# Patent

## Title
Use of chimeric CD4-src protein tyrosine kinases in drug screening and

## Abstract

This application is a continuation of U.S. Ser. No. 07/900,646, filed Jun. 18, 1992, now abandoned.

## Background
This invention relates to processes for diagnosing cancer. In a more specific aspect it relates to a method for detecting melanoma.

Despite improvements in surgical and radiological procedures and an increasingly large arsenal of drugs for treating cancer, cures for the many malignant neoplastic diseases remain elusive. The strides that have been made in reducing mortality have come largely from education programs which have heightened public awareness of the need for routine physical examination, coupled with improved techniques for detecting the presence of neoplastic disease at a sufficiently early stage to provide a favorable prognosis. Such improved techniques include immunoassays for an ever wider array of tumor associated antigens, such as carcinoembryonic antigen (CEA) and alphafetoprotein (AFP), and nucleic acid hybridization techniques. Nucleic acid hybridization can be used, for example, to diagnose the presence of chromosomal translocations associated with forms of cancer such as those identified with chronic myelogenous leukemia (CML), and the like.

Such advances are isolated and, accordingly, there remains a pressing need for better cancer diagnostics. In many cases, particularly those in which histological examination of biopsy material is used, it remains difficult to classify the kind of tumor which may be present. It is also difficult to differentiate malignancies from benign tumors and other non-malignant tissue abnormalities. Of perhaps even greater consequence to the patient, the techniques that are available are so lacking in sensitivity that early stage disease, the most opportune time for therapeutic intervention, is often missed entirely.

Malignant melanoma is an excellent example of a virulent cancer that is treatable by surgical removal with a high rate of success if detected before metastasis. After metastasis, however, the five year mortality rate is a shocking 97-99% using available chemotherapy and radiotherapy techniques (6,500 deaths in the United States in 1991). The shortcomings of modern treatments for melanoma take on heightened significance when it is appreciated that the incidence of this once relatively rare cancer is increasing annually at a rate of 4-10%, with 32,000 new cases in the United States in 1991.

Early detection of melanoma is complicated by the fact that many melanomas presented for diagnosis are either atypical in appearance (i.e., appear identical to non-melanocytic lesions), or are obscured by inflammation. Some melanomas are completely amelanotic (non-pigmented) and others are histologically similar to benign melanocytic growths. Thus, using classical histological techniques, it is frequently impossible to determine whether a lesion is actually a melanoma or whether it is a benign melanocytic growth or other type of carcinoma, for example, a sarcoma.

As the foregoing demonstrates, there clearly exists a pressing need for better techniques to detect and classify neoplastic diseases like melanoma. The present invention satisfies this need and provides related advantages as well.

According to the present invention, methods for detecting the presence of neoplastic disease are provided. The methods involve the detection of a product associated with expression of a gene which encodes a protein receptor for fibroblast growth factor (FGF) in cells or a body fluid, such as, for example, blood, serum or urine. When an assay is performed directly on suspect cells, the target product can be either the messenger RNA (mRNA) transcription product of the gene encoding the receptor or the receptor protein itself. In the case of body fluid and histopathologic analysis, the target expression product will be the receptor protein. Detection of the receptor can be accomplished by any technique which is specific for the protein. For example, immunological techniques employing antibodies to the receptor or a portion thereof that form a detectable antibody/receptor complex can be used. Techniques for detecting mRNA that employ nucleic acid hybridization are also useful for directly assaying suspect cells. These techniques use a nucleic acid probe having a nucleotide sequence that is complementary to that of the receptor. The probe is allowed to bind with the target RNA in a manner that permits detection of the resulting duplex using art established techniques.

Fibroblast growth factor (FGF) is an almost ubiquitous protein that is highly multifunctional and present in many tissues. For this reason, it has generally been thought to act in vivo on the same cell types in which it acts in vitro. Surprisingly, however, it has been found that contrary to all of the predictions made from the results using cells in culture, the receptor for FGF is differentially expressed in vivo. For example, neoplastic tissues and cells after injury have elevated levels of receptor when compared to the low to absent levels in non-malignant (quiescent) tissue. Thus, these significantly higher levels of expression of the receptor gene by select neoplasms can be used to confirm the presence of such neoplasms. The higher levels of expression can be detected either by determining the presence of the receptor or its mRNA in cellular material, or by determining the presence of the receptor in body fluids as a result of sloughing from tumor cells.

The detection of a specific FGF receptor or its mRNA that is associated with malignant melanoma is particularly advantageous since they are both increased in amounts when compared to normal or benign lesions. Furthermore, the expression of FGF has been directly implicated in the transformation from normal melanocytes to melanoma (Halaban, R. et al., "bFGF As An Autocrine Growth Factor For Human Melanomas" Oncogene Res. 3:177-186 (1988) which is incorporated herein by reference). Accordingly, the improved assay of the invention will be described below with particular reference to its use for detecting malignant melanoma. However, the invention is not intended to be limited to malignant melanoma and can be applied to numerous other neoplasms including, but not limited to, subtyping of breast cancer, ovarian cancer, prostate hyperplasia, prostate carcinoma, astrocytoma, glioblastoma and similar diseases that are linked to FGF receptor expression.

