Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-13
2003-03-25
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C530S350000, C530S387100
Reexamination Certificate
active
06537761
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of cytogenetics. In particular it provides new nucleic acid markers useful for detecting chromosomal alterations associated with ovarian and other cancers.
BACKGROUND OF THE INVENTION
Molecular genetic mechanisms responsible for the development and progression of many cancers remain largely unknown. Identification of sites of frequent and recurring allelic deletion or gain is a first step toward identifying some of the important genes involved in the malignant process. Previous studies in retinoblastoma (Friend, et al.
Nature,
323:643-6 (1986)) and other cancers (Cawthon, et al.,
Cell,
62:193-201 (1990); Baker, et al.,
Science,
244:217-21 (1989); Shuin, et al.,
Cancer Res,
54:2832-5 (1994)) have amply demonstrated that definition of regional chromosomal deletions occurring in the genomes of human tumors can serve as useful diagnostic markers for disease and are an important initial step towards identification of critical genes. Similarly, regions of common chromosomal gain have been associated with amplification of specific genes (Visakorpi, et al.,
Nature Genetics,
9:401-6 (1995)). Comparative genomic hybridization (CGH) is a relatively new molecular technique used to screen DNA from tumors for regional chromosomal alterations (Kallioniemi, et al.,
Science,
258:818-21 (1992) and WO 93/18186). Unlike microsatellite or Southern analysis allelotyping studies, which typically sample far less than 0.1% of the total genome, a significant advantage of CGH is that most parts of the test genome are scanned for losses and gains. Moreover, because CGH does not rely on naturally occurring polymorphisms, all regions are informative, whereas polymorphism-based techniques are uninformative in a significant fraction of each tumor studied.
Increases in copy number in the long arm of chromosome 3, in particular 3q25-3qter, has been associated with cancer. Increases in copy number in this area have been seen not only in ovarian tumors (Iwabuchi et al.,
Cancer Research
55:6172-8180 (1995) but also in brain tumors, head and neck cancer, lung cancer, ductal breast cancer, renal cell and other urinary tract cancers, and cervical cancer. Ried et al.,
Genes Chromosomes Cancer
15:234-45 (1996); Yeatman et al.
Clin Exp Metastasis
14:246-52 (1996); Brzoska et al.,
Cancer Res
15:3055-9 (1995); Ried et al.,
Cancer Res
54:1801-6 (1994); and Speicher et al.,
Cancer Res
55:1010-3 (1995).
The identification of narrower regions of genetic alteration or genes associated with cancers such as ovarian cancer would be extremely useful in the early diagnosis or prognosis of these diseases. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for detecting genetic alterations correlated with cancer. The invention can be used to detect alterations in a 2 MB region at 3q26.3 that are associated with a number of cancers. Examples include ovarian cancer, brain cancer, lung cancer, head and neck tumors, renal cell and other urinary tumors, cervical cancer, and ductal breast cancer. The invention is particularly useful for detecting alterations associated with ovarian cancer.
The methods comprise hybridizing a nucleic acid sample from a patient using a probe which binds selectively to a target nucleic acid sequence on 3q26.3 correlated with cancer. In particular, the invention provides sequences from genes encoding the catalytic subunit of phosphatidylinositol kinase type 3 (PIK3CA) or the glucose transporter, GLUT2. The probes of the invention are contacted with the sample under conditions in which the probe binds selectively with the target nucleic acid sequence to form a hybridization complex. The formation of the hybridization complex is then detected. Typically, the number of regions of hybridization are counted. Abnormalities are detected as increases above normal in the number of regions of hybridization.
Alternatively, sample DNA from the patient can be fluorescently labeled and competitively hybridized against normal DNA labeled to fluoresce at a different wavelength to normal arrays of nucleic acid molecules which map to 3q26.3. Alterations in DNA copy number in the sample DNA are then detected as increases in the intensity of signal from the tumor hybridizations relative to that from the normal hybridizations at the 3q26.3 region.
Definitions
A “nucleic acid sample” as used herein refers to a sample comprising DNA in a form suitable for hybridization to a probes of the invention. The nucleic acid may be total genomic DNA, total mRNA, genomic DNA or mRNA from particular chromosomes, or selected sequences (e.g. particular promoters, genes, amplification or restriction fragments, cDNA, etc.) within particular cancer-associated amplifications. The nucleic acid sample may be extracted from particular cells or tissues. The tissue sample from which the nucleic acid sample is prepared may be taken from a patient suspected of having the disease associated with the amplification being detected or from a normal patient to be used as a control. The sample may be prepared such that the nuclei in the tissue sample remain substantially intact and typically comprises interphase nuclei prepared according to standard techniques. A “nucleic acid sample” as used herein may also refer to a substantially intact condensed chromosome (e.g. a metaphase chromosome). Such condensed chromosomes or interphase nuclei are suitable for use as hybridization targets in in situ hybridization techniques (e.g. FISH). The particular usage of the term “nucleic acid sample” (whether as extracted nucleic acid or intact metaphase chromosome) will be readily apparent to one of skill in the art from the context in which the term is used. For instance, the nucleic acid sample can be a tissue or cell sample prepared for standard in situ hybridization methods described below. The sample is prepared such that individual chromosomes remain substantially intact and typically comprises metaphase spreads or interphase nuclei prepared according to standard techniques.
The sample or the probes used in the invention may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose) for use in Southern or dot blot hybridizations and the like. In some embodiments, the sample or probes may compriose an array of nucleic acids as described for instance in WO 96/17958. The prior art also describes techniques capable of producing high density arrays for various applications (see, e.g., Fodor et al.
Science
767-773 (1991) and U.S. Pat. No. 5,143,854). In some cases, the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization. The sample is typically taken from a patient suspected of having cancer associated with the abnormality being detected.
“Nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
“Subsequence” refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
A “probe” or a “nucleic acid probe”, as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a target within a nucleic acid sample can be detected. The probe is often labeled as described below so that its binding to the target can be detected. In some embodiments the probes are attached to a solid surface as an array of nucleic acid molecules. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, for example one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. The probes of the present invention are produced from nucleic acids found in the regions of genetic alteration as described herein. The probe may be processed in so
Gray Joe W.
Shayesteh Laleh
Souaya Jehanne
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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