CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional application no. 61/325,222, filed April 16, 2010 and of U.S. provisional applicaton no. 61/408,427, filed October 29, 2010. Each application is herein incorporated by reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under grant no. R01 CA131524- 01 Al awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The current model of melanoma formation is that melanocytes progress from a normal to malignant state by accumulating mutations in key melanoma genes. See, Meier, F., et al. (1998) Frontiers in Bioscience 3 :D 1005- 1010. Melanoma can arise spontaneously, or within a pre-existing nevus or mole. Nevi possess mutations in known melanoma genes and are therefore a risk factor for developing melanoma. See, e.g., Pollock, P.M., et al, (2003) Nat. Genet. 33(l): 19-20; Kumar, R. et al, (2004) J. Invest. Dermatol. 122(2):342-348; Chin, L., (2003) Nat. Rev. Cancer 3(8):559-570. [0004] The majority of human melanomas and melanocyte nevi have been shown to have activating mutations in the BRAF, NRAS, C-KIT, or HRAS genes. Furthermore, recent studies have demonstrated that melanomas fall into genetically distinct groups having marked differences in the frequency of MAP-kinase pathway activation. See, Curtin, J.A., et al, (2005) NEngl JMed. 353(20):2135-47. One category, uveal melanoma, arises from melanocytes within the choroidal plexus of the eye and is biologically distinct from cutaneous melanoma by characteristic cytogenetic alterations. See, Horsman et al. (1993) Cancer 71(3): 811. The other category are intradermal melanocytic proliferations, which can be congenital or acquired, and present in diverse ways ranging from discrete bluish moles (blue nevi) to large blue-gray patches affecting the conjunctiva and periorbital skin (nevus of Ota), shoulders (nevus of Ito), and the lower back (Mongolain spot). See, Zembowicz, et al.

(2004) Histopathology 45(5):433. These intradermal melanocytic proliferations do not contain either BRAF or NRAS mutations, and thus have a unique eitiology when compared with other nevi and melanoma. See, Ariyanayagam-Baksh SM, et al., (2003) Am J

Dermatopathol. 25(1): p. 21-7.

[0005] Uveal melanoma is a melanocytic neoplasm that arises from melanocytes in the choroidal plexus, ciliary body or iris epithelium of the eye (e.g., Singh, et al, Ophthalmol Clin North Am 18:75-84, viii, 2005). In more aggressive subtypes there are further genetic alterations such as monosomy 3, trisomy 8 and a strong tendency to metastasize to the liver (Singh, et al, Ophthalmol Clin North Am 18:75-84, viii, 2005; Horsman & White, Cancer 71 :811-9, 1993). Uveal melanoma is highly aggressive, with a 5-year disease-specific survival rate of approximately 70% {e.g., Chang et al, Cancer 83: 1664-78, 1998). One risk factor for uveal melanoma is the presence of bluish- grey hyper-pigmentation in the conjunctiva and periorbital dermis, called the naevus of Ota (Singh et al, Ophthalmology 105:195-8, 1998). (1998) Am J Dermatopathol. 20: 109-110).

[0006] Recently, a large-scale mutagenesis screen in mice identified several dark skin (Dsk) mutants. See, Van Raamsdonk CD, et al, (2004) Nat Genet. 36: 961-968. Some of these mutants had a melanocytic phenotype with a sparse cellular proliferation of intradermal melanocytes resembling blue nevi. The mutations were shown to be the result of mutations in G-protein a-subunits.

[0007] G proteins represent a large family of heterotrimeric proteins found in mammals composed of alpha (a), beta (β) and gamma (γ) subunits. See, Wettschureck, N. A. O. S.,

(2005) Physiol. Rev. 85(4): 1159-1204. G-aq, is one of a variety of G-alpha subunits that mediates the stimulation of phospholipase C through the binding and hydrolysis of GTP. See, Markby, D.W., et al, (1993) Science 262(1541): 1895-1901. It has been hypothesized that activation of G-aq promotes the survival of melanocytes in the dermis. See, Van Raamsdonk, C. D., et al, (2004). This is consistent with the observation in mice that hyperactivity of G-aq increases the number of melanoblasts, immature melanocytes, migrating in the dermis without increasing their mitotic rate. See, Van Raamsdonk, C. D., et al, (2004). [0008] Somatic oncogenic mutations in exon 5 of GNAQ, a heterotrimeric G protein alpha subunit, have been identified in various melanocytic neoplasms, including blue nevi and uveal melanomas (WO 2008/098208), among others.

[0009] GNAll is 90% identical to GNAQ at the amino acid level and shares overlapping functions with GNAQ on pigmentation in mice. Mutations in exon 5 of GNAll are also present in various melanocytic neoplasms, including blue nevi and uveal melanomas.

[0010] This invention is based, in part, on the discovery of the occurrence of activating mutations in exon 4 of GNAll and GNAQ in melanocytic neoplasms, including in uveal melanoma. BRIEF SUMMARY OF THE INVENTION

[0011] The current invention provides methods of detecting a melanoma or nevus cell in a biological sample. The methods comprise detecting an activating exon 4 sequence mutation in a GNAll gene or a GNAQ gene in a biological sample comprising the suspected melanoma cell or nevus cell, or a biological sample comprising a cell known to be a melanoma or nevus cell, from a patient. For example, the invention provides methods of detecting melanoma, e.g., either primary or metastatic uveal melanoma; or detecting a nevus, e.g, a blue nevus such as malignant blue nevus, cellular blue nevus, common blue nevus, nevus of Ito, or nevus of Ota; by detecting the presence of an exon 4 mutation in a GNAll or GNAQ gene. The methods can be used for diagnostic and prognostic indications and for identifying melanoma patients that are responsive, or likely to be responsive, to various treatment therapies that target the GNAQ and/or GNAll pathway, such as G-alpha antagonists, or therapies that target downstream signaling components, such as protein kinase C inhibitors. The invention also provides methods of treating a melanoma or nevus comprising administering a GNAQ and/or GNAll inhibitor, e.g., a small molecule, an antibody, or a nucleic acid inhibitor such as a siRNA, to a patient having the melanoma, e.g., uveal melanoma or malignant blue nevus; or nevus, e.g., a blue nevus, arising from a mutation in exon 4 of a GNAQ or GNAll gene.

[0012] Thus, the invention provides a method of detecting a melanocytic neoplasm in a biological sample, e.g., a skin or eye sample, comprising melanoma cells from a patient, e.g., a patient that has, or is suspected of having, melanoma, the method comprising detecting an activating mutation in exon 4 of GNAQ or GNAll in melanoma or nevi cells present in the biological sample, wherein the presence of an activating mutation in exon 4 of GNAQ or GNAll is indicative of the presence of a melanocytic neoplasm. In some embodiments, the melanocytic neoplasm is a uveal melanoma. In other embodiments, the melanocytic neoplasm is a nevus, such as a blue nevus, or a melanoma arising in blue nevus, also known as malignant blue nevus. In some embodiments, the detecting step comprises detecting the presence or absence of an exon 4 mutation in a nucleic acid, e.g., mRNA or genomic DNA. In typical embodiments, such detection steps comprise an amplification reaction that specifically amplifies GNAQ or GNAll, such as PCR or RT-PCR and detection of a mutation using a probe that hybridizes to a target exon 4 GNAQ or GNAll sequence, or detection of the mutation by sequencing the amplified target region in exon 4. In other embodiments, the detecting step comprises detecting the mutation in exon 4 of a GNAQ or GNAll protein. In typical embodiments where the protein is detected, such detecting step comprises the use of antibodies (immunocytochemistry) and/or electrophorectic protein separation {e.g., western blot). Alternatively, the presence of the exon 4 mutation in the protein may be detected using mass spectrometry. In some embodiments the exon 4 mutation is at R183 or VI 82. In some embodiments, the mutation in the codon encoding R183, e.g., in GNAQ, is CGA to CAA (Arginine to Glutamine). In some embodiments, the mutation in the codon encoding Rl 83, e.g., in GNAll, is CGC to TGC (Arginine to Cysteine). In some embodiments, the mutation in the codon encoding VI 82 is GTT to ATT (Valine to Isoleucine). In some embodiments, an exon 4 mutation in a GNAll or GNAQ gene is present in the codon for T175. In some embodiments, the mutation is in the codon for T175, e.g., ACG to AGG (Threonine to Arginine). In some embodiments, the GNAQ or GNAll gene of the melanoma or nevus has two mutations in exon 4, e.g., a mutation at VI 82 and a mutation at T 175.

[0013] In some embodiments, the methods of the invention may comprise an additional step of detecting the presence or absence of an activating mutation in GNAQ or GNA11 in exon 5, e.g., a mutation at the codon encoding Gin 209 of GNAQ or GNA1 1 in a nucleic acid from the biological sample. In some embodiments, the biological sample is from a patient that has uveal melanoma or a malignant blue nevus. In some embodiments, the patient has a melanocytic neoplasm where the melanocytic neoplasm is a blue nevus.

[0014] In some embodiments, the biological sample is from a patient that has, or is suspected of having a melanoma, e.g., uveal melanoma or malignant blue nevus, or metastasis. In other embodiments, the biological sample is from a patient that has, or is suspected of having, a nevus, e.g., a blue nevus such as common blue nevus, cellular blue nevus, nevus of Ito, or nevus of Ota. In some embodiments, the sample is from skin, eye, or from a metastatic site. [0015] The invention also provides a method of monitoring progression of melanoma in a patient subjected to a therapy for treatment of the melanoma arising from a mutation in exon 4 of GNAQ or GNA11. The method comprises detecting a change in the number of cells having an exon 4 mutation in GNAQ orGNAU in a biological sample from a patient, where the change in the number of cells having a mutation is indicative of the patient's response to the therapy; or detecting the presence of additional mutations in genes in the melanoma cells. In some embodiments, the melanoma is uveal melanoma. In some embodiments, the melanoma is a malignant blue nevus. In some embodiments, the sample is from a metastatic site. [0016] In some embodiments, monitoring progression of melanoma in a patient where the melanoma arose from an activating mutation in exon 4 of a GNAQ or GNA11 gene is performed by detecting the mutation in a nucleic acid from the biological sample. In other embodiments, the progression of the melanoma arising from an exon 4 mutation in GNA11 is detected in by evaluating a GNAQ or GNA11 protein present in the biological sample. In some embodiments of the invention, the biological sample is from eye or skin. In other embodiments, the biological sample is from a metastatic site, e.g., liver, lung, blood, lymph node, adrenal gland, or bone.

[0017] Typically, in monitoring melanoma progression in accordance with the invention, the presence of a reduced number of cells having an exon 4 GNAQ or GNA11 mutation in the biological sample taken from a patient after treatment with an agent as compared to the number of cells having an exon 4 mutation in a biological sample taken from the patient before being exposure to the treatment agent is indicative of a positive therapeutic response to the treatment agent.

[0018] In all of the detection methods of the invention the biological sample can be from any source in the body that is suspected of containing primary or metastatic melanoma cells. Thus, the biological sample can be from skin, e.g., eye, e.g., uvea, conjunctiva, or mucosal membranes. In other embodiments, the sample can be from blood, serum, tissue from lymph nodes, or tissue from visceral organs such as adrenal gland, liver or lung; or bone tissue. In some embodiments, for example in monitoring progression of melanoma, the sample is from a readily accessible tissue such as blood.

[0019] In another aspect, the invention provides a method of determining whether a melanoma patient is a candidate for receiving a therapy that inhibits the activity of a Ga subunit, either directly or by inhibiting a protein that is activated by Get. The method comprises determining whether the melanoma cells have an activating mutation in exon 4 of GNAQ or GNA11. Accordingly, the detecting step can comprise detecting the mutation in mRNA, DNA, or protein. In some embodiments, the detecting step can comprise detecting the presence of an exon 4 mutation in a nucleic acid sample from the melanoma or nevus, whereas in other embodiments, the detecting step is from a protein sample from a

melanocytic neoplasm. The nucleic acid sample can be RNA or DNA, e.g., genomic DNA or cDNA made from RNA from the melanocytic neoplasm sample. Often, the detecting step comprises an amplification reaction, such as PCR or RT-PCR. In some embodiments, the melanoma is a uveal melanoma.

