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WO2000061814A1 - Detection de changements dans le nombre de copies d'un chromosome, permettant de distinguer les naevi melanocytiques des melanomes malins - Google Patents

Detection de changements dans le nombre de copies d'un chromosome, permettant de distinguer les naevi melanocytiques des melanomes malins Download PDF

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WO2000061814A1
WO2000061814A1 PCT/US2000/009609 US0009609W WO0061814A1 WO 2000061814 A1 WO2000061814 A1 WO 2000061814A1 US 0009609 W US0009609 W US 0009609W WO 0061814 A1 WO0061814 A1 WO 0061814A1
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probe
sample
nucleic acid
hybridization
detecting
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WO2000061814A9 (fr
WO2000061814A8 (fr
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Boris Bastian
Daniel Pinkel
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority claimed from US09/288,940 external-priority patent/US6261775B1/en
Priority claimed from US09/541,364 external-priority patent/US6551780B1/en
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Priority to JP2000611736A priority Critical patent/JP2002541826A/ja
Priority to CA002368903A priority patent/CA2368903A1/fr
Priority to EP00925923A priority patent/EP1181392A4/fr
Publication of WO2000061814A1 publication Critical patent/WO2000061814A1/fr
Publication of WO2000061814A8 publication Critical patent/WO2000061814A8/fr
Publication of WO2000061814A9 publication Critical patent/WO2000061814A9/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • 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)).
  • 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
  • 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 l ip 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 H-RAS.
  • An additional aspect of typing is the detection of a mutated H-RAS gene present in a tumor sample, which is also associated with, or indicates the presence of a Spitz nevus.
  • 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.
  • the methods comprise detecting the presence of an l ip isochromosome in a tumor sample from a patient.
  • the nucleic acid sample can be extracted from an inte ⁇ hase nucleus.
  • the probe is labeled e.g. with a fluorescent label.
  • the label may be a direct label.
  • a reference probe to a second chromosomal region is used in the methods as an internal control.
  • the second probe is labeled with a fluorescent label distinguishable from the label on the probe that selectively hybridizes to the target polynucleotide sequence.
  • the probe may include repetitive sequences.
  • 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 pu ⁇ ose.
  • the nucleic acid hybridization can be carried out in a number of formats.
  • the hybridization may be an in situ hybridization.
  • the probe is bound to a solid substrate, e.g. as a member of a nucleic acid array.
  • 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.
  • 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.
  • 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.
  • the amplifying step is a PCR reaction, which can be performed, e.g., using oligonucleotides as set out in SEQ ID NOs: 1 and 2, and 3 and 4.
  • the nucleic acid from the sample is preferably genomic DNA or RNA.
  • the invention also includes a method of detecting the presence of a an amplified H-RAS gene by detecting a polypeptide encoded by the H-RAS gene.
  • a polypeptide encoded by the H-RAS gene Preferably the amount of polypeptide is quantified using an immunoassay, e.g., ELISA.
  • the polypeptide is detected using an antibody that selectively binds to a polypeptide encoded by a mutant H-RAS gene.
  • melanoma or "cutaneous melanoma” refer to malignant neoplasms of melanocytes, which are pigment cells present normally in the epidermis and sometimes in the dermis.
  • cutaneous melanoma There are four types of cutaneous melanoma: lentigo maligna melanoma, superficial spreading melanoma (SSM), nodular melanoma, and acral lentiginous melanoma (AM).
  • SSM superficial spreading melanoma
  • AM acral lentiginous melanoma
  • 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.
  • 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”.
  • 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.
  • tumor or “cancer” in an animal refers to the presence of cells possessing characteristics such as atypical growth or mo ⁇ hology, including uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic mo ⁇ hological 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.
  • 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.
  • telomere shortening 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.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions.
  • 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 are sequence dependent, and are different under different environmental parameters.
  • 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).
  • 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.
  • label 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.
  • useful labels include 2 P, 35 S, 3 H, 14 C, 125 1, 13! I; fluorescent dyes (e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense reagents (e.g.
  • 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. DynabeadsTM ), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available.
  • the label can be directly inco ⁇ orated 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).
  • target DNA sequences can be detected by means of the primed in situ labeling technique (PRINS) (Koch et al., Genet. Anal. Tech. Appl. 8: 171-8, (1991)).
  • PRINS primed in situ labeling technique
  • 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)).
