WO2011126740A2 - Hip1 cancer markers - Google Patents

Abstract

The present invention relates to compositions and methods for cancer diagnostics, including but not limited to, HIP1 cancer markers. In particular, the present invention provides compositions and methods of using HIP1 in the diagnosis and treatment of skin cancers (e.g., Merkel Cell Carcinoma). The present invention further provides methods of screening potential therapeutic compounds for HIP1 inhibitory properties.

Description

HIPl CANCER MARKERS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This is invention was made with government support under Grant number

CA098730-01 awarded by the National Cancer Institute. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for cancer diagnostics, including but not limited to, HIPl cancer markers. In particular, the present invention provides compositions and methods of using HIPl in the diagnosis and treatment of skin cancers (e.g., Merkel Cell Carcinoma). BACKGROUND OF THE INVENTION

Most forms of cancer do not have diagnostic screening tests available. For the cancers that do have screening tests available, the tests are frequently invasive, expensive, and lack strong diagnostic utility.

Merkel cell carcinoma (MCC) is an aggressive skin cancer thought to be derived from sensory Merkel cells of the skin (Bichakjian et al. Cancer 2007;1 10: 1-12). Though the overall incidence of this disease is low and affects approximately 1400 patients per year in the United States, this cancer is associated with a poor prognosis, and most patients with metastatic disease do not survive more than 5 years (Bichakjian et al, supra). Clinical management of patients with MCC is limited due to a lack of prognostic markers and effective therapies. These limitations stem from a lack of understanding of the biology of MCC's initiation, maintenance and progression to the metastatic stage.

Currently, surgical excision of the primary tumor and radiation remain the main therapeutics for MCC (Bichakjian et al, supra). Though receptor tyrosine kinases (RTKs), such as c-Kit, have been shown to be increased in expression in MCC, convincing clinical data regarding the effectiveness of the specific c-Kit inhibitor, imatinib, on patient survival is not yet available (Lemos et al., J Invest Dermatol 2007;127:2100-3). Understanding the factors that contribute to the maintenance and progression of MCC is needed to facilitate the development of effective therapies. Thus, development of additional serum and tissue biomarkers specific to cancers such as skin cancers are needed to supplement the currently available screening methods.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for cancer diagnostics, including but not limited to, HIPl cancer markers. In particular, the present invention provides compositions and methods of using HIPl in the diagnosis and treatment of skin cancers (e.g., Merkel Cell Carcinoma).

For example, in some embodiments, the present invention provides a method of detecting merkel cell carcinoma, comprising: detecting expression of Huntingtin interacting protein 1 (HIPl) in a skin sample from a subject; and detecting merkel cell carcinoma in the sample when HIPl expression is elevated in the sample relative the level of expression in normal skin. In some embodiments, detecting comprises detecting an elevated level of HIPl in the sample. In some embodiments, the detecting comprises measuring the level of HIPl in the sample. In some embodiments, the detecting comprises measuring the level of HIPl protein. In some embodiments, the measuring the level of HIPl protein comprises exposing the HIPl protein to an antibody specific to the HIPl protein. In some embodiments, the detecting comprises measuring the level of HIPl mRNA. In some embodiments, the merkel cell carcinoma is metastatic.

Embodiments of the present invention also provide a method of detecting merkel cell carcinoma, comprising: detecting the presence of absence of autoantibodies to HIPl in a serum sample from a subject; and detecting merkel cell carcinoma in the sample when autoantibodies to HIPl are present in the sample. In some embodiments, the merkel cell carcinoma is metastatic merkel cell carcinoma. In some embodiments, the subject is a human female. In some embodiments, the autoantibodies bind to an N- terminal and/or C-terminal portion of HIPl . In some embodiments, the N-terminal portion comprises the lipid-binding ANTH, clathrin binding, and AP2-binding domains. In some embodiments, the N-terminal portion comprises approximately amino acids 1- 430 of SEQ ID NO:5. DESCRIPTION OF THE FIGURES

Figure 1 shows that HIPl is expressed at high levels in primary MCC but not in SCLC and is not required for the development or maintenance of normal Merkel cells.

Figure 2 shows auto-antibody titers against the N-terminal antigen are elevated in metastatic MCC patients. Individual dots represent relative antibody titers from patients with either metastatic MCC or localized MCC. Mean and standard error of the mean for each data set are indicated by horizontal and vertical lines, respectively.

Figure 3 shows that HIPl interacts with c-Kit, a RTK that is expressed at high levels in MCC. A, Association of HIPl with c-Kit in HEK 293T cells was detected by co- immunoprecipitation. This interaction was enhanced by stimulation with the c-kit ligand SCF (lanes 3 and 6). B, HIPl associates with c-Kit in a Merkel cell carcinoma cell line. The MCC565 cell line was treated with SCF for one hour prior to collection. C,

Prolongation of c-Kit' s half life by HIPl was observed in three independent experiments. On the left is a representative western blot demonstrating the stabilization of c-Kit by [ HIPl compared to vector transfected cells after treatment of cells with SCF. * p < 0.05, ** p < 0.01 , Two-tailed t-test.

Figure 4 shows Anti-HIPl antibody testing in MCC patient samples using an additional newly designed antigen. A, Representative serum immunoblots from individual MCC patients (numbers 5-12) for anti HIPl autoantibodies against the new N- terminal and the old C-terminal antigens. B, Percent of MCC patients with anti-HIPl antibodies specific to the N-terminal or C-terminal antigens. Auto-antibodies against a HIPl antigen were considered present if the optical density of the western blot reading was equal to or greater than 20% of an internal positive control. C, Diagram of the functional domains of the HIPl protein that are contained within the C-terminal and N- - terminal antigens. ENTH= Epsin N-terminal homology domain. CHC=clathrin heavy chain-binding domain. AP2 = AP-2 adaptor protein-binding region. CC = coiled-coil domain. CLC = clathrin light chain-binding domain. LZ=leucine zipper. Talin Homology domain = actin-binding region.

Figure 5 shows serum blots for N-terminal and C-terminal HIPl autoantibodies from MCC patients. Blots are shown which were used to calculate serum antibody titer values used in Figure 3 and Table 2. Individual lanes are marked with a patient identification number, a + signs to mark positive tests, and stars to indicate disease stage, as listed in the figure legend. DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

As used herein, the term "immunoglobulin" or "antibody" refer to proteins that bind a specific antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric, and humanized antibodies, Fab fragments, F(ab')₂ fragments, and includes immunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, and secreted immunoglobulins (slg). Immunoglobulins generally comprise two identical heavy chains and two light chains. However, the terms "antibody" and

"immunoglobulin" also encompass single chain antibodies and two chain antibodies.