As indicated above, the present invention provides means for determining the presence of select neoplastic disease. The process of the present invention comprises assaying a suitable sample for the presence of a product indicative of elevated levels of expression of the gene for FGF receptor protein and/or the receptor protein itself. The receptor (hereinafter referred to as "FGFR") is a cell surface protein receptor of which there are numerous subtypes and isoforms, each potentially specific for a particular cell type, more highly expressed in malignant than in non-malignant tissue. The target product of the gene expression to be assayed can either be FGFR itself or the mRNA encoding FGFR. These products can be detected by any suitable means for detection of specific amino acid or nucleic acid sequences. Preferred for this purpose are immunological techniques for detecting FGFR and nucleic acid hybridization techniques for detecting mRNA encoding FGFR.

Immunological and hybridization techniques for assaying proteins and mRNA are generally well known in the art and, accordingly, will not be described in detail. However, immunological techniques that can be used for detecting FGFR in a fluid sample such as blood, serum or urine, for example, include competitive immunoassays that employ an antibody specific for the receptor. Such assays can also typically employ a labeled receptor as a second reagent which competes for the captive antibody with receptor present in the sample. The labeled receptor is typically conjugated with a moiety which permits direct or indirect detection. Among the useful labels are radioactive nuclides such as .sup.35 S enzymatic moieties that cause a color change, or luminescence-generating reactions with suitable substrates. Such enzymes include, for example, luciferase, horseradish peroxidase and alkaline phosphatase. Fluorescent labels can also be used. Alternatively, the labeled receptor can be conjugated with a moiety which is recognized by another reagent which permits detection. For example, the receptor can be bound to biotin which will bind to a detectable avidin derivative, e.g., avidin conjugated with an enzyme as described above.

Another immunological process useful for detecting FGFR in fluid samples is the two-site or "sandwich" assay. This assay typically uses two antibody preparations, one of which is specific for FGFR and is bound, or capable of being bound, to a solid phase to separate FGFR from other material present in the sample. The second antibody preparation also binds FGFR and is labeled to permit detection of the antibody (solid phase):FGFR:antibody (labeled) complex after the appropriate contacting of the sample with the antibody reagents.

A third and presently preferred immunological technique, particularly for detection of melanoma, is the direct immunostaining of sections of biopsy material using anti-FGFR antibodies. Frozen sections or paraffin-fixed sections can be used. The anti-FGFR antibodies can be directly labeled with an enzyme such as peroxidase in order to generate a color reaction when substrate is added to the section. Preferably, however, the section is incubated with the anti-FGFR antibody, treated with a biotinylated antibody against the species of origin of the primary antibody, and then exposed to a complex of avidin and enzyme to increase the amount of enzyme present on the section and thereby increase the sensitivity of the process. A detailed description of this preferred process follows below. However, before doing so it should be noted that the antibodies useful in any of these immunological processes can be either polyclonal or monoclonal in nature and the species of origin, e.g., rabbit, goat, rat or murine, is not a critical feature of the invention. In addition, functional fragments of antibodies such as Fab, (Fab').sub.2 and peptides having an FGFR binding region can be employed. The means for obtaining such antibodies and fragments are also well known to those skilled in the art and will not be described in detail.

An antigen suitable for use in obtaining the antibodies useful in the practice of the present invention is, of course, of the FGF receptors, whose cloning and expression are described, for example, in Isacchi et al., "Complete Sequence of a Human Receptor for Acidic and Basic Fibroblast Growth Factors", Nucleic Acid Res. 18:1906 (1990) and in Kiefer et al., "Molecular Cloning of A Human Basic FGF Receptor cDNA and Expression of a Biologically Active Extracellular Domain in a Baculovirus System", Growth Factors 5:115-119 (1991), which are incorporated herein by reference. Fragments of FGFR may also be used as the antigen for production of either monoclonal or polyclonal antibodies to FGFR(s). One such antigen is the extracellular domain of FGFR. The preparation of polyclonal and monoclonal antibodies is described in Goding, J. W., Monoclonal Antibodies: Principles and Practice, Academic Press, London, NW1 (1986) and Groper, T. G., The Tools of Biochemistry, John Wiley & Sons, New York (1977), which are incorporated herein by reference.

The DNA sequence and encoded amino acids of a full length clone flg (FGFR-1), encoding a human receptor for acidic and basic fibroblast growth factors is set forth in SEQ ID NO. 1. The extracellular domain is included in amino acids 1-376 of SEQ ID NO. 1 and 1-374 of flg5 (Kiefer et al.), which lacks the Arg Met of SEQ ID NO. 1, residues 148-149.

A process for detecting the presence of FGFR in a paraffin-fixed section of biopsy material using a monoclonal or polyclonal antibody to FGFR, the preparation of which is described below, involves the following steps:

1. Incubate the section in 3% hydrogen peroxide for three (3) to five (5) minutes (depending on the antibody used) to quench endogenous peroxidase activity.

