Note: Descriptions are shown in the official language in which they were submitted.
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DETECTION OF CHROMOSOME COPY NUMBER CHANGES TO
DISTINGUISH MELANOCYTIC NEVI FROM MALIGNANT
MELANOMA
BACKGROUND OF THE INVENTION
The melanocyte can give rise to a number of morphologically different
tumors. Most of them are biologically benign and are referred to as
melanocytic nevi.
Examples of melanocytic nevi are congenital nevi, Spitz nevi (including
pigmented
spindle cell nevi, which are regarded as a subtype of Spitz nevi), dysplastic
or Clark's
nevi, blue nevi, lentigo simplex, and deep penetrating nevus.
Spitz nevi are benign melanocytic neoplasms that can have considerable
histological resemblance to melanoma. They were first described as "juvenile
melanoma"
by Sophie Spitz in 1948 and initially regarded as a subset of childhood
melanoma that
follows a benign course (Spitz, S., Am. J. Pathol. 24, 591-609 (1948)). Spitz
nevi are
common and account for about 1% of surgically removed nevi (Casso et al., JAm
Acad
Dermatol., 27, 901-13 (1992)). Although in general the pathological diagnosis
of Spitz
nevus is straightforward, there is a subset of cases in which it is difficult
to impossible to
histologically differentiate Spitz nevi from melanoma because of overlapping
histological
features, such as the presence of melanocytes with abundant cytoplasm and/or
melanocytes with large pleomorphic nuclei. Additionally, mitotic figures,
sometimes
numerous, occur in both neoplasms.
Melanoma refers to malignant neoplasms of melanocytes. Accurate
diagnosis and early treatment is of great importance because, although
advanced
melanoma has a poor prognosis, most melanomas are curable if excised in their
early
stages. Although in general the histopathological diagnosis of melanoma is
straightforward, there is a subset of cases in that it is difficult to
differentiate melanomas
from benign neoplasm of melanocytes (LeBoit, P. E. SIMULANTS OF MALIGNANT
MELANOMA: A ROGUE'S GALLERY OF MELANOCYTIC AND NON-MELANOCYTIC IMPOSTERS,
In Malignant Melanoma and Melanocytic Neoplasms, P. E. Leboit, ed.
(Philadelphia:
Hanley & Belfus), pp. 195-258 (1994)). Even though the diagnostic criteria for
separating the many simulators of melanoma are constantly refined, a fraction
of cases
remains where an unambiguous diagnosis cannot be reached (Farmer et al.,
DISCORDANCE IN THE HISTOPATHOLOGIC DIAGNOSIS OF MELANOMA AND MELANOCYTIC
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NEVI BETWEEN EXPERT PATHOLOGISTS, Human Pathol. 27: 528-31 (1996)). The most
frequent and important diagnostic dilemma is the differential diagnosis
between Spitz
nevus and melanoma.
Misdiagnosis of Spitz nevus as melanoma and vice versa has been
repeatedly reported in the literature (Goldes et al., Pediatr. Dermatol., l:
295-8 (1984);
Okun, M. R. Arch. Dermatol. 115: 1416-1420 (1979); Peters et al.,
Histopathology, 10,
1289-1302 (1986)). A retrospective study of 102 melanomas of childhood found
that
only 60 cases were classified as melanoma by a panel of experts, the majority
of the
remainder being classified as Spitz nevi (Spatz, S., Int. J. Cancer 68, 317-24
(1996)).
The presence of this diagnostic gray zone has even led the authors of a review
article in
the "Continuing Medical Education" section of the Journal of the American
Association
ofDermatology to conclude that Spitz nevus and melanoma may "actually exist on
a
continuum of disease" (Casso et al., J. Am. Acad. Dermatol., 27, 901-13
(1992)). The
authors recommended that "treatment include complete excision of all Spitz
nevi
followed by reexcision of positive margins if present." The need for improved
diagnostics for melanocytic neoplasms has led to numerous attempts to improve
diagnostic accuracy by the use of markers that could be detected by immuno-
histochemistry. While there have been prior efforts aimed at resolving this
problem, none
have been satisfactory. For example, even though tests employing markers such
as S 100,
HMB45 are useful in establishing that a poorly differentiated tumor is of
melanocytic
lineage, adjunctive techniques have been of little help in separating benign
from
malignant melanocytic lesions.
Thus, there exists a great need for improved and accurate diagnostic
methods to distinguish Spitz nevi from malignant melanoma. Furthermore, there
is a
need to distinguish melanocytic neoplasms that fall between Spitz nevi and
malignant and
are difficult to classify. The present invention addresses these and other
needs by
providing methods of typing a melanocytic neoplasm by detecting in a tumor
sample the
presence of an increase in copy number of an l lp chromosome arm,
particularly,
detecting the presence of an 1 lp isochromosome, which indicates the presence
of a Spitz
nevus. Typing can also be performed by determining the presence in a tumor
sample of
an amplification of chromosome 1 lp 15.5, and particularly by detecting the
amplifcation
of H-RAS. An additional aspect of typing is the detection of a mutated H-RAS
gene
present in a tumor sample, which is also associated with, or indicates the
presence of a
Spitz nevus.
