Microphthalmia (Mi) while present in melanocytes, a cells and osteoclast, is not normally present in other cells. We have found that Mi is present in the nucleus of melanoma cells. Melanoma can be diagnosed by contacting a malignant cell with a probe for Mi. If the probe identifies Mi in the nucleus of the cell, the cell is a melanoma.

Description of the Invention

SUMMARY OF THE INVENTION

We have now discovered an improved method that can be used for diagnosis of melanoma. We have discovered that the transcription factor microphthalmia (Mi) is an excellent marker that when present in a malignant tissue is diagnostic of melanoma. Mi is normally present in melanocytes, mast cells, and osteoclasts. However, it is typically not present in other cells. Thus, by obtaining a biological specimen, wherein the specimen is preferably a malignant tissue and measuring for the presence of Mi, by looking at the protein or transcript for Mi, one has a simple method for the diagnosis of melanoma, wherein the presence of Mi is indicative of melanoma.

Additionally, by looking at the quantity of Mi, present in the cell and/or its state, i.e., activated vs. non-activated, one can use Mi prognostically.

Finally, one can take advantage of Mi's correlation with melanoma for a method of treatment. For example, by selectively targeting Mi one is in effect selectively targeting melanoma.

DETAILED DESCRIPTION OF THE INVENTION

Mi is a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells and bone development. Mi is essential to the development and survival of melanocytes.

The gene encoding mouse Mi was cloned in 1993 and found to encode a Myc-related b-HLH-ZIP protein. [Hughes, J. J. et al., J. Biol. Chem. 268:20687-20690 (1993); Hodgkinson, C. A. et al., Cell 74: 395-404 (1993)] Biochemical studies demonstrated a DNA binding specificity for consensus sequence CA(C/T)(G/A)TG and its capacity to heterodimerize in vitro with three structurally related b-HLH-ZIP factors, TFEB, TFE3, and TFEC, but not Myc/Max, USF or other b-HLH-ZIP proteins. [Hemesath, T. J., Genes & Dev. 8: 2770-2780 (1984); Carr, C. S., et al., Mol. Cell Biol., 10: 4384-4388 (1990); Beckman, H., Genes & Dev., 4: 167-179 (1990); Roman, C. A., Mol. Cell Biol. 12(2): 817-827 (1992); Zhao, G. Z., et al., Mol. Cell. Biol., 13: 4505-4512 (1993); Yasumoto, K., et al., Biochimica et. Biophysica Acta, 1353: 23-31 (1997); Blackwood, E. M., Science, 251: 1211-1217 (1991)] Mi/Mi mutant mice display defective eye development (related to pigment cell abnormalities), complete lack of skin melanocytes, deafness related to absent of pigment cells in the inner ear (stria vascularis)), severe defects in mast cells, and osteoporosis. Mutations in human Mi have been detected in the autosomal dominant hereditary deafness and pigmentation condition, Waardenburg syndrome, type 2A [Tassabehji, et al., Nature Genet., 8:251-255 (1994); Hughes, A. E. et al., Nature Genet., 7:509-512 (1994)] (a condition characterized by a white forelock and deafness). The Mi gene (4) encodes a transcription factor (5) which regulates expression of the pigmentation enzymes tyrosinase, TRP1, and TRP2 (5-7). Recent studies have demonstrated that Melanocyte Stimulating Hormone (.alpha.-MSH) upregulates pigmentation through stimulation of Mi expression (8, 9).

While Mi may regulate pigmentation, the complete absence of melanocytes in Mi-deficient mice suggests that Mi is essential for melanocyte development or postnatal survival, or both. One instructive mouse mutant, mi.sup.vit displays nearly normal melanocyte development, but accelerated age-dependent melanocyte death over the first months of life (10). This death is attributable to a mutation within the helix-loop-helix motif of mi (5, 11) and suggests a vital role for Mi in post-natal survival of melanocytes. One potential clue to Mi's survival role comes from evidence that the Steel/Kit cytokine pathway (whose deficiency produces identical absence of melanocytes) regulates MAP kinase-mediated phosphorylation of Mi (12). This produces transcriptional super-activation by Mi protein through selective recruitment of CBP/p300 (13), a family of transcriptional coactivators for Mi (14, 15).