2. Wash section in phosphate-buffered saline (PBS) containing 0.1-0.5% Triton for 15-30 minutes depending on the antibody used as determined by optimization procedures.

3. Preincubate section for 20 minutes with a 1.5% solution in PBS-0.3% Triton (Sigma, St. Louis, Mo.) of a blocking serum comprising non-specific antibody of the same species as the anti-FGFR antibody (e.g., an antibody of murine origin), to saturate non-specific binding sites reactive with the anti-FGFR antibody.

4. Blot excess serum from the section.

5. Incubate section at 4.degree. C. for 16-20 hours with "anti-FGFR antibody solution," prepared as described below.

6. After incubation with anti-FGFR, wash section for fifteen (15) minutes with PBS-0.3% Triton to remove non-specifically bound antibody.

7. Incubate sections with biotinylated anti-mouse antibody (commercially available from Vector Laboratories, Burlingame, Calif.) diluted in PBS-0.3% Triton containing 1.5% of the blocking serum of step 3 for 45 minutes at room temperature.

8. Wash section with PBS-0.3% Triton for fifteen (15) minutes.

9. Incubate section for 30 minutes to one (1) hour with avidin conjugated with peroxidase (ABC complex from Vector), diluted in PBS-0.3% Triton prepared thirty (30) minutes before use.

10. Wash section with PBS-0.3% Triton for fifteen (15) minutes.

11. Incubate section for twenty (20) minutes in peroxidase substrate solution (aminoethylcarbozate from Sigma) in DMSO and 50 mM acetate buffer (pH 5.0), containing 0.01% hydrogen peroxidase.

12. Wash section in deionized water for ten (10) minutes.

13. Counterstain section with hematoxylin (Fisher Chemicals, Tustin, Calif.) and mount slide with crystal mount.

14. Read section under a microscope comparing color versus lack of color presentation. Color development indicates a malignant lesion.

As can be seen in Table 1, malignant melanocytes stain positively using the techniques described above. However, benign melanocytes stain negatively using the above-described method, thus establishing selective expression of the detected protein in the neoplasia. ______________________________________ Antibody to Extracellular Domain of FGF Receptor FGFR LESION CLINICAL.sup.1 STAINING.sup.2 COMMENT ______________________________________ melanoma met, amelanotic +++ melanoma SST, CLIV    +++ melanoma MMSST CLIV   +++ melanoma SST, CLIV, B1.01 +++ melanoma SST          +++       some epidermal staining melanoma SST, halonevus, ++ inflammation melanoma nodular type ++ melanoma SST          ++ melanoma SST          +++       some epidermal staining melanoma (LMM)        ++ melanoma SST          ++ melanoma large inflammatory +++       some epidermal staining halo nevus, minimal deviation melanoma melanoma SST, CLIV    ++ melanoma NT           + melanoma SST, CLIV    +++ melanoma SST, CLIII   ++ melanoma SST          ++        no counterstain melanoma SST          ++        some blood vessel (+) staining melanoma SST, CLII    ++        no counterstain melanoma SST          + melanoma NT, SST      ++ melanoma SST          ++ melanoma SST, NT      ++ melanoma (MM) SST     ++ melanoma SST          +++       some epidermal staining nevi   Bgn intradermal - nevus nevi   Bgn intradermal - nevus nevi   Bgn compound -         some blood vessel nevus                  staining nevi   Bgn compound - nevus nevi   SLIN pigmented - compound nevus nevi   Bgn pigmented - intradermal nevus nevi   Bgn intradermal - nevus nevi   SLIN pigmented -         some epidermal staining compound nevus nevi   Bgn neurotized - nevus nevi   Bgn pigmented - compound nevus nevi   SLIN pigmented - compound nevus nevi   Bgn intradermal +         some nevus staining nevus nevi   Bgn intradermal - nevus nevi   Bgn intradermal -         minimal epidermal nevus                  staining nevi   Bgn compound - nevus nevi   Bgn intradermal - nevus nevi   Bgn compound - nevus nevi   Bgn compound - nevus nevi   blue nevus, heavy - pigment nevi   bgn blue nevus, - heavy pigment nevi   compound bgn - Spitz nevus ______________________________________ .sup.1 Clinical abbreviations: SST = Superficial Spreading MMSST = Malignant Melanoma Superficial Spreading NT = Nodular Type BGN = Benign LMM = Lentigo Malignant Melanoma CL = Clark's Level, e.g., depth of the melanoma from the epidermis SLIN = Slightly Inflamed B = Breslow Level, e.g., actual depth in millimeters from the epidermis .sup.2 Staining Level: - = no staining + = minimal (light) ++ = intermediate +++ = maximal (dark)

The lesions used in this study were obtained from human biopsy samples. The clinical terminology and staining determinations are described in W. F. Lever, Histopathology of the Skin, 7th ed., J. B. Lippincott Company, Philadelphia (1990).