[0020] In another aspect, the invention provides a method of inhibiting growth and/or proliferation of nevus or melanoma cells arising from a somatic exon 4 mutation in GNAQ or GNA11, the method comprising administering a GNAQ or GNA11 antagonist. Although in some embodiments the antagonist may specifically target GNAQ or GNA1 1, e.g., in embodiments in which an siRNA is employed, the antagonist can also target other steps in the pathway. For example, the antagonist may be a small molecule, such as edelfosine, a protein kinase C inhibitor, or the staurosporine analogue CPG41251; an antibody; a peptide; or a nucleic acid. In some embodiments, the inhibitor is siRNA. In some embodiments the siRNA targets both GNA11 and GNAQ nucleic acid sequences. Typically, the nevi or melanoma cells are from e.g., uveal melanoma or a blue nevus. In typical Typically, the nevi or melanoma cells are from e.g., uveal melanoma or a blue nevus.

[0021] The invention also provides a method of determining the risk of progression of a nevus to a melanoma, the method comprising detecting the presence or absence of an exon 4 sequence mutation in a GNAQ or GNA11 gene in a biological sample from the nevus, wherein the presence of the mutation is indicative of increased risk of progression of the nevus to melanoma. In some embodiments, the sequence mutation is at R183 or VI 82; and/or at T175. In some embodiments, the nevus is a blue nevus. In some embodiments, the mutation is detected by evaluating the protein that is encoded by the gene.

[0022] The invention also provides a method of determining the risk of metastasis of a melanoma, the method comprising detecting the presence or absence of an exon 4 sequence mutation in a GNAQ or GNA11 gene in a biological sample from the patient where the biological sample comprises primary melanoma cells and wherein the presence of the mutation is indicative of increased risk of metastasis of the melanoma. In some embodiments the melanoma is uveal melanoma. In some embodiments, the sequence mutation is at R183 or V182; and/or at T175.

[0023] In some embodiments, an exon 4 mutation in GNAQ or GNA11 mutation, e.g., at Rl 83 or VI 82; and/or at T175, is detected in a melanocytic neoplasm such as acral melanoma, acral lentiginous melanoma, chronic sun-induced damaged (CSD) melanoma, non-chronic sun-induced damage (NCSD) melanoma, lentigo maligna melanoma, muscosal melanoma, nodular melanoma, superficial spreading melanoma, desmoplastic melanoma, conjunctival melanoma, recurrent cellular blue nevi, melanoma arising in a congenital nevus, malignant blue nevus, and metastasis. In some embodiments, the exon 4 mutation is detected in melanocytic neoplasms that are nevi. For example, an exon 4 mutation may be detected in a congenital nevus, congenital nevus with nodules, congenital nevus with desmoplastic reaction, giant congenital nevus with atypia, giant congenital nevus with nodules, congenital nevus without specific diagnosis, atypical blue nevus, atypical cellular blue nevus, blue nevus with neurocristic hamartoma, blue nevus without specific diagnosis and deep penetrating nevus without specific diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1. Figure 1 provides an example of data characterizing various uveal melanomas in which GNA11 and GNAQ mutations were identified. Anatomic location and histopathological features and mutation status. A) GNA11 mutations were more common in primary uveal melanomas of ciliochoroidal location. (p=0.048, Fisher's Exact test). Bars: Upper segment, GNAll; middle segment, GNAQ; lower segment, neither

[0025] Figure 2 A-G. Figure 2 A-G provides data showing that GNA 11 ^R183C and

GNA11 ^Q209L and induce tumors in a mouse model. Immortalized mouse melanocytes (melan- a cells) were transduced with GNA 11 ^{Q20 L} , GNA n ^RI83C , GNA 1 l ^wt or β-gal and inj ected bilaterally into the flank of NOD/SCID/interleukin 2 receptor [IL2r] Tnull mice. 6/6 injection sites developed tumors for GNA11 ^Q209L (A), 3/8 for GNA11 ^R183C and none for GNAll ^wt (0/10) or β-gal (0/6) by eleven weeks (B). Graph shows combined results of two independent experiments. Tumors were heavily melanized (C) and comprised of pigmented spindled and epithelioid melanocytes (F). All GNA11 ^Q209L mice developed multiple lung metastases (E) and one mouse developed liver metastases (D). Melan-a cells transduced with GNA HQ ²⁰⁹¹ but not their wild-type counterparts showed activation of the MAP -kinase pathway similar to positive mutant BRAF or NRAS used as positive controls (G).

DETAILED DESCRIPTION OF THE INVENTION

Introduction

[0026] The present invention provides methods, reagents and kits, for detecting melanoma and nevus cells for diagnostic and prognostic uses, and for treating melanomas and nevi. The invention is based, in part, upon the discovery that melanoma and nevi have activating exon 4 mutations in GNAQ or GNA11, i.e., mutations that result in a loss or decrease of GTP hydrolyzing activity of a G-a subunit.

[0027] G-a is the alpha subunit of one of the heterotrimeric GTP -binding proteins that form two subgroups in vertebrates, the widely expressed Ga-q family comprising Gnaq and Gnal 1, and the Gnal4 and Gnal5 family, which show more restricted expression. The Ga-q family mediates stimulation of phospholipase Οβ resulting in the hydrolysis of

bisphosphoinositide (PIP ₂ ) into inositide triphosphate (IP ₃ ) and diacylglycerol (DAG). IP ₃ can stimulate the release of calcium from intracellular storage in the endoplasmic reticulum (ER) leading to downstream calcium-dependent signaling. In parallel, DAG can activate protein kinase C (PKC) and both pathways can then feed into the mitogen activated protein kinase (MAPK) cascade. See, Corbit, K.C., et al, (2000) Mol Cell Biol. 20:5392-5403; Sato, M. et al, (2006) Ann. Rev. Pharm. Toxicol. 46: 151-187.

[0028] The present inventors have discovered that activating mutations in exon 4 of GNAQ or GNA11, e.g., heterozygous, somatic substitution mutations of R183 or V182 in GNAQ or GNA11, are present in several types of melanocytic neoplasms, including nevi such as blue nevi and melanoma, such as uveal melanoma. Further, in some embodiments two activating mutations in exon 4 of GNAQ or GNA11, e.g., at VI 82 and T175, may be present in melanocytic neoplasms, e.g., uveal melanoma.

[0029] In one aspect of the invention, the ability to detect nevi and/or melanoma cells by virtue of detecting an exon 4 somatic mutation in GNAQ or GNA11 that activates the protein is useful for any of a large number of applications. For example, exon 4 mutation detection, alone or in combination with other diagnostic methods, can be used to diagnose melanoma, or a certain type of melanoma, such as uveal melanoma, in the patient. It can also be used to identify particular melanomas that are sensitive to therapeutics, such as therapeutics that target G-proteins or phospho lipase CP or other downstream components of pathways regulated by Gnaq or Gnal 1. In some embodiments, detection of an exon 4 activating GNAQ or GNAll mutations can be employed as a prognostic indicator of more aggressive melanomas that are more likely to lead to metastasis than melanomas that do not have an exon 4 mutation.

[0030] The detection of somatic exon 4 activating mutations in GNAQ or GNAll can also be used to monitor the efficacy of a melanoma treatment. For example, the level of Gnaq or Gnal 1 activity, e.g., Ga activity, or an activity such as phospholipase C that is dependent on Ga activity in exon 4 mutation-positive melanomas, or the numbers of melanocytic cells that have a sequence mutation in exon 4, after an anti-melanoma treatment can be compared to the level before the treatment. A decrease in the level of protein activity, e.g., phospholiapse CP activity, or a reduction in the number of melanoma cells that have mutated exon 4 after the treatment, indicates efficacious treatment. [0031] The level of Gnaq or Gnal 1 protein activity in melanomas that are positive for exon 4 mutations and/or a change in the number of cells having a somatic mutation in exon 4 can also be statistically correlated with the efficacy of particular anti -melanoma therapy or with an observed prognostic outcome, thereby allowing the development of a database on which statistically-based prognosis, or a selection of the most efficacious treatment, can be made in view of a particular level activity or diagnostic presence of an exon 4 mutation.

[0032] Detection of cells having an exon 4 activating mutation in GNAQ or GNAll can be useful to monitor the number or location of melanoma cells in a patient, for example, to monitor the progression of the cancer over time.

[0033] The presence of an exon 4 activating mutation in GNAQ or GNAll can also indicate melanomas that are likely to be responsive to therapeutic agents that target GNAQ and/or GNA1 1 protein(s). Accordingly, the invention also provides methods of treating a melanocytic neoplasm, e.g., uveal melanoma or a blue nevus, that has an activating mutation in exon 4 of GNAQ or GNAll by administering a Ga antagonist, e.g., antibodies, peptides, small molecule inhibitors, such as L-threo-dihydrosphingosine (a PKC specific inhibitor) or other small molecule inhibitors, and nucleic acid inhibitors of GNAQ or GNAll, e.g., GNAll or GNA11/GNAQ siRNA inhibitors, or inhibitors of phospholipase C , or downstream pathways regulated by Gnaq or Gnal 1. Such melanocytic neoplasms can be identified by analyzing for the presence of an exon 4 activating mutation in GNAQ or GNA11 using the methods described herein.

[0034] The presence of an exon 4 activating mutation in nevi often indicates nevi, e.g., conventional types of blue nevi and nevi of Ota, that are at risk for progression to melanoma. Accordingly, a nevus from a patient can be evaluated for the presence of an activating mutation using the methods described herein.

Definitions

[0035] The term "GNAQ" or "Gnaq" refers to the the alpha subunit of a guanine nucleotide binding protein (G-protein). The term encompasses nucleic acid and polypeptide

polymorphic variants, alleles, mutants, and fragments of Gnaq. Gnaq sequences are well known in the art. Examples of human Gnaq sequences are available under the reference sequences NM_002072 in the NCBI nucleotide database (nucleotide sequence) and accession number NP_002063.2 (polypeptide sequence). The sequence NM_002072 is provided as SEQ ID NO: l as an illustrative nucleotide sequence. An illustrative polypeptide sequence is shown in SEQ ID NO:2. As understood in the art, the term "GNAQ" or "gnaq" includes variants, such as polymorphic variants. For example, the SNP database shows that many single nucleotide polymorphisms have been identified in GNAQ genes. Human GNAQ has been localized to 9q21. A human GNAQ gene polymorphic variant is localized to 9q21 and typically encodes a protein that has greater than 97% , 98%, or 99% identity to SEQ ID NO:2.

