Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-31
2003-04-22
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100
Reexamination Certificate
active
06551780
ABSTRACT:
BACKGROUND OF THE INVENTION
The melanocyte can give rise to a number of morphologically different tumors. Most of them are biologically benign and are referred to as melanocytic nevi. Examples of melanocytic nevi are congenital nevi, Spitz nevi (including pigmented spindle cell nevi, which are regarded as a subtype of Spitz nevi), dysplastic or Clark's nevi, blue nevi, lentigo simplex, and deep penetrating nevus.
Spitz nevi are benign melanocytic neoplasms that can have considerable histological resemblance to melanoma. They were first described as “juvenile melanoma” by Sophie Spitz in 1948 and initially regarded as a subset of childhood melanoma that follows a benign course (Spitz, S.,
Am. J. Pathol
. 24, 591-609 (1948)). Spitz nevi are common and account for about 1% of surgically removed nevi (Casso et al.,
J Am Acad Dermatol
., 27, 901-13 (1992)). Although in general the pathological diagnosis of Spitz nevus is straightforward, there is a subset of cases in which it is difficult to impossible to histologically differentiate Spitz nevi from melanoma because of overlapping histological features, such as the presence of melanocytes with abundant cytoplasm and/or melanocytes with large pleomorphic nuclei. Additionally, mitotic figures, sometimes numerous, occur in both neoplasms.
Melanoma refers to malignant neoplasms of melanocytes. Accurate diagnosis and early treatment is of great importance because, although advanced melanoma has a poor prognosis, most melanomas are curable if excised in their early stages. Although in general the histopathological diagnosis of melanoma is straightforward, there is a subset of cases in that it is difficult to differentiate melanomas from benign neoplasm of melanocytes (LeBoit,
P. E. SIMLULANTS OF MALIGNANT MELANOMA: A ROGUE'S GALLERY OF MELANOCYTIC AND NON
-
MELANOCYTIC IMPOSTERS
, In
Malignant Melanoma and Melanocytic Neoplasms
, P. E. Leboit, ed. (Philadelphia: Hanley & Belfus), pp. 195-258 (1994)). Even though the diagnostic criteria for separating the many simulators of melanoma are constantly refined, a fraction of cases remains where an unambiguous diagnosis cannot be reached (Farmer et al.,
DISCORDANCE IN THE HISTOPATHOLOGIC DIAGNOSIS OF MELANOMA AND MELANOCYTIC NEVI BETWEEN EXPERT PATHOLOGISTS, Human Pathol
. 27: 528-31 (1996)). The most frequent and important diagnostic dilemma is the differential diagnosis between Spitz nevus and melanoma.
Misdiagnosis of Spitz nevus as melanoma and vice versa has been repeatedly reported in the literature (Goldes et al.,
Pediatr. Dermatol
., 1: 295-8 (1984); Okun, M. R.
Arch. Dermatol
. 115: 1416-1420 (1979); Peters et al.,
Histopathology
, 10, 1289-1302 (1986)). A retrospective study of 102 melanomas of childhood found that only 60 cases were classified as melanoma by a panel of experts, the majority of the remainder being classified as Spitz nevi (Spatz, S.,
Int. J. Cancer
68, 317-24 (1996)). The presence of this diagnostic gray zone has even led the authors of a review article in the “Continuing Medical Education” section of the
Journal of the American Association of Dermatology
to conclude that Spitz nevus and melanoma may “actually exist on a continuum of disease” (Casso et al.,
J. Am. Acad. Dermatol
., 27, 901-13 (1992)). The authors recommended that “treatment include complete excision of all Spitz nevi followed by reexcision of positive margins if present.” The need for improved diagnostics for melanocytic neoplasms has led to numerous attempts to improve diagnostic accuracy by the use of markers that could be detected by immuno-histochemistry. While there have been prior efforts aimed at resolving this problem, none have been satisfactory. For example, even though tests employing markers such as S100, HMB45 are useful in establishing that a poorly differentiated tumor is of melanocytic lineage, adjunctive techniques have been of little help in separating benign from malignant melanocytic lesions.
