WO2000061814A9 - Detection of chromosome copy number changes to distinguish melanocytic nevi from malignant melanoma - Google Patents

Abstract

The present invention provides for methods of distinguishing melanocytic nevi, such as Spitz nevi, from malignant melanoma. The methods comprise contacting a nucleic acid sample from a patient with a probe which binds selectively to a target polynucleotide sequence on a chromosomal region such as 11p, which is usually amplified in Spitz nevi. The nucleic acid sample is typically from skin tumor cells located within a tumor lesion on the skin of the patient. Using another probe which binds selectively to a chromosomal region such as 1q, 6p, 7p, 9p, or 10q, which usually show altered copy number in melanoma, the method can determine that those tumor cells with no changes in copy number of 1q, 6p, 7p, 9p, or 10q, are not melanoma cells but rather Spitz nevus cells. The finding of amplifications of chromosome 11p, particularly the presence of an 11p isochromosome would be an additional indication of Spitz nevus. An increase in copy number of chromosome 11p can also be determined by detecting the presence of an amplification of the H-RAS gene. The amplified gene can be normal or can be a mutated H-RAS gene.

Description

DETECTION OF CHROMOSOME COPY NUMBER CHANGES TO DISTINGUISH MELANOCYTIC NEVI FROM MALIGNANT

MELANOMA

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 pleomoφhic 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. SIMULANTS 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 SI 00, 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 1 lp chromosome arm, particularly, detecting the presence of an 1 lp isochromosome, which indicates the presence of a Spitz nevus. Typing can also be performed by determining the presence in a tumor sample of an amplification of chromosome 1 lp 15.5, and particularly by detecting the amplifcation of R-RAS. An additional aspect of typing is the detection of a mutated rl-RAS gene present in a tumor sample, which is also associated with, or indicates the presence of a Spitz nevus. SUMMARY OF THE INVENTION The present invention provides for methods of distinguishing melanocytic nevi, such as Spitz nevi, from malignant melanoma. The methods comprise detecting a target polynucleotide sequence, e.g., H-RAS, on a chromosomal region such as 1 lp, particularly 1 lpl5.5, which is frequently amplified in Spitz nevi. The nucleic acid sample is typically taken from skin tumor tissue located within a tumor lesion on the skin of the patient. The methods can also be used to determine whether the tumor cells lack changes in chromosomal regions associated with melanoma (e.g., lq, 6p, 7p, or lOq). Usually, the copy number of the target region is measured.

The methods of the invention further include a method of typing a melanocytic neoplasm from a patient by detecting the presence of an increase in copy number of the 1 lp chromosome arm thereby typing the melanocytic neoplasm as a Spitz nevus. Typically, the methods comprise detecting the presence of an 1 lp isochromosome in a tumor sample from a patient.

The nucleic acid sample can be extracted from an inteφhase 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 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. as a member of a nucleic acid array.

In one embodiment of the invention, a melanocytic neoplasm can be typed as a Spitz nevus by detecting the presence of a mutation in the H-RAS gene. The mutation can be 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 oHgonucleotides 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 an amplified H-RAS gene by detecting a polypeptide encoded by the 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 a polypeptide encoded by a mutant H-RAS gene.

Definitions

To facilitate understanding the invention, a number of terms are defined below.

The terms "melanoma" or "cutaneous melanoma" refer to malignant neoplasms of melanocytes, which are pigment cells present normally in the epidermis and sometimes in the dermis. There are four types of cutaneous melanoma: lentigo maligna melanoma, superficial spreading melanoma (SSM), nodular melanoma, and acral lentiginous melanoma (AM). Melanoma usually starts as a proliferation of single melanocytes at the junction of the epidermis and the dermis. The cells first grow in a horizontal manner and settle an area of the skin that can vary from a few millimeters to several centimeters. As noted above, in most instances the transformed melanocytes produce increased amounts of pigment so that the area involved can easily be seen by the clinician.

The term "melanocytic neoplasm" refers to an accumulation of melanocytes that can undergo a benign, locally aggressive, or malignant course. "Melanocytic neoplasm" encompasses both benign melanocytic neoplasms, "nevi", and malignant melanocytic neoplasms, "melanoma".

The terms "Spitz nevi" or "Spitz nevus" refer to melanocytic neoplasms that can have considerable histological resemblance to melanoma. They generally are benign, but can recur locally, or rarely, spread to the lymph nodes. They were first described as "juvenile melanoma" and initially were thought of as a subset of childhood melanoma that follows a benign course. Spitz nevi are common and account for about 1% of surgically removed nevi.

The terms "tumor" or "cancer" in an animal refers to the presence of cells possessing characteristics such as atypical growth or moφhology, including uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic moφhological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal. "Tumor" includes both benign and malignant neoplasms. The phrase "typing" or "detecting" a neoplasm refers to the determination whether the neoplasm is, or has a high probability of being, a certain class of neoplasm. Classification can be based on whether the neoplasm is benign or. malignant, or type of nevus, e.g., Spitz nevus. "Typing" or "detecting" can also refer to obtaining indirect evidence regarding the likelihood of the presence of a Spitz nevus or melanoma in the patient. Detection of a Spitz nevus versus a melanoma 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 patient.

The terms "hybridizing specifically to", "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, Ch. 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY ("Tijssen"). Generally, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42 °C using standard hybridization solutions (see, e.g., Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed discussion, below), with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72 °C for about 15 minutes. An example of stringent wash conditions is a 0.2X SSC wash at 65 °C for 15 minutes (see, e.g., Sambrook supra.) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 °C for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4X to 6X SSC at 40 °C for 15 minutes.

The term "labeled with a detectable label", as used herein, refers to a nucleic acid attached to a detectable composition, i.e., a label. The detection can be by, e.g., spectroscopic, photochemical, biochemical, immunochemical, physical or chemical means. For example, useful labels include ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵1, ¹³¹I; fluorescent dyes (e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense reagents (e.g. gold), enzymes, e.g., as commonly used in an ELISA (e.g., horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g. colloidal gold), magnetic labels (e.g. Dynabeads™ ), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label can be directly incoφorated into the nucleic acid, peptide or other target compound to be detected, or it can be attached to a probe or antibody that hybridizes or binds to the target. Label can be attached by spacer arms of various lengths to reduce potential steric hindrance or impact on other useful or desired properties. See, e.g., Mansfield, Mol Cell Probes 9: 145-156 (1995). In addition, target DNA sequences can be detected by means of the primed in situ labeling technique (PRLNS) (Koch et al., Genet. Anal. Tech. Appl 8: 171-8, (1991)). The sensitivity of the detection can be increased by using chemical amplification procedures, e.g., by using tyramide (Speel et al., J. Histochem. Cytochem. 45:1439-46, (1997)).

The term "paired hybridization signals" or a "hybridization signal pair" refers to a spatial pattern of hybridization signals wherein two signals are consistently identified in close proximity. Isochromosomes are typically characterized by the presence of "paired hybridization signals" from a single probe. For example, in a sample with many cells, a "hybridization signal pair" is a consistent occurrence of two signals in close proximity that is clearly not due to an artifact or a random event.

The term "nucleic acid" as used herein refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oHgonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the puφoses desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for the puφoses desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. DNA backbone analogues provided by the invention include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, moφholino carbamate, and peptide nucleic acids (PNAs); see OHgonucleotides and Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford University Press (1991); Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) j. Med. Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197. Other synthetic backbones encompasses by the term include methylphosphonate linkages or alternating methylphosphonate and phosphodiester linkages (Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate linkages (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.

The term a "nucleic acid array" as used herein is a plurality of target elements, each target element comprising one or more nucleic acid molecules (probes) immobilized on one or more solid surfaces to which sample nucleic acids can be hybridized. The nucleic acids of a target element can contain sequence(s) from specific genes or clones, e.g. from the regions identified here. Other target elements will contain, for instance, reference sequences. Target elements of various dimensions can be used in the arrays of the invention. Generally, smaller, target elements are preferred. Typically, a target element will be less than about 1 cm in diameter. Generally element sizes are from 1 μm to about 3 mm, preferably between about 5 μm and about 1 mm. The target elements of the arrays may be arranged on the solid surface at different densities. The target element densities will depend upon a number of factors, such as the nature of the label, the solid support, and the like. One of skill will recognize that each target element may comprise a mixture of nucleic acids of different lengths and sequences. Thus, for example, a target element may contain more than one copy of a cloned piece of DNA, and each copy may be broken into fragments of different lengths. The length and complexity of the nucleic acid fixed onto the target element is not critical to the invention. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization procedure, and to provide the required resolution among different genes or genomic locations. In various embodiments, target element sequences will have a complexity between about 1 kb and about 1 Mb, between about 10 kb to about 500 kb, between about 200 to about 500 kb, and from about 50 kb to about 150 kb.

The terms "nucleic acid sample" or "sample of human nucleic acid" as used herein refers to a sample comprising human DNA or RNA in a form suitable for detection by hybridization or amplification. Typically, it will be prepared from a skin tissue sample from a patient who has or is suspected of having melanocytic tumor that may be difficult to classify. The sample will most usually be prepared from tissue taken from the tumor. In many instances, the nucleic acid sample will 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 prepared according to standard techniques. Alternatively, the nucleic acid may be isolated, cloned or amplified. It may be, e.g., genomic DNA, mRNA, or cDNA from a particular chromosome, or selected sequences (e.g. particular promoters, genes, amplification or restriction fragments, cDNA, etc.) within particular amplicons or deletions disclosed here.