Various abnormalities in Mi have been connected to pigmentation deafness and osteoporosis as stated above. However, its correlation with melanoma cells has heretofore been unknown. For example, Mi is involved in a signaling pathway linked to Kit signaling. However, the presence of Kit does not correlate with melanoma, despite the fact that Kit is an oncogene. We have now discovered that there is a high correlation between the presence of Mi in a malignant cell and that cell being a melanoma. We have been able to determine that Mi is specific for melanoma. For example, we have looked at numerous malignant tissues including many brain tissues and found that Mi was not present. The negative Mi staining tumors include basal cell carcinoma, squamous cell carcinoma, atypical fibroxanthoma, granular cell tumor, Schwannoma and neurofibroma. Thus, by looking for the presence of Mi one is able to determine the origin of the cell and use such information to determine the course of treatment.

Mi staining in melanomas produces a nuclear pattern which has some theoretical advantages over cytoplasmic immunostains. It may be difficult to distinguish background staining from positivity for cytoplasmic antibodies, especially with weak signal. Furthermore, cellular architecture is not obscured with nuclear staining which aids in the preservation of the tissue structure being examined. For pigmented lesions it may be difficult to distinguish cytoplasmic stains from pigmentation, although such lesions are less likely to require special stains.

In one series of experiments, Mi was expressed in 8/8 histologic amelanotic melanomas. Based on its recognition of the M box promoter element (5), Mi is thought to regulate transcription of the pigmentation enzymes tyrosinase, TRP1 and TRP2 (5-7). Its persistent expression in the amelanotic melanomas examined here suggests that factors downstream of Mi may downregulate pigmentation. One such mechanism is the proteolytic degradation of tyrosinase, recently described for human melanoma cells (25). These findings suggest that downregulation of pigmentation is beneficial to melanoma cells and that rather than loss of Mi itself, mechanisms downstream of Mi may more commonly occur. Of note, nondetection of Mi expression at the RNA level has been observed in a murine amelanotic melanoma cell line (15).

Mi is a sensitive marker for this clinical entity, which can represent a diagnostic challenge. Due to its propensity for vertical growth, malignant melanoma may metastasize at an early stage, even before a primary cutaneous lesion is identified (as occurs in 5-14% of cases(18-22)). Moreover since a significant fraction of metastatic melanomas are amelanotic, such lesions may be difficult to classify on simple morphologic grounds, certainly to the non-specialist, and could represent a variety of undifferentiated or poorly differentiated tumors such as epithelial tumors, sarcomas, lymphoid neoplasms or germ cell tumors (38). Combined detection of S-100 and Keratin may help rule in or out the possibility a melanoma. S-100 is sensitive for melanoma, but commonly stains other tumors in this differential including breast adenocarcinomas, lung carcinomas, teratomas, neurogenic tumors, and others (23, 29) (39-42), whereas Keratin expression is atypical in melanomas (43).

2 of the breast cancer specimens produced cytoplasmic Mi staining. Mi is expressed in osteoclasts (37), and many breast cancers express genes involved in bone resorption such as PTH-rp, cathepsin K, IL-6, IL-1, TGF, and collagenases (44, 45). Mi may upregulate osteoclast-like genes such as cathepsin K, a resorption factor recently detected in breast tumor lines (46). As such, Mi expression may play a role in bone metastasis of breast cancer, perhaps even predicting osteotrophism. Accordingly, targetting Mi expression may also be useful in treating and/or diagnosing breast cancer.

Mi was not detected in 9 desmoplastic/neurotropic melanomas. Desmoplastic melanomas account for less than 1% of melanomas and often arise in association with lentigo maligna (47). About 20-30% of these tumors lack an in situ component. Desmoplastic tumors tend to grow as a fibrous nodule, frequently track along nerves, and have a distinct clinical behavior compared with other melanomas. HMB-45 is often negative in desmoplastic melanomas, but S-100 is usually positive. While this tumor is classified as a melanoma, there is some debate as to the origin and true biology of the spindle cells (48-50), and lack of Mi is believed to be notable.

Metastatic melanoma tissue can be present throughout the body and such locations typically include lymph glands, liver, bones, brain, lung, adrenal glands, spinal cord and vertebrae. However, malignant tissues present in such sites can be from numerous types of cancers. Thus, obtaining a biological sample and looking for the presence of Mi is important in being able to diagnose the tissue as a melanoma. Since Mi is normally present in melanocytes, mast cells and osteoclasts, the biological specimen preferably does not include those cells.

Standard detection techniques well known in the art for detecting proteins, RNA, DNA, and peptides can readily be applied to detect Mi or its transcript.