As used herein, the term "antigen binding protein" refers to proteins that bind to a specific antigen. "Antigen binding proteins" include, but are not limited to,

immunoglobulins, including polyclonal, monoclonal, chimeric, and humanized antibodies; Fab fragments, F(ab')₂ fragments, and Fab expression libraries; and single chain antibodies.

The term "epitope" as used herein refers to that portion of an antigen that makes contact with a particular immunoglobulin.

When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as "antigenic determinants". An antigenic determinant may compete with the intact antigen (i.e., the "immunogen" used to elicit the immune response) for binding to an antibody.

The terms "specific binding" or "specifically binding" when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A," the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

As used herein, the terms "non-specific binding" and "background binding" when used in reference to the interaction of an antibody and a protein or peptide refer to an interaction that is not dependent on the presence of a particular structure (i.e., the antibody is binding to proteins in general rather that a particular structure such as an epitope).

As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and

"patient" are used interchangeably herein in reference to a human subject.

As used herein, the term "subject suspected of having cancer" refers to a subject that presents one or more symptoms indicative of a cancer (e.g., a noticeable lump or mass) or is being screened for a cancer (e.g., during a routine physical). A subject suspected of having cancer may also have one or more risk factors. A subject suspected of having cancer has generally not been tested for cancer. However, a "subject suspected of having cancer" encompasses an individual who has received a preliminary diagnosis (e.g., a CT scan showing a mass) but for whom a confirmatory test (e.g., biopsy and/or histology) has not been done or for whom the stage of cancer is not known. The term further includes people who once had cancer (e.g., an individual in remission). A "subject suspected of having cancer" is sometimes diagnosed with cancer and is sometimes found to not have cancer.

As used herein, the term "subject diagnosed with a cancer" refers to a subject who has been tested and found to have cancerous cells. The cancer may be diagnosed using any suitable method, including but not limited to, biopsy, x-ray, blood test, and the diagnostic methods of the present invention. A "preliminary diagnosis" is one based only on visual (e.g., CT scan or the presence of a lesion) and antigen tests.

As used herein, the term "initial diagnosis" refers to a test result of initial cancer diagnosis that reveals the presence or absence of cancerous cells (e.g., using a biopsy and histology). An initial diagnosis does not include information about the stage of the cancer.

As used herein, the term "identifying the risk of said tumor metastasizing" refers to the relative risk (e.g., the percent chance or a relative score) of a tumor (e.g., skin cancer tumor tissue) metastasizing.

As used herein, the term "identifying the risk of said tumor recurring" refers to the relative risk (e.g., the percent chance or a relative score) of a tumor (e.g., skin cancer) recurring in the same organ as the original tumor.

As used herein, the term "subject at risk for cancer" refers to a subject with one or more risk factors for developing a specific cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental expose, and previous incidents of cancer, preexisting non-cancer diseases, and lifestyle.

As used herein, the term "characterizing cancer in subject" refers to the identification of one or more properties of a cancer sample in a subject, including but not limited to, the presence of benign, pre-cancerous or cancerous tissue and the stage of the cancer. Cancers may be characterized by the identification of HIP1 in tumor tissues.

As used herein, the term "characterizing tissue in a subject" refers to the identification of one or more properties of a tissue sample (e.g., including but not limited to, the presence of cancerous tissue, the presence of pre-cancerous tissue that is likely to become cancerous, and the presence of cancerous tissue that is likely to metastasize). In some embodiments, tissues are characterized by the identification of the expression of HIP1.

As used herein, the term "reagent(s) capable of specifically detecting HIP1 expression" refers to reagents used to detect the expression of HIP 1. Examples of suitable reagents include but are not limited to, nucleic acid probes capable of specifically hybridizing to HIP1 mRNA or cDNA, and antibodies (e.g., monoclonal antibodies).

As used herein, the term "detecting a decrease in viability" refers to a decrease in the number of living cells in a culture. In preferred embodiments, the decrease is due to the induction of programmed cell death (e.g., apoptosis) in some or all of the cells in a population. As used herein, the term "induces cell death" refers to a molecule (e.g., a test compound or a drug) that induces a programmed cell death (e.g., apoptosis).

As used herein, the term "drug that inhibits HIP1 biological activity" refers to a drug that inhibits one or more biological activities of HIP 1 (e.g., ligand binding and signaling). Preferred drugs are those that inhibit polyphosphoinositol lipid binding and subsequent signaling.

As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor, whether the tumor has spread to other parts of the body and where the cancer has spread (e.g., within the same organ or region of the body or to another organ).

As used herein, the term "providing a prognosis" refers to providing information regarding the impact of the presence of cancer (e.g., as determined by the diagnostic methods of the present invention) on a subject's future health (e.g., expected morbidity or mortality, the likelihood of getting cancer, and the risk of metastasis).

As used herein, the term "non-human animals" refers to all non-human animals including, but are not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.

As used herein, the term "nucleic acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,

1 - methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine,

2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,

7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil,

2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl- 2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and

2,6-diaminopurine.

The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rR A, tRNA). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non- translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA

(hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA

polymerase), and for protein encoding genes, into protein through "translation" of mRNA. Gene expression can be regulated at many stages in the process. "Up- regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C- A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.

The term "homology" refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid is "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target that is substantially non-complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized."

As used herein, the term "T_m" is used in reference to the "melting temperature."

The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81 .5 + 0.41 (% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization [1985]). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T_m.

As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Under "low stringency conditions" a nucleic acid sequence of interest will hybridize to its exact complement, sequences with single base mismatches, closely related sequences (e.g., sequences with 90% or greater homology), and sequences having only partial homology (e.g., sequences with 50-90% homology). Under 'medium stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely relation sequences (e.g., 90% or greater homology). Under "high stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement, and (depending on conditions such a temperature) sequences with single base mismatches. In other words, under conditions of high stringency the temperature can be raised so as to exclude hybridization to sequences with single base mismatches.

The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, nonisolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double-stranded).

"Amino acid sequence" and terms such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

The term "native protein" as used herein to indicate that a protein does not contain amino acid residues encoded by vector sequences; that is, the native protein contains only those amino acids found in the protein as it occurs in nature. A native protein may be produced by recombinant means or may be isolated from a naturally occurring source.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals

(including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for cancer diagnostics, including but not limited to, HIPl cancer markers. In particular, the present invention provides compositions and methods of using HIPl in the diagnosis and treatment of skin cancers (e.g., Merkel Cell Carcinoma). The present invention further provides methods of screening potential therapeutic compounds for HIPl inhibitory properties.