The nucleic acid sample may be extracted from particular cells or tissues, e.g. melanocytes. Methods of isolating cell and tissue samples are well known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, needle biopsies, and the like. Frequently the sample will be a "clinical sample" which is a sample derived from a patient, including sections of tissues such as frozen sections or paraffin sections taken for histological puφoses. The sample can also be derived from supematants (of cells) or the cells themselves from cell cultures, cells from tissue culture and other media in which it may be desirable to detect chromosomal abnormalities or determine amplicon copy number. In some cases, the nucleic acids may be amplified using standard techniques such as PCR, prior to the hybridization. The sample may be isolated nucleic acids immobilized on a solid. The term "probe" or "nucleic acid probe", as used herein, is defined to be a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected. The probe may be unlabeled or labeled as described below so that its binding to the target or sample can be detected. The probe is produced from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., 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 described herein. A probe that is "adjacent to the centromere" refers to a probe that hybridize to regions adjacent to the centromere bind to sequences at 1 lpl 1.1 to 1 lpl 1.2 or l lql l .l to l lql l.2.

An "Up chromosome arm" is defined cytogeneticallya s encompassing the chromosome from band 1 lpl 1 to 1 lpter.

The probe or genomic nucleic acid sample may be processed in some manner, e.g., by blocking or removal of repetitive nucleic acids or enrichment with unique nucleic acids. The word "sample" may be used herein to refer not only to detected nucleic acids, but to the detectable nucleic acids in the form in which they are applied to the target, e.g., with the blocking nucleic acids, etc. The blocking nucleic acid may also be referred to separately. What "probe" refers to specifically is clear from the context in which the word is used. The probe may also be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. In some embodiments, the probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this puφose (see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124; U.S. Patent No. 5,143,854). One of skill will recognize that the precise sequence of the particular probes described herein can be modified to a certain degree to produce probes that are "substantially identical" to the disclosed probes, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived (see discussion above). Such modifications are specifically covered by reference to the individual probes described herein.

The term "immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background, more typically more than 10 to 100 times background, and do not substantially bind in a significant amount to other proteins present in the sample. The term "immunoassay" is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background, more typically more than 10 to 100 times background, and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under some conditions may require an antibody that is selected for its specificity for a particular H-RAS protein. For example, an antibody that selectively binds to a polypeptide encoded by a mutated H-RAS gene binds to mutated, but not normal H-RAS.

"Providing a nucleic acid sample" means to obtain a biological sample for use in the methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g. isolated by another person), or by performing the methods of the invention in vivo.

"Tissue biopsy" refers to the removal of a biological sample for diagnostic analysis. In a patient with cancer, tissue may be removed from a tumor, allowing the analysis of cells within the tumor.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the summary of chromosome copy number changes in 32 primary cutaneous melanomas. Chromosomal gains are shown as lines to the right of the chromosome ideogramms, losses are shown as lines to the left. Thick lines to the right indicate amplifications, thick lines to the left summarize losses in 10 cases (Bastian et al., Cancer Res 58: 2170-5, 1998).

Fig. 2 shows the summary of chromosome copy number changes in 17 Spitz nevi. Chromosomal gains are shown as lines to the right of the chromosome ideogramms. Thick lines indicate amplifications.

Fig. 3 shows the average ratio profiles of fluorescence intensity of tumor vs. reference DNA in the four Spitz nevi that had abnormal CGH profiles. The dotted lines indicate the 1.2 and 0.8 ratio thresholds that were used for defining aberrations, n indicates the number of chromosomes measured for the respective profile. Fig. 4 shows the frequency distribution of hybridization signals after dual- target hybridization of probe RMC11B022 for chromosome 1 lp (black bars) and RMC11P008 for chromosome 1 lq (white bars). Three cases of Spitz nevi are shown. Case 2 (A, B) showed no chromosomal aberrations by CGH, Case 13 (C, D) had an gain of chromosome 1 lp by CGH, Case 15 (E, F) did not show aberrations by CGH, it had a subpopulation of tumor cells with large nuclei. Charts A, C, E show signal distribution in tumor cells; Charts B, D, F show signal distribution in keratinocytes of the corresponding lesions.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Introduction

The present invention provides for unique and accurate methods for distinguishing Spitz nevus, from malignant melanoma. This invention is based upon the observation that chromosomal regions that have frequently altered copy numbers in melanoma such as lq, 6p, 7p, 9p, or lOq, are rarely changed in Spitz nevi. In addition, Spitz nevi cells show a single amplification of chromosomal region 1 lp, particularly 1 lpl 5.5, and more particularly, the H-RAS gene, which is localized to 1 lpl 5.5, as shown by the increase of its copy number, a phenomenon that is exceedingly rare in melanoma. The amplification of chromosome 1 lp typically occurs via amplification of the lip chromosome arm, and is characterized by the presence of an 1 lp isochromosome. This difference in pattern of chromosomal aberrations between Spitz nevi and melanoma can lead to more accurate diagnostic distinction of Spitz nevi from melanoma.

The present invention further provides methods of typing a melanocytic neoplasm by detecting in a skin tumor sample the presence of a mutated H-RAS gene that is associated with the diagnosis of a Spitz nevus.

The frequency of chromosomal aberrations among melanoma cells, including primary and metastatic melanoma has been studied using CGH (Bastian et al, Cancer Res 58, 2170-5 (1998). One of the findings of this experiment was the frequent loss of chromosome 9 and chromosome 10 that occurred in 81% and 63% of the tumors, respectively. By comparing the frequency of occurrence in thin and thick tumors, and comparing parts of tumors that were in different phases of tumor progression, it was discovered that losses of chromosomes 9 and 10 occurred early in tumorigenesis.

Another set of experiments was performed, extending the data set to 70 tumors. Results from the second set of experiments confirmed that losses of chromosomes 9 and 10 are the most frequent changes in primary melanomas of the skin. In these 70 melanomas only four exhibited no changes by CGH. Results of these experiments performed with melanoma cells are shown in Fig. 1.

There have been several studies of ploidy in Spitz nevi using measurement of nuclear DNA content by image cytometry or flow cytometry (How at et al, Cancer 63, 474-8 (1989); LeBoit et al, J Invest Dermatol 88, 753-7 (1987); Otsuka et al., Clin Exp Dermatol 18, 421-4 (1993); Vogt et al, Am J Dermatopathol 18, 142-50 (1996)). However, routine application of these techniques has been hampered by the complexity of the procedure and its lack of sensitivity. Recently, molecular cyotgenetic analysis has shown that Spitz nevi, particularly a subset of Spitz nevi, exhibit amplification of chromosome 1 lp (see, e.g., Bastian et al, J. Invest. Dermatol. 113:1065-1069, 1999; and Bastian et al. , Cancer Res. 58:2170-2175, 1998), including the l lpl5.5 region. As disclosed herein, amplification with or without mutations of the H-RAS gene, which is localized to 1 lpl5.5, are also present in Spitz nevi. As H-RAS is rarely mutated in melanoma (see, e.g., Jiveskog et al, J. Invest. Dermatol. 111 :757-761, 1998; van Elsas et al, Am J. Pathol. 149:883-893, 1996), but is mutated in Spitz nevi, mutations in H-RAS can be used to further distinguish a Spitz nevus from melanoma. Accordingly, the present invention provides methods for determining the presence of an amplified H-RAS gene and/or a mutation in an H-RAS gene in a melanocytic neoplasm in order to determine if the neoplasm is a Spitz nevus.

It has been determined that a subset of Spitz nevi recur following excision. The subset typically is characterized by an amplification of the entire 1 lp chromosome arm, in particular, by the presence of an 1 lp isochromosome. Amplification of the entire arm of chromosome li p has not been observed in melanoma. Thus, the present invention also provides methods of typing or classifying a melanocytic neoplasm as a Spitz nevus by detecting the presence of an increase in copy number of the entire arm of 1 lp, in particular detecting the presence of an 1 lp isochromosome.

General methods for measuring chromosomal abnormality Genomic instability is a hallmark of solid tumors, and virtually no solid tumor exists which does not show major alterations of the genome. With the vast majority of tumors this instability is expressed at the level of the chromosomal complement, and thus is detectable by cytogenetic approaches (Mitelman, F., Catalog of chromosome aberrations in cancer, 5th Edition (New York: Wiley-Liss) (1994)). However, aneuploidy per se is not indicative of malignancy and many benign tumors can have an aberrant karyotype (Mitelman, 1994). To efficiently take advantage of aneuploidy as a marker, it is mandatory to know characteristic aberrations of the tumors that are to be differentiated.

Several techniques that permit the study of chromosomal complement in post-fixation tissue have been developed. Fluorescence in-situ hybridization (FISH) can be used to study copy numbers of individual genetic loci in inteφhase nuclei (Pinkel et al, Proc. Natl Acad. Sci. U.S.A. 85, 9138-42 (1988)) and comparative genomic hybridization (CGH) (Kallioniemi et al. Science 258, 818-2 1 (1992)) has proven a useful technique (Houldsworth et al. Am J Pathol 145, 1253-60 (1994)) to probe the entire genome for copy number changes of chromosomal regions.

The application of FISH as an adjunctive diagnostic technique for the differentiation of Spitz nevi from melanomas has been suggested previously (De Wit et al, J Pathol. 173, 227-33 (1994)). The investigators used a centromeric probe for chromosome 1 and found a significant difference in the number of cells with an aberrant number of signals between 15 melanoma and 15 Spitz nevi. At this point no detailed knowledge about chromosomal changes in primary melanomas of the skin was available and chromosome 1 was selected based on its frequent numerical change in melanoma metastasis (Thompson et al, Cancer Genet Cytogenet 83, 93-104 (1995)). It is to be expected that a selection of a panel of chromosomal markers of regions that are frequently involved in primary melanomas could increase sensitivity and specificity to a level that would allow the application of FISH as a routine method. To achieve this goal, it is essential to know the pattern of aberrations in melanomas as well as its benign counteφarts.

Detection of Copy Number

Methods of evaluating the copy number of a particular gene or chromosomal region are well known to those of skill in the art. In this invention, the presence or absence of chromosomal gain or loss can be evaluated simply by a determination of copy number of the regions identified here. Typically, the regions evaluated are lq, 6p, 7p, 9p, lOq, and l ip.