As pointed out above, mRNA transcribed from the receptor gene is an alternative target to the receptor itself when an assay is performed directly on suspected cancer cells. In such assays, a nucleic acid probe having a nucleotide sequence complementary to the target mRNA is added to the cells in a manner which permits the probe to hybridize to the mRNA encoding the receptor. The length of the probe can, of course, be any length adequate to permit a diagnostic result when high stringency hybridization conditions are used. As used herein, "high stringency conditions" means conditions of low salt concentration and elevated temperature whereby the cRNA probe for FGFR will anneal to the RNA encoding FGFR alone and not non-specifically to other mRNA. Accordingly, the length of the probe is preferably at least about 20 nucleotides, and more preferably at least about 50 nucleotides, and will not normally exceed about 500 nucleotides.

The probe can be labeled to permit its direct detection. While a variety of labels are useful, since the sample to be assayed will typically be a paraffin-embedded section from a tissue biopsy, it is preferred that the label be one which can generate a color reaction when a suitable substrate is added to the section after any unbound probe has been removed by washing. Among suitable labels, therefore, are enzymes which, in the presence of a suitable substrate, generate a color. These include various peroxidases, such as horseradish peroxidase, alkaline phosphatase and the like in a particular embodiment the probe is labeled with digoxin.

Although direct labeling can be used, it is preferred to label the probe with biotin and to treat the hybridized probe with an avidin/enzyme complex as described above for the immunostaining process. This procedure can conveniently be done by synthesizing the probe using a nucleotide which is biotinylated. Biotinylated uracil triphosphate is preferred for this purpose.

A preferred process for detecting mRNA encoding FGFR in a standard paraffin-fixed section of biopsy material using, for example, a biotinylated probe of a sequence derived for example from a sequence set forth in Kiefer et al., Isacchi et al., Keegan et al., "Characterization of the FGFR-3 Gene and its Gene Product", Ann. N.Y. Acad. Sci. 638:400-402 (1991), Hou et al. "Fibroblast Growth Factor Receptors From Liver Vary in Three Structural Domains", Science 251:665-668 (1991) or Partanen et al., "FGFR-4, A Novel Acidic Fibroblast Growth Factor Receptor With Distinct Expression Pattern", EMBO J. 10:1345-1354 (1991) or other related sequences corresponding to other parts of the many isoforms and subtypes of the FGF receptor, involves the following steps:

1. Prior to hybridization, wash the slide with the following reagents:

2. Dilute probe to 800 ng/ml with buffer (recipe steps 2a-2f below) and apply to slide. Seal the coverslips and incubate overnight at 55.degree. C. on a warm plate.

3. Rinse with 4.times. SSPE for 20 minutes to remove coverslip followed by 3.times.10-minute rinses.

4. Rinse with 50% formamide/2.times. SSPE, 2.times. for 15 minutes at 48.degree.-50.degree. C.

5. Rinse with 2.times. SSPE buffer, 2.times. (10 min.)

6. Incubate with RNase, 37.degree. C. (30 min.)

7. Rinse with 2.times. SSPE buffer, 2.times. (10 min.)

8. Rinse with 50% formamide/2.times. SSPE at 48.degree.-50.degree. C. (15 min.)

9. Rinse with 2.times. SSPE buffer (5 min.)

10. Rinse with 0.1M Tris Saline (5 min.)

11. Incubate section with avidin complex with alkaline phosphatase (Vector) for 1 hour at room temperature.

12. Rinse section with 0.1M Tris saline for 5 minutes (3.times.).

13. Incubate section with Fast Red solution for 8 hours (optionally with Levamisole, an inhibitor of endogenous alkaline phosphatase).

14. Rinse with deionized water 3.times..

15. Counterstain with hematoxylin (optional).

16. Rinse with deionized water.

17. Mount section with crystal mount.

18. Read section under a microscope and look for color change.

As shown in Table 2, malignant melanocytes stain positively using the techniques described above. Benign melanocytes stain negatively using the same techniques, thus establishing that there is selective expression of the detected mRNAs in neoplastic cells. The lesions used in this study were obtained from human biopsy samples. ______________________________________ IN SITU HYBRIDIZATION OF FGFR mRNA PROBE (BIOTINYLATED) LESION    CLINICAL.sup.1 STAINING.sup.2 COMMENT ______________________________________ melanoma  SST       ++ melanoma  SST       ++        grainy melanoma  SST       ++ nevi (intradermal)  - nevi (compound)     - nevi (intradermal)  -         some epidermal staining ______________________________________ .sup.1 Clinical abbreviations: SST = Superficial Spreading .sup.2 Staining Level: - = no staining + = minimal (light) ++ = intermediate +++ = maximal (dark)