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SUMMARY OF THE INVENTION
The present invention provides for methods of distinguishing melanocytic
nevi, such as Spitz nevi, from malignant melanoma. The methods comprise
detecting a
target polynucleotide sequence, e.g., H-RAS, on a chromosomal region such as l
lp,
particularly 11p15.5, which is frequently amplified in Spitz nevi. The nucleic
acid
sample is typically taken from skin tumor tissue located within a tumor lesion
on the skin
of the patient. The methods can also be used to determine whether the tumor
cells lack
changes in chromosomal regions associated with melanoma (e.g., lq, 6p, 7p, or
lOq).
Usually, the copy number of the target region is measured.
The methods of the invention further include a method of typing a
melanocytic neoplasm from a patient by detecting the presence of an increase
in copy
number of the l lp chromosome arm thereby typing the melanocytic neoplasm as a
Spitz
nevus. Typically, the methods comprise detecting the presence of an 1 lp
isochromosome
in a tumor sample from a patient.
The nucleic acid sample can be extracted from an interphase nucleus.
Typically, the probe is labeled e.g. with a fluorescent label. The label may
be a direct
label. Usually, a reference probe to a second chromosomal region is used in
the methods
as an internal control. In these embodiments, the second probe is labeled with
a
fluorescent label distinguishable from the label on the probe that selectively
hybridizes to
the target polynucleotide sequence.
In some embodiments, the probe may include repetitive sequences. In this
case, the methods may further comprising the step of blocking the
hybridization capacity
of repetitive sequences the probe Unlabeled blocking nucleic acids comprising
repetitive
sequences (e.g. Cot-1 DNA) can be contacted with the sample for this purpose.
The nucleic acid hybridization can be carned out in a number of formats.
For instance, the hybridization may be an in situ hybridization. In some
embodiments,
the probe is bound to a solid substrate, e.g. as a member of a nucleic acid
array.
In one embodiment of the invention, a melanocytic neoplasm can be typed
as a Spitz nevus by detecting the presence of a mutation in the H-RAS gene.
The
mutation can be detected by amplifying a nucleic acid that encodes H-RAS or a
fragment,
and sequencing the amplified product to determine whether the sequence
contains a
mutation relative to a normal H-RAS sequence. Amplification is typically
performed
using PCR. Primers for the PCR reaction include those set out in SEQ ID NOs: l
and 2,
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and SEQ ID NOs: 3 and 4. The nucleic acid that is amplified can be genomic DNA
or
RNA.
In another aspect of the invention, the presence of a mutation in the H-RAS
gene is detected by contacting a nucleic acid from a skin tumor sample with a
probe that
selectively hybridizes to a target nucleic acid comprising an H-RAS gene to
form a stable
hybridization complex. The probe is contacted under condition in which the
probe binds
selectively to the target nucleic acid that includes the H-RAS gene. In one
embodiment,
the probe binds selectively to a mutated H-RAS gene. The method can further
include a
step of amplifying the nucleic acid from the sample. Preferably, the
amplifying step is a
PCR reaction, which can be performed, e.g., using oligonucleotides as set out
in SEQ ID
NOs: 1 and 2, and 3 and 4. The nucleic acid from the sample is preferably
genomic DNA
or RNA.
The invention also includes a method of detecting the presence of a an
amplified H-RAS gene by detecting a polypeptide encoded by the H-RAS gene.
Preferably the amount of polypeptide is quantified using an immunoassay, e.g.,
ELISA.
In one embodiment, the polypeptide is detected using an antibody that
selectively binds to
a polypeptide encoded by a mutant H-RAS gene.
Definitions
To facilitate understanding the invention, a number of terms are defined
below.
The terms "melanoma" or "cutaneous melanoma" refer to malignant
neoplasms of melanocytes, which are pigment cells present normally in the
epidermis and
sometimes in the dermis. There are four types of cutaneous melanoma: lentigo
maligna
melanoma, superficial spreading melanoma (SSM), nodular melanoma, and acral
lentiginous melanoma (AM). Melanoma usually starts as a proliferation of
single
melanocytes at the junction of the epidermis and the dermis. The cells first
grow in a
horizontal manner and settle an area of the skin that can vary from a few
millimeters to
several centimeters. As noted above, in most instances the transformed
melanocytes
produce increased amounts of pigment so that the area involved can easily be
seen by the
clinician.
The term "melanocytic neoplasm" refers to an accumulation of
melanocytes that can undergo a benign, locally aggressive, or malignant
course.
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"Melanocytic neoplasm" encompasses both benign melanocytic neoplasms, "nevi",
and
malignant melanocytic neoplasms, "melanoma".
The terms "Spitz nevi" or "Spitz nevus" refer to melanocytic neoplasms
that can have considerable histological resemblance to melanoma. They
generally are
benign, but can recur locally, or rarely, spread to the lymph nodes. They were
first
described as "juvenile melanoma" and initially were thought of as a subset of
childhood
melanoma that follows a benign course. Spitz nevi are common and account for
about
1 % of surgically removed nevi.
The terms "tumor" or "cancer" in an animal refers to the presence of cells
possessing characteristics such as atypical growth or morphology, including
uncontrolled
proliferation, immortality, metastatic potential, rapid growth and
proliferation rate, and
certain characteristic morphological features. Often, cancer cells will be in
the form of a
tumor, but such cells may exist alone within an animal. "Tumor" includes both
benign
and malignant neoplasms.