Thus, there exists a great need for improved and accurate diagnostic methods to distinguish Spitz nevi from malignant melanoma. Furthermore, there is a need to distinguish melanocytic neoplasms that fall between Spitz nevi and malignant and are difficult to classify. The present invention addresses these and other needs by providing methods of typing a melanocytic neoplasm by detecting in a tumor sample the presence of an increase in copy number of an 11 p chromosome arm, particularly, detecting the presence of an 11p isochromosome, which indicates the presence of a Spitz nevus. Typing can also be performed by determining the presence of a mutated H-RAS gene, which is also associated with, or indicates the presence of a Spitz nevus.
SUMMARY OF THE INVENTION
The present invention provides for methods of typing a melanocytic neoplasm by detecting the presence of a mutated H-RAS gene in a patient sample, whereby the presence of the mutation in the H-RAS gene indicates the presence of a Spitz nevus, or classification as a Spitz nevus. Frequently, the mutation in the H-RAS gene is at codon 12, 13, or 61.
Typically, the copy number of the entire chromosome 11p including the H-RAS gene is increased relative to normal in the patient sample. The increase in gene copy number is frequently due to the presence of an 11p isochromosome.
In one embodiment of the invention, the presence of a mutation in the H-RAS gene is detected by amplifying a nucleic acid that encodes H-RAS or a fragment, and sequencing the amplified product to determine whether the sequence contains a mutation relative to a normal H-RAS sequence. Amplification is typically performed using PCR. Primers for the PCR reaction include those set out in SEQ ID NOs: 1 and 2, and SEQ ID NOs: 3 and 4. The nucleic acid that is amplified can be genomic DNA or RNA.
In another aspect of the invention, the presence of a mutation in the H-RAS gene is detected by contacting a nucleic acid from a skin tumor sample with a probe that selectively hybridizes to a target nucleic acid comprising an H-RAS gene to form a stable hybridization complex. The probe is contacted under condition in which the probe binds selectively to the target nucleic acid that includes the H-RAS gene. In one embodiment, the probe binds selectively to a mutated H-RAS gene. The method can further include a step of amplifying the nucleic acid from the sample. Preferably, the amplifying step is a PCR reaction, which can be performed, e.g., using oligonucleotides as set out in SEQ ID NOs: 1 and 2, and 3 and 4. The nucleic acid from the sample is preferably genomic DNA or RNA.
The invention also includes a method of detecting the presence of a mutated H-RAS gene by detecting a polypeptide encoded by the mutant H-RAS gene. Preferably the amount of polypeptide is quantified using an immunoassay, e.g., ELISA. In one embodiment, the polypeptide is detected using an antibody that selectively binds to the polypeptide encoded by the mutant H-RAS gene.
The methods of the invention further include a method of typing a patient melanocytic neoplasm from a patient by detecting the presence of an increase in copy number of the 11p chromosome arm whereby the presence of the increase in copy number of the 11p arm is indicates the presence of a Spitz nevus, or diagnosis of Spitz nevus. Typically, the methods comprise detecting the presence of an 11p isochromosome in a sample from a patient.
In one embodiment, the 11p isochromosome is detected by hybridizing a nucleic acid from the skin tumor sample with a probe that selectively hybridizes to sequences on chromosome 11p that are adjacent to the centromere and detecting the presence of one or more pairs of hybridization signals compared to normal. Additionally, the method can include hybridization of the nucleic acid sample with a second probe that is labeled with a second label distinguishable from the first and selectively hybridizes to a target nucleic acid sequence at chromosome 11q adjacent to the centromere; and detecting the presence of at least one pair of hybridization signals that consists of a signal from each of the probes
Bastian Boris
Pinkel Daniel
Goldberg Jeanine
Jones W. Gary
The Regents of University of California
Townsend and Townsend / and Crew LLP
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