Although the invention has been described with reference to the presently-preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2469 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION: 1...2466 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATGTGGAGCTGGAAGTGCCTCCTCTTCTGGGCTGTGCTGGTCACAGCC48 MetTrpSerTrpLysCysLeuLeuPheTrpAlaValLeuValThrAla 151015 ACACTCTGCACCGCTAGGCCGTCCCCGACCTTGCCTGAACAAGCCCAG96 ThrLeuCysThrAlaArgProSerProThrLeuProGluGlnAlaGln 202530 CCCTGGGGAGCCCCTGTGGAAGTGGAGTCCTTCCTGGTCCACCCCGGT144 ProTrpGlyAlaProValGluValGluSerPheLeuValHisProGly 354045 GACCTGCTGCAGCTTCGCTGTCGGCTGCGGGACGATGTGCAGAGCATC192 AspLeuLeuGlnLeuArgCysArgLeuArgAspAspValGlnSerIle 505560 AACTGGCTGCGGGACGGGGTGCAGCTGGCGGAAAGCAACCGCACCCGC240 AsnTrpLeuArgAspGlyValGlnLeuAlaGluSerAsnArgThrArg 65707580 ATCACAGGGGAGGAGGTGGAGGTGCAGGACTCCGTGCCCGCAGACTCC288 IleThrGlyGluGluValGluValGlnAspSerValProAlaAspSer 859095 GGCCTCTATGCTTGCGTAACCAGCAGCCCCTCGGGCAGTGACACCACC336 GlyLeuTyrAlaCysValThrSerSerProSerGlySerAspThrThr 100105110 TACTTCTCCGTCAATGTTTCAGATGCTCTCCCCTCCTCGGAGGATGAT384 TyrPheSerValAsnValSerAspAlaLeuProSerSerGluAspAsp 115120125 GATGATGATGATGACTCCTCTTCAGAGGAGAAAGAAACAGATAACACC432 AspAspAspAspAspSerSerSerGluGluLysGluThrAspAsnThr 130135140 AAACCAAACCGTATGCCCGTAGCTCCATATTGGACATCCCCAGAAAAG480 LysProAsnArgMetProValAlaProTyrTrpThrSerProGluLys 145150155160 ATGGAAAAGAAATTGCATGCAGTGCCGGCTGCCAAGACAGTGAAGTTC528 MetGluLysLysLeuHisAlaValProAlaAlaLysThrValLysPhe 165170175 AAATGCCCTTCCAGTGGGACCCCAAACCCCACACTGCGCTGGTTGAAA576 LysCysProSerSerGlyThrProAsnProThrLeuArgTrpLeuLys 180185190 AATGGCAAAGAATTCAAACCTGACCACAGAATTGGAGGCTACAAGGTC624 AsnGlyLysGluPheLysProAspHisArgIleGlyGlyTyrLysVal 195200205 CGTTATGCCACCTGGAGCATCATAATGGACTCTGTGGTGCCCTCTGAC672 ArgTyrAlaThrTrpSerIleIleMetAspSerValValProSerAsp 210215220 AAGGGCAACTACACCTGCATTGTGGAGAATGAGTACGGCAGCATCAAC720 LysGlyAsnTyrThrCysIleValGluAsnGluTyrGlySerIleAsn 225230235240 CACACATACCAGCTGGATGTCGTGGAGCGGTCCCCTCACCGGCCCATC768 HisThrTyrGlnLeuAspValValGluArgSerProHisArgProIle 245250255 CTGCAAGCAGGGTTGCCCGCCAACAAAACAGTGGCCCTGGGTAGCAAC816 LeuGlnAlaGlyLeuProAlaAsnLysThrValAlaLeuGlySerAsn 260265270 GTGGAGTTCATGTGTAAGGTGTACAGTGACCCGCAGCCGCACATCCAG864 ValGluPheMetCysLysValTyrSerAspProGlnProHisIleGln 275280285 TGGCTAAAGCACATCGAGGTGAATGGGAGCAAGATTGGCCCAGACAAC912 TrpLeuLysHisIleGluValAsnGlySerLysIleGlyProAspAsn 290295300 CTGCCTTATGTCCAGATCTTGAAGACTGCTGGAGTTAATACCACCGAC960 LeuProTyrValGlnIleLeuLysThrAlaGlyValAsnThrThrAsp 305310315320 AAAGAGATGGAGGTGCTTCACTTAAGAAATGTCTCCTTTGAGGACGCA1008 LysGluMetGluValLeuHisLeuArgAsnValSerPheGluAspAla 325330335 GGGGAGTATACGTGCTTGGCGGGTAACTCTATCGGACTCTCCCATCAC1056 GlyGluTyrThrCysLeuAlaGlyAsnSerIleGlyLeuSerHisHis 340345350 TCTGCATGGTTGACCGTTCTGGAAGCCCTGGAAGAGAGGCCGGCAGTG1104 SerAlaTrpLeuThrValLeuGluAlaLeuGluGluArgProAlaVal 355360365 ATGACCTCGCCCCTGTACCTGGAGATCATCATCTATTGCACAGGGGCC1152 MetThrSerProLeuTyrLeuGluIleIleIleTyrCysThrGlyAla 370375380 TTCCTCATCTCCTGCATGGTGGGGTCGGTCATCGTCTACAAGATGAAG1200 PheLeuIleSerCysMetValGlySerValIleValTyrLysMetLys 385390395400 AGTGGTACCAAGAAGAGTGACTTCCACAGCCAGATGGCTGTGCACAAG1248 