Huntingtin interacting protein 1 (HIPl) is a highly conserved protein that interacts with endocytic machinery, including 3-phosphoinositides, clathrin and AP-2 (Engqvist- Goldstein et al., J Cell Biol 1999;147: 1503-18; Engqvist-Goldstein et al., J Cell Biol 2001 ; 154: 1209-23; Metzler et al., J Biol Chem 2001 ;276:39271-6; Mishra et al., J Biol Chem 2001 ;276:46230-6; Rao et al., Mol Cell Biol 2001 ;21 :7796-806; Waelter et al., Hum Mol Genet 2001 ; 10:1807-17; Hyun et al., Trends Mol Med 2004;10: 194-9). All of these molecules are involved in the clathrin-mediated internalization of surface receptors. Unlike AP-2 (Mitsunari et al., Mol Cell Biol 2005;25:9318-23), HIPl is not necessary for embryogenesis or early post natal development, but young adult mice deficient for HIPl do develop a degenerative phenotype (Oravecz- Wilson et al., Hum Mol Genet

2004; 13:851-67). In addition, HIPl over-expression transforms fibroblasts (Rao et al., Cancer Cell 2003;3:471-82) and prostate epithelial cells (Wang et al., Neoplasia

2008; 10:847-56) and HIPl transgenic mice develop plasma cell neoplasms (Bradley et al., Cancer Res 2007;67:8923-31).

In addition to its transforming activity, high HIPl expression is associated with a variety of human cancers, including prostate, colon, brain, and lymphoid cancers (Bradley et al. Cancer Res 2007;67:8923-31 ; Rao et al, J Clin Invest 2002;1 10:351-60; Bradley et al. Cancer Res 2007;67:3609-15; US 7,429,450 and US 2009001 1412). ΗΙΡΓ expression in prostate tumors is associated with a poor prognosis (Bradley et al. Cancer Res 2005;65:4126-33). Anti-HIPl antibodies have been detected in the sera of patients with prostate, lymphoid, and brain cancers more frequently than cancer-free individuals (Bradley et al. Cancer Res 2007;67:8923-31 ; Bradley et al. Cancer Res 2007;67:3609- 15; Bradley et al. Cancer Res 2005;65:4126-33; US 20040265929). Further investigation into the role of HIPl in tumorigenesis has demonstrated an association between the presence of this oncoprotein and enhanced RTK expression (Rao et al. Cancer Cell 2003;3:471 -82; Bradley et al., Cancer Res 2007;67:3609-15). HIPl over expression in tumors associated with the over expression of RTKs, and ΗΓΡ1- mediated transformation can be blocked with tyrosine kinase inhibitors (Rao et al., Cancer Cell 2003;3:471 -82). It was found that co-expression of HIPl with the EGFR increases the half life of the EGFR upon ligand stimulation (Hyun et al., J Biol

Chem 2004;279: 14294-306) and that HIPl physically associates with the EGFR (Bradley et al., Cancer Res 2007;67:3609-15). Others have found that HIPl also stabilizes and associates with the fibroblast growth factor receptor 4 (FGFR4) (Wang et al., Neoplasia 2008; 10:847-56). These interactions together with HIPl 's over-expression in multiple cancers indicate that HIPl -mediated transformation may occur via increases in multiple, parallel RTK signals.

Experiments conducted during the course of development of embodiments of the present invention determined that HIPl could serve as a marker for MCC. MCC tissue samples were evaluated and vastly elevated HIPl protein levels compared to normal surrounding skin tissue were observed. High levels of anti-HIPl antibodies were detected in sera from MCC patients and patients with metastatic disease exhibited higher levels of anti-HIPl antibodies than MCC patients with localized disease. It was also discovered that HIPl physically associates with and stabilizes c-Kit, a RTK specifically over- expressed in MCC (Sattler et al., Leuk Res 2004 ;28 Suppl 1 :S 1 1-20; Su et al., Am J Dermatopathol 2002;24:289-93).

MCC is a rare cancer, for which investigation of the molecular mechanisms of its cause and maintenance, to guide the development of better treatment regimens, has only recently received significant attention. Patients with MCC have a poor prognosis similar to patients with other neuroendocrine tumors, such as SCLC. In contrast to SCLC (Socinski et al., J Clin Oncol 2007;25:4137-45), MCC patients suffer from a lack of therapies and prognostic markers (Bichakjian et al., supra). It was demonstrated not only that HIPl is a useful immunohistochemical marker for MCC but also that auto-antibodies against a novel HIPl antigen in patient sera predict the presence of metastatic disease. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that the discovery of high HIP1 levels in the tumors of MCC indicates that HIP 1 , which acts as an oncogene when expressed at high levels, contributes to the mechanism(s) of MCC development, maintenance or progression. It is contemplated that the over expression of HIP 1 leads to elevated RTK signaling through its prevention of receptor degradation and thus, increasing cellular proliferation and/or survival signals ultimately leading to transformation of Merkel cells.

The expression levels of HIP 1 in the cytoplasm of MCC tumors serve as immunogens in MCC patients more frequently than in tumor-free individuals, leading to the specificity of a positive anti-HIPl antibody test to cancer patients (Bradley et al.,

Cancer Res 2005;65:4126-33). Experiments conducted during the course of development of embodiments of the present invention employed both a new amino-terminal HIP1 test antigen and the previously described carboxy-terminal HIP1 test antigen for anti-HIPl antibody analysis (Bradley et al., Cancer Res 2005;65:4126-33). Antibodies against the amino-terminus of HIP1 are present more often in patients with metastatic MCC than in patients with localized MCC. This was not the case for the carboxyterminal antigen which yielded many more positive test results. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that this difference is due, in part, to the possibility that there is differential expression of antigenically distinct isoforms of HIP1 in cancer and normal cells.

In some embodiments, the prognostic results associated with the new HIP1 N- terminal antigen blood test are used to evaluate patients with metastatic ovarian, prostate, or colon cancer for higher titers of antibodies against the N-terminal antigen compared to patients with localized disease. For example, HIP1 over expression in prostate cancer tumors is associated with poor prognosis (Rao et al., J Clin Invest 2002; 1 10:351 -60). Because the decision to resect prostate tumors is often difficult due to potential urologic side effects, the value of a blood test that can predict which tumors are likely to metastasize finds use in determining treatment courses of action in such cancers. In some embodiments, a combination test for both HIP1 N-terminal and C-terminal antibodies is utilized. In some embodiments, such combination tests are more sensitive and specific than either test alone.