  • 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.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form.
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides which have similar or improved binding properties, for the pu ⁇ oses 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 pu ⁇ oses desired.
  • nucleic-acid-like structures with synthetic backbones are examples of 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, mo ⁇ holino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides 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 (NY AS 1992); Milligan (1993) J. Med. Chem.
  • 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).
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and amplification product.
  • nucleic acid array 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 ⁇ m to about 3 mm, preferably between about 5 ⁇ m 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.
  • each target element may comprise a mixture of nucleic acids of different lengths and sequences.
  • 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.
  • 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.
  • 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.
  • 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 purposes.
  • 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.
  • 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.
  • 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 llpl l.l to llpll.2 or l lql l .l to l lql l.2.
  • l ip 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.
  • 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.
  • 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 pu ⁇ ose (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).
  • 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.
  • 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 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.
  • 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.
  • 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 RMCl 1B022 for chromosome 1 lp (black bars) and RMCl 1P008 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.
  • 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.
  • Spitz nevi cells show a single amplification of chromosomal region 1 lp, particularly 1 lpl5.5, and more particularly, the H-RAS gene, which is localized to 1 lpl5.5, 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 1 lp 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.
  • 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 1 lpl5.5 region.
  • amplification with or without mutations of the H-RAS gene which is localized to 1 lpl 5.5, are also present in Spitz nevi.
  • H-RAS is rarely mutated in melanoma (see, e.g., Jiveskog et al, J. Invest. Dermatol. I l l :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.
  • the present invention provides methods for determining the presence of an amplified H-i 4S 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.
  • 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.
  • 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)).
  • cytogenetic approaches Mitsubishi, F., Catalog of chromosome aberrations in cancer, 5th Edition (New York: Wiley-Liss) (1994).
  • 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.
  • Fluorescence in-situ hybridization can be used to study copy numbers of individual genetic loci in inte ⁇ hase 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.
  • FISH Fluorescence in-situ hybridization
  • FISH FISH as an adjunctive diagnostic technique for the differentiation of Spitz nevi from melanomas
  • 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.
  • chromosome 1 was selected based on its frequent numerical change in melanoma metastasis (Thompson et al., Cancer Genet Cytogenet 83, 93-104 (1995)).
  • 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).
  • direct probe methods
  • CGH Comparative Genomic Hybridization
  • 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.
  • substrate e.g. membrane or glass
  • In situ hybridization assays are well known (e.g., Angerer (1987) Meth.
  • 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.
  • cells are fixed to a solid support, typically a glass slide.
  • 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
  • the targets 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.
  • human genomic DNA or Cot-1 DNA is used to block non-specific hybridization.
  • Comparative Genomic Hybridization methods a first collection of
  • sample nucleic acids e.g. from a possible tumor
  • control nucleic acids e.g. from a healthy cell/tissue
  • 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.
  • 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.
  • 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.
  • an isochromosome is detected using two probes, each labeled with a distinct compound, e.g., different fluorescent labels with distinguishable colors.
  • 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.
  • the H-RAS gene is located at l lpl5.5, 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.
  • Onco genie 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.
  • 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 oligonucleotides (ASO), dot blot analysis, PCR-SSCP, and allele-specific PCR.
  • CFLP-cleavase fragment length polymo ⁇ hism Another method known in the art is CFLP-cleavase fragment length polymo ⁇ hism. 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.
  • TMHC temperature modulation heteroduplex chromatography
  • 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.
  • a probe that specifically hybridizes to t eH-RAS gene can be used to detect the presence of mutations.
  • 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 oligonucleotides (see, e.g.,