SerGlyThrLysLysSerAspPheHisSerGlnMetAlaValHisLys 405410415 CTGGCCAAGAGCATCCCTCTGCGCAGACAGGTAACAGTGTCTGCTGAC1296 LeuAlaLysSerIleProLeuArgArgGlnValThrValSerAlaAsp 420425430 TCCAGTGCATCCATGAACTCTGGGGTTCTTCTGGTTCGGCCATCACGG1344 SerSerAlaSerMetAsnSerGlyValLeuLeuValArgProSerArg 435440445 CTCTCCTCCAGTGGGACTCCCATGCTAGCAGGGGTCTCTGAGTATGAG1392 LeuSerSerSerGlyThrProMetLeuAlaGlyValSerGluTyrGlu 450455460 CTTCCCGAAGACCCTCGCTGGGAGCTGCCTCGGGACAGACTGGTCTTA1440 LeuProGluAspProArgTrpGluLeuProArgAspArgLeuValLeu 465470475480 GGCAAACCCCTGGGAGAGGGCTGCTTTGGGCAGGTGGTGTTGGCAGAG1488 GlyLysProLeuGlyGluGlyCysPheGlyGlnValValLeuAlaGlu 485490495 GCTATCGGGCTGGACAAGGACAAACCCAACCGTGTGACCAAAGTGGCT1536 AlaIleGlyLeuAspLysAspLysProAsnArgValThrLysValAla 500505510 GTGAAGATGTTGAAGTCGGACGCAACAGAGAAAGACTTGTCAGACCTG1584 ValLysMetLeuLysSerAspAlaThrGluLysAspLeuSerAspLeu 515520525 ATCTCAGAAATGGAGATGATGAAGATGATCGGGAAGCATAAGAATATC1632 IleSerGluMetGluMetMetLysMetIleGlyLysHisLysAsnIle 530535540 ATCAACCTGCTGGGGGCCTGCACGCAGGATGGTCCCTTGTATGTCATC1680 IleAsnLeuLeuGlyAlaCysThrGlnAspGlyProLeuTyrValIle 545550555560 GTGGAGTATGCCTCCAAGGGCAACCTGCGGGAGTACCTGCAGGCCCGG1728 ValGluTyrAlaSerLysGlyAsnLeuArgGluTyrLeuGlnAlaArg 565570575 AGGCCCCCAGGGCTGGAATACTGCTACAACCCCAGCCACAACCCAGAG1776 ArgProProGlyLeuGluTyrCysTyrAsnProSerHisAsnProGlu 580585590 GAGCAGCTCTCCTCCAAGGACCTGGTGTCCTGCGCCTACCAGGTGGCC1824 GluGlnLeuSerSerLysAspLeuValSerCysAlaTyrGlnValAla 595600605 GGAGGCATGGAGTATCTGGCCTCCAAGAAGTGCATACACCGAGACCTG1872 GlyGlyMetGluTyrLeuAlaSerLysLysCysIleHisArgAspLeu 610615620 GCAGCCAGGAATGTCCTGGTGACAGAGGACAATGTGATGAAGATAGCA1920 AlaAlaArgAsnValLeuValThrGluAspAsnValMetLysIleAla 625630635640 GACTTTGGCCTCGCACGGGACATTCACCACATCGACTACTATAAAAAG1968 AspPheGlyLeuAlaArgAspIleHisHisIleAspTyrTyrLysLys 645650655 ACAACCAACGGCCGACTGCCTGTGAAGTGGATGGCACCCGAGGCATTA2016 ThrThrAsnGlyArgLeuProValLysTrpMetAlaProGluAlaLeu 660665670 TTTGACCGGATCTACACCCACCAGAGTGATGTGTGGTCTTTCGGGGTG2064 PheAspArgIleTyrThrHisGlnSerAspValTrpSerPheGlyVal 675680685 CTCCTGTGGGAGATCTTCACTCTGGGCGGCTCCCCATACCCCGGTGTG2112 LeuLeuTrpGluIlePheThrLeuGlyGlySerProTyrProGlyVal 690695700 CCTGTGGAGGAACTTTTCAAGCTGCTGAAGGAGGGTCACCGCATGGAC2160 ProValGluGluLeuPheLysLeuLeuLysGluGlyHisArgMetAsp 705710715720 AAGCCCAGTAACTGCACCAACGAGCTGTACATGATGATGCGGGACTGC2208 LysProSerAsnCysThrAsnGluLeuTyrMetMetMetArgAspCys 725730735 TGGCATGCAGTGCCCTCACAGAGACCCACCTTCAAGCAGCTGGTGGAA2256 TrpHisAlaValProSerGlnArgProThrPheLysGlnLeuValGlu 740745750 GACCTGGACCGCATCGTGGCCTTGACCTCCAACCAGGAGTACCTGGAC2304 AspLeuAspArgIleValAlaLeuThrSerAsnGlnGluTyrLeuAsp 755760765 CTGTCCATGCCCCTGGACCAGTACTCCCCCAGCTTTCCCGACACCCGG2352 LeuSerMetProLeuAspGlnTyrSerProSerPheProAspThrArg 770775780 AGCTCTACGTGCTCCTCAGGGGAGGATTCCGTCTTCTCTCATGAGCCG2400 SerSerThrCysSerSerGlyGluAspSerValPheSerHisGluPro 785790795800 CTGCCCGAGGAGCCCTGCCTGCCCCGACACCCAGCCCAGCTTGCCAAT2448 LeuProGluGluProCysLeuProArgHisProAlaGlnLeuAlaAsn 805810815 CGGGGACTCAAACGCCGCTGA2469 ArgGlyLeuLysArgArg 820 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 822 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetTrpSerTrpLysCysLeuLeuPheTrpAlaValLeuValThrAla 