The phrase "typing" or "detecting" a neoplasm refers to the determination
whether the neoplasm is, or has a high probability of being, a certain class
of neoplasm.
Classification can be based on whether the neoplasm is benign or. malignant,
or type of
nevus, e.g., Spitz nevus. "Typing" or "detecting" can also refer to obtaining
indirect
evidence regarding the likelihood of the presence of a Spitz nevus or melanoma
in the
patient. Detection of a Spitz nevus versus a melanoma can be accomplished
using the
methods of this invention alone, or in combination with other methods or in
light of other
information regarding the state of health of the patient.
The terms "hybridizing specifically to", "specific hybridization", and
"selectively hybridize to," as used herein refer to the binding, duplexing, or
hybridizing of
a nucleic acid molecule preferentially to a particular nucleotide sequence
under stringent
conditions. The term "stringent conditions" refers to conditions under which a
probe will
hybridize preferentially to its target subsequence, and to a lesser extent to,
or not at all to,
other sequences. A "stringent hybridization" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization (e.g., as in array,
Southern or
Northern hybridizations) are sequence dependent, and are different under
different
environmental parameters. An extensive guide to the hybridization of nucleic
acids is
found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and
Molecular
Biology -- Hybridization with Nucleic Acid Probes part l, Ch. 2, "Overview of
principles
of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY
("Tijssen")
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Generally, highly stringent hybridization and wash conditions are selected to
be about 5
°C lower than the thermal melting point (Tm) for the specific sequence
at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at
which 50% of the target sequence hybridizes to a perfectly matched probe. Very
stringent conditions are selected to be equal to the Tm for a particular
probe. An example
of stringent hybridization conditions for hybridization of complementary
nucleic acids
which have more than 100 complementary residues on an array or on a filter in
a
Southern or northern blot is 42 °C using standard hybridization
solutions (see, e. g.,
Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Yol. I-3,
Cold
Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed
discussion,
below), with the hybridization being carned out overnight. An example of
highly
stringent wash conditions is 0.15 M NaCI at 72 °C for about 15 minutes.
An example of
stringent wash conditions is a 0.2X SSC wash at 65 °C for 15 minutes
(see, e.g.,
Sambrook supra.) for a description of SSC buffer). Often, a high stringency
wash is
preceded by a low stringency wash to remove background probe signal. An
example
medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx
SSC at 45
°C for 15 minutes. An example of a low stringency wash for a duplex of,
e.g., more than
100 nucleotides, is 4X to 6X SSC at 40 °C for 15 minutes.
The term "labeled with a detectable label", as used herein, refers to a
nucleic acid attached to a detectable composition, i.e., a label. The
detection can be by,
e.g., spectroscopic, photochemical, biochemical, immunochemical, physical or
chemical
means. For example, useful labels include 32p, 355, 3H, ~aC, Izsh ~3~I;
fluorescent dyes
(e.g., FITC, rhodamine, lanthanide phosphors, Texas red), electron-dense
reagents (e.g.
gold), enzymes, e.g., as commonly used in an ELISA (e.g., horseradish
peroxidase, beta-
galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g.
colloidal gold),
magnetic labels (e.g. DynabeadsTM ), biotin, digoxigenin, or haptens and
proteins for
which antisera or monoclonal antibodies are available. The label can be
directly
incorporated into the nucleic acid, peptide or other target compound to be
detected, or it
can be attached to a probe or antibody that hybridizes or binds to the target.
Label can be
attached by spacer arms of various lengths to reduce potential steric
hindrance or impact
on other useful or desired properties. See, e.g., Mansfield, Mol Cell Probes
9: 145-156
(1995). In addition, target DNA sequences can be detected by means of the
primed in situ
labeling technique (PRINS) (Koch et al., Genet. Anal. Tech. Appl. 8: 171-8,
(1991)). The
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sensitivity of the detection can be increased by using chemical amplification
procedures,
e.g., by using tyramide (Speel et al., J. Histochem. Cytochem. 45:1439-46,
(1997)).
The term "paired hybridization signals" or a "hybridization signal pair"
refers to a spatial pattern of hybridization signals wherein two signals are
consistently
identified in close proximity. Isochromosomes are typically characterized by
the
presence of "paired hybridization signals" from a single probe. For example,
in a sample
with many cells, a "hybridization signal pair" is a consistent occurrence of
two signals in
close proximity that is clearly not due to an artifact or a random event.
The term "nucleic acid" as used herein refers to a deoxyribonucleotide or
ribonucleotide in either single- or double-stranded form. The term encompasses
nucleic
acids, i.e., oligonucleotides, containing known analogues of natural
nucleotides which
have similar or improved binding properties, for the purposes desired, as the
reference
nucleic acid. The term also includes nucleic acids which are metabolized in a
manner
similar to naturally occurring nucleotides or at rates that are improved for
the purposes
desired. The term also encompasses nucleic-acid-like structures with synthetic
backbones. DNA backbone analogues provided by the invention include
phosphodiester,
phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate,
alkyl
phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-
carbamate,
morpholino carbamate, and peptide nucleic acids (PNAs); see Oligonucleotides
and
Analogues, a Practical Approach, edited by F. Eckstein, IRL Press at Oxford
University
Press (1991); Antisense Strategies, Annals of the New York Academy of
Sciences,
Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med.