[0036] The term "GNAl 1 " or "Gnal l" refers to the the alpha subunit of a guanine nucleotide binding protein (G-protein). The term encompasses nucleic acid and polypeptide polymorphic variants, alleles, mutants, and fragments of GNAl 1. GNAl 1 sequences are well known in the art. Illlustrative human GNAl 1 sequences are available under the reference sequences NM_002067 in the NCBI nucleotide database (nucleotide sequence) and accession number NP_002068 (polypeptide sequence). The sequence NM_002067 is provided as SEQ ID NO:3 as an example nucleotide sequence. An example of a polypeptide sequence is shown in SEQ ED NO:4. As understood in the art, the term "GNAl 1" or "gnal l" includes variants. For example, the SNP database shows that many single nucleotide polymorphisms have been identified in GNA11 genes. Human GNA11 is localized to chromosome region 19pl3.3. A human GNA11 gene variant is localized to chromosome region 19pl3.3 and typically encodes a protein that has greater than 97%, 98%, or 99% identity to SEQ ID NO:4. [0037] An "exon 4 mutation" as used herein refers to a mutation in exon 4 of a GNAQ or GNAll gene. In some embodiments, the mutation is at a codon that encodes a valine at position 182 or a codon that encodes an arginine at position 183 of the Gnaq or Gnal 1 protein sequence, which mutation results in a change of protein sequence at those positions. In some embodiments, the mutation in exon 4 is at a codon encoding positon 175 of the Gnaq or Gnal 1 protein sequence. The positions of the mutations are indicated with reference to the Gnaq and Gna 11 protein sequences, including the start methionine. Exon 4 of GNAQ corresponds to nucleotides 518 to 646 of SEQ ID NO: l and encodes the region of the protein shown in SEQ ID NO:2 from about amino acid 159 to aabout amino acid 202. Exon 5 corresponds to nucleotides 647-776 of SEQ ID NO: l, which encodes the region of the protein sequence shown in SEQ ID NO:2 from about amino acid 202 to about 245. Exon 4 of GNAll corresponds to nucleotides 719 to 847 of SEQ ID NO:3 and encodes the region of the protein shown in SEQ ID NO:4 from about amino acid 159 to aabout amino acid 202. Exon 5 corresponds to nucleotides 848-977 of SEQ ID NO:3, which encodes the region of the protein sequence shown in SEQ ID NO:4 from about amino acid 202 to about 245.

[0038] A "Gnaq-dependent melanoma" as used in the context of this application refers to a melanocytic neoplasm comprising melanoma cells that have a mutation in Gnaq that activates Gnaq, i.e., has an "activating" mutation, in comparison to melanocytes that do not have the mutation, and leads to a loss or decrease of GTP hydrolyzing activity of the mutant G-oc subunit. The defect in Gnaq can involve a mutation, e.g., a substitution mutation, that results in constitutive activity of the protein. The "Gnaq-dependent melanoma cells" may have one or more of such mutations, e.g, the cells may have a somatic substitution mutation involving R183 as well as other mutations. A "Gnaq-dependent melanoma" of the present invention can arise, e.g., from sun exposed skin sites, a nevus (e.g., a blue nevus) or the eye (e.g., the uvea). A "Gnaq-dependent melanoma" may also have mutations in genes other than Gnaq.

[0039] A "Gnal 1 -dependent melanoma" as used in the context of this application refers to a melanocytic neoplasm comprising melanoma cells that have a mutation in Gnal 1 that results in mutation that activates GNA11 , i.e., has an "activating" mutation, in comparison to melanocytes that do not have the mutation, and leads to a loss or decrease of GTP

hydrolyzing activity of the mutant G-cc subunit. The defect in GNA11 can involve a mutation, e.g., a substitution mutation, that results in constitutive activity of the protein. The "GNA 1 1 -dependent melanoma cells" may have one or more of such mutations, e.g, the cells may have a somatic substitution mutation involving exon 4, e.g., a mutation at R183 as well as other mutations. A "GNAl 1 -dependent melanoma" of the present invention can arise, e.g., from a nevus {e.g., a blue nevus) or the eye (e.g., the uvea). A "GNAl 1 -dependent melanoma" may also have mutations in genes other than GNAl 1.

[0040] The term "mucosal melanoma" refers to tumors arising on mucosal membranes; "ocular melanoma" as used herein is melanoma that arises from the eye. "Ocular melanoma" includes uveal and conjunctival melanoma. "Conjunctival melanoma" refers to a melanoma that arises on the conjunctiva, while "uveal melanoma" refers to a melanoma of the pigmented tract of the eye.

[0041] "CSD melanoma" as used herein refers to melanoma arising from skin with chronic sun-induced damage; and "NCSD melanoma" as used herein refers to melanoma arising from skin without chronic sun-induced damage. The distinction between the "CSD" and "NCSD" groups in the instant application is based on a microscopic determination of the presence or absence of marked solar elastosis of the dermis surrounding the melanomas. In all but a few cases, melanomas associated with chronic sun-induced damage (CSD) occur on the face and distal extremities such as the forearms, dorsal hands, shins and calfs. These melanomas typically occur in individuals older than 50 years of age, and microscopically, have an intraepidermal component in which melanocytes are arranged as solitary units rather than nests. In addition, these melanomas tend to have an atrophic epidermis with the effacement of the rete ridges. A subset of the CSD melanomas is lentigo maligna melanomas. By contrast melanomas that were not associated with chronic sun-induced damage (NCSD) occur on the trunk and proximal extremities such as thighs and upper arms. The NCSD melanomas typically show an intraepidermal component in which melanocytes are arranged as nests rather than solitary units and display considerable upward scatter (pagetoid spread). Many of the NCSD melanomas are superficial spreading melanomas. [0042] Chronic sun-induced damage is defined as having a CSD score greater than CSD 2. The scores are obtained by determining the degree of solar elastosis on hematoxylin-and- eosin (H&E) stained sections of normal skin surrounding the melanomas at 100-200x magnification using the following system (Landi et αί, Science 313: 521 - 522, 2006), examples of which are provided in Landi et al.

CSD 0: absence of elastotic fibers; CSD 0+: rare elastotic fibers discernible only at

200x magnification; CSD 1: scattered elastotic fibers lying as individual units, not as bushels, between collagen bundles; "-" or "+" classifiers were used to indicate whether the elastotic fibers were scarcely or densely scattered.

CSD 2: densely scattered elastotic fibers distributed predominantly as bushels rather than individual units; The "-"classifier was used to indicate that bushels were present, but elastotic fibers distributed as individual units predominated; The "+" classifier was used when larger aggregates of bushels formed, but preserving the outline of individual bushels instead of forming amorphous deposits;

CSD 3: amorphous deposits of blue-gray material with lost fiber texture; "-" only focal formation of amorphous deposits; "+" very large agglomerates of diffuse basophilic material.

[0043] As used herein, the term "determining that the melanoma arose from" a site, e.g., uvea, mucosa, conjunctiva, acral skin, skin having chronic sun-induced damage, or skin that that does not have chronic sun-induced damage, refers to identifying the site of origin of a melanoma. Such a determination can be performed by visual inspection of a patient or by a pathology evaluation of the melanoma.

[0044] The terms "tumor" or "cancer" in an animal refers to the presence of cells possessing characteristics such as atypical growth or morphology, including uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone within an animal. "Tumor" includes both benign and malignant neoplasms. The term "neoplastic" refers to both benign and malignant atypical growth.

[0045] The term "melanocytic neoplasm" as used herein refers to an area of

hyperpigmentation relative to the surrounding tissue. Melanocytic neoplasms include both nevi and primary melanoma as well as melanoma that has metastasized to anywhere in the body. Thus, "melanocytic neoplasm" as used here include benign neoplasms. Similarly, the term "melanocyte" refers to both neoplastic and normal melanocytes. Typcially, melanocytic neoplasms occur on skin, mucosal membranes, and the eye. Non-limiting melanocytic neoplasms include melanomas, e.g., acral lentiginous melanoma, CSD melanoma, NCSD melanoma, lentigo maligna melanoma, muscosal melanoma, nodular melanoma, superficial spreading melanoma, desmoplastic melanoma, uveal melanoma, conjunctival melanoma, recurrent cellular blue nevi, melanoma arising in a congenital nevus, malignant blue nevus, and metastasis. Melanocyte neoplasms as used herein also include nevi. For example, non- limiting examples of nevus melanocyte neoplasms as used herein can include congenital nevus, congenital nevus with nodules, congenital nevus with desmoplastic reaction, giant congenital nevus with atypia, giant congenital nevus with nodules, congenital nevus without specific diagnosis, blue nevus, atypical blue nevus, atypical cellular blue nevus, blue nevus with neurocristic hamartoma, blue nevus without specific diagnosis and deep penetrating nevus without specific diagnosis.

[0046] The term "blue nevus" or "blue nevi" as used herein refers to an intradermal, i.e., within the dermal layer of the skin, melanocyte proliferation that exhibits increased pigmentation such that the nevus typically has a bluish color. A blue nevus, which can be congenital or acquired, may present in diverse ways ranging from discrete bluish moles (blue nevi) to large blue-gray patches affecting the conjunctiva and periorbital skin (nevus of Ota), shoulders (nevus of Ito), and the lower back (Mongolian spot). In some embodiments a "blue nevus" may be a "malignant blue nevus", i.e., a melanoma that arose within a blue nevus or of which certain portions resemble a blue nevus histopathologically.

[0047] "Biological sample" as used herein refers to a sample obtained from a patient suspected of having, or having a melanoma. In some embodiments, the sample may be a tissue biopsy, which refers to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc. The sample typically comprises a tissue sample, e.g., from skin or eye harboring the melanocyte neoplasm or melanocytes suspected of being a melanocyte neoplasm, although the biological sample may also be derived from another, site, e.g., a site to which a melanoma may metastasize, or from the blood. In some cases, the biological sample may also be from a region adjacent to the melanocyte neoplasm or suspected melanocyte neoplasm. [0048] "Providing a biological sample" means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from a patient, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, can also be used.

[0049] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid

chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene. The term "purified" in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Preferably, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. "Purify" or "purification" in other embodiments means removing at least one contaminant from the composition to be purified. In this sense, purification does not require that the purified compound be homogenous, e.g., 100% pure.

[0050] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer.

[0051] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.

[0052] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. [0053] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid. One of skill will recognize that in certain contexts each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, often silent variations of a nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0054] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. typically conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). [0055] "Nucleic acid" or "oligonucleotide" or "polynucleotide" or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non- ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds.. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose- phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip.

Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

[0056] A variety of references disclose such nucleic acid analogs, including, for example, phosphoramidate (Beaucage et al, Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al, J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al, Chemica Scripta 26: 141 91986)), phosphorothioate (Mag et al, Nucleic Acids Res. 19: 1437 (1991); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111 :2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 1 14: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31 : 1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34: 17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins et al, Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. [0057] Other analogs include peptide nucleic acids (PNA) which are peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (T _m ) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4°C drop in T _m for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9°C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, PNAs are not degraded by cellular enzymes, and thus can be more stable. [0058] The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. "Transcript" typically refers to a naturally occurring RNA, e.g., a pre- mRNA, hnRNA, or mRNA. As used herein, the term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus, e.g. the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.

[0059] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ³² P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. The labels may be incorporated into the KIT nucleic acids, proteins and antibodies at any position. Any method known in the art for conjugating the antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0060] A "labeled nucleic acid probe or oligonucleotide" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. Alternatively, method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.

[0061] As used herein a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not functionally interfere with hybridization. Thus, e.g. , probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence. Diagnosis or prognosis may be based at the genomic level, or at the level of RNA or protein expression. [0062] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. Similarly, a

"recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.

[0063] The phrase "selectively (or specifically) hybridizes to" refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a mixture (e.g., total cellular or library DNA or RNA, an amplification reaction), such that the binding of the molecule to the particular nucleotide sequence is determinative of the presence of the nucleotide sequence is the mixture.

[0064] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T _m ) for the specific sequence at a defined ionic strength pH. The T _m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T _m , 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Illustrative stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length. For high stringency PCR amplification, a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).

[0065] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Illustrative "moderately stringent

hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al.

[0066] "Percent identity" can be determined using methods well known in the art, e.g., the BLAST algorithm set to default parameters. An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.

[0067] The phrase "functional effects" in the context of assays for testing compounds that inhibit activity of a Gnaq or Gnal 1 protein includes the determination of a parameter that is indirectly or directly under the influence of the GNAQ or GNA1 1 protein or nucleic acid, e.g., a functional, physical, or chemical effect, such as the ability to decrease tumori genesis, or alter GTP hydrolase activity. Activities or functional effect of GNAQ or GNA11 can include protein-protein interaction activity, e.g., the ability of GNAQ or GNA1 1 to bind an antibody or other protein with which it interacts; GTP hydrolase activity, the ability of GNAQ or GNA11 to bind GTP and/or GDP; contact inhibition and density limitation of growth; cellular proliferation; cellular transformation; changes in pigmentation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo, including measurement of tumor growth and tumor "take" in a model system; mRNA and protein expression in cells, including those undergoing metastasis, and other characteristics of cancer cells. "Functional effects" include in vitro, in vivo, and ex vivo activities.