Such techniques may include detection with nucleotide probes or may comprise detection of the protein by, for example, antibodies or their equivalent. Preferably, the nucleotide probes may be any that will selectively hybridize to Mi. For example, it will hybridize to Mi transcript more strongly than to other naturally occurring transcription factor sequences. Types of probes include cDNA, riboprobes, synthetic oligonucleotides and genomic probe. The type of probe used will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example. Detection of the Mi encoding gene, per se, will be useful in screening for conditions associated with enhanced expression. Other forms of assays to detect targets more readily associated with levels of expression--transcripts and other expression products will generally be useful as well. The probes may be as short as is required to differentially recognize Mi mRNA transcripts, and may be as short as, for example, 15 bases, more preferably it is at least 17 bases. Still more preferably the Mi probe is at least 20 bases.

A probe may also be reverse-engineered by one skilled in the art from the amino acid sequence of Mi. However use of such probes may be limited, as it will be appreciated that any one given reverse-engineered sequence will not necessarily hybridize well, or at all with any given complementary sequence reverse-engineered from the same peptide, owing to the degeneracy of the genetic code. This is a factor common in the calculations. of those skilled in the art, and the degeneracy of any given sequence is frequently so broad as to yield a large number of probes for any one sequence.

The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, .sup.32p and .sup.35S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases. Other forms of labeling may include enzyme or antibody labeling such as is characteristic of ELISA.

Detection of RNA transcripts may be achieved by Northern blotting, for example, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabelled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.

In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense cRNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples may be stained with haematoxylon to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows up the developed emulsion. Non-radioactive labels such as digoxigenin may also be used.

Immunohistochemistry is preferably used to detect expression of human Mi in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by enzyme, such as peroxidase, avidin or by radiolabelling. Chromogenic labels are generally preferable, as they can be detected under a microscope. Mi is a nuclear protein and provides a good staining pattern.

More generally preferred is to detect the protein by immunoassay, for example by ELISA or RIA, which can be extremely rapid. Thus, it is generally preferred to use antibodies, or antibody equivalents, to detect Mi.

It may not be necessary to label the substrate, provided that the product of the enzymatic process is detectable and characteristic in its own right (such as hydrogen peroxide for example). However, if it is necessary to label the substrate, then this may also comprise enzyme labeling, labeling with radioisotopes, antibody labeling, fluorescent marker labeling or any other suitable form which will be readily apparent to those skilled in the art.

Antibodies may be prepared as described below, and used in any suitable manner to detect expression of Mi. Antibody-based techniques include ELISA (enzyme linked immunosorbent assay) and RIA (radioimmunoassay). Any conventional procedures may be employed for such immunoassays. The procedures may suitably be conducted such that: a Mi standard is labeled with a radioisotope such as .sup.125I or .sup.35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase and, together with the unlabelled sample, is brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first and radioactivity or the immobilized enzyme assayed (competitive assay); alternatively, Mi in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-Mi antibody is allowed to react with the system and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.

For example using a monoclonal antibody to Mi resulted in strong nuclear staining within melanocytes, nevi, dysplastic nevi, melanoma in situ, and 100% of 76 consecutively acquisitioned melanomas, including amelanotic and metastatic tumors. In side by side comparisons Mi stained tumors which were negative for HMB-45 or S100. Among nonmelanoma tumors, Mi stained cytoplasms in two of 81 cases, and no cases exhibited nuclear staining. Thus, Mi is a sensitive and specific marker for melanoma.

The above techniques may be conducted essentially as a "one-step" or "two-step" assay. The "one-step" assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. The "two-step" assay involves washing before contacting the mixture with labeled antibody. Other conventional methods may also be employed as suitable.

Enzymatic and radio-labeling of Mi and/or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.

Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

Other techniques may be used to detect Mi according to preference. One such technique is Western blotting (Towbin et at., Proc. Nat Acad. Sci 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-Mi antibodies (unlabelled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including .sup.125I, horseradish peroxidase and alkaline phosphatase).

Samples for diagnostic purposes may be obtained from any number of sources. A sample obtained directly from the tumor, such as the stroma or cytosol, may be used to determine the origin of the tumor. It may also be appropriate to obtain a sample from other biological specimens, where Mi is present. Such diagnosis may be of particular importance in monitoring progress of a patient, such as after surgery to remove a tumor. If a reference reading is taken after the operation, then another taken at regular intervals, any rise could be indicative of a relapse, or possibly a more severe metastasis. Preferably, the sample is from the tumor itself.