  • 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.
  • 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 oligonucleotides 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)).
  • 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).
  • 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.
  • the 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.
  • 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.
  • 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.
  • substances that form gels can be used. Such materials include, e.g., proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides.
  • various pore sizes may be employed depending upon the nature of the system.
  • a plurality of different materials may be employed, particularly as laminates, to obtain various properties.
  • proteins e.g., bovine serum albumin
  • macromolecules e.g., Denhardt's solution
  • 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.
  • Target elements of various sizes ranging from 1 mm diameter down to 1 ⁇ m 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.
  • Such small array target elements are typically used in arrays with densities greater than 10 4 /cm 2 .
  • Relatively simple approaches capable of quantitative fluorescent imaging of 1 cm 2 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).
  • 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.
  • 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 autofluorescence than glass, can also be silanized.
  • probes can also be immobilized on commercially available coated beads or other surfaces.
  • 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.
  • paramagnetic particles such as ferric oxide particles, with or without avidin coating, can be used.
  • 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.
  • DMSO dimethylsulfoxide
  • Small capillaries tubes can be used to "spot" the probe mixture.
  • nucleic acid hybridization formats are known to those skilled in the art.
  • 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.
  • such binding occurs through ligand and anti-ligand interactions as between a ligand-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.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • 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.
  • hybrid duplexes e.g., DNA:DNA, RNA:RNA, or RNA:DNA
  • 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.
  • 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.
  • 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.
  • 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.
  • a detergent e.g., C-TAB
  • a blocking reagent e.g., sperm DNA, cot-1 DNA, etc.
  • the hybridization is performed in the presence of about 0.1 to about 0.5 mg/ml DNA (e.g., cot-1 DNA).
  • 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.
  • label e.g., fluorescence
  • 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 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.
  • 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.
  • the hybridized nucleic acids are detected by detecting one or more labels attached to the sample or probe nucleic acids.
  • the labels may be inco ⁇ orated 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 label e.g., a fluorophore
  • linkers for the attachment of labels to nucleic acids are also known.
  • 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., DynabeadsTM), 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., 3 H, 125 I, 35 S, 14 C, or 32 P), 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 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropy
  • 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.
  • 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.
  • fluorescers should absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the abso ⁇ tion 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.
  • 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.
  • ESR electron spin resonance
  • 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.
  • direct labels are detectable labels that are directly attached to or inco ⁇ orated into the target (sample) nucleic acid prior to hybridization.
  • indirect labels are joined to the hybrid duplex after hybridization.
  • the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization.
  • 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.
  • an antibody that is labeled with a fluorochrom, or an enzyme such as horseradish peroxidase or alkaline phosphatase.
  • Fluorescent labels are easily added during an in vitro transcription reaction.
  • fluorescein labeled UTP and CTP can be inco ⁇ orated into the RNA produced in an in vitro transcription.
  • the labels can be attached directly or through a linker moiety.
  • the site of label or linker-label attachment is not limited to any specific position.
  • 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.
  • certain Label-ON Reagents from Clontech provide for labeling interspersed throughout the phosphate backbone of an oligonucleotide and for terminal labeling at the 3' and 5' ends.
  • 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 pu ⁇ ose 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.
  • 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.
  • 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).
  • 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 can be used to measure copy number.
  • the nucleic acid sequences act as a template in an amplification reaction (e.g. Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • 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.).
  • LCR ligase chain reaction
  • 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.
  • gene transcript e.g. mRNA
  • 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.
