Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-17
2002-10-15
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S174000, C435S283100, C435S287200, C536S023100, C536S024300, C536S024330, C536S026600
Reexamination Certificate
active
06465180
ABSTRACT:
BACKGROUND OF THE INVENTION
Melanoma refers to malignant neoplasms of melanocytes. Its proper diagnosis and early treatment by complete excision is of great importance because advanced melanoma has a poor prognosis and most melanomas are curable if excised in their early stages. In most instances the transformed melanocytes produce increased amounts of pigment so that the area involved can easily be seen by the clinician. When the excision margins of a melanoma are identified based on this macroscopic appearance and no margin of seemingly uninvolved skin is excised, melanoma has the risk of local recurrence.
This has led to the recommendation to remove a safety margin of normal skin that varies from 0.5 to 3 cm depending on the thickness of the primary tumor (Wingo, P. A. et al.,
Cancer
82:1197-207 (1998); Rigel, D. S. et al.,
J Am Acad Dermatol
34:839-47 (1996); McGovern, V. J. et al.,
Cancer
32:1446-57 (1973)). It is obvious that the resulting defect inflicted by the excision can be considerable. If a melanoma measuring 2 cm in diameter that has a thickness of >4 mm is to be excised under the current guidelines, the resulting defect would be 8 cm (2+3+3 cm) in diameter. The closure of excisions with 2-3 cm margins usually require skin grafting and have the potential of adverse consequences such as unsatisfactory cosmetic result, increased morbidity and costs, and sometimes permanent functional impairment. Even with “adequate” safety margins, the melanoma can recur locally.
Obviously, it would be desirable if the margins could be tailored to the needs of the individual patient's tumor. Unfortunately, so far, no technique exists that is able to detect the extent of a tumor accurately. In some types of melanomas the horizontally expanding portion of the tumor mainly consists of single melanocytes along the basal layer of the epidermis. These melanoma types are referred to as lentiginous melanomas. In these, the amount of atypical cells often gradually diminishes towards the margins so that it can be difficult or impossible for the pathologist to determine the border of the melanoma. However, current thinking implies that in most instances, the extent of a melanoma can be assessed by pathology. The fact that the removal of a margin of “healthy” skin reduces the recurrence rate, however, suggests that this skin is actually not healthy but contains residual melanoma which is undetectable by current methods.
The identification of useful means by which morphologically normal premalignant cells that have the capacity to form melanomas can be identified. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides methods of screening for the presence of premalignant melanocytes in a sample from a patient. The methods comprise contacting a nucleic acid sample from a biological sample from the patient with a probe which binds selectively to a target polynucleotide sequence on a chromosomal region which is amplified in melanoma cells. Usually, the copy number of the target sequence is determined. The nucleic acid sample is typically from morphologically normal cells adjacent to a melanoma lesion in the patient.
In the methods, the probe is contacted with the sample under conditions in which the probe binds selectively with the target polynucleotide sequence to form a stable hybridization complex and the formation of a hybridization complex is detected. The target sequence is selected from the group consisting of 11p15, 11q13, 22q12, 7p, 6p, 1q, 12q14, and 5p.
The nature of the nucleic acid sample is not critical to the invention. In some embodiments, the nucleic acid sample is a metaphase spread or an interphase nucleus. Typically, the probe is labeled e.g. with a fluorescent label. The label may be a direct label. Usually, a reference probe to a second chromosomal region (e.g. a centromere) is used in the methods as an internal control. In these embodiments, the second probe is labeled with a fluorescent label distinguishable from the label on the probe that selectively hybridizes to the target polynucleotide sequence.
In some embodiments, the probe may include repetitive sequences. In this case, the methods may further comprising the step of blocking the hybridization capacity of repetitive sequences the probe Unlabeled blocking nucleic acids comprising repetitive sequences (e.g. Cot-1 DNA) can be contacted with the sample for this purpose.
The nucleic acid hybridization can be carried out in a number of formats. For instance, the hybridization may be an in situ hybridization. In some embodiments, the probe is bound to a solid substrate e.g. in as a member of a nucleic acid array.
Definitions
To facilitate understanding 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 melanomas.
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 melanomas, 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 the presence or absence of cancer in an animal, in this case, melanoma cells or premalignant melanocytes. “Detecting a cancer” can also refer to obtaining indirect evidence regarding the likelihood of the presence of cancerous cells in the animal or to the likelihood or predilection to development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, or in combination with other methods or in light of other information regarding the state of health of the animal.
The terms “hybridizing specifically to” and “specific hybridization” and “selectively hybridize to,” as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. A “stringent hybridization” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization (e.g., as in array, Southern or Northern hybridizations) are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I, chapt
2, “
Overview of principles of hybridization
Bastian Boris
Pinkel Daniel
Forman B J
Jones W. Gary
The Regents of the University of California
Townsend and Townsend / and Crew LLP
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