[0068] As used herein, "inhibitors" or "antagonists" of GNAQ or GNA11 (e.g. "GNA11 antagonists" or "GNAQ antagonists") refer to modulatory molecules or compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of GNAQ or GNA1 1 protein, phospholipase CP, or downstream molecules regulated by GNAQ or GNA11, e.g., protein kinase C (PKC). Inhibitors can include siRNA or antisense RNA, e.g., siRNA or antisense RNA to target GNAQ nucleic acids or GNAll nucleic acids, or siRNA or antisense RNA that targets both GNAQ and GNAll nucleic acids; genetically modified versions of GNAQ or GNA1 1 protein, e.g., versions with altered activity, as well as naturally occurring and synthetic GNAQ or GNA11 antagonists, antibodies, small chemical molecules and the like. GNAQ or GNA11 inhibitors for use in the invention are known in the art. For example, non- limiting examples of inhibitors suitable for use with the present invention can include inhibitors of PKC, for example the the relatively nonspecific PKC inhibitor staurosporine, the staurosporie analogue CPG41251, bryostatin-1, KAI-9803, 7-hydroxystaurosporine, L-threo- dihydrosphingosine (safmgol), AHT956 and AEB071, the non-selective PKC inhibitor (PKC412), ilmofosine (BM 41 440), indolcarbazole G56796 which is a more specific inhibitor of the classical PKC isoforms including Ρ Χϋμ, the PKC-alpha antisense inhibitor LY900003, and the PKC-beta inhibitors LY333531, LY317615 (Enzastaurin). An examples of an antisense molecule suitable for use in depleting PKC-alpha mRNA is 5'-

GTTCTCGCTGGTGAGTTTCA-3'. Non-limiting illutrative inhibitors of phospholipase CP can include edelfosine and fluvirusin B[2]. Assays for identifying other inhibitors can be performed in vitro or in vivo, e.g., in cells, or cell membranes, by applying test inhibitor compounds, and then determining the functional effects on activity. [0069] In some embodiments, samples or assays comprising GNAQ or GNA11 proteins that are treated with a potential inhibitor are compared to control samples without the inhibitor, to examine the effect on activity. Typically, control samples, e.g., melanoma cells, that have a GNAQ or GNA11 activating mutation and that are untreated with inhibitors are assigned a relative protein activity value of 100%. Inhibition of GNAQ or GNA11 is achieved when the activity value relative to the control is changed at least 20%, preferably 50%, more preferably 75-100%, or more. In some embodiments, an inhibitor will activate a particular activity, such as GTP hydrolysis, however, the net effect will be a decrease in the activity of GNAQ or GNA11, e.g., in comparison to controls that have activated GNAQ or GNA1 1. [0070] The phrase "changes in cell growth" refers to any change in cell growth and proliferation characteristics in vitro or in vivo, such as formation of foci, anchorage independence, semi-solid or soft agar growth, changes in contact inhibition and density limitation of growth, loss of growth factor or serum requirements, changes in cell

morphology, gaining or losing immortalization, gaining or losing tumor specific markers, ability to form or suppress tumors when injected into suitable animal hosts, and/or immortalization of the cell. See, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique pp. 231-241 (3 ^rd ed. 1994).

[0071] As used herein, "antibody" includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). The term "antibody" also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab') ₂ , Fab, Fv and rlgG. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL). See also, e.g., Kuby, J., Immunology, 3 ^rd Ed., W.H. Freeman & Co., New York (1998). The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al.. (1992) J Immunol 148: 1547, Pack and Pluckthun (1992) Biochemistry 31 : 1579, Hollinger et al, 1993, supra, Gruber et al. (1994) J Immunol :5368, Zhu et al. (1997) Protein Sci 6:781 , Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[0072] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen.

[0073] Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). Light and heavy chain variable regions contain four framework" regions interrupted by three hypervariable regions, also called complementarity-determining regions (CDRs).

[0074] References to "VH" or a "VH" refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv , or Fab. References to "V _L " or a "VL" refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv , dsFv or Fab. [0075] A "chimeric antibody" is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. [0076] A "humanized antibody" is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature

321 :522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol 2:593-596 (1992)). Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature 321 :522-525 (1986); Riechmann et al, Nature 332:323-327 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

[0077] The term "fully human antibody" refers to an immunoglobulin comprising human hypervariable regions in addition to human framework and constant regions. Such antibodies can be produced using various techniques known in the art. For example in vitro methods involve use of recombinant libraries of human antibody fragments displayed on

bacteriophage {e.g., McCafferty et al, 1990, Nature 348:552-554; Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); and Marks et al, J. Mol. Biol. 222:581 (1991)), yeast cells (Boder and Wittrup, 1997, Nat Biotechnol 15:553-557), or ribosomes (Hanes and Pluckthun, 1997, Proc Natl Acad Sci US A 94:4937-4942). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Patent Nos. 6,150,584, 5,545,807; 5,545,806;

5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: (e.g., Jakobavits, Adv Drug Deliv Rev. 31 :33-42 (1998), Marks et al, Bio/Technology 10:779- 783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature

Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

General recombinant methods

[0078] This invention relies in part on routine techniques in the field of recombinant genetics, e.g., for methods used in detecting mutations in GNAQ or GNAl l, or for the preparation of GNAQ or GNAl 1 polypeptides and nucleic acids. Basic texts disclosing the general methods of use in this invention include Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); and Current Protocols in Molecular Biology, Ausubel, 1994-1999, including supplemental updates through April 2010). Identification of a GNAQ or GNAll sequence mutation in a sample from a patient

[0079] In one aspect of the invention, the presence of an activating mutation in exon 4 of a GNAQ or GNAl 1 polynucleotide, e.g., mRNA or genomic DNA and/or the presence of an exon 4 sequence mutation in a Gnaq or Gnal 1 protein, is determined in biological samples suspected of comprising nevus, such as blue nevus, and/or melanoma, e.g., uveal melanoma or malignant blue nevus, cells.

[0080] In typical embodiments, nucleic acids from a biological sample that has, or is suspected of having a malignant neoplasm, such as uveal melanoma, are analyzed for the presence of exon 4 mutations in GNAQ or GNAl 1. GNAQ and GNA 11 nucleic acid sequences are well known. Accordingly, the presence of exon 4 mutations can be readily determined.

[0081] "Sequence mutation" as used in this application refers to changes in a

polynucleotide sequence that result in changes to protein activity. Mutations can be nucleotide substitutions, such as single nucleotide substitutions, insertions, or deletions. In some embodiments, exon 4 GNAQ or GNAl 1 mutations in melanocytic neoplasms in accordance with the present invention are activating mutations at amino acid positions 182 or 183 that lead to constituitive activation of GNAQ or GNAl 1 activity. In some embodiments, an exon 4 mutation occurs at postion 175. In some embodiments, a melnocytic neoplasm may have mutations at two positions in exon 4, e.g., at position 175 and position 182 of GNAQ.

[0082] In the present invention, an exon 4 sequence mutation is detected for the diagnosis (or for prognostic indications) of melanocytic neoplasms, e.g., for the diagnosis of subtypes of melanoma such as uveal melanoma and nevi such as blue nevi. Thus, biological samples obtained from patients that have or are suspected of having a melanocytic neoplasm can be analyzed for mutations in the sequence of exon 4 of GNAQ or GNAl 1 mRNA, genomic DNA, or protein. [0083] In some embodiments, the methods of the invention may further comprise evaluating a biological sample from a patient for the presence or absence of an activating mutation in exon 5 of GNAQ or GNAl 1, for example, evaluating the biological sample for a mutation at the codon encoding Gin 209 of GNAQ or GNAl 1 in a nucleic acid from the biological sample. In some embodiments, the biological sample is from a patient that has uveal melanoma or a malignant blue nevus. In some embodiments, the patient has a melanocytic neoplasm where the melanocytic neoplasm is a blue nevus. Methods of detecting activating mutations in exon 5 of GNAQ are known (see WO 2008/098208).

Similar methodology is employed to detect activating mutations in exon 5 of GNAl 1.

[0084] Exon 4 mutations in GNAQ or GNAl 1 are typically present in melanocytic neoplasms, e.g., uveal melanomas, that do not have an exon 5 mutations such as a Gin 209 mutation and vice versa. Similarly, exon 4 mutations in GNAQ are typically present in melanocytic neoplasms, e.g., uveal melanomas, that do not have mutations in GNAl 1, and vice versa.

Detection of Sequence Mutations in GNAQ or GNA11 [0085] In one embodiment, diagnostic and prognostic detection of a sequence mutation in GNAQ or GNA11 is performed by evaluating nucleic acids to determine the presence of an exon 4 sequence mutation in GNAQ or GNAl J. Methods of evaluating the sequence of a particular gene are well known to those of skill in the art, and include, inter alia,

hybridization and amplification based assays. [0086] In some embodiments, an exon 4 sequence mutation in GNA11 in the instant invention can be determined using a probe that selectively hybridizes to the mutant sequence. [0087] In some embodiments, the presence of an exon 4 mutant GNAQ or GNA11 allele can be conveniently determined using DNA sequencing, such as sequencing-by-synthesis methods including dideoxy sequence, pyrosequencing, and methods of sequencing by cleavage or other known sequencing techniques. Other detection methods include single- stranded conformational polymorphism or restriction fragment length polymorphism detection methods and denaturing gradient gel electrophoresis analysis.

[0088] In some embodiments, an exon 4 sequence mutation in a biological sample is determined by hybidzation of sample DNA or RNA to a probe that specifically hybridizes to a GNAQ or GNAl 1 sequence. The probes used in such applications specifically hybridize to the region of the GNAQ or GNAl 1 sequence harboring the mutation. Preferred probes are sufficiently long, e.g., from about 10, 15, or 20 nucleotides to about 50 or more nucleotides, so as to specifically hybridize with the target nucleic acid(s) under stringent conditions.

[0089] In some embodiments, a probe may be used to hybridize to the regions of GNAl 1 or GNAQ nucleic acid that encodes position 183 of GNA11 or GNAQ. [0090] Any of a number hybridization-based assays can be used to detect a sequence mutation in GNAQ or GNAl 1 in the cells of a biological sample. For example, dot blots, array-based assays and the like can be used to determine GNAQ or GNAl 1 sequence mutations.

[0091] In some embodiments, amplification-based assays are used to detect sequence mutations in GNAQ or GNAl 1. In such an assay, a target GNAQ and/or GNAl 1 nucleic acid sequence is specifically amplified in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR). Examples of amplification-based assays include RT-PCR methods well known to the skilled artisan (see, e.g., Ausubel et al., supra). Detailed protocols for PCR of DNA and RNA, including quantitative amplification methods,are known (see, e.g., Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.; and Ausubel and Russell & Sambrook, both supra). The known nucleic acid sequences for GNAQ (e.g., SEQ ID NO: l) and GNAl 1 (e.g., SEQ ID NO:3) are sufficient to enable one of skill to routinely select primers to specifically amplify the desired region. Suitable primers for amplification of specific sequences can be designed using principles well known in the art (see, e.g., Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)).

[0092] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see, Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc. [0093] The presence of mutations in GNAQ or GNA11 DNA or RNA can also be determined using known techniques such as allele-specific oligonucleotide hybridization, which relies on distinguishing a mutant from a normal nucleic acid sequence using an oligonucleotide that specifically hybridizes to the mutant or normal nucleic acid sequence. This method typically employs short oligonucleotides, e.g., 15-20 nucleotides, in length, that are designed to differentially hybridize to the normal or exon 4 mutant allele. Guidance for designing such probes is available in the art. The presence of a mutant allele is determined by measuring the amount of allele-specific oligonucleotide that hybridizes to the sample.

