Title: Use of microphthalmia
for diagnosis, prognosis and/or treatment of melanoma
United States Patent: 7,338,767
Issued: March 4, 2008
Inventors: Fisher; David E.
Cancer Institute, Inc. (Boston, MA)
Appl. No.: 09/229,283
Filed: January 13, 1999
Executive MBA in Pharmaceutical Management, U. Colorado
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
Description of the
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
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
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
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
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
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
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
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
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 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
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
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
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