Hybridization-based Assays

Preferred hybridization-based assays include, but are not limited to, traditional "direct probe" methods such as Southern Blots or In Situ Hybridization (e.g., FISH), and "comparative probe" methods such as Comparative Genomic Hybridization (CGH). The methods can be used in a wide variety of formats including, but not limited to substrate (e.g. membrane or glass) bound methods or array-based approaches as described below. In situ hybridization assays are well known (e.g., Angerer (1987) Meth.

Enzymol 152: 649). Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and the conditions for use vary depending on the particular application. In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained.

The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters. The preferred size range is from about 200 bp to about 1000 bases, more preferably between about 400 to about 800 bp for double stranded, nick translated nucleic acids.

In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, human genomic DNA or Cot-1 DNA is used to block non-specific hybridization. In Comparative Genomic Hybridization methods a first collection of

(sample) nucleic acids (e.g. from a possible tumor) is labeled with a first label, while a second collection of (control) nucleic acids (e.g. from a healthy cell/tissue) is labeled with a second label. The ratio of hybridization of the nucleic acids is determined by the ratio of the two (first and second) labels binding to each fiber in the array. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number.

Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, NJ (1994), etc. In one particularly preferred embodiment, the hybridization protocol of Pinkel et al. (1998) Nature Genetics 20: 207-211 or of Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.

Detection of isochromosomes

Changes in copy number of a particular gene or chromosomal region can be due to a number of mechanisms, including the presence of an isochromosome, in which one of the arms of a chromosome is duplicated, thus increasing the copy nubmer of the sequences located on the duplicated arm. Methods of evaluating the copy number of a particular gene or chromosomal region, and particularly analyzing for the presence of an isochromosome, are well known to those of skill in the art. An increase in copy number of the whole arm of chromosome 11 can be detected, e.g., using procedures described in co-pending application U.S.S.N. 09/288,940. Copy number changes, and particularly, isochromosomes, are typically detected using hybridzation-based assays, such as FISH. The presence of an isochromosome can be detected using a single probe to that hybridizes to a region on the duplicated chromosomal arm. Typically, the probe will be localized to a regions of the chromosomal arm that is adjacent to the centromere. Normal cells have two randomly positioned signals in their nucleus. Cells that possess an isochromosome will have one to several pairs of signals present in the nucleus.

Preferably, an isochromosome is detected using two probes, each labeled with a distinct compound, e.g., different fluorescent labels with distinguishable colors. Usually, the analysis employs two probes that hybridize to nucleic acid sequences close to the centromere. One of the probes hybridizes to target sequences on the p arm that are adjacent to the centromere, e.g., sequences localized to 1 lpl 1.1 or 1 lpl 1.2. The second probe hybridizes to target sequences on the q arm adjacent to the centromer, i.e., 1 lql 1.1 or 1 lql 1.2. An isochromosome is detected by determining the presence of hybridization regions that occur as pairs of the same color compared to a normal situation in which the visualized pairs contain two colors.

Detection of Mutations in H-RAS

The H-RAS gene is located at 1 lpl 5.5, a region which has been shown to be amplified in a subset of Spitz nevi. (Bastian et al, J. Invest. Dermatol. 113, 1065- 1069, 1999 and co-pending U.S.S.N. 09/288,940). Melanocytic neoplasms that are to be typed can be analyzed for the presence of an amplified H-RAS gene as described and further, may be analyzed for the presence of additional mutations in the H-RAS gene. Oncogenic mutations of H-RAS typically involve codons 12, 13, and 61. However, other mutations such as point mutations occurring at any region within the structural gene or regulatory regions of H-RAS, insertions, and deletions can also be detected using the methods of the invention.

There are many methods known in the art for detecting mutations in a given gene. Useful techniques include, but are not limited to, FISH, direct DNA sequencing, Southern blot analysis, single stranded conformation analysis (SSCP), denaturing gradient gel electrophoresis, RNAse protection assays, allele-specific oHgonucleotides (ASO), dot blot analysis, PCR-SSCP, and allele-specific PCR.

Another method known in the art is CFLP-cleavase fragment length polymoφhism. This method involves amplifying the gene of interest, here H-RAS, followed by digestion with cleavase I, which cuts the DNA at sites dependent on secondary structure. Results are resolved on agarose gels and different patterns of cleavage digestion products are obtained for wild-type and mutant samples.

A further method known in the art is temperature modulation heteroduplex chromatography (TMHC). The method involves amplification of the H-RAS gene followed by denaturing of the PCR products and then slowly cooling, to a predetermined temperature based on the composition of the sample. While cooling, the PCR products renature to form hetero- and homoduplexes which are resolved from one another using TMHC. The resolution can be performed using a WAVE® DNA fragment analysis system (Transgenomic,Inc, San Jose, CA). Mutations in the gene can be found directly by amplifying the gene, e.g., using PCR, in a biological sample, such as a skin tumor sample, and sequencing the amplified product. Alternatively, a probe that specifically hybridizes to t eH-RAS gene can used to detect the presence of mutations. Further, a probe that specifically hybridizes to a mutated H-RAS gene, but not the normal gene, e.g., an allele-specific oligonucleotide, can be used to determine the presence of a specific mutation. A probe such as an allele- specific oligonucleotide may be used directly as a probe or as a primer in an amplification reaction in which a product is obtained only if the mutation is present.

Mutations in the H-RAS gene can be detected by a variety of hybridization analyses. Detection of single base mutations can be conveniently accomplished by differential hybridization techniques using allele-specific oHgonucleotides (see, e.g.,

Suggs et al, Proc. Natl. Acad. Sci. 78: 6613-6617 (1981); Conner et al, Proc. Natl Acad. Sci. 80: 278-282 (1983); Saiki et al, Proc. Natl. Acad. Sci. 86: 6230-6234 (1989)). Mutations can be diagnosed on the basis of the higher thermal stability of the perfectly matched probes as compared to the mismatched probes. The hybridization reactions can, for example, be carried out in a filter-based format, in which the target nucleic acids are immobilized on nitrocellulose or nylon membranes and probed with oligonucleotide probes. Any of the known hybridization formats may be used, including Southern blots, slot blots, "reverse" dot blots, solution hybridization, solid support based sandwich hybridization, bead-based, silicon chip-based and microtiter well-based hybridization formats.

An alternative strategy involves detection mutations in the H-RAS gene by sandwich hybridization methods. In this strategy, the mutant and normal target nucleic acids are separated from non-homologous DNA/RNA using a common capture oligonucleotide immobilized on a solid support and detected by specific oligonucleotide probes tagged with reporter labels. The capture oHgonucleotides can be immobilized on microtitre plate wells or on beads (Gingeras et al, J. Infect. Dis. 164: 1066-1074 (1991); Richman et al., Proc. Natl. Acad. Sci. 88: 11241-11245 (1991)).

Nucleic acid arrays

The methods of this invention are particularly well suited to array-based hybridization formats. For a description of one preferred array-based hybridization system see Pinkel et al. (1998) Nature Genetics, 20: 207-211. Arrays are a multiplicity of different "probe" or "target" nucleic acids (or other compounds) attached to one or more surfaces (e.g., solid, membrane, or gel). In a preferred embodiment, the multiplicity of nucleic acids (or other moieties) is attached to a single contiguous surface or to a multiplicity of surfaces juxtaposed to each other.

In an array format a large number of different hybridization reactions can be run essentially "in parallel." This provides rapid, essentially simultaneous, evaluation of a number of hybridizations in a single "experiment". Methods of performing hybridization reactions in array based formats are well known to those of skill in the art (see, e.g., Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature Biotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958. Arrays, particularly nucleic acid arrays can be produced according to a wide variety of methods well known to those of skill in the art. For example, in a simple embodiment, "low density" arrays can simply be produced by spotting (e.g. by hand using a pipette) different nucleic acids at different locations on a solid support (e.g. a glass surface, a membrane, etc.). This simple spotting approach has been automated to produce high density spotted arrays (see, e.g., U.S. Patent No: 5,807,522). This patent describes the use of an automated systems that taps a microcapillary against a surface to deposit a small volume of a biological sample. The process is repeated to generate high density arrays. Arrays can also be produced using oligonucleotide synthesis technology. Thus, for example, U.S. Patent No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092 teach the use of light-directed combinatorial synthesis of high density oligonucleotide arrays.

In another embodiment the array., particularly a spotted array, can include genomic DNA, e.g. overlapping clones that provide a high resolution scan of the amplicon corresponding to the region of interest. Amplicon nucleic acid can be obtained from, e.g., MACs, YACs, BACs, PACs, Pis, cosmids, plasmids, inter- Alu PCR products of genomic clones, restriction digests of genomic clone, cDNA clones, amplification (e.g., PCR) products, and the like. In various embodiments, the array nucleic acids are derived from previously mapped libraries of clones spanning or including the target sequences of the invention, as well as clones from other areas of the genome, as described below. The arrays can be hybridized with a single population of sample nucleic acid or can be used with two differentially labeled collections (as with an test sample and a reference sample). Many methods for immobilizing nucleic acids on a variety of solid surfaces are known in the art. A wide variety of organic and inorganic polymers, as well as other materials, both natural and synthetic, can be employed as the material for the solid surface. Illustrative solid surfaces include, e.g., nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, and cellulose acetate. In addition, plastics such as polyethylene, polypropylene, polystyrene, and the like can be used. Other materials which may be employed include paper, ceramics, metals, metalloids, semiconductive materials, cermets or the like. In addition, substances that form gels can be used. Such materials include, e.g., proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may be employed, particularly as laminates, to obtain various properties. For example, proteins (e.g., bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt's solution) can be employed to avoid non-specific binding, simplify covalent conjugation, enhance signal detection or the like. If covalent bonding between a compound and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature.

For example, methods for immobilizing nucleic acids by introduction of various functional groups to the molecules is known (see, e.g., Bischoff (1987) Anal. Biochem., 164: 336-344; Kremsky (1987) Nucl Acids Res. 15: 2891-2910). Modified nucleotides can be placed on the target using PCR primers containing the modified nucleotide, or by enzymatic end labeling with modified nucleotides. Use of glass or membrane supports (e.g., nitrocellulose, nylon, polypropylene) for the nucleic acid arrays of the invention is advantageous because of well developed technology employing manual and robotic methods of arraying targets at relatively high element densities. Such membranes are generally available and protocols and equipment for hybridization to membranes is well known.