Chem.
36:1923-1937; Antisense Research and Applications (1993, CRC Press). PNAs
contain
non-ionic backbones, such as N-(2-aminoethyl) glycine units. Phosphorothioate
linkages
are described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl.
Pharmacol.
144:189-197. Other synthetic backbones encompasses by the term include methyl-
phosphonate linkages or alternating methylphosphonate and phosphodiester
linkages
(Strauss-Soukup (1997) Biochemistry 36: 8692-8698), and benzylphosphonate
linkages
(Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156). The term nucleic
acid is
used interchangeably with gene, cDNA, mRNA, oligonucleotide primer, probe and
amplification product.
The term a "nucleic acid array" as used herein is a plurality of target
elements, each target element comprising one or more nucleic acid molecules
(probes)
immobilized on one or more solid surfaces to which sample nucleic acids can be
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hybridized. The nucleic acids of a target element can contain sequences) from
specific
genes or clones, e.g. from the regions identified here. Other target elements
will contain,
for instance, reference sequences. Target elements of various dimensions can
be used in
the arrays of the invention. Generally, smaller, target elements are
preferred. Typically,
a target element will be less than about 1 cm in diameter. Generally element
sizes are
from 1 ~.m to about 3 mm, preferably between about 5 ~,m and about 1 mm. The
target
elements of the arrays may be arranged on the solid surface at different
densities. The
target element densities will depend upon a number of factors, such as the
nature of the
label, the solid support, and the like. One of skill will recognize that each
target element
may comprise a mixture of nucleic acids of different lengths and sequences.
Thus, for
example, a target element may contain more than one copy of a cloned piece of
DNA, and
each copy may be broken into fragments of different lengths. The length and
complexity
of the nucleic acid fixed onto the target element is not critical to the
invention. One of
skill can adjust these factors to provide optimum hybridization and signal
production for a
given hybridization procedure, and to provide the required resolution among
different
genes or genomic locations. In various embodiments, target element sequences
will have
a complexity between about 1 kb and about 1 Mb, between about 10 kb to about
500 kb,
between about 200 to about S00 kb, and from about 50 kb to about 150 kb.
The terms "nucleic acid sample" or "sample of human nucleic acid" as
used herein refers to a sample comprising human DNA or RNA in a form suitable
for
detection by hybridization or amplification. Typically, it will be prepared
from a skin
tissue sample from a patient who has or is suspected of having melanocytic
tumor that
may be difficult to classify. The sample will most usually be prepared from
tissue taken
from the tumor.
In many instances, the nucleic acid sample will be a tissue or cell sample
prepared for standard in situ hybridization methods described below. The
sample is
prepared such that individual chromosomes remain substantially intact prepared
according to standard techniques. Alternatively, the nucleic acid may be
isolated, cloned
or amplified. It may be, e.g., genomic DNA, mRNA, or cDNA from a particular
chromosome, or selected sequences (e.g. particular promoters, genes,
amplification or
restriction fragments, cDNA, etc.) within particular amplicons or deletions
disclosed here.
The nucleic acid sample may be extracted from particular cells or tissues,
e.g. melanocytes. Methods of isolating cell and tissue samples are well known
to those of
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skill in the art and include, but are not limited to, aspirations, tissue
sections, needle
biopsies, and the like. Frequently the sample will be a "clinical sample"
which is a
sample derived from a patient, including sections of tissues such as frozen
sections or
paraffin sections taken for histological purposes. The sample can also be
derived from
supernatants (of cells) or the cells themselves from cell cultures, cells from
tissue culture
and other media in which it may be desirable to detect chromosomal
abnormalities or
determine amplicon copy number. In some cases, the nucleic acids may be
amplified
using standard techniques such as PCR, prior to the hybridization. The sample
may be
isolated nucleic acids immobilized on a solid.
The term "probe" or "nucleic acid probe", as used herein, is defined to be a
collection of one or more nucleic acid fragments whose hybridization to a
sample can be
detected. The probe may be unlabeled or labeled as described below so that its
binding to
the target or sample can be detected. The probe is produced from a source of
nucleic
acids from one or more particular (preselected) portions of the genome, e.g.,
one or more
clones, an isolated whole chromosome or chromosome fragment, or a collection
of
polymerise chain reaction (PCR) amplification products. The probes of the
present
invention are produced from nucleic acids found in the regions described
herein.
A probe that is "adj acent to the centromere" refers to a probe that
hybridize to regions adjacent to the centromere bind to sequences at l lp11.1
to l 1p11.2
or llqll.l to 11q11.2.
An "1 lp chromosome arm" is defined cytogeneticallya s encompassing the
chromosome from band l lpl l to 1 lpter.