The mechanisms of how HIP1 transforms cells remain a subject of investigation. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that HIP1 inhibits the degradation of active RT s during the process of receptor-mediated endocytosis (Hyun et al., Trends Mol Med 2004; 10: 194-9) as its over expression stabilizes RTKs following receptor activation (Hyun et al., J Biol Chem 2004;279: 14294-306). Cells transformed by HIP1 over-expression have elevated EGFR levels and EGFR inhibitors inhibit the transformed phenotype, indicating that this receptor stabilization is an important element of HIP 1 -mediated transformation (Rao et al., Cancer Cell 2003;3 :471 -82). Prior reports have shown that HIP1 also physically interacts with EGFR (Bradley et al., Cancer Res 2007;67:3609-15) and FGFR4 (Wang et al., Neoplasia 2008; 10:847-56). Neither of these receptors is known to be expressed in MCC, indicating that the tiimorigenic function of HIP1 in MCC may be mediated through stabilization of a different RTK. For example, the SCF receptor c-Kit is often over expressed in MCC as well as other tumor types such as breast tumors, SCLC, colorectal cancers, and gastrointestinal stromal tumors (GIST), where it is a pharmacological target of imatinib (Sattler et al., Leuk Res 2004;28 Suppl 1 :S 1 1-20; Su et al., Am J

Dermatopathol 2002;24:289-93). HIPl 's ability to interact, directly or indirectly through its role in endocytosis, and to, therefore, increase the levels of c-Kit may promote tumorigenesis in MCC.

Interactions between HIP 1 and ionotropic glutamate receptors have been observed. Since HIP1 interacts with and stabilizes the AMPA ionotropic glutamate receptor (Metzler et al., Embo J 2003;22:3254-66), which activates the mitogen-activated kinase pathway (MAPK) and may have a role in glioma proliferation (de Groot et al., Journal of Neuro-Oncology 2008;88: 121 -33). This receptor is expressed in normal Merkel cells, due to their role in sensory signaling. I. HIP1 as a Marker for Cancer The present invention relates to compositions and methods for cancer diagnostics, including but not limited to, HIP1 cancer markers. In particular, the present invention provides markers (e.g., HIP1 ) whose expression is specifically altered in cancerous tissues (e.g., Merkel Cell Carcinoma). Such markers find use in the detection, screening, diagnosis and characterization of cancer.

In some embodiments, the present invention provides methods for detection of HIP1 . In preferred embodiments, tlie presence of HIP 1 protein or mRNA is measured directly. In some embodiments, HIP1 mRNA or protein is detected in tissue samples (e.g., biopsy samples). In other embodiments, HIP1 mRNA or protein is detected in bodily fluids (e.g., serum, plasma, or urine). The present invention further provides kits for the detection of HIP1 . In preferred embodiments, the presence of HIP1 is used to provide a diagnosis or prognosis to a subject.

In some embodiments, MCC is detected using an assay of embodiments of the present invention (e.g., assaying for serum autoantibodies or elevated levels of HIP 1 expression in skin samples). The diagnosis is then confirmed using additional diagnostic methods (e.g., biopsy, staining, examination by a pathologist, etc.). Thus, in some embodiments, the detecting serves as a screening assay that identifies a likelihood of cancer but not does not provide a diagnosis.

In some embodiments, an elevated level of expression of HIP1 polypeptide or nucleic acid is indicative of an increased likelihood or diagnosis of MCC. For example, in some embodiments, an increase in HIP1 expression of at least 10%, 20%, 50%, 100%, 500%, or 100% over the level of expression in subjects not diagnosed with cancer is indicative of an increased likelihood or diagnosis of MCC.

In some embodiments, HIP1 protein is detected. Protein expression may be detected by any suitable method. In some embodiments, proteins are detected by binding of an antibody specific for the protein. The present invention is not limited to a particular antibody. Any antibody (monoclonal or polyclonal) that specifically detects HIP1 may by utilized. Methods for the generation of antibodies are described below.

Antibody binding is detected by techniques known in the art. For example, in some embodiments where HIP1 protein is detected in bodily fluids, antibody binding is detected using a suitable technique, including but not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays,

immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays. In other embodiments, where HIP1 protein is detected in tissue samples,

immunohistochemistry is utilized for the detection of antibody binding.

In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include, but are not limited to, those described in U.S. Patents 5,885,530, 4,981 ,785, 6, 159,750, and 5,358,691 , each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a diagnosis and/or prognosis based on the presence or absence of a series of proteins corresponding to cancer markers is utilized.

In other embodiments, the immunoassay described in U.S. Patents 5,599,677 and 5,672,480, each of which is herein incorporated by reference, is utilized. In other embodiments, proteins are detected by immunohistochemistry.

In other embodiments, HIP1 is detected at the level of HIP1 RNA. In some embodiments, HIP 1 RNA is detected by measuring the expression of corresponding m A in a tissue sample (e.g., skin sample). mR A expression may be measured by any suitable method, including but not limited to, those disclosed below.

In some embodiments, RNA is detected by Northern blot analysis. Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe. Methods for Northern blot analysis are well known in the art. In other embodiments, RNA expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g., U.S. Patent Nos. 5,846,717, 6,090,543; 6,001 ,567; 5,985,557; and 5,994,069; each of which is herein incorporated by reference). The INVADER assay detects specific nucleic acid (e.g., RNA) sequences by using structure-specific enzymes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes.

In still further embodiments, RNA (or corresponding cDNA) is detected by hybridization to an oligonucleotide probe. A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, in some embodiments, TaqMan assay (Applied Biosystems, Foster City, CA; See e.g., U.S. Patent Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference) is utilized. The assay is performed during a PCR reaction. The TaqMan assay exploits the 5'-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probe consisting of an oligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye) and a 3'- quencher dye is included in the PCR reaction. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. In some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in U.S. Patents 5,639,606, 5,643,765, and 5,876,978 (each of which is herein

incorporated by reference) is utilized.

In some embodiments, the presence of autoantibodies to a HIP1 antigen is detected. In some embodiments, autoantibodies to an N-terminal HIP1 antigen (e.g., the antigen described herein) or a C-terminal HIP1 antigen are detected. In some embodiments, N-terminal antigens comprise approximately amino acids 1-430 of SEQ ID NO:5. In some embodiments, both C and N-terminal antigens are utilized. For example, in some embodiments, patients that exhibit autoantibodies to N-terminal but not C- terminal antigens are found to have cancers that are metastatic. This approach to diagnosing and typing tumors is particularly suited to tumor antigens that are present, but not immunogenic, in normal cells and immunogenic in tumor cells. For example, in some embodiments, antibodies (e.g., monoclonal or polyclonal) are generated to the autoantibodies identified during the development f the present invention. Such antibodies are then used to detect the presence of autoantibodies using any suitable technique, including but not limited to, those described above.