Target elements of various sizes, ranging from 1 mm diameter down to 1 μm can be used. Smaller target elements containing low amounts of concentrated, fixed probe DNA are used for high complexity comparative hybridizations since the total amount of sample available for binding to each target element will be limited. Thus it is advantageous to have small array target elements that contain a small amount of concentrated probe DNA so that the signal that is obtained is highly localized and bright. Such small array target elements are typically used in arrays with densities greater than 10⁴/cm². Relatively simple approaches capable of quantitative fluorescent imaging of 1 cm² areas have been described that permit acquisition of data from a large number of target elements in a single image (see, e.g., Wittrup, Cytometry 16: 206-213, 1994). Arrays on solid surface substrates with much lower fluorescence than membranes, such as glass, quartz, or small beads, can achieve much better sensitivity. Substrates such as glass or fused silica are advantageous in that they provide a very low fluorescence substrate, and a highly efficient hybridization environment. Covalent attachment of the target nucleic acids to glass or synthetic fused silica can be accomplished according to a number of known techniques (described above). Nucleic acids can be conveniently coupled to glass using commercially available reagents. For instance, materials for preparation of silanized glass with a number of functional groups are commercially available or can be prepared using standard techniques (see, e.g., Gait (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Wash., D.C.). Quartz cover slips, which have at least 10-fold lower auto fluorescence than glass, can also be silanized.

Alternatively, probes can also be immobilized on commercially available coated beads or other surfaces. For instance, biotin end-labeled nucleic acids can be bound to commercially available avidin-coated beads. Streptavidin or anti-digoxigenin antibody can also be attached to silanized glass slides by protein-mediated coupling using e.g., protein A following standard protocols (see, e.g., Smith (1992) Science 258: 1122-

1126). Biotin or digoxigenin end-labeled nucleic acids can be prepared according to standard techniques. Hybridization to nucleic acids attached to beads is accomplished by suspending them in the hybridization mix, and then depositing them on the glass substrate for analysis after washing. Alternatively, paramagnetic particles, such as ferric oxide particles, with or without avidin coating, can be used.

In one particularly preferred embodiment, probe nucleic acid is spotted onto a surface (e.g., a glass or quartz surface). The nucleic acid is dissolved in a mixture of dimethylsulfoxide (DMSO) and nitrocellulose and spotted onto amino-silane coated glass slides. Small capillaries tubes can be used to "spot" the probe mixture.

A variety of other nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL Press;

Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969)

Nature 223: 582-587.

Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution.

The sample will provide the target nucleic acid. The "capture" nucleic acid and "signal" nucleic acid probe hybridize with the target nucleic acid to form a "sandwich" hybridization complex. To be most effective, the signal nucleic acid should not hybridize with the capture nucleic acid. Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids.

Typically, such binding occurs through ligand and anti-ligand interactions as between a

Hgand-conjugated probe and an anti-ligand conjugated with a signal. The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga, Ontario) and Q Beta Replicase systems.

Nucleic acid hybridization simply involves providing a denatured probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids, or in the addition of chemical agents, or the raising of the pH. Under low stringency conditions (e.g., low temperature and/or high salt and/or high target concentration) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches. One of skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency. In a preferred embodiment, hybridization is performed at low stringency to ensure hybridization and then subsequent washes are performed at higher stringency to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25 X SSPE-T at 37 °C to 70 °C) until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide. Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present.

In general, there is a tradeoff between hybridization specificity (stringency) and signal intensity. Thus, in a preferred embodiment, the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity. Thus, in a preferred embodiment, the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular probes of interest.

In a preferred embodiment, background signal is reduced by the use of a detergent (e.g., C-TAB) or a blocking reagent (e.g., sperm DNA, cot-1 DNA, etc.) during the hybridization to reduce non-specific binding. In a particularly preferred embodiment, the hybridization is performed in the presence of about 0.1 to about 0.5 mg/ml DNA (e.g., cot-1 DNA). The use of blocking agents in hybridization is well known to those of skill in the art (see, e.g., Chapter 8 in P. Tijssen, supra.) Methods of optimizing hybridization conditions are well known to those of skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier, N.Y.).

Optimal conditions are also a function of the sensitivity of label (e.g., fluorescence) detection for different combinations of substrate type, fluorochrome, excitation and emission bands, spot size and the like. Low fluorescence background membranes can be used (see, e.g., Chu (1992) Electrophoresis 13:105-114). The sensitivity for detection of spots ("target elements") of various diameters on the candidate membranes can be readily determined by, e.g., spotting a dilution series of fluorescently end labeled DNA fragments. These spots are then imaged using conventional fluorescence microscopy. The sensitivity, linearity, and dynamic range achievable from the various combinations of fluorochrome and solid surfaces (e.g., membranes, glass, fused silica) can thus be determined. Serial dilutions of pairs of fluorochrome in known relative proportions can also be analyzed. This determines the accuracy with which fluorescence ratio measurements reflect actual fluorochrome ratios over the dynamic range permitted by the detectors and fluorescence of the substrate upon which the probe has been fixed.

Probes useful in the methods described here are available from a number of sources. For instance, PI clones are available from the DuPont PI library (Shepard, et al., Proc. Natl Acad. Sci. USA, 92: 2629 (1994), and available commercially from Genome Systems. Various libraries spanning entire chromosomes are also available commercially (Clonetech, South San Francisco, CA), or from the Los Alamos National Laboratory. Labeling and Detection of Nucleic Acids.

In a preferred embodiment, the hybridized nucleic acids are detected by detecting one or more labels attached to the sample or probe nucleic acids. The labels may be incoφorated by any of a number of means well known to those of skill in the art. Means of attaching labels to nucleic acids include, for example nick translation or end- labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore). A wide variety of linkers for the attachment of labels to nucleic acids are also known. In addition, intercalating dyes and fluorescent nucleotides can also be used.

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40 -80 ran diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. The nucleic acid samples can all be labeled with a single label, e.g., a single fluorescent label. Alternatively, in another embodiment, different nucleic acid samples can be simultaneously hybridized where each nucleic acid sample has a different label. For instance, one target could have a green fluorescent label and a second target could have a red fluorescent label. The scanning step will distinguish cites of binding of the red label from those binding the green fluorescent label. Each nucleic acid sample (target nucleic acid) can be analyzed independently from one another. Suitable chromogens which can be employed include those molecules and compounds which absorb light in a distinctive range of wavelengths so that a color can be observed or, alternatively, which emit light when irradiated with radiation of a particular wave length or wave length range, e.g., fluorescers.

Desirably, fluorescers should absorb light above about 300 ran, preferably about 350 ran, and more preferably above about 400 ran, usually emitting at wavelengths greater than about 10 ran higher than the wavelength of the light absorbed. It should be noted that the absoφtion and emission characteristics of the bound dye can differ from the unbound dye. Therefore, when referring to the various wavelength ranges and characteristics of the dyes, it is intended to indicate the dyes as employed and not the dye which is unconjugated and characterized in an arbitrary solvent. Fluorescers are generally preferred because by irradiating a fluorescer with light, one can obtain a plurality of emissions. Thus, a single label can provide for a plurality of measurable events.

Detectable signal can also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and can then emit light which serves as the detectable signal or donates energy to a fluorescent acceptor. Alternatively, luciferins can be used in conjunction with luciferase or lucigenins to provide bioluminescence. Spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like. Exemplary spin labels include nitroxide free radicals.

The label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization. So called "direct labels" are detectable labels that are directly attached to or incoφorated into the target (sample) nucleic acid prior to hybridization. In contrast, so called "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected. The nucleic acid probe may also be labeled with digoxigenin and then detected with an antibody that is labeled with a fluorochrom, or an enzyme such as horseradish peroxidase or alkaline phosphatase. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

Fluorescent labels are easily added during an in vitro transcription reaction. Thus, for example, fluorescein labeled UTP and CTP can be incoφorated into the RNA produced in an in vitro transcription.

The labels can be attached directly or through a linker moiety. In general, the site of label or linker-label attachment is not limited to any specific position. For example, a label may be attached to a nucleoside, nucleotide, or analogue thereof at any position that does not interfere with detection or hybridization as desired. For example, certain Label-ON Reagents from Clontech (Palo Alto, CA) provide for labeling interspersed throughout the phosphate backbone of an oligonucleotide and for terminal labeling at the 3' and 5' ends. As shown for example herein, labels can be attached at positions on the ribose ring or the ribose can be modified and even eliminated as desired. The base moieties of useful labeling reagents can include those that are naturally occurring or modified in a manner that does not interfere with the puφose to which they are put. Modified bases include but are not limited to 7-deaza A and G, 7-deaza-8-aza A and G, and other heterocyclic moieties.

It will be recognized that fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like. Thus, for example, CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281 : 2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281 : 2016-2018).

Amplification-based Assays.

In another embodiment, amplification-based assays can be used to measure copy number. In such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction (e.g. Polymerase Chain Reaction (PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate (e.g. healthy tissue) controls provides a measure of the copy number of the desired target nucleic acid sequence. Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).

Other suitable amplification methods include, but are not limited to ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), and self- sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874).

Detection of Gene Expression

As indicated below, a number of oncogenes, particularly H-RAS, are found in the regions of amplification disclosed here. Thus, oncogene activity can be detected by, for instance, measuring levels of the gene transcript (e.g. mRNA), or by measuring the quantity of translated protein.

Detection of Gene Transcripts. Methods of detecting and/or quantifying t gene transcripts using nucleic acid hybridization techniques are known to those of skill in the art (see Sambrook et al. supra). For example , a Northern transfer may be used for the detection of the desired mRNA directly. In brief, the mRNA is isolated from a given cell sample using, for example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane. As with the Southern blots, labeled probes are used to identify and/or quantify the target mRNA.