The probe or genomic nucleic acid sample may be processed in some
manner, e.g., by blocking or removal of repetitive nucleic acids or enrichment
with
unique nucleic acids. The word "sample" may be used herein to refer not only
to detected
nucleic acids, but to the detectable nucleic acids in the form in which they
are applied to
the target, e.g., with the blocking nucleic acids, etc. The blocking nucleic
acid may also
be referred to separately. What "probe" refers to specifically is clear from
the context in
which the word is used. The probe may also be isolated nucleic acids
immobilized on a
solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as
in an array. In
some embodiments, the probe may be a member of an array of nucleic acids as
described,
for instance, in WO 96/17958. Techniques capable of producing high density
arrays can
also be used for this purpose (see, e.g., Fodor (1991) Science 767-773;
Johnston (1998)
Curr. Biol. 8: 8171-8174; Schummer (1997) Biotechniques 23: 1087-1092; Kern
(1997)
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Biotechniques 23: 120-124; U.S. Patent No. 5,143,854). One of skill will
recognize that
the precise sequence of the particular probes described herein can be modified
to a certain
degree to produce probes that are "substantially identical" to the disclosed
probes, but
retain the ability to specifically bind to (i.e., hybridize specifically to)
the same targets or
samples as the probe from which they were derived (see discussion above). Such
modifications are specifically covered by reference to the individual probes
described
herein.
The term "immunoassay" is an assay that uses an antibody to specifically
bind an antigen. The immunoassay is characterized by the use of specific
binding
properties of a particular antibody to isolate, target, and/or quantify the
antigen.
The phrase "specifically (or selectively) binds" to an antibody or
"specifically (or selectively) immunoreactive with," when refernng to a
protein or
peptide, refers to a binding reaction that is determinative of the presence of
the protein in
a heterogeneous population of proteins and other biologics. Thus, under
designated
immunoassay conditions, the specified antibodies bind to a particular protein
at least two
times the background, more typically more than 10 to 100 times background, and
do not
substantially bind in a significant amount to other proteins present in the
sample. The
term "immunoassay" is an assay that uses an antibody to specifically bind an
antigen.
The immunoassay is characterized by the use of specific binding properties of
a particular
antibody to isolate, target, and/or quantify the antigen.
The phrase "specifically (or selectively) binds" to an antibody or
"specifically (or selectively) immunoreactive with," when referring to a
protein or
peptide, refers to a binding reaction that is determinative of the presence of
the protein in
a heterogeneous population of proteins and other biologics. Thus, under
designated
immunoassay conditions, the specified antibodies bind to a particular protein
at least two
times the background, more typically more than 10 to 100 times background, and
do not
substantially bind in a significant amount to other proteins present in the
sample. Specific
binding to an antibody under some conditions may require an antibody that is
selected for
its specificity for a particular H-RAS protein. For example, an antibody that
selectively
binds to a polypeptide encoded by a mutated H-RAS gene binds to mutated, but
not
normal H-RAS.
"Providing a nucleic acid sample" means to obtain a biological sample for
use in the methods described in this invention. Most often, this will be done
by removing
a sample of cells from an animal, but can also be accomplished by using
previously
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isolated cells (e.g. isolated by another person), or by performing the methods
of the
invention in vivo.
"Tissue biopsy" refers to the removal of a biological sample for diagnostic
analysis. In a patient with cancer, tissue may be removed from a tumor,
allowing the
analysis of cells within the tumor.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the summary of chromosome copy number changes in 32
primary cutaneous melanomas. Chromosomal gains are shown as lines to the right
of the
chromosome ideogramms, losses are shown as lines to the left. Thick lines to
the right
indicate amplifications, thick lines to the left summarize losses in 10 cases
(Bastian et al.,
Cancer Res 58: 2170-5, 1998).
Fig. 2 shows the summary of chromosome copy number changes in 17
Spitz nevi. Chromosomal gains are shown as lines to the right of the
chromosome
1 S ideogramms. Thick lines indicate amplifications.
Fig. 3 shows the average ratio profiles of fluorescence intensity of tumor
vs. reference DNA in the four Spitz nevi that had abnormal CGH profiles. The
dotted
lines indicate the 1.2 and 0.8 ratio thresholds that were used for defining
aberrations. n
indicates the number of chromosomes measured for the respective profile.
Fig. 4 shows the frequency distribution of hybridization signals after dual-
target hybridization of probe RMC11B022 for chromosome l lp (black bars) and
RMC 11 P008 for chromosome 11 q (white bars). Three cases of Spitz nevi are
shown.
Case 2 (A, B) showed no chromosomal aberrations by CGH, Case 13 (C, D) had an
gain
of chromosome l lp by CGH, Case 15 (E, F) did not show aberrations by CGH, it
had a
subpopulation of tumor cells with large nuclei. Charts A, C, E show signal
distribution in
tumor cells; Charts B, D, F show signal distribution in keratinocytes of the
corresponding
lesions:
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Introduction
The present invention provides for unique and accurate methods for
distinguishing Spitz nevus, from malignant melanoma. This invention is based
upon the
observation that chromosomal regions that have frequently altered copy numbers
in
melanoma such as 1 q, 6p, 7p, 9p, or 1 Oq, are rarely changed in Spitz nevi.
In addition,
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Spitz nevi cells show a single amplification of chromosomal region 1 lp,
particularly
l 1p15.5, and more particularly, the H-RAS gene, which is localized to l
1p15.5, as shown
by the increase of its copy number, a phenomenon that is exceedingly rare in
melanoma.