In other embodiments, HIP1 antigens are attached to a solid surface. The presence of autoantibodies is identified by contacting the solid surface (e.g., microarray) with serum from the subject and detecting binding to a tumor marker. One exemplary method for performing such an assay is described in the experimental section below.

In some embodiments, the present invention provides kits for the detection and characterization of cancer (e.g., Merkel Cell Carcinoma). In some embodiments, the kits contain antibodies specific for HIP1 , in addition to detection reagents and buffers. In other embodiments, the kits contain reagents specific for the detection of HIP 1 mRNA or cDNA (e.g., oligonucleotide probes or primers). In some embodiments, kits contain HIP1 antigens for use in detecting serum autoantibodies (e.g., HIP1 C-terminal and/or N- terminal antigens). In some embodiments, the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results. II. Antibodies

The present invention provides isolated antibodies. In preferred embodiments, the present invention provides monoclonal antibodies that specifically bind to an isolated polypeptide comprised of at least five amino acid residues of HIP 1. These antibodies find use in the diagnostic methods described herein.

An antibody against a protein of the present invention may be any monoclonal or polyclonal antibody, as long as it can recognize the protein. Antibodies can be produced by using a protein of the present invention as the antigen according to a conventional antibody or antiserum preparation process.

The present invention contemplates the use of both monoclonal and polyclonal antibodies. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present invention, including but not limited to, those disclosed herein. For example, for preparation of a monoclonal antibody, protein, as such, or together with a suitable carrier or diluent is administered to an animal (e.g., a mammal) under conditions that permit the production of antibodies. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times. Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animal whose antibody titer has been confirmed (e.g., a mouse) is selected, and 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma. Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled protein, as described hereinafter and antiserum and then measuring the activity of the labeling agent bound to the antibody. The cell fusion can be carried out according to known methods, for example, the method described by oehler and Milstein (Nature 256:495 [1975]). As a fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1 , P3U1 , SP2/0, AP-1 and the like. The proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 : 1 to about 20: 1. PEG (preferably PEG 1000-PEG 6000) is preferably added in concentration of about 10% to about 80%. Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C for about 1 minute to 10 minutes.

Various methods may be used for screening for a hybridoma producing the antibody (e.g., against HIP1 ). For example, where a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase. Alternately, a supernatant of the hybridoma is added to a solid phase to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.

Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT

(hypoxanthine, aminopterin, thymidine) are added is employed. Any selection and growth medium can be employed as long as the hybridoma can grow. For example, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used.

Normally, the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2 gas. The antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against HIP1) can be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purification of immunoglobulins, for example, salting-out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen (an antigen against the protein) and a carrier protein is prepared and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation. A material containing the antibody against is recovered from the immunized animal and the antibody is separated and purified.

As to the complex of the immunogen and the carrier protein to be used for immunization of an animal, any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently. For example, bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.

In addition, various condensing agents can be used for coupling of a hapten and a carrier. For example, glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention. The condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method. The antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to any particular type of immunogen. For example, HIP1 protein (further including a gene having a nucleotide sequence partly altered) can be used as the immunogen. Further, fragments of the protein may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the gene, enzymatic processing of the protein, chemical synthesis, and the like.

In some embodiments, antibodies (e.g., monoclonal antibodies) are humanized. Humanized antibodies are altered in order to make them less immunogenic to humans, e.g., by constructing chimeric antibodies in which a mouse antigen-binding variable domain is coupled to a human constant domain. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Methods for humanizing antibodies are well known in the art and include but are not limited to, those disclosed in U.S. patents 6,054,297, 4,816,567, 6,180,377, 5,871,907, 5,585,089, and 6, 180,370, each of which is herein incorporated by reference.

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example 1

Methods

MCC and small cell lung cancer (SCLC) tissue samples. Archived formalin fixed and paraffin-embedded MCC and SCLC tissue samples were obtained from the Pathology Department at the University of Michigan Medical Center. Diagnoses were determined by morphology and the site of the primary tumor (listed in Table I).

Immunohistochemical staining. Immunohistochemical staining for HIP1 was performed as described previously (Bradley et al., Cancer Res 2007;67:3609-15) with appropriate negative (no primary antibody) and positive (glioblastoma) controls. Staining for Merkel cells in mouse skin was performed using the mouse monoclonal antibody s20.8 (ThermoScientific). Photomicrographs of the immunohistochemical staining were taken with a model BX41 Olympus microscope.

Patients. The study of patients with MCC and serum levels of anti-HIPl levels was approved by the University of Michigan Internal Review Board. Serum from 40 patients was collected at the University of Michigan Merkel Cell Carcinoma clinic, aliquoted into multiple 20 portions for single use to avoid freeze thaw cycles, and stored at -80°C until use. The ages, gender distribution, and tumor stages of these patients are illustrated in Table II.

Quantitative real-time polymerase chain reaction (qPCR). First-strand cDNA - for real-time PCR was generated using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). Random hexamers were used for reverse transcription of cDNA. Four micrograms of total RNA extracted from Merkel cell tumors or normal skin tissue were used to generate cDNA. The cDNA concentrations were determined using a ND- 1000 Nanodrop spectrophotometer. Samples were diluted to a final concentration of 100 ng^L. A total of 100 ng of cDNA was used for each reaction. Reactions were performed in triplicate. The real-time PCR reactions were performed using a Mastercycler ep realplex (Eppendorf) according to the manufacturer's instructions. The RNA content of samples subjected to qPCR was normalized based on the amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The primers used in these reactions were designed to generate amplicons in the range of 150 to 200 base pairs with the exception of GAPDH amplicon (294 bp). Primer efficiencies were determined prior to data analysis, and these efficiencies were used to calculate relative expression using the Pfaffl method (Pfaffl, Nucleic Acids Res

2001 ;29:e45). The following primers were used: GAPDH forward, 5'-CTG GTG CTG AGT ATG TCG TG-3'; (SEQ ID NO: 1) GAPDH reverse, 5'- CAG TCT TCT GAG TGG CAG TG-3*; (SEQ ID NO:2) total HIP1 forward, 5'-GCT GGG GAG CCA CTG TCA T-3*; (SEQ ID NO:3) and total HIP1 reverse, 5'-GGT TGG GGC TGT CCT TAT CA-3' (SEQ ID NO:4).