[0094] In other embodiments, the presence of a normal or mutant exon 4 GNAQ or GNA11 nucleic acid can be detected using allele-specific amplification or primer extension methods. These reactions typically involve use of primers that are designed to specifically target a normal or mutant allele via a mismatch at the 3' end of a primer. The presence of a mismatch effects the ability of a polymerase to extend a primer when the polymerase lacks error- correcting activity. The amount of amplified product can be determined using a probe or by directly measuring the amount of DNA present in the reaction. [0095] Detection of levels of GNAQ or GNA11 nucleic acids, e.g., levels of normal or exon 4 mutant GNA11 polynucleotides, or the presence of an exon 5 GNAQ or GNA11 mutation can also be performed using a quantitative assay such as a 5 '-nuclease activity (also referred to as a "TaqMan®" assay), e.g., as described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al, 1988, Proc. Natl. Acad. Sci. USA 88:7276-7280. In such an assay, labeled detection probes that hybridize within the amplified region are added during the amplification reaction. In some embodiments, the hybridization probe can be an allele- specific probe that discriminates a normal or mutant allele. Alternatively, the method can be performed using an allele-specific primer and a labeled probe that binds to amplified product.

[0096] Another method for determining the presence of an exon 4 mutation in a GNA11 or GNAq gene is based on mass spectrometry. Mass spectrometry takes advantage of the unique mass of each of the four nucleotides of DNA. The allele can be unambiguously genotyped by mass spectrometry by measuring the differences in the mass of nucleic acids having alternative GNAQ or GNAl 1 alleles. MALDI-TOF (Matrix Assisted Laser

Desorption Ionization - Time of Flight) mass spectrometry technology is preferred for extremely precise determinations of molecular mass, such as single nucleotide mutations. Preferred mass spectrometry-based methods of single nucleotide mutation assays include primer extension assays, which can also be utilized in combination with other approaches, such as traditional gel-based formats and microarrays.

Detection of GNAQ or GNA11 polypeptide sequences

[0097] Ex on 4 mutations in GNAQ or GNAl 1 may also be detected by detecting mutant protein. For example, detection of the presence of GNAQ or GNAl 1 proteins that have an ex on 4 mutation can be used for diagnostic purposes or in screening assays. In some embodiments, the presence of a normal or mutant GNAQ or GNAl 1 polypeptide in a sample is conveniently determined using immunological assays using reagents, e.g., an antibody, that specifically detects exon 4 mutations. The following section discusses immunological detection of GNAQ or GNAl 1 The section also relates to generation and engineering of antibodies that can be used, e.g., in therapeutic applications.

Immunological detection GNAQ or GNAl I

[0098] Antibodies can be used to detect GNAQ or GNAl 1 or can be assessed in the methods of the invention for the ability to inhibit GNAQ or GNAl 1. The detection and/or quantification of GNAQ or GNAl 1 can be accomplished using any of a number of well recognized immunological binding assays. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988) and Harlow & Lane, Using Antibodies (1999). Other resources include see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991, and Current Protocols in Immunology (Coligan, et al. Eds, John C. Wiley, 1999-present). Immunological binding assays can use either polyclonal or monoclonal antibodies. In some embodiments, antibodies that specifically detect mutant GNAQ or GNAl 1 molecules may be employed.

[0099] Commonly used assays include noncompetitive assays (e.g., sandwich assays) and competitive assays. Commonly used assay formats include immunoblots, which are used to detect and quantify the presence of protein in a sample. Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers, which are then detected according to standard techniques (see Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)).

[0100] Antibodies to GNAQ and GNA1 lare commercially available. In some

embodiments, mutations to GNA11 or GNAQ can be detected using antibodies that specifically bind a mutant form, thus immunoassays can also be used to detect mutant GNAQ or GNA1 1 proteins.

[0101] GNAQ or GNA11, or a fragment thereof, e.g., the portion of the peptide frequently containing a sequence mutation, may be used to produce antibodies specifically reactive with GNAQ or GNA11 using techniques known in the art (see, e.g., Coligan; Harlow & Lane, both supra). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989)). Such antibodies can be used for diagnostic or prognostic applications, e.g., in the detection of melanomas or for other cancers that exhibit increased expression or activity of GNA11.

[0102] Typically, polyclonal antisera with a titer of 10 ⁴ or greater are selected and tested for cross reactivity against non-GNAQ or non-GNAl 1 proteins or even other related proteins from other organisms, using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a Kd of at least about 0.1 mM, more usually at least about 1 μΜ, optionally at least about 0.1 μΜ or better, and optionally 0.01 μΜ or better.

[0103] In some embodiments, a GNAq or GNA1 1 antibody may be used for therapeutic applications. For example, in some embodiments, such an antibody may used to reduce or eliminate a biological function of a GNAQ or GNA11 having an exon 4 mutations is described below. That is, the addition of anti-GNAQ or anti- GNA11 antibodies (either polyclonal or preferably monoclonal) to a melanocyte neoplasm (or a cell population containing cancererous cells) may reduce or eliminate the neoplasm. Generally, at least a 25% decrease in activity, growth, size or the like is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred. [0104] Often, antibodies to the GNAQ or GNA1 1 proteins for therapeutic applications are humanized antibodies (e.g., Xenerex Biosciences, Mederex, Inc., Abgenix, Inc., Protein Design Labsjnc). Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom & Winter, J. Mol. Biol. 227:381 (1991); Marks et al, J. Mol. Biol. 222:581 (1991)). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol 147(l):86-95 (1991)). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous

immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al, Bio/Technology 10:779- 783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51 (1996); Neuberger, Nature

Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Detection of activity

[0105] As appreciated by one of skill in the art, GNAQ or GNAl 1 activity can be detected to evaluate expression levels or for identifying inhibitors of activity. The activity can be assessed using a variety of in vitro and in vivo assays, including GTP and GDP binding activity, GTP-hydrolase activity, or measurement of phospho lipase Cp. In some

embodiments GNAQ or GNAl 1 activity can be evaluated using additional endpoints, such as those associated with transformation or pigmentation. Such assays are described in greater detail in the examples and section detailing methods of identifying additional GNAQ or GNAl 1 inhibitors. Typically, GNAQ or GNAl 1 activity is determined by measuring the ability to bind a protein to which it interacts, e.g., an antibody, ligand, or other protein, such as signaling molecules.

Disease diagnosis/prognosis

[0106] GNAQ or GNAl 1 nucleic acid and polypeptide sequences can be used for diagnosis or prognosis of a melanocyte neoplasm, e.g., a blue nevus, uveal melanoma, or malignant blue nevus, in a patient. For example, as described above, the sequence of GNAQ or GNA11 in a melanocytic neoplasm sample from a patient can be determined, wherein an activating mutation in exon 4, e.g., at position 182, 183, or both 175 and 182, of Gnaq or Gnal l indicates the presence or the likelihood of a melanocytic neoplasm. [0107] The methods of the present invention can be used to determine the optimal course of treatment in a patient with cancer. For example, the presence of an exon 4 activating mutation in GNAQ or GNA11, can indicate that certain therapeutics, such as those that target GNAQ or GNAl 1, phospholipase CP, or downstream pathways regulated by GNAQ or GNAl 1 will be beneficial to those patients. In addition, a correlation can be readily established between the number of melanocytic neoplasm cells having the exon 4 mutation in GNAQ or GNAl 1, and the relative efficacy of one or another anti-melanoma agent. Such analyses can be performed, e.g., retrospectively, i.e., by analyzing for an activating mutation in samples taken previously from patients that have subsequently undergone one or more types of anti-cancer therapy, e.g., therapies that target G-proteins or phospholipase Cp, or other downstream pathways regulated by GNAQ or GNAl 1, and correlating the number of melanocytic neoplasm cells having the mutation with the known efficacy of the treatment.

[0108] Often, such methods will be used in conjunction with additional diagnostic methods, e.g., detection of other melanoma indicators, e.g., cell morphology, and the like. In other embodiments, a tissue sample known to contain melanoma cells, e.g., from a tumor, will be analyzed for GNAQ or GNAl 1 exon 4 activating mutations to determine information about the cancer, e.g., the efficacy of certain treatments, such as therapeutics that target GNAQ or GNAl 1 , or downstream pathways regulated by GNAl 1.

[0109] In some embodiments, analysis of melanoma cells for the presence of GNAQ or GNAl 1 exon 4 activating mutations can be used to determine the prognosis of a patient with melanoma, e.g., uveal melanoma or malignant blue nevus, or for determining progression of the disease. A "diagnostic presence" refers to the presence of one or more exon 4 activating sequence mutations in GNAQ or GNAl 1.

[0110] Any biological sample suspected of containing melanoma cells can be evaluated to determine progression. For example, tissues from visceral organs, such as liver or lung, blood, lymph nodes, bone and the like can be analyzed for the presence of exon 4 GNAQ or GNAl 1 sequence mutations, as well as tissues such as skin or eye.

[0111] In some embodiments, the methods of the invention involve recording the presence or absence of an exon 4 activating mutation in GNAQ or GNAl 1. This information may be stored in a computer readable form. Such a computer system typically comprises major subsystems such as a central processor, a system memory (typically RAM), an input/output (I/O) controller, an external device such as a display screen via a display adapter, serial ports, a keyboard, a fixed disk drive via a storage interface and the like. Many other devices can be connected, such as a network interface connected via a serial port.

[0112] The computer system also be linked to a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable (coax or lOBaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal

transmission medium, whereby at least one network device (e.g., computer, disk array, etc.) comprises a pattern of magnetic domains (e.g., magnetic disk) and/or charge domains (e.g., an array of DRAM cells) composing a bit pattern encoding data acquired from an assay of the invention.

Inhibitors or modulators of GNAQ or GNA11

[0113] In another aspect, this invention includes methods of inhibiting the proliferation of melanoma cells that have an exon 4 activating mutation in GNAQ or GNA11 where the method comprises administering an inhibitor or Gnaq or Gnal 1 antagonist to a patient that has such a melanoma, e.g., uveal melanoma. Inhibitors and GNAQ and GNA11 antagonists are known. For example, non- limiting examples of inhibitors suitable for use with the present invention can include specific and nonspecific inhibitors of PKC and various PKC isoforms including ΡΚΟμ and PKCs. Other examples of inhibitors suitable for use with the present invention include staurosporine, the staurosporine analogue CPG41251, bryostatin-1, KAI-9803, 7-hydroxystaurosporine, L-threo-dihydrosphingosine (safmgol), the non-selective PKC inhibitor (PKC412), ilmofosine (BM 41 440), Go6976, which is an indolcarbazole that more specifically inhibits the classical isoforms of PKC, including ΡΟΚμ, the PKC-alpha antisense inhibitor LY900003, and the PKC-beta inhibitors LY333531, LY317615

(Enzastaurin). Non-limiting examples of inhibitors of phospholipase Οβ can include edelfosine and fluvirusin B[2], which are also suitable for use in the present invention.

[0114] Other inhibitors include inhibitors such as antibodies, peptide, nucleic acids, e.g., siRNA, and the like. As used herein, a Gnaq inhibitor can be a molecule that modulates Gnaq nucleic acid expression and/or Gnaq protein activity, or in some embodiments, downstream pathways regulated by Gnaq. Similarly, a Gnal 1 inhibitor can be a molecule that modulates Gnal 1 nucleic acid expression and/or Gnal 1 protein activity, or in some embodiments, downstream pathways regulated by Gnal 1. In some embodiments, a GNA11 or GNAQ inhibitor is an inhibitory RNA molecule that targets GNA11 nucleic acid sequences or GNAQ nucleic acid sequences. In some embodiments, an inhibitory RNA molecule may target both GNA11 and GNAQ nucleic acid sequences.