The amplification of chromosome 1 lp typically occurs via amplification of the
1 lp
chromosome arm, and is characterized by the presence of an 1 lp isochromosome.
This
difference in pattern of chromosomal aberrations between Spitz nevi and
melanoma can
lead to more accurate diagnostic distinction of Spitz nevi from melanoma.
The present invention further provides methods of typing a melanocytic
neoplasm by detecting in a skin tumor sample the presence of a mutated H-RAS
gene that
is associated with the diagnosis of a Spitz nevus.
The frequency of chromosomal aberrations among melanoma cells,
including primary and metastatic melanoma has been studied using CGH (Bastian
et al.,
Cancer Res 58, 2170-5 (1998). One of the findings of this experiment was the
frequent
loss of chromosome 9 and chromosome 10 that occurred in 81% and 63% of the
tumors,
respectively. By comparing the frequency of occurrence in thin and thick
tumors, and
comparing parts of tumors that were in different phases of tumor progression,
it was
discovered that losses of chromosomes 9 and 10 occurred early in
tumorigenesis.
Another set of experiments was performed, extending the data set to 70
tumors. Results from the second set of experiments confirmed that losses of
chromosomes 9 and 10 are the most frequent changes in primary melanomas of the
skin.
In these 70 melanomas only four exhibited no changes by CGH. Results of these
experiments performed with melanoma cells are shown in Fig. 1.
There have been several studies of ploidy in Spitz nevi using measurement
of nuclear DNA content by image cytometry or flow cytometry (Howat et al.,
Cancer 63,
474-8 (1989); LeBoit et al., Jlnvest Dermatol 88, 753-7 (1987); Otsuka et al.,
Clin Exp
Dermatol 18, 421-4 (1993); Vogt et al., Am JDermatopathol 18, 142-50 (1996)).
However, routine application of these techniques has been hampered by the
complexity of
the procedure and its lack of sensitivity. Recently, molecular cyotgenetic
analysis has
shown that Spitz nevi, particularly a subset of Spitz nevi, exhibit
amplification of
chromosome l lp (see, e.g., Bastian et al., J. Invest. Dermatol. 113:1065-
1069, 1999; and
Bastian et al., Cancer Res. 58:2170-2175, 1998), including the l 1p15.5
region. As
disclosed herein, amplification with or without mutations of the H-RAS gene,
which is
localized to 1 lp15.5, are also present in Spitz nevi. As H-RAS is rarely
mutated in
melanoma (see, e.g., Jiveskog et al., J. Invest. Dermatol. 111:757-761, 1998;
van Elsas et
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al., Am J. Pathol. 149:883-893, 1996), but is mutated in Spitz nevi, mutations
in H-RAS
can be used to further distinguish a Spitz nevus from melanoma. Accordingly,
the present
invention provides methods for determining the presence of an amplified H-RAS
gene
and/or a mutation in an H-RAS gene in a melanocytic neoplasm in order to
determine if
the neoplasm is a Spitz nevus.
It has been determined that a subset of Spitz nevi recur following excision.
The subset typically is characterized by an amplification of the entire 1 lp
chromosome
arm, in particular, by the presence of an 1 lp isochromosome. Amplification of
the entire
arm of chromosome 11 p has not been observed in melanoma. Thus, the present
invention also provides methods of typing or classifying a melanocytic
neoplasm as a
Spitz nevus by detecting the presence of an increase in copy number of the
entire arm of
l lp, in particular detecting the presence of an l lp isochromosome.
General methods for measuring chromosomal abnormality
Genomic instability is a hallmark of solid tumors, and virtually no solid
tumor exists which does not show major alterations of the genome. With the
vast
majority of tumors this instability is expressed at the level of the
chromosomal
complement, and thus is detectable by cytogenetic approaches (Mitelman, F.,
Catalog of
chromosome aberrations in cancer, 5th Edition (New York: Wiley-Liss) (1994)).
However, aneuploidy per se is not indicative of malignancy and many benign
tumors can
have an aberrant karyotype (Mitelman, 1994). To efficiently take advantage of
aneuploidy as a marker, it is mandatory to know characteristic aberrations of
the tumors
that are to be differentiated.
Several techniques that permit the study of chromosomal complement in
post-fixation tissue have been developed. Fluorescence in-situ hybridization
(FISH) can
be used to study copy numbers of individual genetic loci in interphase nuclei
(Pinkel et
al., Proc. Natl. Acad. Sci. U.S.A. 85, 9138-42 (1988)) and comparative genomic
hybridization (CGH) (Kallioniemi et al.. Science 258, 818-2 1 (1992)) has
proven a useful
technique (Houldsworth et al.. Am J Pathol 145, 1253-60 (1994)) to probe the
entire
genome for copy number changes of chromosomal regions.