Preparation of HIP1 antigen. Glutathione S-transferase(GST) HIP1 (3' and 5') fusion cDNAs were used to generate C-terminal and N-terminal recombinant antigens, respectively. The C-terminal antigen has been previously described (Bradley et al.,

Cancer Res 2005;65:4126-33). The N terminal antigen was generated by sub-cloning an in-frame GST fusion protein to the 5' end of the region of HIP 1 that terminates at the internal EcoRI site in the HIP1 sequence. The antigen was produced in bacteria and purified as previously described for the C terminal antigen (Bradley et al., Cancer Res 2005;65:4126-33). Immunoblot analysis of anti-HIPl antibodies in MCC patient serum. Twenty micrograms of N-terminal or C-terminal antigen were separated on a preparative 10% SDS-PAGE mini-gel, transferred to nitrocellulose, and blocked overnight in 5% milk and 5% donkey serum in Tris-buffered saline with 0.1% Tween-20 (TBST). Patient serum was then applied to the blots using a Miniblotter 28 dual unit system (Immunetics, Inc., Cambridge, MA) as previously described (Bradley et al., Cancer Res 2005 ;65:4126- 33). The optical density reflective of serum antibodies bound to the HIP1 antigen was measured using the program ImageJ (Bradley et al., Cancer Res 2007;67:8923-31). Lanes that had an optical density of at least 20% of the internal positive control (a patient with ALL; Bradley et al., Cancer Res 2007;67:8923-31), was used for the C-terminal antigen and a patient with MCC in this cohort for the N-terminal antigen were considered positive.

Statistics. Data were analyzed using GraphPad Prism 5 statistical software and Image J densitomety analysis software. Statistical significance values for Table I and Table II were calculated using Pearson χ2 analysis. Statistical significance values in Figure 4B and C were calculated using a Student's t test. ^J

Immunoprecipitation. HE 293T cells were grown to 70% confluence and transfected with 20 μg of full-length HIP1 and c-Kit (Origene) cDNA in pcDNA3 and pCMV6, respectively. After 24 hours, transfected cells were lysed using an all-purpose non-denaturing lysis buffer (50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% Triton X- 100, 1.5 mmol/L MgC12, 5 mmol/L EGTA, 10% glycerol, complete EDTA-free protease inhibitor tablets (Roche), 30 mmol L sodium pyrophosphate, 50 mmol/L NaF, and 100 μηιοΙ/L sodium orthovandate). To ensure maximum protein extraction, cells were manually ground 50 times with plastic pestles and then incubated for 1 hour at 4°C with rotation. The mixture was centrifuged for 15 minutes at 4°C at 13,200 rpm, and the supernatant stored at -80°C until use. Biorad's Protein Assay (cat# 500-0006) was used to determine the protein concentrations of the lysates.

One milligram of protein lysate was pre-cleared by incubating with a 50:50 slurry of the Protein G sepharose beads in lysis buffer (20 μί) for 30 minutes at 4°C with rotation. After centrifugation, the cleared supernatant was incubated with 20 μί, of rabbit non-immune serum or the appropriate polyclonal antibodies overnight at 4°C. One hundred microliters of a 50:50 slurry of beads were added to the protein-antibody mixture and incubated with rotation for 1 hour at room temperature. These beads were then precipitated via centrifugation for five minutes at 13,200 x g. The pellet, which contained bead-protein complexes, was then washed three times with lysis buffer. The pellet was then dissolved in 20 μΐ, of 6X Laemmli buffer, boiled for 5 minutes, and centrifuged. The supernatant was used for western blot analysis.

To test if c-Kit immunoprecipitation co-precipitated HIP1, a cDNA that expresses a c-terminal V5 tagged c-Kit receptor was constructed. To do this, the full length c-Kit cDNA was PCR amplified with primers that were compatible for ligation with a Hindlll and Xhol digested pcDNA3.1-V5-His vector. The resultant pcDNA3.1/c-Kit-V5-His construct was co-expressed with the pcDNA3.1/HIPl-myc construct in 293T cells, lmg of protein lysate was pre-cleared with Protein G sepharose beads as above and c-Kit-V5 was immunoprecipitated by incubating the lysate with 10 μΐ-, of anti-V5 conjugated beads (Sigma) and prepared for western blot as described above. Protein lysates from cells expressing HIPl -myc, but not c-Kit-V5, served as a negative control.

c-Kit receptor stabilization assay. HEK-293T cells were grown to 70% confluence in 6-well plates and then transfected with 2 μg of c-Kit cDNA in pCMV6 and either 2 μg full-length HIP1 cDNA in pcDNA3 or 2 μg empty pcDNA3 vector (total 4 μg DNA). Twenty-four hours after transfection, cells were starved for 16 h in serum-free media. Cells were then treated with 100 mg/mL cycloheximide for 30 minutes followed by the addition of 150 ng/mL c-Kit ligand SCF. Cells were collected at 0, 0.5, 1, and 2 hours in RIPA lysis buffer (150 mM NaCl; 50 mM Tris, pH 8.0; 1% NP-40; 0.5% DOC (deoxycholic acid); 0.1% SDS; complete EDTA-free protease inhibitor tablets (Roche); 30 mmol/L sodium pyrophosphate; 50 mmol/L NaF; and 100 μιηοΙ/L sodium

orthovandate).

Western blot. Whole cell lysates and immunoprecipitated pellets were separated with 6% or 10% SDS-PAGE and transferred to nitrocellulose. The nitrocellulose membranes were incubated in 20 mL of blocking solution (TBST with 5% non-fat dry milk) one hour at room temperature with rocking. Membranes were then incubated with the appropriate primary antibody overnight at 4°C with rocking. The blots were then washed three times with TBST, incubated with HRP-conjugated secondary antibody for 1 hour at room temperature, and then washed three times with TBST. Development of a chemiluminescent substrate (ECL, Thermo Scientific) was used to visualize the blot. Primary antibodies for western blot analysis included HIP1/4B 10 (anti-HIPl mouse monoclonal), HIP1 UM354 (anti-HIPl rabbit polyclonal), anti-c-Kit (Cell Signaling, mouse monoclonal), anti-myc (Upstate Biotechnology, rabbit polyclonal), anti-V5 (Invitrogen, mouse monoclonal) and anti-actin (Sigma, mouse monoclonal).

Assay for steady state levels of c-Kit. Two 10 cm dishes of HEK-293T cells were co-transfected with equal (10 μg) amounts of pcDNA3.1/cKit and pcDNA3.1/Hipl- IRES-GFP or pcDNA3.1/IRES-GFP. Cells were transfected with the Superfect

(Invitrogen) reagent in serum free DMEM for four hours. After 4 hours serum free medium was replaced with DMEM containing 10% FBS. Twenty four hours later cells were trypsinized and suspended in sterile 2% FBS-HBSS at a concentration of lxl06/ml. To control for variability in transfection efficiency, GFP positive and negative cells were separated using a FACS-Vantage machine. GFP positive cells were analyzed by western blot using antibodies against HIPl , c-Kit and actin as described above.