[0115] The ability to inhibit Gnaq or Gnal 1 can be evaluated using appropriate assays, e.g., by assaying activity, e.g., GTP binding or GTP hydrolase activity and comparing the amount of activity to controls that are not treated with the inhibitor.

[0116] In another embodiment, mRNA and/or protein expression levels can be measured to assess the effects of a test compound on Gnaq or Gnal 1 expression levels. A host cell expressing Gnaq or Gna 11 is contacted with a test compound for a sufficient time to effect any interactions, and then the level of mRNA or protein is measured. The amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of expression as a function of time. The amount of expression may be measured by using any method known to those of skill in the art to be suitable.

[0117] The amount of expression is then compared to the amount of expression in the absence of the test compound. A substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. A difference in the amount of expression indicates that the test compound has in some manner altered Gnaq or Gnal 1 levels.

[0118] In some assays to identify Gnaq or Gnal 1 inhibitors, samples that are treated with a potential inhibitor are compared to control samples to determine the extent of modulation. Control samples without the mutation and untreated with candidate inhibitors are assigned a relative activity value of 100. Inhibition of GNA11 is achieved when the activity value relative to the control is about 80%, optionally 50%, optionally 25-0%.

[0119] Gnaq or Gnal 1 inhibitors can be any small chemical compound, or a biological entity, e.g., a macromolecule such as a protein, sugar, nucleic acid or lipid.

[0120] In some embodiments, GNAQ or GNA11 inhibitors are small molecules that have a molecular weight of less than 1,500 daltons, and in some cases less than 1,000, 800, 600, 500, or 400 daltons. The relatively small size of the agents can be desirable because smaller molecules have a higher likelihood of having physiochemical properties compatible with good pharmacokinetic characteristics, including oral absorption than agents with higher molecular weight. For example, agents less likely to be successful as drugs based on permeability and solubility were described by Lipinski et al. as follows: having more than 5 H-bond donors (expressed as the sum of OHs and NHs); having a molecular weight over 500; having a LogP over 5 (or MLogP over 4.15); and/or having more than 10 H-bond acceptors (expressed as the sum of Ns and Os). See, e.g., Lipinski et al. Adv Drug Delivery Res 23:3- 25 (1997). Compound classes that are substrates for biological transporters are typically exceptions to the rule.

Inhibiton of expression

[0121] As noted above, nucleic acid inhibitors may also be used to inhibit GNAQ or GNA11. Therefore, a nucleotide sequence that specifically interferes with expression of GNAQ or GNA11 at the transcriptional or translational level can be used to treat a melanoma or nevus. In some embodiments, such a nucleic acid inhibitor may target GNA11 and GNAQ nucleotide sequences that are sufficiently identical so as to interfere with expression of both GNA1 1 and GNAQ. An inhibitory nucleic acid approach may utilize, for example, siRNA and/or antisense oligonucleotides to block transcription or translation of the GNAQ or GNA11 (or both) mRNA, either by inducing degradation of the mRNA with a siRNA or by masking the mRNA with an antisense nucleic acid.

[0122] An "siRNA" or "RNAi" refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. "siRNA" thus refers to the double stranded RNA formed by the complementary strands. The

complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. [0123] "Silencing" or "downregulation" refers to a detectable decrease of transcription and/or translation of a target sequence, i.e., the sequence targeted by the siRNA, or a decrease in the amount or activity of the target sequence or protein in comparison to the normal level that is detected in the absence of the interfering RNA or other nucleic acid sequence. A detectable decrease can be as small as 5 % or 10 %, or as great as 80 %, 90 % or 100 %. More typically, a detectable decrease ranges from 20 %, 30 %, 40 %, 50 %, 60 %, or 70 %.

[0124] A DNA molecule that transcribes dsRNA or siRNA (for instance, as a hairpin duplex) also provides RNAi. For example, dsRNA oligonucleotides that specifically hybridize to a GNAQ or GNAll nucleic acid sequence such as SEQ ID NO:l or SEQ ID NO: 3 can be used in the methods of the present invention.

[0125] Antisense oligonucleotides that specifically hybridize to GNAQ or GNA11 nucleic acid sequences can also be used to silence the transcription and/or translation of GNAQ or GNA11, and thus treat melanoma, e.g., uveal melanoma, or a nevus such as a blue nevus. Methods of designing antisense nucleic acids (either DNA or RNA molecules) are well known in the art. Antisense nucleic acids may comprise naturally occurring nucleotides or modified nucleotides such as, e.g., phosphorothioate, methylphosphonate, and -anomeric sugar-phosphate, backbone-modified nucleotides. [0126] The ability of an inhibitor to modulate the expression of GNAQ or GNA11 can be evaluated using known methods. Such methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing GNAQ or GNA11 and then detecting a decrease in expression (either transcript or translation product).

Melanoma treatment and administration of pharmaceutical compositions

[0127] Inhibitors of GNAQ or GNA11 can be administered to a patient for the treatment of a melanocytic neoplasm having an exon 4 activating sequence mutation in GNAQ or GNAll.

As described in detail below, the inhibitors are administered in any suitable manner, optionally with pharmaceutically acceptable carriers. In some embodiments, inhibitors of

PKC or phospholipase C are administered. Protocols for the administration of inhibitors are known and can be further optimized for melanoma patients based on principles known in the pharmacological arts (see, e.g.. Remington: The Science and Practice of Pharmacy, 21st

Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005).

[0128] The inhibitors can be administered to a patient at therapeutically effective doses to prevent, treat, or control a melanocytic neoplasm. The compounds are administered to a patient in an amount sufficient to elicit an effective protective or therapeutic response in the patient. An effective therapeutic response is a response that at least partially arrests or slows the symptoms or complications of the disease. An amount adequate to accomplish this is defined as "therapeutically effective dose." The dose will be determined by the efficacy of the particular GNAQ or GNA11 inhibitor employed and the condition of the subject, as well as the body weight or surface area of the area to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject.

[0129] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD ₅ 0/ED ₅ 0.

Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects. [0130] The data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ (the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.

[0131] siRNA can be delivered to the subject using any means known in the art, including by injection, inhalation, or oral ingestion of the siRNA. Another suitable delivery system for siRNA is a colloidal dispersion system such as, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. Nucleic acids, including RNA and DNA within liposomes and be delivered to cells in a biologically active form (Fraley, et al, Trends Biochem. Sci., 6:77, 1981). Liposomes can be targeted to specific cell types or tissues using any means known in the art. [0132] Delivery of antisense polynucleotides specific for GNAQ or GNA11 can be achieved using any means known in the art including, e.g., direct injection, inhalation, or ingestion of the polynucleotides. In addition, antisense polynucleotides can be delivered using a recombinant expression vector (e.g., a viral vector based on an adenovirus, a herpes virus, a vaccinia virus, or a retrovirus) or a colloidal dispersion system (e.g., liposomes). [0133] A treatment that targets GNAQ or GNA11 can be administered with other melanoma therapeutics, either concurrently or before or after treatment with another melanoma thereapeutic agent.

[0134] Pharmaceutical compositions for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. The compounds and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, parenterally (e.g., intravenously, intraperitoneally, intravesically or intrathecally) or rectally.

[0135] For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, including binding agents, for example, pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose; fillers, for example, lactose, microcrystalline cellulose, or calcium hydrogen phosphate; lubricants, for example, magnesium stearate, talc, or silica; disintegrants, for example, potato starch or sodium starch glycolate; or wetting agents, for example, sodium lauryl sulphate. Tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound.

[0136] For administration by inhalation, the compounds may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromefhane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0137] The compounds can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents, for example, suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. [0138] The compounds can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.

[0139] Furthermore, the compounds can be formulated as a depot preparation. Such long- acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0140] The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, for example, a blister pack. The pack or dispenser device can be accompanied by instructions for administration. Kits for Use in Diagnostic and/or Prognostic Applications

[0141] The invention also provides kits for diagnostic or therapeutic applications. For diagnostic/prognostic applications, such kits may include any or all of the following: assay reagents, buffers, GNAQ and/or GNAl 1 probes, primers, antibodies, or the like that can be used to identify the presence of an exon 4 activating mutation. In some embodiments, a kit that comprises GNAl 1 and/or GNAQ diagnostic reagents for exon 4 may also comprise diagnostic reagents for detecting mutations in exon 5, e.g., at position 209, of GNAQ and/or GNAl 1. Such a kit may, for example, comprise a primer set to specifically amplify GNAl 1, or a subregion comprising nucleic acid sequences encoding exon 4, either alone or with exon 5, and a primer set to specifically amplify GNAQ or a subregion comprising nucleic acid sequences encoding exon 4, either alone or with exon 5. Further, a kit may comprise a probe that detects an exon 4 mutation, such as a mutation at position 182 or 183 of GNAQ or GNAl 1 ; and optionally, one or more probes for detecting the codon encoding position 209 of GNAl 1 and/or GNAQ. In some embodiments, the kit may comprise a probe specific for GNAl 1 and a probe specific for GNAQ, as well as probes that discriminate between mutant and wildtype alleles. Optionally, the kit may further comprises amplification primers for GNAl l and GNAQ.

[0142] In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

Example 1- Examination of melanoma and nevus samples for presence of GNAQ and GNAll exon 4 sequence mutations.

[0143] To further determine whether specific mutations in exon 4 in GNAQ and/or GNAll play a role in human melanocyte neoplasia, the coding regions of GNAQ and GNAll were sequenced in uveal melanomas and blue nevi. The GNAQ and GNAll coding region was also sequenced in normal surrounding tissue from selected biopsies. Methodology

Tissues

[0144] Archival, paraffin-embedded biopsies were used. For each sample, DNA was extracted from sections of 5-20 μπι thickness from which tumor-bearing tissue had been manually microdissected.

Sequencing

[0145] Sample DNA was amplified using PCR. The reaction conditions were 0.25 mM each dNTPs, 0.4X BSA (New England Biolabs), 1 U Hotstar Taq (Qiagen), IX Hotstar Taq buffer (Qiagen), and 0.5 μΜ forward and reverse primers. PCR reactions were purified using columns and then used as templates for sequencing reactions using Big Dye (ABI).

Sequencing was performed in both directions. Samples identified with mutations in both sequencing directions were replicated at least twice. For samples with mutations, DNA was sequenced from the adjacent normal tissue, to determine whether the mutations were somatically acquired. Results

[0146] The results are summarized in Table 1. We identified mutational hotspots in GNAQ and GNAl 1 in uveal melanoma. We sequenced exons 4 and 5 of GNAQ and GNAl 1 in 123 uveal melanomas (primary or metastasic) and discovered five melanoma samples that had mutations in R183 and one melanoma sample that had a mutation in VI 82 in exon 4 of GNAQ (=5%), as well as three melanoma samples that had mutations in Rl 83 of GNAl 1 (=2%). The melanoma that had a mutation at VI 82 of exon 4 of GNAQ also had a mutation at T175.

[0147] The uveal melanomas without mutations at Rl 83 or VI 82 had mutations at GNAQ Q209 in 49/123 (40%) and in GNAl 1 Q209 42/123 (34%) of the time. All mutations were found to be mutually exclusive, i.e. none of the uveal melanomas harbored more than one of these mutations with the exception of the melanoma sample that had the exon 4 mutations at V182 and T175.

[0148] We also identified an exon 4 mutation in GNA11 in a blue nevus sample.

[0149] The identified mutated arginine residues are highly conserved across the entire family of G alpha proteins. Mutations in the corresponding arginine of G alpha S and G alpha I were discovered in various human endocrine tumors (pituitary, thyroid, ovary and adrenal) and named gsp and gip2 oncogenes et al, Nature 340:692-696 (1989); Lyons, et al, Science 249:655-659 (1990)). The mutated oncoproteins were demonstrated to have lost their GTPase activity being constitutionally activated in a GTP -bound state (e.g., Conklin, et al, J. Biol. Chem 267;31-34 (1992); Kalinec, et al, Mol. Cell Biol 12:4687-4693 (1992)). No human neoplasms carrying Rl 83 mutations have been previously described.

