Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-19
2002-09-17
Whisenant, Ethan C. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06451529
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of cytogenetics. In particular it provides new diagnostic nucleic acid markers for prostate cancer.
BACKGROUND OF THE INVENTION
Molecular genetic mechanisms responsible for the development and progression of prostate cancer 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)). Additionally, definition of the full spectrum of common allelic changes in prostate cancer may lead to the association of specific changes with clinical outcome, as indicated by recent studies in colon cancer and Wilms' tumor (Jen, et al.,
N. Engl. J. Med.,
331:213-21 (1994); Grundy, et al.,
Cancer Res,
54:2331-3 (1994)).
Prostate cancer allelotyping studies (Carter, et al.,
Proc Natl Acad Sci USA,
87:8751-5 (1990); Kunimi, et al.,
Genomics,
11:530-6 (1991)) designed to investigate one or two loci on many chromosomal arms have revealed frequent loss of heterozygosity (LOH) on chromosomes 8p (50%), 10p (55%), 10q (30%), 16q (31-60%) and 18q (17-43%). Recently, several groups have performed more detailed deletion mapping studies in some of these regions. On 8p, the high frequency of allelic loss has been confirmed, and the regions of common deletion have been narrowed (Bova, et al.,
Cancer Res,
53:3869-73 (1993); MacGrogan, et al.,
Genes Chromosom Cancer,
10:151-159 (1994); Bergerheim, et al.,
Genes Chromosom Cancer,
3:215-20 (1991); Chang, et al., Am T Pathol, 144:1-6 (1994); Trapman, et al.,
Cancer Res,
54:6061-4 (1994); Suzuki, et al.,
Genes Chromosom Cancer,
13:168-74 (1995)). Similar efforts also served to narrow the region of common deletion on chromosome 16q (Bergerheim, et al.,
Genes Chromosom Cancer,
3:215-20 (1991); Cher, et al.,
J Urol,
153:249-54 (1995)). Other prostate cancer allelotyping studies utilizing a smaller number of polymorphic markers have not revealed new areas of interest (Phillips, et al.,
Br J Urol,
73:390-5 (1994); Sake, et al.,
Cancer Res,
54:3273-7 (1994); Latil, et al.,
Genes Chromosom Cancer,
11:119-25 (1994); Massenkeil, et al.,
Anticancer Res,
14:2785-90 (1994)). At present, allelotyping studies are limited by the low number of loci studied, low case numbers, heterogeneous groups of patients, the use of tumors of low or unclear purity, and lack of standardization of experimental techniques. For these reasons, it has been difficult to compare frequencies of alterations between studies, and we have yet to gain an overall view of regional chromosomal alterations occurring in this disease.
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 all chromosome arms 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 limited by homozygous (uninformative) alleles among a fraction of tumors studied at every locus.
CGH can detect and map single copy losses and gains in prostate cancer with a high degree of accuracy when compared with the standard techniques of allelotyping (Cher, et al.,
Genes Chromosom Cancer,
11:153-162 (1994)). Copy-number karyotype maps have been generated for prostate cancer showing several recurrently altered regions of the genome (Cher, et al.,
Genes Chromosom Cancer,
11:153-162 (1994); Visakorpi, et al.,
Cancer Res,
55:342-347 (1995)).
Although previous studies have begun to reveal a genome-wide view of chromosomal alterations occurring in primary and recurrent prostate cancer, metastatic prostate cancer has not been examined in depth. The present invention addresses these and other needs in the prior art.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods of detecting a genetic alterations correlated with prostate cancer. The methods comprise contacting a nucleic acid sample from a patient with a probe which binds selectively to a target polynucleotide sequence correlated with prostate cancer. The invention provides the following chromosomal regions which are deleted in prostate cancer cells: 2q, 4q, 5q, 6q, 10p, and 15q. Regions which show increases in copy number in prostate cancer cells are: 1q, 2p, 3q, 3p, 4q, 6p, 7p, 7q, 9q, 11p, 16p, and 17q.
The probes of the invention are contacted with the sample under conditions in which the probe binds selectively with the target polynucleotide sequence to form a hybridization complex. The formation of the hybridization complex is then detected.
Alternatively, sample DNA from the patient can be fluorescently labeled and competitively hybridized against fluorescently labeled normal DNA to normal lymphocyte metaphases. Alterations in DNA copy number in the sample DNA are then detected as increases or decreases in sample DNA as compared to normal DNA.
The chromosome abnormality is typically a deletion or an increase in copy number. The methods can be used to detect both metastatic prostate cancers and in androgen independent prostate cancer.
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. 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 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 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 a prostate 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 can be detected. The probe is labeled as described below so that its binding to the target can be detected. 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 some manner, for example, by blocking or removal of repetitive nucleic acids or enrichment with un
Cher Michael L.
Jensen Ronald H.
The Regents of the University of California
Townsend & Townsend & Crew LLP
Whisenant Ethan C.
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