The application of FISH as an adjunctive diagnostic technique for the
differentiation of Spitz nevi from melanomas has been suggested previously (De
Wit et
al., JPathol. 173, 227-33 (1994)). The investigators used a centromeric probe
for
chromosome 1 and found a significant difference in the number of cells with an
aberrant
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number of signals between 1 S melanoma and 15 Spitz nevi. At this point no
detailed
knowledge about chromosomal changes in primary melanomas of the skin was
available
and chromosome 1 was selected based on its frequent numerical change in
melanoma
metastasis (Thompson et al., Cancer Genet Cytogenet 83, 93-104 (1995)). It is
to be
expected that a selection of a panel of chromosomal markers of regions that
are frequently
involved in primary melanomas could increase sensitivity and specificity to a
level that
would allow the application of FISH as a routine method. To achieve this goal,
it is
essential to know the pattern of aberrations in melanomas as well as its
benign
counterparts.
Detection of Copy Number
Methods of evaluating the copy number of a particular gene or
chromosomal region are well known to those of skill in the art. In this
invention, the
presence or absence of chromosomal gain or loss can be evaluated simply by a
determination of copy number of the regions identified here. Typically, the
regions
evaluated are lq, 6p, 7p, 9p, lOq, and l lp.
Hybridization-based Assay
Preferred hybridization-based assays include, but are not limited to,
traditional "direct probe" methods such as Southern Blots or In Situ
Hybridization (e.g.,
FISH), and "comparative probe" methods such as Comparative Genomic
Hybridization
(CGH). The methods can be used in a wide variety of formats including, but not
limited
to substrate (e.g. membrane or glass) bound methods or array-based approaches
as
described below.
In situ hybridization assays are well known (e.g., Angerer ( 1987) Meth.
Enzymol 152: 649). Generally, in situ hybridization comprises the following
major steps:
(1) fixation of tissue or biological structure to be analyzed; (2)
prehybridization treatment
of the biological structure to increase accessibility of target DNA, and to
reduce
nonspecific binding; (3) hybridization of the mixture of nucleic acids to the
nucleic acid
in the biological structure or tissue; (4) post-hybridization washes to remove
nucleic acid
fragments not bound in the hybridization and (5) detection of the hybridized
nucleic acid
fragments. The reagent used in each of these steps and the conditions for use
vary
depending on the particular application.
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In a typical in situ hybridization assay, cells are fixed to a solid support,
typically a glass slide. If a nucleic acid is to be probed, the cells are
typically denatured
with heat or alkali. The cells are then contacted with a hybridization
solution at a
moderate temperature to permit annealing of labeled probes specific to the
nucleic acid
sequence encoding the protein. The targets (e.g., cells) are then typically
washed at a
predetermined stringency or at an increasing stringency until an appropriate
signal to
noise ratio is obtained.
The probes are typically labeled, e.g., with radioisotopes or fluorescent
reporters. The preferred size range is from about 200 by to about 1000 bases,
more
preferably between about 400 to about 800 by for double stranded, nick
translated nucleic
acids.
In some applications it is necessary to block the hybridization capacity of
repetitive sequences. Thus, in some embodiments, human genomic DNA or Cot-1
DNA
is used to block non-specific hybridization.
In Comparative Genomic Hybridization methods a first collection of
(sample) nucleic acids (e.g. from a possible tumor) is labeled with a first
label, while a
second collection of (control) nucleic acids (e.g. from a healthy cell/tissue)
is labeled with
a second label. The ratio of hybridization of the nucleic acids is determined
by the ratio of
the two (first and second) labels binding to each fiber in the array. Where
there are
chromosomal deletions or multiplications, differences in the ratio of the
signals from the
two labels will be detected and the ratio will provide a measure of the copy
number.
Hybridization protocols suitable for use with the methods of the invention
are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988)
Proc.
Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular
Biology, Yol. 33: In Situ Hybridization Protocols, Choo, ed., Humana Press,
Totowa, NJ
(1994), etc. In one particularly preferred embodiment, the hybridization
protocol of
Pinkel et al. (1998) Nature Genetics 20: 207-211 or of Kallioniemi (1992)
Proc. Natl
Acad Sci USA 89:5321-5325 (1992) is used.
Detection of isochromosomes
Changes in copy number of a particular gene or chromosomal region can
be due to a number of mechanisms, including the presence of an isochromosome,
in
which one of the arms of a chromosome is duplicated, thus increasing the copy
nubmer of
the sequences located on the duplicated arm. Methods of evaluating the copy
number of a
CA 02368903 2001-10-05
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particular gene or chromosomal region, and particularly analyzing for the
presence of an
isochromosome, are well known to those of skill in the art. An increase in
copy number
of the whole arm of chromosome 11 can be detected, e.g., using procedures
described in
co-pending application U.S.S.N. 09/288,940. Copy number changes, and
particularly,
isochromosomes, are typically detected using hybridzation-based assays, such
as FISH.
The presence of an isochromosome can be detected using a single probe to
that hybridizes to a region on the duplicated chromosomal arm. Typically, the
probe will
be localized to a regions of the chromosomal arm that is adjacent to the
centromere.
Normal cells have two randomly positioned signals in their nucleus. Cells that
possess an
isochromosome will have one to several pairs of signals present in the
nucleus.
Preferably, an isochromosome is detected using two probes, each labeled
with a distinct compound, e.g., different fluorescent labels with
distinguishable colors.
Usually, the analysis employs two probes that hybridize to nucleic acid
sequences close to
the centromere. One of the probes hybridizes to target sequences on the p arm
that are
adjacent to the centromere, e.g., sequences localized to l lp11.1 or l 1p11.2.