Mi binds E box-type enhancer elements and may heterodimerize with the related family members TFEB, TFEC and TFE3 (Hemesath, T. J., et al., Genes Dev. 8, 2770-80 (1994)). Mutations in c-Kit or its ligand SI (stem cell factor, mast cell growth factor) similarly result in animals lacking melanocytes and functional mast cells, along with defects in hematopoiesis and germ cell development (Russell, E., Adv. Genet. 20, 357-459 (1979). 3. Witte, O. Steel, Cell 63, 5 (1990)). This striking phenotypic overlap has led to suggestions that SI, c-Kit, and Mi function in a common growth/differentiation pathway (Steingrimsson, E., et al., Nature Genet. 8, 256-63 (1994); Dubreuil, P., et al., Proc. Natn. Acad. Sci. U.S.A. 88, 2341-2345 (1991)). Germline mutations at loci encoding the transcription factor Mi, the cytokine receptor c-Kit, and its ligand Steel factor (SI) result in strikingly similar defects in mast cell and melanocyte development (Moore, K. J., Trends Genet. 11, 442-8 (1995); Russell, E., Adv. Genet. 20, 357-459 (1979); Witte, O. Steel, Cell 63, 5 (1990).

We found a biochemical Link between Kit signaling and the activity of Mi. Stimulation of melanoma cells with SI results in activation of MAP kinase, which in turn phosphorylates Mi at a consensus target serine. This phosphorylation upregulates Mi transactivation of the tyrosinase pigmentation gene promoter. In addition to modulating pigment production, such signaling may regulate the expression of genes essential for melanocyte survival and development. The pathway represents a novel use of the general MAP kinase machinery to transduce a signal between a tissue-specific receptor at the cell surface and a tissue-specific transcription factor in the nucleus.

The antibodies may be raised against either a peptide of Mi or the whole molecule. Such a peptide may be presented together with a carrier protein, such as an KLH, to an animal system or, if it is long enough, say 25 amino acid residues, without a carrier. Preferred peptides include regions unique to Mi.

Polyclonal antibodies generated by the above technique may be used direct, or suitable antibody producing cells may be isolated from the animal and used to form a hybridoma by known means (Kohler and Milstein, Nature 256:795. (1975)). Selection of an appropriate hybridoma will also be apparent to those skilled in the art, and the resulting antibody may be used in a suitable assay to identify Mi.

This invention also provides a convenient kit for detecting human Mi levels. This kit includes a probe for Mi such as antibodies or antibody fragments which selectively bind human Mi or a set of DNA oligonucleotide primers that allow synthesis of cDNA encoding human Mi. Preferably, the primers comprise at least 10 nucleotides, more preferably at least about 20 nucleotides, and hybridizes under stringent conditions to a DNA fragment having the human Mi sequence nucleotide. The kit will contain instructions indicating how the probe can be used diagnostically or prognostically. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% homology between the sequences.

Homology is measured by means well known in the art. For example % homology can be determined by any standard algorithm used to compare homologies. These include, but are not limited to BLAST 2.0 such as BLAST 2.0.4 and i. 2.0.5 available from the NIH (Altschul, S. F., et al. Nucleic Acids Res. 25: 3389-3402 (1997)) and DNASIS (Hitachi Software Engineering America, Ltd.). These programs should preferably be set to an automatic setting such as the standard default setting for homology comparisons. As explained by the NIH, the scoring of gapped results tends to be more biologically meaningful than ungapped results.

One can also take advantage of Mi's correlation with melanoma to treat melanoma. Thus, one can screen for and select compounds, preferably small molecules that selectively react with Mi. These compounds can then be used to provide selective targeting of the melanoma. For example, the small molecule could be cytotoxic. In another embodiment, the compound, e.g. a cytotoxic compound such as ricin could bind to Mi and be activated so that the molecule serves as a target or catalyst for a second compound that it used to treat a melanoma.
 

Claim 1 of 5 Claims

1. A method for screening for melanoma using immunohistochemistry to determine whether melanocyte microphthalmia (Mi) is expressed which comprises: (a) contacting in vitro a biological specimen containing malignant cells with a monoclonal antibody that selectively binds to an epitope in the N-terminus Taq-Sac fragment of human melanocyte Mi; and (c) determining whether melanocyte Mi is being expressed in the specimen by the binding of the antibody to melanocyte Mi, wherein the expression of melanocyte Mi in a malignant cell is indicative of melanoma.

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