  • amplification e.g. PCR
  • polypeptides encoded by regions of the chromosome that are amplified 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.
  • 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.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like
  • various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectro- phoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluor
  • 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.
  • the labeling agent in detecting H-RAS, the labeling agent may be a labeled H-RAS polypeptide or a labeled anti-H-RAS antibody.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a detectable moiety such as biotin
  • streptavidin another molecule specifically binds
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LOA liposome immunoassays
  • kits are also provided by the invention.
  • 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.
  • 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.
  • DNA Preparation Paraffin material 30 ⁇ m sections were cut, with a 5 ⁇ m section for H & E every 5 sections. The unstained 30 ⁇ m 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, ImM EDTA, 0.5% Tween 20 buffer.
  • proteinase K Life Technologies, Inc., Gaithersburg, MD
  • 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 12 ⁇ g. Comparative Genomic Hybridization (CGH) and Digital Image Analysis
  • 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 ⁇ g human Cot- 1 DNA (Life Technologies, Inc., Gaithersburg, MD) dissolved in 10 ⁇ l 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.
  • 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 (RMCl 1B022 and RMC 11 PO 14) and the long arm (RMC 11P008) 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). 6 ⁇ m sections were mounted on positively charged glass slides (Fisher Scientific, Pittsburgh, PA), deparaffmized, 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 20 ⁇ g Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD) were dissolved in lO ⁇ l hybridization buffer (50% formamide, 10% dextrane sulfate, and 2 x SSC, pH 7.0) and denatured for 10 min at 72°C.
  • lO ⁇ l hybridization buffer 50% formamide, 10% dextrane sulfate, and 2 x SSC, pH 7.0
  • 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 XI 00 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.
  • 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.
  • 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 l ip 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 lpl 5.5, clone RMCl 1B022) and a reference probe mapping to chromosome 1 lq23 (clone RMCl 1P008).
  • keratinocytes of the epidermis adjacent to the lesion were used as internal controls.
  • the hybridization was carried out on sections of 6 ⁇ m thickness, many nuclei were not fully represented in the slide.
  • 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).
  • a diagnostic test for spitzoid melanocytic neoplasms might include copy number detection of chromosomes 1 lp, 9, and 10. Gains of chromosome l ip could be inte ⁇ reted as in favor of Spitz nevus, and losses of chromosomes 9 and/or 10 as in favor of melanoma.
  • 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 l ip represent a recurrent aberration in a subset of lesions, (III) Spitz nevi are probably clonal neoplasms, (TV) 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.
  • Pl-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.
  • FISH is capable of detecting homozygous and heterozygous deletions in tissue sections.
  • 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.
  • 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)).
  • 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.
  • Tissue arrays were constructed according to Kononen et. al, (Nat. Med. 4:844-847, 1998).
  • 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 ⁇ m 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.
  • 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).
  • BAC clone RMCl 1B022
  • RMCl 1P008 RMCl 1P008
  • 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 lO ⁇ l hybridization buffer (50% formamide, 10% dextran sulfate, and 2 x SSC, pH 7.0, 20 ⁇ g Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD)).
  • DNA Sequence Analysis DNA was extracted from 30 ⁇ m 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 ⁇ l reaction volumes.
  • Each PCR reaction contained 3.5 mM MgCl 2 , 0.2 mM dNTP, 0.625 U Taq Gold Polymerase (Perkin Elmer), IX PCR Buffer II, 0.5 ⁇ M 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.
  • PCR products 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 ⁇ l 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 ⁇ l of gel loading buffer (83% deionized formamide, 17% gel loading dye) (PE Applied Biosystems).
  • 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.
  • amplification of 1 lp was found in 12 (11.8%) cases.
  • 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.
  • 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) .
  • 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 1 lp had wild-type sequences of both exons of H- RAS.
  • 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 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.
  • the percentages in the n column refere to the total number of 102 cases.
  • the ot er percentages refer to the number of cases that have the respective biological feature.
  • ⁇ v ⁇ 3 integrin The expression of the cellular adhesion molecule ⁇ v ⁇ 3 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 ⁇ 3 integrin was performed to determine the level of ⁇ v ⁇ integrin expression.
  • 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 2 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- 1156cl3, 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.

