectal tumors. For instance, deletions of p53 genes on chromosome 17p are often late events associated with the transition from the benign (adenoma) to the malignant (carcinoma) state. See Vogelstein et al.,
New England Journal of Medicine
, 319:525 (1988), Fearon and Vogelstein,
Cell
, 61:759-767 (1990) and Baker et al.
Cancer Res
. 50:7717-22 (1990). More recently, comparative genomic hybridization has shown that specific patterns of chromosomal gains and losses take place during colorectal carcinogenesis (see, e.g. Schlegel, et al.
Cancer Research
. 55, 6002-6005 (1995); Ried, et al.
Genes, Chromosomes
&
Cancer
15, 234-245 (1996); and Nakao et al.,
Jpn. J. Surg
. 28, 567-569 (1998). These changes included overrepresentation (amplification) of large portion of chromosome 20 material.
Because carcinomas are often lethal, while the precursor adenomas are uniformly curable, the early detection of chromosomal changes associated with this transition are of considerable importance. The identification of regions of chromosomal abnormalities in other cancers is obviously great use in diagnosis, prognosis and treatment of these diseases. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods of screening for colon carcinoma cells in a sample. The methods comprise providing a nucleic acid sample from a premalignant lesion in colorectal tissue from a human patient and contacting the sample with a nucleic acid probe that selectively hybridizes to a chromosomal region on 20q. The formation of a hybridization complex is then detected and the presence or absence of increased copy number at 20q (usually 20q13.2) is determined.
The sample used can be, for instance, one suitable for in situ hybridization techniques, such as a metaphase spread or an interphase nucleus. Usually, the probe is labeled, e.g. with fluorescent label, which can be attached directly or indirectly to the probe. In some embodiments, a second probe that selectively hybridizes to a second chromosomal region is used as a reference. In these embodiments, the second probe is labeled with a fluorescent label distinguishable from the label on the probe that selectively hybridizes to a chromosomal region on 20q.
The sample can be derived from any premalignant colorectal tissue suspected of containing cancer cells. Often, the premalignant tissue is an ademomatous polyp or tissue showing high grade dysplasia.
In some embodiments the probe may comprise repetitive sequences. In this the methods may further comprise the step of blocking the hybridization capacity of repetitive sequences in the probe. This can be done by, for example, including unlabeled blocking nucleic acids with the labeled probe. The unlabeled blocking nucleic acids can be,f for example, Cot-1 DNA.
A number of hybridization formats can be used. In some formats, the probe is bound to a solid substrate as a member of an array of probes. In these embodiments, the probe is not labeled and the sample nucleic acids are labeled.
Definitions
To facilitate understanding of the invention, a number of terms are defined below.
The term “amplicon” as used herein refers to a region of genomic nucleic acid which, when present in altered copy number, is associated with cancer. For example, the invention provides nucleic acid sequences which, when present in aberrant copy number, are associated with colon cancer.
The term “20q13.2 amplicon” refers to a region on the q arm of human chromosome 20 at about band 13.2 that has been identified in cancer cells. This amplicon has been extensively analyzed (see, e.g., WO 98/02539) in breast cancer cells. A 1.5 megabase (Mb) wide amplified region within 20q13.2 was identified (Stokke (1995)
Genomics
26: 134-137); Tanner (1994)
Cancer Res
. 54:4257-4260). Interphase FISH revealed low-level (>1.5×) and high level (>3×) 20q13.2 sequence amplification in 29% and 7% of breast cancers, respectively (Tanner (1995)
Clin. Cancer Res
. 1: 1455-1461). High level amplification was associated with an aggressive tumor phenotype (Tanner (1995) supra; Courjal (1996)
Br. J. Cancer
74: 1984). Another study, using FISH to analyze 14 loci along chromosome 20q in 146 uncultured breast carcinomas, identified three independently amplified regions, including RMC20C001 region at 20q13.2 (highly amplified in 9.6% of the cases), PTPN1 region 3 Mb proximal (6.2%), and AIB3 region at 20q11 (6.2%) (Tanner (1996) Cancer Res. 56:3441-3445).
An “animal” refers to a member of the kingdom Animalia, characterized by multicellularity, the possession of a nervous system, voluntary movement, internal digestion, etc. An “animal” can be a human or other mammal. Preferred animals include humans, non-human primates, and other mammals. Thus, it will be recognized that the methods of this invention contemplate veterinary applications as well as medical applications directed to humans.
A “cancer” in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreas cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testis cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and the like.
The phrase “detecting a cancer” refers to the ascertainment of t
Pauletti Giovanni E.
Slamon Dennis J.
Arthur Lisa B.
Goldberg Jeanine
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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