Results

MCC specimens demonstrate high levels of HIPl staining. To determine whether HIPl is a suitable marker for MCC, paraffin-embedded tissue samples from MCC tumors and the similar neuroendocrine tumor, SCLC, were immunostained for ' HIP l expression. These tumors were evaluated for expression level and for cellular localization of HIPl staining. HIPl is not expressed at high levels in normal skin with the exception of vascular endothelium (Rao et al., J Clin Invest 2002; 1 10:351-60). Tumor tissue was visually scored for HIPl expression on a scale of 0-3, in which a score of 3 represented the highest HIPl staining and a score of zero indicated a lack of staining (Rao et al, J Clin Invest 2002; 1 10:351-60).

Eighty nine percent (n=25/28; 89%) of MCC tissue samples exhibited HIPl expression (Table I). MCC tissue samples exhibited both diffuse cytoplasmic staining and perinuclear dot patterns of staining. HIPl expression occurred much more frequently in MCC tumors than in SCLC tumors (n=5/12; 42%). In particular, frequency of HIPl expression was elevated in metastatic MCC as compared to metastatic SCLC (Table I). This finding is clinically significant since MCC and SCLC are often difficult to distinguish from one another in metastatic tissue sections.

To determine if this elevated expression was due to altered message levels, expression arrays were evaluated for relative levels of HIPl mRNA in MCC samples compared to squamous cell carcinomas. On average a 6-fold increase in HIPl message was detected in MCC tumors where 29 out of the 30 tumors displayed an increase compared to squamous cell carcinomas (Fig. 1). Squamous cell carcinoma was used as a negative control as this is a tumor that does not display elevated HIPl protein levels. In contrast, HIPl-related, HIPl 's only known mammalian relative was not elevated at the message level.

HIPl tumor staining was also compared to the known MCC markers, C 20 and c- Kit. A separate cohort of 14 MCC patients represented on a tissue microarray (TMA) was used for this comparison. As is evident in the top row, HIPl staining for MCC was very strong, diffuse and sensitive (100% positive; n=14/14). In comparison, CK20 staining was less reliable and positive in only 64% of the patients (row 3 vs. the top row; n=9/14). This frequency of CK20 staining is lower than previous reports where it has been found to be positive in up to 80% of MCC tumors. This difference is likely due to the use of a TMA rather than the entire slide for testing each patient. Because CK20 staining is not as uniformly distributed in the tumor cells as HIPl staining is, it is possible that a positive tumor could test falsely negative for CK20 due to there being less tissue represented on a TMA. These data nevertheless indicate that the chance of misdiagnosing a MCC when staining for CK20 is greater than when staining for HIPl . Because the HIPl antibody stained every tumor, the HIPl test was positive in all tumors that tested positive for CK20 (third row vs. top row) and another important MCC marker c-Kit (second row vs. top row).

HIPl does not affect the development or maintenance of normal Merkel cells. In order to determine whether HIPl is necessary for the normal development of Merkel cells, the skin of wild-type and HIPl-null mice was analyzed (Oravecz- Wilson et al., Hum Mol Genet 2004; 13:851 -67). Anti-cytokeratin 20 (C 20) antibodies were used to identify mature Merkel cells in the mouse tail skin and vibrissae, where Merkel cells generally congregate on the periphery of hair follicles. No visible changes in the abundance of mature Merkel cells were observed in the HIPl -null mouse skin as compared to wild-type littermate skin. These data indicate that HIP1 is not necessary for the development or maintenance of normal Merkel cells.

MCC patients harbor anti-HIPl autoantibodies in their blood. To detect the presence of anti-HIPl antibodies in MCC patient blood, patient sera were tested for immune- reactivity to recombinant HIP1 antigens as described previously (Bradley et al., Cancer Res 2005;65:4126-33). Initially, serum samples were screened against the previously described C-terminal HIP1 recombinant antigen (Bradley et al., Cancer Res

2007;67:8923-31 ; Bradley et al., Cancer Res 2007;67:3609-15; Bradley et al., Cancer Res 2005;65:4126-33). Eighty-five percent of MCC patients (n=34/40; 85%) exhibited the presence of autoantibodies (Fig. AA and 5). This result was similar to the proportion previously found in patients with glioblastoma multiforme (Bradley et al., Cancer Res 2007;67:3609-15). Patient sera were also tested for reactivity against a different HIP1 recombinant antigen that encoded the amino terminus. This antigen contains the lipid- binding ANTH, clathrin binding, and AP2-binding domains (Fig. AC). Only thirty percent of MCC patients (n=12/40; 30%) harbored antibodies against the HIP1 N-terminal antigen (Fig. AA and AB). This proportion was infrequent enough that intergroup comparisons for clinical correlations were possible (see below).

Sera from patients with metastatic MCC exhibited high titers of antibodies against the HIP1 N-terminus. To examine the correlation of anti-N-terminal HIP1 antibody titers in MCC patient sera with a biological outcome, the titers of the antibodies against the N-terminus of HIP1 in MCC patients were compared with a number of clinical parameters. These parameters included tumor size, disease status at time of blood draw, presence of metastasis at time of blood draw, survival 2.5 years after blood draw, age, and gender. Of these parameters, metastasis had the greatest association with a high titer of anti-HIPl antibodies. Hence, a positive test for HIPl serum antibodies marked the presence of metastatic disease (Fig. 2).

Patients with metastatic MCC exhibited anti-HIPl N-terminal antigen antibodies much more frequently than patients with localized primary tumors (Table II; Fig. 3; 46% versus 0%; p < 0.005; Pearson χ2). This test showed great specificity (specificity = 1.0). Additionally, a non-significant trend was also observed (Table II) between autoantibody presence and extensive metastatic disease compared to microscopic and local lymph node metastases (Table II). The presence of antibodies against the C-terminal HIPl antigen in patient sera was not significantly associated with metastasis. The high percentage of patients with auto-antibodies against the HIPl C-terminus indicate that this antigen does not serve as a metastasis biomarker, since 62% of patients localized disease also tested positive for antibodies against the HIPl C-terminal.