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Abstract

La présente invention concerne des méthodes permettant de distinguer les naevi mélanocytiques, par exemple les naevi de Spitz, des mélanomes malins. Ces méthodes consistent à mettre en contact un échantillon d'acide nucléique d'un patient avec une sonde se fixant de manière sélective sur une séquence polynucléotidique cible sur une région chromosomique, par exemple 11p, généralement amplifiée dans les naevi de Spitz. L'échantillon d'acide nucléique provient en général de cellules tumorales cutanées d'une lésion tumorale cutanée du patient. Au moyen d'une autre sonde se fixant de manière sélective sur une région chromosomique, par exemple 1q, 6p, 7p, 9p, ou 10q, présentant en général un changement dans le nombre de copies du mélanome, la méthode de l'invention permet de déterminer que les cellules tumorales ne présentant aucun changement dans le nombre de copies de 1q, 6p, 7p, 9p, ou 10q ne sont pas des cellules de mélanome mais bien des cellules de naevus de Spitz. La découverte d'amplifications du chromosome 11p et, en particulier, la présence d'un isochromosome 11p, constituerait une indication supplémentaire de la présence du naevus de Spitz. On peut également déterminer une augmentation du nombre de copies du chromosome 11p grâce à la détection de la présence d'une amplification du gène H-RAS. Le gène amplifié peut être normal ou peut être un gène H-RAS mutant.
PCT/US2000/009609 1999-04-09 2000-04-10 Detection de changements dans le nombre de copies d'un chromosome, permettant de distinguer les naevi melanocytiques des melanomes malins Ceased WO2000061814A1 (fr)

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CA002368903A CA2368903A1 (fr) 1999-04-09 2000-04-10 Detection de changements dans le nombre de copies d'un chromosome, permettant de distinguer les naevi melanocytiques des melanomes malins
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EP1929044A4 (fr) * 2005-09-02 2009-08-19 Univ California Methodes et combinaisons de sondes pour la detection de melanomes
US7597152B2 (en) 2003-11-25 2009-10-06 Baker Hughes Incorporated Swelling layer inflatable
US7960110B2 (en) 2006-11-15 2011-06-14 The Regents Of The University Of California Detection of chromosomal region copy number changes to diagnose melanoma
CN113817777A (zh) * 2021-10-27 2021-12-21 上海交通大学医学院附属第九人民医院 来源于人的先天性巨大黑痣良性肿瘤细胞系及其构建方法

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AU647741B2 (en) * 1989-12-01 1994-03-31 Regents Of The University Of California, The Methods and compositions for chromosome-specific staining

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US6465180B1 (en) * 1999-03-17 2002-10-15 The Regents Of The University Of California Detection of premalignant melanocytes

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DE WITT ET. AL.: "DNA in situ hybridization as a diagnostic tool in the discrimination of melanoma and spitz naevus", J. OF PATHOLOGY, vol. 173, 1994, pages 227 - 233, XP002929827 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005525786A (ja) * 2001-09-27 2005-09-02 スペクトラル ジェノミクス、インク． アレイを用いた遺伝子モザイクの検出方法
US7597152B2 (en) 2003-11-25 2009-10-06 Baker Hughes Incorporated Swelling layer inflatable
EP1929044A4 (fr) * 2005-09-02 2009-08-19 Univ California Methodes et combinaisons de sondes pour la detection de melanomes
EP2458016A1 (fr) * 2005-09-02 2012-05-30 The Regents of the University of California Procédés et combinaisons de sonde pour détecter un mélanome
US11441194B2 (en) 2005-09-02 2022-09-13 Abbott Molecular Inc. Methods and probe combinations for detecting melanoma
US7960110B2 (en) 2006-11-15 2011-06-14 The Regents Of The University Of California Detection of chromosomal region copy number changes to diagnose melanoma
US10119170B2 (en) 2006-11-15 2018-11-06 The Regents Of The University Of California Detection of chromosomal region copy number changes to diagnose melanoma
CN113817777A (zh) * 2021-10-27 2021-12-21 上海交通大学医学院附属第九人民医院 来源于人的先天性巨大黑痣良性肿瘤细胞系及其构建方法

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