151015 ThrLeuCysThrAlaArgProSerProThrLeuProGluGlnAlaGln 202530 ProTrpGlyAlaProValGluValGluSerPheLeuValHisProGly 354045 AspLeuLeuGlnLeuArgCysArgLeuArgAspAspValGlnSerIle 505560 AsnTrpLeuArgAspGlyValGlnLeuAlaGluSerAsnArgThrArg 65707580 IleThrGlyGluGluValGluValGlnAspSerValProAlaAspSer 859095 GlyLeuTyrAlaCysValThrSerSerProSerGlySerAspThrThr 100105110 TyrPheSerValAsnValSerAspAlaLeuProSerSerGluAspAsp 115120125 AspAspAspAspAspSerSerSerGluGluLysGluThrAspAsnThr 130135140 LysProAsnArgMetProValAlaProTyrTrpThrSerProGluLys 145150155160 MetGluLysLysLeuHisAlaValProAlaAlaLysThrValLysPhe 165170175 LysCysProSerSerGlyThrProAsnProThrLeuArgTrpLeuLys 180185190 AsnGlyLysGluPheLysProAspHisArgIleGlyGlyTyrLysVal 195200205 ArgTyrAlaThrTrpSerIleIleMetAspSerValValProSerAsp 210215220 LysGlyAsnTyrThrCysIleValGluAsnGluTyrGlySerIleAsn 225230235240 HisThrTyrGlnLeuAspValValGluArgSerProHisArgProIle 245250255 LeuGlnAlaGlyLeuProAlaAsnLysThrValAlaLeuGlySerAsn 260265270 ValGluPheMetCysLysValTyrSerAspProGlnProHisIleGln 275280285 TrpLeuLysHisIleGluValAsnGlySerLysIleGlyProAspAsn 290295300 LeuProTyrValGlnIleLeuLysThrAlaGlyValAsnThrThrAsp 305310315320 LysGluMetGluValLeuHisLeuArgAsnValSerPheGluAspAla 325330335 GlyGluTyrThrCysLeuAlaGlyAsnSerIleGlyLeuSerHisHis 340345350 SerAlaTrpLeuThrValLeuGluAlaLeuGluGluArgProAlaVal 355360365 MetThrSerProLeuTyrLeuGluIleIleIleTyrCysThrGlyAla 370375380 PheLeuIleSerCysMetValGlySerValIleValTyrLysMetLys 385390395400 SerGlyThrLysLysSerAspPheHisSerGlnMetAlaValHisLys 405410415 LeuAlaLysSerIleProLeuArgArgGlnValThrValSerAlaAsp 420425430 SerSerAlaSerMetAsnSerGlyValLeuLeuValArgProSerArg 435440445 LeuSerSerSerGlyThrProMetLeuAlaGlyValSerGluTyrGlu 450455460 LeuProGluAspProArgTrpGluLeuProArgAspArgLeuValLeu 465470475480 GlyLysProLeuGlyGluGlyCysPheGlyGlnValValLeuAlaGlu 485490495 AlaIleGlyLeuAspLysAspLysProAsnArgValThrLysValAla 500505510 ValLysMetLeuLysSerAspAlaThrGluLysAspLeuSerAspLeu 515520525 IleSerGluMetGluMetMetLysMetIleGlyLysHisLysAsnIle 530535540 IleAsnLeuLeuGlyAlaCysThrGlnAspGlyProLeuTyrValIle 545550555560 ValGluTyrAlaSerLysGlyAsnLeuArgGluTyrLeuGlnAlaArg 565570575 ArgProProGlyLeuGluTyrCysTyrAsnProSerHisAsnProGlu 580585590 GluGlnLeuSerSerLysAspLeuValSerCysAlaTyrGlnValAla 595600605 GlyGlyMetGluTyrLeuAlaSerLysLysCysIleHisArgAspLeu 610615620 AlaAlaArgAsnValLeuValThrGluAspAsnValMetLysIleAla 625630635640 AspPheGlyLeuAlaArgAspIleHisHisIleAspTyrTyrLysLys 645650655 ThrThrAsnGlyArgLeuProValLysTrpMetAlaProGluAlaLeu 660665670 PheAspArgIleTyrThrHisGlnSerAspValTrpSerPheGlyVal 675680685 LeuLeuTrpGluIlePheThrLeuGlyGlySerProTyrProGlyVal 690695700 ProValGluGluLeuPheLysLeuLeuLysGluGlyHisArgMetAsp 705710715720 LysProSerAsnCysThrAsnGluLeuTyrMetMetMetArgAspCys 725730735 TrpHisAlaValProSerGlnArgProThrPheLysGlnLeuValGlu 740745750 AspLeuAspArgIleValAlaLeuThrSerAsnGlnGluTyrLeuAsp 755760765 LeuSerMetProLeuAspGlnTyrSerProSerPheProAspThrArg 770775780 SerSerThrCysSerSerGlyGluAspSerValPheSerHisGluPro 785790795800 LeuProGluGluProSerLeuProArgHisProAlaGlnLeuAlaAsn 805810815 ArgGlyLeuLysArgArg 820 __________________________________________________________________________