Survival after a positive test for the anti-N-terminal HIPl antibodies was poor. In total, 5 out of 10 patients (50%) with metastatic disease and a high titer of auto-antibodies against the HIPl N-terminus (Figure 2; cohort 1) were deceased from disease after a 2.5 year observation period. This contrasts with the fact that only 25% (3/12) of the patients with metastatic disease and negative anti-N-terminal HIPl antibody tests were dead at 2.5 years (below the line in Figure 2; cohort 2). An even more striking contrast to the 50% death rate in the patients with antibody positive metastatic disease was a 100% survival of those patients with localized disease and a low anti-N-terminal HIPl antibody titer (right hand side of Figure 2; cohort 3). Two of the original patients in cohorts 1 and 2 were lost to follow-up.

A disproportionate number of female patients had elevated anti-HIPl antibody titers in their blood. Approximately 45% of female patients had anti-HIPl N-terminal antibodies, while only 1 1 % of male patients had N-terminal anti-HIPl auto-antibodies. The positive association between anti-HIPl antibodies and female gender was significant (p < 0.05, Pearson χ2). The increase in anti-HIPl antibodies remained associated with metastasis within the female population. In fact, when males were excluded, the association between metastasis and the presence of N-terminal antibodies increased significantly in women (p < 0.001). In addition, no association between the presence of anti-HIPl antibodies and age was identified in this relatively elderly population (Table II).

HIP1 interacts physically and functionally with the c-Kit RTK. MCC tumors express significant levels of several RTKs (Brunner et al., Mod Pathol 2008); however, expression of the receptors previously reported to interact with HIP1 , including EGFR (Bradley et al., Cancer Res 2007;67:3609-15) and FGFR4 (Wang et al., Neoplasia 2008; 10:847-56), has not been found in MCC tissue. Thus, the ability of HIP1 to physically interact with c-Kit, an RTK that is frequently expressed at high levels in MCC (Brunner et al., Mod Pathol 2008) was assayed. Since c-Kit is rarely expressed in normal adult tissues other than progenitors of the hematopoietic system such as rare

hematopoietic stem cells (Bernex et al., Development 1996;122:3023-33), the cDNA for c-Kit was co-expressed with the cDNA for HIP1 in HEK 293T cells to obtain enough material for analysis.

HI PI was immunoprecipitated from the whole cell lysate using rabbit polyclonal antibodies (UM410 or UM323). Western blot analysis of the immunoprecipitate showed that c-Kit specifically co-immunoprecipitated with HIP1 (Fig. 3 A, left hand panel). In addition, when reverse immunoprecipitation was performed by precipitating c-Kit, it was found that HIP1 was present in the immunoprecipitate (Fig. 3 A, right hand panel).

It was hypothesized that if this interaction was functionally related to endocytosis, it may be dependent on activation of the receptor. Addition of SCF, the c-Kit ligand, one hour prior to cell collection for immunoprecipitation did indeed enhance the detected interaction between c-Kit and HIP1 (Fig. 3A, lanes 3 and 6). Western blot analysis of whole cell lysates from these cells showed no differences in c-Kit expression in those cells treated with SCF as compared to untreated cells. An interaction between

endogenous HIP1 and c-Kit in a MCC cell line (MCC565) when SCF was added to the cell media 1 hour prior to cell harvest was observed (Fig. 3B). This interaction was not observed in the absence of SCF. It was also examined whether HIP1 over expression has the ability to inhibit the degradation of the c-Kit receptor similar to the effect of HIP 1 on EGFR and FGFR4 levels. Indeed, HIP1 stabilized c-Kit following SCF stimulation of starved and cycloheximide treated cells. The receptor levels were significantly higher one and two hours after stimulation when HIP1 was over expressed with c-Kit (Fig. 3Q. These data together indicatea that the interaction of HIP 1 with c-Kit is functionally important.

Table I. HIF1 immunosraining of paraffin-fixed .\ ICC and SCLC tissue

Tumor type 3+ 2+ 1+ No staining HIP1 positiviry (%)

MCC Total 3 8 14 3 S9«*

Primary 2 4 9 1 94

Metastatic 1 4 5 2 83*

SCLC To;al 0 1 4 7 42

Primary 0 0 3 2 60

Metastatic 0 1 1 5 29

"Significant difference compared with patients with SCLC p < 0.0025, χ² analysis).

*Significaat difference compared with patients with metastatic SCLC (p 0.025, χ^* analysis).

Table Π. Frequency of positive anri-HTPl ( -terminal) antibody blood test in metastatic MCC.

Patient status Positive Negative Frequency Age (years) ±SD Male (%)

Ail MCC patients 13' 27 0.33 69 ± 12 45 To metastases 0 13 0.07 71 ± 12 54

Metastatic disease 12 14 0.46*- 67 ± 11 42

Extensive Metastases 6 3 0.67 72 ± 12 33

Local Metastases 3 4 0.38 63 ± 10 50

MicTometastases 3 6 0.33 66 ± 9 44

•Significant difference compared with patients without metastatic disease (p ■ 0.005, χ¹ analysis)

One patient was lost to fol!ow-up and metastatic status could not be determined

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS We claim:

1. A method of detecting merkel cell carcinoma, comprising:

a) detecting Huntingtin interacting protein 1 (HIP1) expression in a skin sample from a subject; and

b) detecting merkel cell carcinoma in said sample when HIP1 expression is elevated in said sample relative the level of expression in normal skin.

2. The method of claim 1, wherein said detecting comprises detecting an elevated level of HIP 1 in said sample.

3. The method of claim 1, wherein said detecting comprises measuring the level of HIP 1 in said sample.

4. The method of claim 1, wherein said detecting comprises measuring the level of HIP 1 protein.

5. The method of claim 4, wherein said measuring the level of HIP 1 protein comprises exposing said HIP1 protein to an antibody specific to said HIP1 protein.

6. The method of claim 1, wherein said detecting comprises measuring the level of HIP 1 mR A.

7. The method of claim 1, wherein said merkel cell carcinoma is metastatic.

8. A method of detecting merkel cell carcinoma, comprising:

a) detecting the presence of absence of autoantibodies to HIP1 in a serum sample from a subject; and

b) detecting merkel cell carcinoma in said sample when autoantibodies to HIP1 are present in said sample.

8. The method of claim 8, wherein said merkel cell carcinoma is metastatic merkel cell carcinoma.

9. The method of claim 8, wherein said autoantibodies bind to an N-terminal portion of HIP 1.

10. The method of claim 9, wherein said N-terminal portion comprises the lipid-binding ANTH, clathrin binding, and AP2 -binding domains.

11. The method of claim 8, wherein said N-terminal portion comprises amino acids 1-430 of SEQ ID NO:5.

The method of claim 8, wherein said patient is a female human.