In another preferred embodiment, the gene transcript can be measured using amplification (e.g. PCR) based methods as described above for directly assessing copy number of the target sequences.

Detection of Expressed Protein

The presence of polypeptides encoded by regions of the chromosome that are amplified, e.g, H-RAS can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectro- phoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like. For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).

Immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra. Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent may itself be one of the moieties comprising the antibody/antigen complex. For example, in detecting H-RAS, the labeling agent may be a labeled H-RAS polypeptide or a labeled anti-H-RAS antibody. Alternatively, the labeling agent may be a third moiety, such a secondary antibody, that specifically binds to the antibody/H-RAS complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, may also be used as the label agent. These proteins exhibit a strong nonimmunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al, J. Immunol. 111 :1401-1406 (1973); Akerstrom et al, J. Immunol. 135:2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.

Immunoassays for detecting polypeptides in a sample may be either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of antigen is directly measured. In one preferred "sandwich" assay for detecting, for example, H-RAS, the anti-H-RAS antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture the H-RAS protein present in the test sample. The H-RAS thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second or third antibody is typically modified with a detectable moiety, such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety. In competitive assays, the amount of a polypeptide present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) protein displaced (competed away) from an anti-polypeptide antibody by the unknown polypeptide present in a sample. In one competitive assay, for example, a known amount of H-RAS protein is added to a sample and the sample is then contacted with an antibody that specifically binds to the H-RAS protein. The amount of exogenous H-RAS protein bound to the antibody is inversely proportional to the concentration of H-RAS protein present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of H-RAS bound to the antibody may be determined either by measuring the amount of H-RAS present in a H-RAS/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein. The amount of H-RAS may be detected by providing a labeled H-RAS molecule.

A hapten inhibition assay is another preferred competitive assay. In this assay, the known protein is immobilized on a solid substrate. A known amount of antibody to the protein is added to the sample, and the sample is then contacted with the immobilized protein. The amount of antibody bound to the known immobilized protein is inversely proportional to the amount of protein present in the sample. Again, the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.

Western blot (immunoblot) analysis is used to detect and quantify the presence of a polypeptide in a sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the polypeptide, e.g., H-RAS, and/or antibodies that specifically bind to mutated versions of the polypeptide. The polypeptide antibodies specifically bind to the polypeptide on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies.

Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al, Amer. Clin. Prod. Rev. 5:34-41 (1986)).

Kits for Use in Diagnostic and/or Prognostic Applications.

For use in diagnostic, research ,and therapeutic applications suggested above, kits are also provided by the invention. In the diagnostic and research applications such kits may include any or all of the following: assay reagents, buffers, nucleic acids for detecting the target sequences and other hybridization probes and/or primers. A therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base. In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES

Example One: CGH and FISH studies of Spitz nevi cells

This example demonstrates that using CGH studies, Spitz nevi cells are shown to have a gain in chromosomal region 11. Seventeen (17) cases of Spitz nevi were studied using CGH. The procedures of CGH were performed following standard protocols as described as follows.

Material and Methods Tumor Material

Formalin- fixed, paraffin-embedded tissue from Spitz nevi from 17 patients were retrieved from the archives of the Department of Dermatology (University Hospital, Wϋrzburg, Germany) and the Dermatopathology Section, Departments of Pathology and Dermatology (University of California, San Francisco). We selected lesions that had an extensive and densely cellular dermal component that allowed the collection of mostly melanocytes and had at most a sparse lymphocytic infiltrate, so that lymphocyte DNA would not obscure aberrations in the neoplastic cells.

DNA Preparation Paraffin material: 30μm sections were cut, with a 5μm section for H & E every 5 sections. The unstained 30 μm sections were placed on glass slides and an area of interest was microdissected without de-paraffinizing.

Microdissection was carried out manually under a dissecting microscope. Depending on the size of the tumor 20-60 unstained sections were used and regions with a high density of tumor cells were separated from normal cells. The dissected tumor parts were collected in tubes and de-paraffinized by washing with xylene and ethanol. DNA extraction and labeling was performed as published by Isola et al. (8). Briefly, tissue was incubated until complete digestion (3 days) with proteinase K (Life Technologies, Inc., Gaithersburg, MD) in a 50 mM Tris pH8.5, lmM EDTA, 0.5% Tween 20 buffer. DNA was extracted with phenol-chloroform-isoamylalcohol (25:24:1, v/v), precipitated with 7.5 M ammonium acetate and 100% ethanol, and resuspended in water. The amount of DNA obtained ranged from 2 to 12μg. Comparative Genomic Hybridization (CGH) and Digital Image Analysis

All tumors were measured both with the tumor DNA labeled with fluorescein- 12-dUTP (DuPont, Inc., Boston, MA), and reference DNA with Texas red-5- dUTP ("standard" labeling), and with the labeling reversed. Labeling was performed by Nick translation. Nick translation conditions were adjusted so that the mean probe fragment size after labeling ranged between 800 and 1500 bp. The hybridization mixture consisted of 200-1000 ng of labeled tumor DNA, 200 ng inversely labeled sex-matched normal human reference DNA from peripheral blood lymphocytes, and 25 μg human Cot- 1 DNA (Life Technologies, Inc., Gaithersburg, MD) dissolved in 10 μl hybridization buffer (50%) formamide, 10%> dextrane sulfate, and 2 X SSC, pH 7.0). Hybridization was carried out for 2-3 days at 37°C to normal metaphases (9). All samples were investigated with a single batch of metaphase slides. Slides were washed three times in a washing solution (50% formamide in 2 X SSC, pH) at 45°C, once in PN buffer (0.1 M NaH PO₄, 0.1 M Na₂HPO₄, and 0.1 % Nonidet P40, pH 8.0), and once in distilled water (both 10 minutes at room temperature). Slides were counterstained with 4,6-diamino-2- phenylindole in an anti-fade solution. Hybridization quality was evaluated as published previously (7). Digital images were collected from five metaphases with a Photometries CCD camera (Microimager 1400, Xillix Technologies, Vancouver, British Columbia, Canada) on a standard fluorescence microscope. The average tumor/reference fluorescence ratios along each chromosome were calculated with custom CGH analysis software. Measurements were made on at least 4 copies of each autosome.

Controls and Threshold Definitions Normal DNA and DNA from tumor cell lines with known aberrations were used as controls. We regarded a region as aberrant when 1) either the standard labeling or the reverse labeling resulted in a tumoπreference fluorescent ratios <0.80 or > 1.2 or 2) both the standard and the reverse labeling resulted in a tumor:reference fluorescent ratios <0.85 or >1.15. Results of this experiment showed that 13 tumors did not show any chromosomal aberrations. One case had an isolated gain of the distal part of chromosome 7, 7q21-qter. Three cases showed a single high level gain of the entire short arm of chromosome 11 (Fig. 2). This phenomenon of a gain in chromosome 1 lp of Spitz nevi cells is not seen among melanoma cells, as shown in Fig. 1. Fluorescence in-situ hybridization (FISH)

Dual-color FISH was carried out on tissue sections of the cases in which tissue was left after CGH (14/17). Probes mapping to the short arm (RMC11B022 and RMC 11 PO 14) and the long arm (RMC 11 P008) of chromosome 11 were obtained from the resource of the laboratory. Probes were labeled by nick translation with Cy3 (Amersham, Arlington Heights, IL) or Digoxigenin (Boehringer Mannheim, Indianapolis IN). 6μm sections were mounted on positively charged glass slides (Fisher Scientific, Pittsburgh, PA), deparaffinized, and hydrated by decreasing strength ethanol. Sections were incubated for 2-4 min in 1M sodium thiocyanate at 80°C , in 4 mg/ml Pepsin in 0.2 N HC1 at 37°C for 4-8 min, dehydrated by increasing strength ethanol and air-dried. Slides were denatured in 70%> formamide, 2x SSC pH 7.0 for 5 min at 72°C, and dehydrated again in a graded ethanol series. 2.5 to 25 ng of each of the labeled probes together with 20μg Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD) were dissolved in lOμl hybridization buffer (50%> formamide, 10%o dextrane sulfate, and 2 x SSC, pH 7.0) and denatured for 10 min at 72°C. Hybridization was carried out for 48-72 hours at 37°C. Slides were washed three times in washing solution (50%> formamide in 2 x SSC, pH 7.0) at 45°C, once in 2x SSC at 45°C, once in 2x SSC at room temperature (RT), and once in 0.1% Triton X100 in 4 x SSC/ at RT. Subsequently, sections were incubated with 10% BSA in 4 x SSC in a moist chamber at 37 °C, and then with a FITC labeled anti- digoxigenin antibody (Boehringer Mannheim, Indianapolis IN) diluted in 4 x SSC with 10% BSA. Sections were counterstained with 4,6-diamino-2-phenylindole (Sigma, St. Louis, MS) in an anti-fade solution. The two-tailed student's t-test was used for the comparison of FISH signals for the locus of interest and the reference probe.

Results

Table 1 shows the clinical information of the Spitz nevi patients, and aberrations found by CGH and FISH. Patient age ranged from 3-45 years (mean 18 years). Follow-up was available from most patients. The follow-up time was 1.2-9 years (mean 4.9 years). All patients with available follow-up were free of disease by the end of the follow-up interval. In one case (case 16) 2 recurrences prior to the final excision of the lesion that entered the study occurred, possibly because the tumor was curetted twice. Recut sections of all cases represented typical Spitz nevi by histopathological examination. 13 of the 17 tumors (76%) showed no DNA copy number changes by CGH. Three cases (18%>) showed gain of the entire short arm of chromosome 11 as the sole abnormality. (Fig. 3). One case showed gain of chromosome 7q21-qter as the only abnormality (Fig. 3).