[0150] Various in vitro studies have shown the Q209 mutations to be more potent than the R183 mutations. While the proteins mutated at glutamine 209 are 100% GTP-bound, arginine 183 mutants are only 50% GTP bound, whereas wild type proteins are virtually exclusively GDP-bound(Kleuss, et al, Proc. Natl Acad. Sci. U.S.A 91 :9828-9831 (1994)). Correspondingly, GNAQ Q209 mutants show higher phospholipase c beta 1 binding, inositol phosphate induction and serum response factor mediated gene transcription than R183 mutants (Orth, et al, Proc. Natl. Acad. Sci. U.S.A 106: 7179-7184 (2009); Evanko, et al, Cell Signal 17: 1218-1228 (2005); Takasaki, et al. J. of Biol Chem. 279:47438-47445 (2004)). The GNAQ inhibitor YM-254890 and the activator PMT (Pasteurella multicida toxin) still respectively dampen or augment the function of the R183 mutation, however they have no influence on Q209L mutants (Orth et al, and Takasaki et al, supra).

[0151] Not to be bound by theory, the weaker nature of the R183 mutations suggests that uveal melanomas harboring this mutation may behave differently, e.g., additional genetic alterations may allow them to become as active as Q209 mutants. The ability to alter the activation state of R183 mutations, e.g., with small molecules, may allow the development of treatment options radically different from those of Q209 mutant tumors. Further, the mutation at VI 82 that occurred with the mutation and T175 are probably additive.

Example 2. Additional analyses detecting GNAQ, GNA11 mutations [0152] Paraffin-embedded biopsies from specimen archives were retrieved after obtaining approval of the institutional review boards at the participating institutions. DNA was extracted and used to sequence GNAQ and GNA11 and to perform array comparative genomic hybridization (CGH) on a subset of cases. Tumongenicity experiments were carried out in NOD/SCID/interleukin 2 receptor " null mice using melan-a cells (immortalized, non- tumori genie mouse melanocytes) transduced with wildtype or constitutively active GNA1 1 or GNAQ expression constructs or a β-galactosidase control vector. Sequencing results

[0153] We found mutations affecting Q209 in GNA11 in the same specific subsets of melanocyte tumors (Tables 2 and 3) that were previously described for GNAQ (see, e.g.. Van Raamsdonk, et al., Natur 457:599-602, 2009). The mutation frequency of GNA11 at the codon encoding Q209 increased progressively from blue nevi (7%), to primary uveal melanomas (32%), to uveal melanoma metastases (57%), a pattern inverse to the distribution of GNAQ Q209 mutations, which are most common in blue nevi (55%) and least common in uveal melanoma metastases (22%) (p=<0.001) (Table 2). Mutations affecting codon 209 in GNA11 were CAOCTG (94.5%), CAG>CCG (2.7%), CAG>CTA (1.4%) and CAOCTT (1.4%). These mutations indicate substitution by leucine in 97.3% of samples analyzed, and by proline in 2.7% of these samples.

[0154] We also identified mutations in GNAQ and GNA11 in exon 4 at arginine 183, which is analogous to R201 and R179 in GNAS and GNAI2, respectively (Table 3) (Lyons et al. Science 249:655-9, 1990). Mutations affecting R183 in either GNAQ or GNA11 were present in 2% of blue nevi and 6% of uveal melanomas. Most mutations affecting R183 in GNA11 resulted in a substitution to cysteine, either caused by a single (CGC>TGC) or a double (GTC CGC>GTT TGC) cytosine-to-thymine transition. This double mutation results in a silent mutation in codon 182. A single blue nevus had a guanine-to-adenine transition

(CGC>CAC) at codon 183, predicting a substitution with histidine. This tumor also harbored a concomitant GNA11 Q209L mutation and was the only tumor that we analyzed that harbored mutations at both codons 183 and 209. In all other samples, mutations at codons 183 and 209 were mutually exclusive; in fact, none had concomitant GNAQ and GNA11 mutations (p<0.0001). In GNAQ all mutations in codon 183 were CGA to CAA transitions, indicating substitution with glutamine. A single uveal melanoma had mutations in codons 175 (ACG>AGG) and 182 (GTT>ATT).

[0155] All 1 1 tumors with mutations at Rl 83 showed a cytosine-to-thymine transition on either the forward or reverse strand. Cytosine-to-thymine transitions on the reverse strand appear as guanine to adenine transitions on the forward strand. Furthermore, three tumors harbored a CC>TT transition. These alterations are characteristic mutational patterns induced by ultraviolet light (Hocker & Tsao, Hum Mutat 2007;28:578-88, 2007; Pfeifer & You, Mutat Res 571 : 19-31, 2005) and are found with markedly increased frequency in cutaneous melanomas arising on sun-exposed skin (Pleasance et al, Nature 2010;463: 191-6, 2010). At codon Q209, in both GNAQ and GNAll, the predominant mutation is an adenine to thymine (A>T) transversion). Interestingly, single C>T transitions in the forward strand at Q209 would result in stop codons rather than in activating mutations (CAA>TAA and CAG>TAG, for GNAQ and GNAll, respectively). Similarly, a C>T transition on the reverse strand in GNAll at 209 would generate a silent mutation (CAG>CAA). Thus, Q209 does not have a base pair composition that would reveal a known UV "signature". However, we found two instances of tandem base mutations at Q209 that included C>T transitions (CAA>TTA and CAG>CTA, for GNAQ and GNAll, respectively).

[0156] The total fraction of combined types of blue nevi affected by mutations in GNAQ or GNAll was 63% (Table 2 and Table 3). Because the segmental melanocytoses, Nevus of Ito and the Nevus of Ota, are sparsely cellular 26, it is possible we missed mutations in some samples. If the Nevus of Ito and Ota are excluded, 75% of blue nevi have a mutation in GNAQ or GNAll. Altogether, 83% of all uveal melanomas that we examined had oncogenic mutations in GNA Q or GNA 11. Correlation with prognostic indicators and survival

[0157] For the 1 18 cases of uveal melanoma in which it was possible to determine the location of the primary tumor, lesions arising in the ciliochoroidal regions had a higher frequency of GNAll mutations (Figure 1). Mutations of GNAQ and GNAll were found more commonly in primary tumors with epithelioid cells or a mixture of epitheliod and spindled cells when compared to samples comprises of only spindled cells, but this was not statistically significant. Using Comparative Genomic Hybridization (CGH), we attempted to determine whether prognostically relevant chromosomal aberrations {e.g., White et al., Cancer 83:354-359, 1998) were present in 36 primary uveal melanoms and observed no association beween their presence and mutation status of GNAQ and GNAll. However, this analysis was very limited, as none of the 35 samples was without a mutation for comparison.

[0158] Examination of overall survival and disease-free survival for the 81 cases for which we had the requisite data did not reveal a significant difference between those with tumors bearing a GNAQ mutation and those with tumors bearing a GNAll mutations. A trend towards longer survival in those tumor carrying a GNAll mutatios, when compared with those tumor carrying a GNAQ mutation, or no GNAQ or GNAll mutations, was observed, however. Functional validation of GNA11

[0159] To validate GNA11 ^R,S3C (and GNA11 ^Q209L ) as oncogenes, we transduced

immortalized mouse melanocytes (melan-a cells) with one or the other of these mutant genes, injected the transduced cells into immunocompromised mice, and monitored the mice for the formation of tumors. Mice injected with melanocytes transduced with non-mutated GNAllov lacZ, encoding β-galactosidase, served as negative controls (Figure 2A-F). Rapidly growing tumors developed at each of the six injection sites for GNA11 ^Q209L melanocytes. Consistent with a report of its lesser potency (Conklin et al., J Biol Chem 1992;267:31-4, 1992), transduction with the GNA11 ^R,83C mutant resulted in tumors with an increased latency (when compared with tumors arising from GNA1 i ^e20¾ -transduced melanocytes) in three of eight injection sites during the time of observation. We observed no tumors at the injection sites of the control groups (Pgalactosidase: n=6; GNAllwt: n=10). All three mice injected with GNA1 ]® ^209L -transduced melan-a cells developed metastases; three developed metastases in the lung and one developed metastases in the liver. Western blot analyses of GNA1 i ^O209L . transduced melanocytes revealed MAP-kinase activation (Figure 2G).

[0160] The level of activation of GaqR183 mutants is lower than that of GaqQ209 mutants in vitro, raising the possibility that R183-mutated oncoproteins may be less potent (Conklin et ah, supra). Our finding of an increased latency and lower penetrance in the tumorigenicity assay is consistent with this notion. It is noted that it has been observed that that G qRl 83, but not GaqQ209, can be inhibited with YM-254890, a naturally occurring toxin from Chromobacterium (Takasaki et al, J Biol Chem 279:47438-45, 2004).

[0161] All publications, patents, accession numbers, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0162] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Table 1

Overview of cases in which exons 4 and 5 were sequenced for GNAQ and GNAll

(all mutations were mutually exclusive)

Exon 4 Exon 5

GNAQ GNAl l GNAQ GNAl l unknown overall

Uveal melanoma samples 6 3 49 42 23 123

Percentages 4.9 2.4 39.8 34.2 18.7 100

Other samples screened

Exon 4

Number of

samples GNAQ GNAl l

conjunctival melanoma 4 0 0

Blue Nevi 75 0 1(?)

Spitz Nevi 10 0 0

Pigmented Epitheloid

Melanocytoma 1 0 0

Spitzoid Tumors of Childhood 10 0 0

Individual listing of tumors with Exon 4 mutations

Uveal Melanoma

Coexistent mutations of

Amino acid GNAl l or GNAQ (exon 4 or

Exon 4 GNAQ chanae Codon chanae 5}

183Q CGA to CAA no

R183Q CGA to CAA no

R183Q CGA to CAA no

R183Q CGA to CAA no

R183Q CGA to CAA no

T175R + V182I ACG to AGG + GT Γ to ATT no

Exon 4 GNAl l

R183C CGC to TGC no

R183C CGC to TGC no

R183C CGC to TGC no

Blue nevus

Exon 4 GNAl l

R183H (?) CGC to CAC Yes, GNAl l Q209L Grand

Categories Subtypes GNAll GNAQ Neither

Total

Amelanotic blue

0 0.0% 7 70.0% 3 30.0% 10

Blue nevi nevus

Cellular blue nevus 3 8.3% 26 72.2% 7 19.4% 36

Common blue nevus 4 6.7% 39 65.0% 17 28.3% 60

Nevus of Ito 0 0.0% 0 0.0% 7 100.0% 7

Nevus of Ota 1 5.0% 2 10.0% 17 85.0% 20

Malignant blue

1 16.7% 2 33.3% 3 50.0% 6 nevus

Total 9 6.5% 76 54.7% 54 38.8% 139

Ocular

melanocytic Conjunctival melanoma 0 0.0% 0 0.0% 9 100.0% 9 tumors

52 31.9% 73 44.8% 38 23.3% 163

Uveal melanoma, primary

Uveal melanoma, 13 56.5% 5 21.7% 5 21.7% 23 metastasis

0 0.0% 1 100.0% 0 0.0% 1

Uveal nevus

Total 65 33.2% 79 40.3% 52 26.5% 196

Other nevi Common nevus 0 0.0% 0 0.0% 22 100.0% 22

Congenital nevus 0 0.0% 0 0.0% 17 100.0% 17

Deep penetrating nevus 0 0.0% 0 0.0% 27 100.0% 27

Spitz nevus 0 0.0% 0 0.0% 19 100.0% 19

Atypical Spitz tumor 0 0.0% 0 0.0% 20 100.0% 20

Total 0 0.0% 0 0.0% 105 100.0% 105

Extraocular Acral 0 0.0% 0 0.0% 47 100.0% 47 melanomas

CSD 0 0.0% 1 1.4% 73 98.6% 74

Mucosal 0 0.0% 0 0.0% 62 100.0% 62

NonCSD 0 0.0% 0 0.0% 90 100.0% 90

Total 0 0.0% 1 0.4% 272 99.6% 273

Grand Total 713

Table 2. Mutation frequency of Q209 in exons 5 of GNAQ and GNAll in melanocytic

neoplasms. CSD, melanoma located on chronically sun damaged skin; NonCSD, melanomas on skin without microscopic signs of chronic sun-induced damage . Categories Subtypes GNA11 GNAQ Neither Grand Total