## Claims

1. A process for detecting malignant melanoma in a subject, comprising detecting, in a sample that contains melanocytes obtained from the subject, a first product indicative of elevated expression of a fibroblast growth factor receptor gene or a second product indicative of elevated amounts of a fibroblast growth factor receptor (FGFR), wherein detection of said first or second product in elevated expression or amount, respectively, compared to a control sample containing normal or benign melanocytes indicates the presence of malignant melanoma in said subject.

2. A process for detecting malignant melanoma in a subject, comprising detecting, in a sample that contains melanocytes obtained from the subject, a first product indicative of elevated expression of a fibroblast growth factor receptor gene or a second product indicative of elevated amounts of a fibroblast growth factor receptor (FGFR), wherein detection of said first or second product in elevated expression or amount, respectively, compared to a control sample containing normal or benign melanocytes indicates the presence of malignant melanoma in said subject; and detection is effected by contacting the sample obtained from the subject with an agent that binds to the extracellular domain of an FGFR or with a nucleic acid probe that includes a sequence of at least about 20 nucleotides that hybridizes under conditions of high stringency to nucleic acid encoding the extracellular domain of an FGFR.

3. The process of claim 2, wherein the detection of said product is accomplished by: detection of the binding of said agent indicates the presence of malignant melanoma.

4. The agent of claim 3, wherein said process is an antibody or a functional fragment thereof.

5. The agent of claim 3, wherein said process is a nucleic acid probe.

6. The agent of claim 3, wherein said process is labelled with a detectable marker.

7. A process according to claim 3, wherein the product is a receptor protein.

8. A process according to claim 2, wherein an immunological process is used to detect the receptor protein.

9. The process of claim 8, wherein the sample is biopsied tissue and the immunological process comprises fixing the sample in paraffin; and treating the paraffin-fixed section of biopsy material with an antibody having specific reactivity with the receptor protein, removing unbound antibody from the section and detecting the antibody bound to receptor protein present in the section, wherein the detection of antibody bound to said receptor protein indicates the presence of malignant melanoma in said subject.

10. A process according to claim 9, wherein the bound antibody is detected by: first antibody, wherein said second antibody is biotinylated; malignant malanoma.

11. A process according to claim 3, wherein the product is mRNA encoding the receptor protein.

12. A process according to claim 11, wherein the mRNA is detected by hybridization with a detectable nucleic acid probe having a nucleotide sequence sufficiently complementary to at least a portion of the mRNA to hybridize to said mRNA.

13. A process according to claim 12, wherein the probe is biotinylated.

14. A process according to claim 12, wherein the probe is labeled with digoxin.

15. A process according to claim 13 or 14, wherein hybridization of the probe to mRNA in said sample is detected by: phosphatase; phosphatase; malignant melanoma.

16. The process of claim 2, wherein the product is mRNA.

17. The process of claim 2, wherein the FGFR is FGFR-1.

18. The process of claim 12, wherein the FGFR is FGFR-1.

19. The process of claim 7, wherein the receptor is FGFR-1.

20. The process of claim 11, wherein the sample is biopsied tissue.

21. A process for detecting malignant melanoma in a subject, comprising detecting, in a sample that contains melanocytes obtained from the subject, a first product indicative of elevated expression of a fibroblast growth factor receptor gene or a second product indicative of elevated amounts of a fibroblast growth factor receptor (FGFR), wherein detection of said first or second product in elevated expression or amount, respectively, compared to a control sample containing normal or benign melanocytes indicates the presence of malignant melanoma in said subject; and detection is effected by contacting the sample obtained from said subject with an agent that binds to FGFR-1 or with a nucleic acid probe that includes a sequence of at least about 20 nucleotides that hybridizes under conditions of high stringency to nucleic acid encoding FGFR-1.

22. The process of claim 21, wherein the detection of the product is accomplished by: detection of the binding of the agent indicates the presence of malignant melanoma.

23. The process of claim 21, wherein the agent is an antibody or a functional fragment thereof.

24. The process of claim 21, wherein the agent is a nucleic acid probe.