FISH measurements were performed to tissue sections in order to study the histopathologic distribution of the recurrent gain on chromosome 1 lp and to find potential minor populations of cells with this aberration in the cases with normal CGH profiles. A test probe was selected that mapped to the distal part of chromosome 1 lp (1 lpl5.5, clone RMCl 1B022) and a reference probe mapping to chromosome 1 lq23 (clone RMCl 1P008). In all experiments keratinocytes of the epidermis adjacent to the lesion were used as internal controls. As the hybridization was carried out on sections of 6μm thickness, many nuclei were not fully represented in the slide. For counting hybridization signals, we selected nuclei that appeared minimally truncated when the focus of the microscope was slightly altered. The nuclear signal counts in keratinocytes for the q-arm and the p-arm probe ranged from mean values of 1.6- 1.9 and 1.7-1.9, respectively (Fig. 4b, 4d, 4f). A mean of 2.0 is expected if all counted nuclei are intact and the hybridization efficiency is 100%. The numbers of p-arm signals tended to be slightly higher than that of the q-arm, which can be explained by the larger size of the p- arm probe, resulting in slightly higher hybridization efficiencies.

However, this difference was not statistically significant. As the variance of the signal number was low (0.16-0.24) in this control population of supposedly normal cells, counting 20 cells per tumor was sufficient to establish the success of the hybridization. When analyzing the neoplastic cells, 20 cells of each moφhologically distinct subpopulation were counted. The three cases that had a gain of chromosome 1 lp by CGH showed a mean of 3.5-5.3 signals with the p-arm probe compared to a mean of 1.5-2.1 counts for the q-arm probe (Fig. 4c,). This difference was highly significant (p<0.00001). The counts for the q-arm (control) probes were not statistically different from signal counts in keratinocytes (normal cells) of the respective lesions. The ratio of p-arm signals to q-arm signals in the cases with increased copies of chromosome 1 lp ranged from 1.8-3.0. The increased signal number of the p-arm probe was present in virtually every cell of each the nevi. From the 14 tumors that had no gain of chromosome 1 lp by CGH twelve could be studied by FISH. In the other two cases the paraffin blocks were exhausted. Of these twelve cases, eleven had no significant differences in signal distribution of the probes for p-arm and the q-arm of chromosome 11 (Fig. 4a, 4b). One case (case 5) had 2.4 p-arm signals vs. 1.9 q-arm signals, a difference which was statistically significant (p=0.01). In two cases (cases 3 and 15) a subpopulation of cells was present that had increased numbers of both the q-arm and the p-arm signal (Fig. 4e). These cells mostly had considerably larger nuclei than the tumor cells with 1-2 signals, and are thus likely to be polyploid. As illustrated in Fig. 3, the area of chromosome 11 that was found by CGH to be gained in three cases seems to be identical. The profiles of case 13 and case 16 suggest the highest increase of DNA copy number towards the p-telomere. However, the profiles of the CGH measurement in which the labeling was reversed showed a decrease of red:green fluorescence ratio toward the telomere, indicating that the p-telomeric ratio increase is artifactual. To confirm this, FISH experiments were performed with a different probe for the p-arm that mapped more proximally to 1 lpl 4 (RMCl IPO 14). The number of signals in the nuclei of the tumor cells with this probe was similar to that found with the probe for 1 lpl 5.

One probe mapped to the distal part of the p-arm of chromosome 11 and the second probe mapped to 1 lq. Of the three cases that showed a gain of chromosome 1 lp, 6-10 signals of the 1 lp probe per nucleus were detected, whereas the probe that mapped to the q-arm only gave two signals. Interestingly, the signal number was virtually constant over the entire lesion, suggesting a clonal nature of the neoplasms. Among the other Spitz nevi studied which showed no indication of 1 lp gain in CGH, only one additional case showed an amplification of 1 lp. All other cases had two signals of both markers. Exceptions were cells with large nuclei that occurred in three tumors. Those cells had up to 10 signals of both markers. These findings suggest that gain of chromosome 1 lp is a recurrent aberration in Spitz nevi. The cells of Spitz nevi are diploid, with the exception of cells with large nuclei that can be polyploid. Spitz nevi are clonal neoplasms.

Table 1 : Clinical information of the Spitz nevi and aberrations found by CGH and FISH. (PSCT = pigmented spindle cell tumor, FOD = free of disease, NA = not available). Table 1i

Discussion

These results show that the majority of Spitz nevi have a normal chromosomal complement, but that a subset may have abnormalities. We detected gains of chromosome 1 lp in 4/17 cases. Thus Spitz nevi is one of the many benign lesions that contain genetic abnormalities at the chromosomal level. The pattern of chromosomal aberrations in Spitz nevi shows clear differences from that observed in primary cutaneous melanoma. In the latter only a small minority of cases does show no aberrations when analyzed by CGH. In this example, CGH measurements were performed on 102 melanomas and only 5 cases did not show changes. The 5 cases without detectable aberrations had considerable contamination of normal cells in the tumor that may account for at least a part for these findings. In the Spitz nevi of the present study cases with an inflammatory component were excluded, so that contamination of normal cells could not have accounted for the high frequency of negative findings. The finding of an increased copy number of chromosome 1 lp in four out of 17 lesions indicates that this aberration represents a recurrent change in Spitz nevi. It suggests that increased dosage of genes of chromosome 1 lp has relevance in the pathogenesis of this tumor. As the gained genomic fragment is large, additional studies are warranted to refine the extent of the region as a first step toward identifying the critical gene(s). It may well be that in the Spitz nevi without l ip gain, those genes, or the pathways they belong to, are activated by a different mechanism than increase of gene copy number.

A gain of chromosome 1 lp was not seen in any of the 102 primary melanomas we have analyzed by CGH. Gains including 1 lp were found in only two cases of the 239 published karyotypes, mostly from metastatic melanomas (11, 12). However, in these two cases the gain extended far on to the q-arm (12).

Furthermore, none of the most frequent findings in primary melanomas, losses of chromosomes 9 and 10, was found in any of the Spitz nevi. However, chromosome 7 was gained in 50%> of our melanoma cases (7), and one of the Spitz nevi showed a gain of chromosome 7q23-pter. Thus, even though future studies will be required to determine the full spectrum and frequency of aberrations in Spitz nevi, the current data clearly shows that there is a clear difference in the pattern of chromosomal gains and losses in melanoma and Spitz nevi. These differences raise the possibility of defining genetic markers that can be used for diagnostic puφoses. Cytogenetic studies have been of great help in the classification of soft tissue tumors and can provide pivotal diagnostic information (13). A diagnostic test for spitzoid melanocytic neoplasms might include copy number detection of chromosomes 1 lp, 9, and 10. Gains of chromosome 1 lp could be inteφreted as in favor of Spitz nevus, and losses of chromosomes 9 and/or 10 as in favor of melanoma.

It is indeed remarkable that the chromosomal alterations most frequently found in primary melanomas are absent in Spitz nevi. And this absence of aberrations frequently found in melanoma may be a difference that may indeed offer diagnostic opportunities. Previous CGH studies on 32 primary melanomas showed losses of chromosome 9p in 82%>, chromosome 10 in 63%, and 6q in 28%> of the cases (Bastian et al. 1998). Frequent gains in melanoma involved chromosome 7p (50%), 8q (34%), and 6p (28%ι). None of these changes was found in our series of Spitz nevi. Note that one study found interstitial deletions of chromosome 9p in two out of 27 Spitz nevi indicating that losses of 9p are not exclusive to melanoma (Healy E, et al., ALLELOTYPES OF PRIMARY CUTANEOUS MELANOMA AND BENIGN MELANOCYTIC NEVI, Cancer Res 56: 589, 1996). It may thus be that the determination of copy number of other chromosomal regions such as lq, 6p, 7p, and lOq, may prove to be more helpful in the differential diagnosis. The efficacy of such a test needs to be evaluated through the analysis of a larger set of tumors with the inclusion of cases that have conflicting histopatho logic criteria but have known follow-up. This will permit determination of the sensitivity and specificity under clinically relevant conditions.

FISH measurements not only confirmed the CGH findings but also allowed some interesting insight into the ploidy and clonality of Spitz nevi. Since almost all cells in the nevi had 2 copies of the control locus on 1 lq by FISH and CGH showed no aberrant copy numbers for that locus, the large majority of the cells in these nevi are diploid, which is consistent with previous flow cytometry studies (10). Two cases had a subpopulation of cells with large nuclei. Those cells elevated copy number had elevated FISH signals for the two loci tested, indicating that the increased nuclear size is most likely due to polyploidy. These data also show that Spitz nevi are probably comprised of a monoclonal population of melanocytes. This can be concluded from the three cases with a gained 1 lp, because the increased copy number of this chromosomal arm was present in all cells of the lesions

In summary, this example shows that in Spitz nevi, (I) the majority of cases have a normal chromosomal complement at the level of CGH resolution, (II) gains of chromosome 1 lp represent a recurrent aberration in a subset of lesions, (III) Spitz nevi are probably clonal neoplasms, (IV) the majority of the melanocytes of a Spitz nevus are diploid with the exception of cells with large nuclei which can be polyploid, and (V) the clear differences in the location and frequencies of the cytogenetically detectable aberrations in primary cutaneous melanoma and Spitz nevi make CGH and FISH promising techniques for refining diagnostic accuracy of this difficult differential diagnosis.

Example Two: FISH study of melanocytic tumor using chromosome 9 probes This example demonstrates FISH experiments using chromosome 9 probes in detecting primary melanoma cells.

Pi-clones for chromosome 9 were similarly used for FISH studies of sections of primary melanomas. Loss of chromosome 9 was the most frequent finding in the CGH-study of melanoma. The FISH experiments showed that in most cases of melanoma 0-1 signals per nucleus with a probe for chromosome 9p was detected, whereas a simultaneously hybridized reference locus revealed more than 2 signals per nucleus.

This indicates that FISH is capable of detecting homozygous and heterozygous deletions in tissue sections.

The selection of hybridization probes will thus be based on the following criteria: (a) the corresponding chromosomal regions should show frequent aberration in one neoplasm and not in the other (e.g. lq, 6p, 7p, 9p, lOq, and 1 lp), (b) probes should give strong and reproducible hybridization signals.