Blue nevi Amelanotic blue nevus 0 0.0% 0 0.0% 9 100.0% 9

Cellular blue nevus 0 0.0% 0 0.0% 25 100.0% 25

Common blue nevus 1 2.4% 0 0.0% 40 97.6% 41

Nevus of Ito 0 0.0% 0 0.0% 7 100.0% 7

Nevus of Ota 0 0.0% 1 9.1% 10 90.9% 11

Malignant blue nevus 0 0.0% 0 0.0% 3 100.0% 3

Total 1 1.0% 1 1.0% 94 97.9% 96

Ocular

melanocytic 0 0.0% 0 0.0% 6 100.0% 6 tumors Conjunctival melanoma

Uveal melanoma,

3 2.1% 4 2.8% 138 95.2% 145 primary

Uveal melanoma,

1 5.9% 1 5.9% 15 88.2% 17 metastasis

Uveal nevus 0 0.0% 0 0.0% 1 100.0% 1

Total 4 2.4% 5 3.0% 160 94.7% 169

Other nevi Deep penetrating nevus 0 0.0% 0 0.0% 14 100.0% 14

Spitz nevus 0 0.0% 0 0.0% 8 100.0% 8

Atypical Spitz tumor 0 0.0% 0 0.0% 8 100.0% 8

Total 0 0.0% 0 0.0% 30 100.0% 30

Extra-ocular

0 0.0% 0 0.0% 18 100.0% 18 melanomas Acral

CSD 0 0.0% 0 0.0% 49 100.0% 49

Mucosal 0 0.0% 0 0.0% 38 100.0% 38

NonCSD 0 0.0% 0 0.0% 53 100.0% 53

Total 0 0.0% 0 0.0% 158 100.0% 158

Grand Total 453

Table 3. Mutation frequency of R183 in exons 4 of GNAQ and GNA11 in melanocytic

neoplasms. *One primary uveal melanoma sample had concomitant T175R, V182I mutations in GNAQ, which are of unknown functional significance, and is not included in this table. CSD, melanoma located on chronically sun-damaged skin; NonCSD, on skin without microscopic signs of chronic sun-induced damage. Illustrative GNAQ sequences:

SEQ ID NO:l Accession Number NM 002072 human guanine nucleotide bindmg protein (G protein) q (GNAQ), mRNA

1 agggggtgcc ggcggggctg cagcggaggc actttggaag aatgactctg gagtccatca 61 tggcgtgctg cctgagcgag gaggccaagg aagcccggcg gatcaacgac gagatcgagc

121 ggcagctccg cagggacaag cgggacgccc gccgggagct caagctgctg ctgctcggga 181 caggagagag tggcaagagt acgtttatca agcagatgag aatcatccat gggtcaggat 241 actctgatga agataaaagg ggcttcacca agctggtgta tcagaacatc ttcacggcca 301 tgcaggccat gatcagagcc atggacacac tcaagatccc atacaagtat gagcacaata 361 aggctcatgc acaattagtt cgagaagttg atgtggagaa ggtgtctgct tttgagaatc 421 catatgtaga tgcaataaag agtttatgga atgatcctgg aatccaggaa tgctatgata 481 gacgacgaga atatcaatta tctgactcta ccaaatacta tcttaatgac ttggaccgcg 541 tagctgaccc tgcctacctg cctacgcaac aagatgtgct tagagttcga gtccccacca 601 cagggatcat cgaatacccc tttgacttac aaagtgtcat tttcagaatg gtcgatgtag 661 ggggccaaag gtcagagaga agaaaatgga tacactgctt tgaaaatgtc acctctatca 721 tgtttctagt agcgcttagt gaatatgatc aagttctcgt ggagtcagac aatgagaacc 781 gaatggagga aagcaaggct ctctttagaa caattatcac atacccctgg ttccagaact 841 cctcggttat tctgttctta aacaagaaag atcttctaga ggagaaaatc atgtattccc 901 atctagtcga ctacttccca gaatatgatg gaccccagag agatgcccag gcagcccgag 961 aattcattct gaagatgttc gtggacctga acccagacag tgacaaaatt atctactccc 1021 acttcacgtg cgccacagac accgagaata tccgctttgt ctttgctgcc gtcaaggaca 1081 ccatcctcca gttgaacctg aaggagtaca atctggtcta attgtgcctc ctagacaccc 1141 gccctgccct tccctggtgg gctattgaag atacacaaga gggactgtat ttctgtggaa 1201 aacaatttgc ataatactaa tttattgccg tcctggactc tgtgtgagcg tgtccacaga 1261 gtttgtagta aatattatga ttttatttaa actattcaga ggaaaaacag aggatgctga 1321 agtacagtcc cagcacattt cctctctatc ttttttttag gcaaaacctt gtgactcagt 1381 gtattttaaa ttctcagtca tgcactcaca aagataagac ttgtttcttt ctgtctctct 1441 ctctttttct tttctatgga gcaaaacaaa gctgatttcc cttttttctt cccccgctaa 1501 ttcatacctc cctcctgatg tttttcccag gttacaatgg cctttatcct agttccattc 1561 ttggtcaagt ttttctctca aatgatacag tcaggacaca tcgttcgatt taagccatca 1621 tcagcttaat ttaagtttgt agtttttgct gaaggattat atgtattaat acttacggtt 1681 ttaaatgtgt tgctttggat acacacatag tttctttttt aatagaatat actgtcttgt 1741 ctcactttgg actgggacag tggatgccca tctaaaagtt aagtgtcatt tcttttagat 1801 gtttaccttc agccatagct tgattgctca gagaaatatg cagaaggcag gatcaaagac 1861 acacaggagt cctttctttt gaaatgccac gtgccattgt ctttcctccc ttctttgctt 1921 ctttttctta ccctctcttt caattgcaga tgccaaaaaa gatgccaaca gacactacat 1981 taccctaatg gctgctaccc agaacctttt tataggttgt tcttaatttt tttgttgttg 2041 ttgttcaagc ttttcctttc ttttttttct tagtgtttgg gccacgattt taaaatgact 2101 tttattatgg gtatgtgttg ccaaagctgg ctttttgtca aataaaatga atacgaactt 2161 aaaaaataaa aaaaaaaaaa aaaaaaaa SEQ ID NO:2 Accession Number NP 002063.2 guanine nucleotide binding protein (G protein), q polypeptide [Homo sapiens] The positions of exon 4 mutations that occur in melanoma are bolded and shown in enlarged font.

1 mtlesimacc lseeakearr indeierqlr rdkrdarrel kllllgtges gkstfikqmr 61 iihgsgysde dkrgftklvy qniftamqam iramdtlkip ykyehnkaha qlvrevdvek

121 vsafenpyvd aikslwndpg iqecydrrre yqlsdstkyy lndldrvadp aylptqqdvl

181 rVXvpttgii eypfdlqsvi frmvdvggqr serrkwihcf envtsimflv alseydqvlv

241 esdnenrmee skalfrtiit ypwfqnssvi lflnkkdlle ekimyshlvd yfpeydgpqr 301 daqaarefil kmfvdlnpds dkiiyshftc atdtenirfv faavkdtilq lnlkeynlv Illustrative GNA1 1 cDNA and protein sequences:

SEQ ID O:3 Accession Number NM 002067 GNA11, mRNA, CDS243..1322

1 gctgcggcgg cggcgcgggc tgagtgcggc cgcgcgggag tccgcggctg gcgcggcccg

61 agcggggacc cggcggctcg ccaggcggcg gccgaggcgg ggcgggccgg cccggggccg

121 agggccggtg gccgaggccg gagggccgcg gcgggcggcg gccgaggcgg ctccggccag

181 ggccgggccg ggggccgggg ggcggcggcg ggcaggcggc cgcgtcggcc ggggccggga

241 cgatgactct ggagtccatg atggcgtgtt gcctgagcga tgaggtgaag gagtccaagc

301 ggatcaacgc cgagatcgag aagcagctgc ggcgggacaa gcgcgacgcc cggcgcgagc

361 tcaagctgct gctgctcggc acgggcgaga gcgggaagag cacgttcatc aagcagatgc

421 gcatcatcca cggcgccggc tactcggagg aggacaagcg cggcttcacc aagctcgtct

481 accagaacat cttcaccgcc atgcaggcca tgatccgggc catggagacg ctcaagatcc

541 tctacaagta cgagcagaac aaggccaatg cgctcctgat ccgggaggtg gacgtggaga

601 aggtgaccac cttcgagcat cagtacgtca gtgccatcaa gaccctgtgg gaggacccgg

661 gcatccagga atgctacgac cgcaggcgcg agtaccagct ctccgactct gccaagtact

721 acctgaccga cgttgaccgc atcgccacct tgggctacct gcccacccag caggacgtgc

781 tgcgggtccg cgtgcccacc accggcatca tcgagtaccc tttcgacctg gagaacatca

841 tcttccggat ggtggatgtg gggggccagc ggtcggagcg gaggaagtgg atccactgct

901 ttgagaacgt gacatccatc atgtttctcg tcgccctcag cgaatacgac caagtcctgg

961 tggagtcgga caacgagaac cggatggagg agagcaaagc cctgttccgg accatcatca

1021 cctacccctg gttccagaac tcctccgtca tcctcttcct caacaagaag gacctgctgg

1081 aggacaagat cctgtactcg cacctggtgg actacttccc cgagttcgat ggtccccagc

1141 gggacgccca ggcggcgcgg gagttcatcc tgaagatgtt cgtggacctg aaccccgaca

1201 gcgacaagat catctactca cacttcacgt gtgccaccga cacggagaac atccgcttcg

1261 tgttcgcggc cgtgaaggac accatcctgc agctcaacct caaggagtac aacctggtct

1321 gagcgcccag gcccagggag acgggatgga gacacggggc aggaccttcc ttccacggag 1381 cctgcggctg ccgggcgggt ggcgctgccg agtccgggcc ggggcctctg cccgcgggag

1441 gagatttttt tttttcatat ttttaacaaa tggtttttat ttcacagtta tcaggggatg

1501 tacatctctc cctccgtaca cttcgcgcac cttctcacct tttgtcaacg gcaaaggcag

1561 cctttttctg gccttgactt atggctcgct tttttctaaa aaaaaaaaaa aaaaa

SEQ ID NO:4 GNA11 Protein Sequence

Accession Numbers: UniProtKB P29992-1 ; NP_002058

The positions of exon 4 mutations that occur in melanoma are bolded and shown in enlarged font.

1 MTLES MACC LSDEVKESKR INAEIEKQLR RDKRDARREL KLLLLGTGES GKSTFIKQMR 61 IIHGAGYSEE D RGFTKLVY QNIFTAMQAM IRAMETLKIL YKYEQNKA A LLIREVDVEK

121 VTTFEHQYVS AIKTLWEDPG IQECYDRRRE YQLSDSAKYY LTDVDRIATL GYLPTQQDVL

181 RVRVPTTGII EYPFDLENII FRMVDVGGQR SERRKWIHCF EMVTSIMFLV ALSEYDQVLV 241 ESDNENRMEE SKALFRTIIT YPWFQNSSVI LFLNKKDLLE DKILYSHLVD YFPEFDGPQR 301 DAQAAREFIL KMFVDLMPDS DKIIYSHFTC ATDTENIRFV FAAVKDTILQ LNLKEYNLV