Example Three: Tissue Hybridization Protocols This example demonstrates the use of tissue hybridization protocols in studying the difference in signal ratios per chromosome locus between melanoma cells and Spitz nevi cells.

A hybridization protocol is adapted from Thompson et al, Cancer Genet Cytogenet 83, 93-104 (1995). Briefly, tissue sections are mounted on positively charged slides. The slides are heated at 55°C for about 30 minutes and deparaffinized with xylene, and ethanol dehydrated. They are then sequentially incubated in NaSCN, followed by Pepsin. After being denatured in formamide, they are hybridized using standard techniques. Probes will be labeled directly with Cy-3 and indirectly with digoxigenin that will later be detected with FITC-labeled anti-digoxigenin antibodies. Alternative labeling approaches may be employed so as to be able to detect three differentially labeled probes in one hybridization.

Based on previous studies, it is expected that counting signals of each hybridization probe in a total of 25 tumor cell nuclei and 25 nuclei of normal tissue cells will suffice. One parameter for decision making will be the ratio of average number of signals per locus per tumor cell compared to the average number of signals per locus in normal cells within the tissue (e.g. keratinocytes of the epidermis or epidermal appendages). According to the preliminary studies, the ratio is expected to be less than one for loci frequently lost in melanoma and more than one for loci gained in Spitz nevi. The second parameter will be the variance of the signal number per tumor cell. Based on previous studies and experience of others, the variance is expected to be significantly higher in malignant tumors than in benign tumors (De Wit et al., J Pathol 173, 227-33 (1994)).

Example Four: Identification of H-RAS mutations

This example demonstrates that a mutated H-RAS gene is associated with Spitz nevus. The procedures to identify H-RAS mutations were performed following standard protocols as described as follows.

Selection of cases: Paraffin blocks of Spitz nevi were retrieved randomly from the archives of the Dermatopathology section of the Departments of Dermatology and Pathology at the University of California, San Francisco. We performed a computer search of the database of the Dermatopathology Section with the following criteria: select all cases from 1/1/98 to 12/31/98 that were assigned a main diagnosis of one 30 different descriptive variants of Spitz nevus that are used in our laboratory. Cases sent in as a slide in consultation were excluded in order to avoid a bias towards unusual Spitz nevi. The request yielded 144 cases from which blocks were available.

In addition to these cases, 22 cases of the Department of Dermatology University of Wϋrzburg, Germany were included in the study. These cases had originally been retrieved for comparative genomic hybridization and only included Spitz nevi with at least 1 mm thickness.

Assembly of tissue arrays: Tissue arrays were constructed according to Kononen et. al, (Nat. Med. 4:844-847, 1998). In brief, a tissue arraying instrument (Beecher Instruments, Silver Spring, MD) was used to punch 0.8 mm biopsy cores of the most cellular areas of the nevi. The biopsy cores were arrayed in recipient paraffin blocks, according to the manufacturer's instructions. Multiple sections of 6 μm thickness were cut with a microtome using an adhesive-coated tape sectioning system (Instrumedics, Hackensack, NJ). H&E sections were used for the histological examination of the biopsy cores. Only cases with at least one area with a cohesive population of neoplastic melanocytes were included in the analysis.

FISH to formalin-fixed tissue microarray sections: Dual-color FISH was carried out on tissue sections of the array as described previously (Bastian et al, J. Invest. Dermatol. 113:1065-1069, 1999). We used a BAC clone (RMCl 1B022) that contained H-RAS for the detection of amplifications of chromosome 1 lp, and a reference PI clone (RMCl 1P008) for the q-arm of chromosome 11. Probes were labeled with Cy3 (Amersham, Arlington Heights, IL) or with digoxigenin (Boehringer Mannheim, Indianapolis IN) by nick-translation. Tissue sections were deparaffinized, hydrated, and pre-treated for 2-4 min in 1M sodium thiocyanate at 80°C, in 4 mg/ml Pepsin in 0.2 N HC1 at 37°C for 4-8 min. After dehydration, sections were denatured in 70% formamide, 2x SSC pH 7.0 for 5 min at 72°C, and hybridized over 48-72 h at 37°C in lOμl hybridization buffer (50% formamide, 10% dextran sulfate, and 2 x SSC, pH 7.0, 20μg Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD)). Slides were washed three times in washing solution (50% formamide in 2 x SSC, pH 7.0) at 45°C, once in 2x SSC at 45°C, once in 2x SSC at room temperature (RT), and once in 0.1 %> TritonXlOO in 4 x SSC/ at RT. Subsequently, sections were incubated with 10% BSA in 4 x SSC in a moist chamber at 37 °C, and then with a FITC labeled anti-digoxigenin antibody (Boehringer Mannheim, Indianapolis IN) diluted in 4 x SSC with 10%) BSA. Sections were counterstained with 4,6-diamino-2-phenylindole (Sigma, St. Louis, MS) in an anti-fade solution. FISH signals were scored with a fluorescence microscope Zeiss (Jena, Germany) using a 63X objective. Criteria for amplification were: at least three times more test probe signals than reference signals in at least 30%> of the tumor cells.

DNA Sequence Analysis: DNA was extracted from 30 μm sections from which the tumor- bearing areas were dissected manually with a scalpel under a dissecting microscope. Two to three sections were collected in a 0.5 ml tube and after washing with xylene and ethanol were incubated at 55 °C with 0.4mg/ml proteinase K (Life Technologies, Inc., Gaithersburg, MD) in PCR buffer (Perkin Elmer) containing 0.5%> Tween 20 for three days. Fresh proteinase K was added every 24h to a final concentration of 0.4mg/ml. HRAS codon 12 primers were 5 '- AGGAGACCCTGTAGGAGGA-3 ' (SEQ ID NO:l) (forward) and 5 '-CGCTAGGCTCACCTCTATAGTG-3 ' (SEQ ID NO:2) (reverse) and codon 61 primers were 5 '-CTGCAGGATTCCTACCGGA-3 ' (SEQ ID NO:3) and 5 '- ACTTGGTGTTGTTGATGGCA-3' (SEQ ID NO:4). PCR was carried out in a Gene Amp PCR System 9700 Thermal Cycler (Perkin Elmer) in 25 μl reaction volumes. Each PCR reaction contained 3.5 mM MgCl₂, 0.2 mM dNTP, 0.625 U Taq Gold Polymerase (Perkin Elmer), IX PCR Buffer II, 0.5 μM each of forward and reverse primer, and 50- 300 ng of genomic DNA. PCR cycling conditions were as follows: 95 °C for 15 min followed by 35 cycles of 95 °C for 15 sec, 55 °C for 30 seconds, and 72 °C for 60 seconds, and a final hold at 72 °C for 10 minutes.

Prior to sequencing, PCR products were purified using the PCR product Pre-sequencing kit (Amersham, Arlington Heights, IL) to remove excess primers and nucleotides. Fluorescent DNA sequencing was carried out using Big Dye terminator sequencing chemistry (PE Applied Biosystems). Briefly, 30-50 ng of purified PCR product and 3.2 pmol of sequencing primer were used for sequencing in a 15 μl reaction according to the manufacturer's instructions. The sequencing products were purified using a Sephadex G50 column, dried in a vacuum concentrator and resuspended in 3 μl of gel loading buffer (83%> deionized formamide, 17%> gel loading dye) (PE Applied Biosystems). 0.5 μl of the sample was then loaded on a denaturing sequence gel on an ABI automated DNA sequencer. All samples were sequenced in both forward and reverse directions to confirm the presence/absence of mutations. The data were analyzed using the Sequencher software (Gene Codes, Ann Arbor, MI).

FISH analysis of chromosome 1 lp copy number using tissue arrays

High-quality hybridizations of cases in which tumor cells could be definitively identified were obtained from 102 cases. This yield of 61.4% is relatively low compared to arrays that we have constructed from melanomas. Most of the cases that could not be analyzed were very small Spitz nevi that consisted only of single cells or small nests of junctional melanocytes so that the neoplastic melanocytes could not be reliably recognized in the array. Thirty nine cases (38.2%) were from male and 61 (59.8%) from female patients, in two cases the gender was not known. The mean age was 30.0 years. 52 (51%) of the cases had features of the pigmented spindle cell nevus variant of Spitz (PSCN).

The hybridization efficiency could be assessed by counting the hybridization signals in normal epidermis that was present in many of the biopsies. The average copy numbers for test and reference probes in normal keratinocytes were 1.7 and 1.6, respectively. Hybridizations were analyzed of three separate sections of the array, and counts from two or more sections were available for 47 (46.1%) cases. In 45 (95.7%.) of these, the result of the separate counts were identical, in one case a definitive amplification was seen in one analysis, and was not found in the cells present in the other section. Amplifications were only scored if more than 30%> of the tumor cells had at least 3-fold increased signals of 1 lp when compared to the reference probe on 1 lq. According to these criteria, amplification of 1 lp was found in 12 (11.8%>) cases. The average thickness of cases with amplifications was significantly greater than the thickness of cases with normal copy number of 1 lp (1.1 mm vs. 0.6 mm, p=0.01). The amplification frequency within the randomly retrieved set of cases was 6/84 (7.1%), whereas of the 18 cases that had been selected for thickness, 6 (33.3%) showed amplifications of chromosome 1 lp.

H-RAS mutations

Oncogenic mutations of H-RAS typically involve codons 12, 13 in exon 1 and codon 61 in exon 2 (Barbacid, M., Annu. Rev. Biochem: 56:779-827, 1987) . We sequenced exons 1 and 2 of H-RAS of 9 cases in which FISH detected an amplification of chromosome 1 lp, and in 13 cases in which FISH showed normal copy numbers of chromosome 1 lp. Five of nine cases (56%>) with 1 lp amplification had HRAS mutations, significantly more (p=0.002) than in the cases with normal 1 lp copy numbers, in which only one (8%>) had a mutation. Three mutations were 61Gln->Leu (fig. IE), two 61 Gln- >Arg, and one 12 Gly->Arg.

Additionally, we sequenced H-RAS in 11 Spitz nevi used for our previous CGH analysis (Bastian et al. , J. Invest. Dermatol. 113:1065- 1069, 1999). H-RAS mutations were identified in all three cases (100%>) in which CGH detected increased copies of chromosome 1 lp. All of these involved codon 61; two cases had a transition of glutamine to arginine, and the other to leucine. The seven cases in which CGH found normal copy numbers of chromosome l ip had wild-type sequences of both exons of H- RAS.

Of the total of 33 Spitz nevi in which H-RAS was sequenced, 8/12 cases (67%) with amplified l ip had H-RAS mutations, significantly more (pθ.0001) than in the cases with normal copies of chromosome 1 lp (1/21 or 5%).

Histologic features of cases with amplified chromosome l ip

Amplifications were most common in compound or predominantly intradermal Spitz nevi (11/47 or 23.4%), and only rarely occurred in the pigmented spindle cell variant of Spitz nevus (1/52 or 1.9%>; p=0.0007). The cases with amplification of 1 lp frequently showed several histological features that occurred infrequently in the cases with normal copy number of chromosome 1 lp (Table 2). The tumors commonly showed single cells splayed between collagen bundles at the base resulting in a pattern of haphazardly arranged collagen and marked desmoplasia (8/12, p=0.0005). Cells typically had vesicular nuclei with delicate nuclear membranes, and ample amphophilic cytoplasm. Cells at the base frequently (5/12) seemed to be surrounded by thin, eosinophilic membranes), that were only seen in 3 of the 87 cases that had normal copy numbers of 1 lp (p<0.0001). These membranes stained positive with a reticulin stain (not shown). Cases with l ip amplifications were also notably more pleomoφhic (p<0.0001) with nuclei varying in sizes and shapes and staining intensity and in some cases intranuclear inclusions. These features were also present in the three cases that had shown amplification of chromosome 1 lp by comparative genomic hybridization in a previous study (Bastian et al, J. Invest. Dermatol. 113:1065-1069, 1999). No association of 1 lp amplification with patient age or sex was found.

Table 2. Histological features associated with amplification of chromosome 1 lp in Spitz nevi.

The percentages n the n co umn refere to the total number of 102 cases. The other percentages refer to the number of cases that have the respective biological feature.

Cell proliferation using a Ki-67 antibody was assessed in all 12 cases with amplifications of 1 lp and 24 cases with normal copy numbers of chromosome 1 lp. Immunostains were performed on sections of the original blocks to allow inspection of sufficient numbers of cells. In 11/12 (92%) of the cases with 1 lp amplification and 20/24 (83%) with normal 1 lp copy numbers the number of labeled nuclei ranged from 0 to maximal 1%>. A total of five cases had higher labeling rates: Four had normal 1 lp copy numbers and a labeling index of at most 5%>, mostly affecting melanocytes at the demo- epidermal junction. One case that had an average of 6 copies of chromosome 1 lp by FISH analysis and a 61G->A mutation had a labeling index of 10%. It was a superficial biopsy, so that the proliferation rate in deeper areas of the nevus could not be assessed.

The expression of the cellular adhesion molecule α_vβ integrin is correlated with tumor progression and invasion in melanoma (Albelda et al, Cancer Res. 1990, 50:6757-6764) and has recently been reported to be expressed in Spitz nevus (Van Belle et al, Hum. Pathol. 1999, 30:562-567). The pattern of single cells between collagen bundles leading to a considerable remodelling of collagen frequently found in cases with 1 lp amplification was indicative of a marked invasive capacity of the cells. Immunohistochemistry to detect β3 integrin was performed to determine the level of α_vβ integrin expression. This analysis showed that of a total of 80 informative cases, 29 (41.4%) showed expression (1+ and above) of α_vβ integrin. α_vβ₃ integrin expression was significantly (pO.OOOl) associated with amplification of HRAS. Of 29 cases which expressed α_vβ integrin, 9 had amplifications of HRAS. Of 41 cases without expression of α_vβ integrin, only two cases had an amplification of chromosome 1 lp. In all cases with 1 lp amplification the expression pattern of α_vβ₃ integrin was membranous.

In summary, this example shows that H-RAS mutations are present in a subset of Spitz nevus and can be used as a target in typing tumor samples to assist in the differential diagnosis of a Spitz nevus.

Example Five: Identification of 1 lp isochromosomes

Ten Spitz nevi samples that showed amplification of H-RAS (see, e.g., Example 1) were analyzed for the presence of an 1 lp isochromosome using FISH. Two probes were employed for the analysis. The first probe RPCI- 1156c 13, which was labeled with FITC and detected as a green fluorescent signal, maps to chromosome 1 lp to a region adjacent to the centromere, 1 lpl 1.2. The second probe RPCI-11135h08, which was labeled with Cy3 and detected as a red fluorescent label, hybridizes to sequences on the q arm of chromosome 11 adjacent to the centromere at 1 lql 1. Normal control tissue showed only the presence of paired hybridization signals containing two colors, i.e., the pair of signals included one red signal and one green signal. The results from all of the 10 samples from Spitz nevi indicated the additional presence of paired hybridization signals of the same color (green). These results demonstrate the presence of an l ip isochromosome.

It is understood that the examples and embodiments described herein are for illustrative puφoses only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incoφorated by reference in their entirety for all puφoses. Sequence Listing:

SEQ ID NO: 1 HRAS codon 12 forward primer 5 '- AGGAGACCCTGTAGGAGGA-3 '

SEQ ID NO: 2 HRAS codon 12 reverse primer 5 '-CGCTAGGCTCACCTCTATAGTG-3 '

SEQ ID NO: 3 HRAS codon 61 forward primer 5'-CTGCAGGATTCCTACCGGA-3'

SEQ ID NO:4 HRAS codon 61 reverse primer 5 '-ACTTGGTGTTGTTGATGGCA-3 '

Claims

WHAT IS CLAIMED IS:

1. A method of screening for the presence of a melanocytic nevus in a skin tumor sample from a patient, the method comprising detecting the presence of an increase of copy number at 1 lp, or an absence of change in copy number at lq, 6p, 7p, or 1 Oq in a nucleic acid sample from the patient.

2. The method of claim 1, further comprising detecting the absence of change in copy number at 9p in the nucleic acid sample from the patient.

3. The method of claim 1 , wherein the method is carried out by:

a) contacting a nucleic acid sample from the patient with a probe which binds selectively to a target polynucleotide sequence on a chromosomal region selected from the group consisting of lq, 6p, 7p, 9p, lOq and 1 lp, wherein 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;

b) detecting the formation of the hybridization complex; and

c) detecting the presence of an increase of copy number at 1 lp, or an absence of change in copy number at lq, 6p, 7p, 9p, or lOq, thereby determining the presence of the melanocytic nevus.

4. The method of claim 3, wherein the target nucleic acid is H-RAS.

5. The method of claim 1 , wherein the nucleic acid sample is an inteφhase nucleus.

6. The method of claim 1 , wherein the probe is labeled with a detectable composition.

7. The method of claim 6, wherein the detectable composition is selected from the group consisting of a direct label and an indirect label.

8. The method of claim 7, wherein the direct label is Cy3.

9. The method of claim 7, wherein the indirect label is selected from a group consisting of a digoxigenin and a biotin.

10. The method of claim 7, wherein the indirect label is detected by a fluorescent dye.

11. The method of claim 10, wherein the fluorescent dye is FITC.

12. The method of claim 1, further comprising contacting the sample with a reference probe to a second chromosomal region.

13. The method of claim 12, wherein the reference probe is labeled with a fluorescent label distinguishable from the label on the probe that selectively hybridizes to the target polynucleotide sequence.

14. The method of claim 1, wherein the step of detecting the hybridization complex comprises determining the copy number of the target polynucleotide sequence.

15. The method of claim 1, further comprising the step of blocking the hybridization capacity of repetitive sequences in the first and second collections of nucleic acids.

16. The method of claim 15, wherein unlabeled blocking nucleic acids comprising repetitive sequences are contacted with the sample.

17. The method of claim 16, wherein the unlabeled blocking nucleic acids are Cot-1 DNA.

18. The method of claim 1 , wherein probe is bound to a solid substrate.

19. The method of claim 18, wherein the probe is a member of an array.

20. A method of typing a melanocytic neoplasm in a sample from a patient, the method comprising detecting an increase in copy number of an 1 lp chromosome arm, thereby typing the neoplasm as a Spitz nevus.

21. The method of 20 comprising detecting the presence of an 1 lp isochromosome.

22. The method of claim 21, wherein the detecting step comprises: hybridizing a nucleic acid from the sample with a first probe labeled with a detectable label, wherein the probe selectively hybridizes to a target nucleic acid sequence on chromosome 1 lp adjacent to the centromere, and detecting the presence of paired hybridization signals from the first probe.

23. The method of claim 22, wherein the detecting step further comprises hybridizing the nucleic acid from the sample with a second probe labeled with a second detectable label distinguishable from the first, wherein the second probe selectively hybridizes to a target nucleic acid sequence on chromosome 1 lq adjacent to the centromere; and further comprising detecting the second probe adjacent to the first.

24. The method of claim 23, wherein the first label is a fluorescent label and the second label is a fluorescent label that is a different color from the first.

25. A method of typing a melanocytic neoplasm in a patient sample, the method comprising detecting the presence of a mutant H-RAS gene in the sample, thereby typing the neoplasm as a Spitz nevus.

26. The method of claim 25, wherein the mutant H-RAS gene copy number is increased in the patient sample relative to normal.

27 The method of claim 25, wherein the mutant H-RAS gene is mutated at a codon of the H-RAS gene selected from the group consisting of codons 12, 13, and 61.

28. The method of typing a melanocytic neoplasm in a sample from a patient, the method comprising detecting an amplification in the H-RAS gene, wherein the step of detecting comprises detecting a polypeptide encoded by the H-RAS gene.

29. The method of claim 28, wherein the polypeptide is detected with an antibody.

30. The method of claim 29, wherein the amount of the polypeptide is quantified by immunoassay.