The second
probe hybridizes to target sequences on the q arm adj acent to the centromer,
i. e., 11 q 11.1
or l lql 1.2. An isochromosome is detected by determining the presence of
hybridization
regions that occur as pairs of the same color compared to a normal situation
in which the
visualized pairs contain two colors.
Detection of Mutations in H RAS
The H-RAS gene is located at 11p15.5, a region which has been shown to
be amplified in a subset of Spitz nevi. (Bastian et al., J. Invest. Dermatol.
113, 1065-
1069, 1999 and co-pending U.S.S.N. 09/288,940). Melanocytic neoplasms that are
to be
typed can be analyzed for the presence of an amplified H-RAS gene as described
and
further, may be analyzed for the presence of additional mutations in the H-RAS
gene.
Oncogenic mutations of H-RAS typically involve codons 12, 13, and 61. However,
other
mutations such as point mutations occurring at any region within the
structural gene or
regulatory regions of H-RAS, insertions, and deletions can also be detected
using the
methods of the invention.
There are many methods known in the art for detecting mutations in a
given gene. Useful techniques include, but are not limited to, FISH, direct
DNA
sequencing, Southern blot analysis, single stranded conformation analysis
(SSCP),
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denaturing gradient gel electrophoresis, RNAse protection assays, allele-
specific
oligonucleotides (ASO), dot blot analysis, PCR-SSCP, and allele-specific PCR.
Another method known in the art is CFLP-cleavase fragment length
polymorphism. This method involves amplifying the gene of interest, here H
RAS,
followed by digestion with cleavase I, which cuts the DNA at sites dependent
on
secondary structure. Results are resolved on agarose gels and different
patterns of
cleavage digestion products are obtained for wild-type and mutant samples.
A further method known in the art is temperature modulation heteroduplex
chromatography (TMHC). The method involves amplification of the H-RAS gene
followed by denaturing of the PCR products and then slowly cooling, to a
predetermined
temperature based on the composition of the sample. While cooling, the PCR
products
renature to form hetero- and homoduplexes which are resolved from one another
using
TMHC. The resolution can be performed using a WAVE~ DNA fragment analysis
system (Transgenomic,Inc., San Jose, CA).
1 S Mutations in the gene can be found directly by amplifying the gene, e.g.,
using PCR, in a biological sample, such as a skin tumor sample, and sequencing
the
amplified product. Alternatively, a probe that specifically hybridizes to theH-
RAS gene
can used to detect the presence of mutations. Further, a probe that
specifically hybridizes
to a mutated H-RAS gene, but not the normal gene, e.g., an allele-specific
oligonucleotide,
can be used to determine the presence of a specific mutation. A probe such as
an allele-
specific oligonucleotide may be used directly as a probe or as a primer in an
amplification
reaction in which a product is obtained only if the mutation is present.
Mutations in the H-RAS gene can be detected by a variety of hybridization
analyses. Detection of single base mutations can be conveniently accomplished
by
differential hybridization techniques using allele-specific oligonucleotides
(see, e.g.,
Suggs et al., Proc. Natl. Acad. Sci. 78: 6613-6617 (1981); Conner et al.,
Proc. Natl. Acad.
Sci. 80: 278-282 (1983); Saiki et al., Proc. Natl. Acad. Sci. 86: 6230-6234
(1989)).
Mutations can be diagnosed on the basis of the higher thermal stability of the
perfectly
matched probes as compared to the mismatched probes. The hybridization
reactions can,
for example, be carned out in a filter-based format, in which the target
nucleic acids are
immobilized on nitrocellulose or nylon membranes and probed with
oligonucleotide
probes. Any of the known hybridization formats may be used, including Southern
blots,
slot blots, "reverse" dot blots, solution hybridization, solid support based
sandwich
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hybridization, bead-based, silicon chip-based and microtiter well-based
hybridization
formats.
An alternative strategy involves detection mutations in the H-RAS gene by
sandwich hybridization methods. In this strategy, the mutant and normal target
nucleic
acids are separated from non-homologous DNA/RNA using a common capture
oligonucleotide immobilized on a solid support and detected by specific
oligonucleotide
probes tagged with reporter labels. The capture oligonucleotides can be
immobilized on
microtitre plate wells or on beads (Gingeras et al., J. Infect. Dis. 164: 1066-
1074 (1991);
Richman et al., Proc. Natl. Acad. Sci. 88: 11241-11245 (1991)).
Nucleic acid arrays
The methods of this invention are particularly well suited to array-based
hybridization formats. For a description of one preferred array-based
hybridization
system see Pinkel et al. (1998) Nature Genetics, 20: 207-211.
Arrays are a multiplicity of different "probe" or "target" nucleic acids (or
other compounds) attached to one or more surfaces (e.g., solid, membrane, or
gel). In a
preferred embodiment, the multiplicity of nucleic acids (or other moieties) is
attached to a
single contiguous surface or to a multiplicity of surfaces juxtaposed to each
other.
In an array format a large number of different hybridization reactions can
be run essentially "in parallel." This provides rapid, essentially
simultaneous, evaluation
of a number of hybridizations in a single "experiment". Methods of performing
hybridization reactions in array based formats are well known to those of
skill in the art
(see, e.g., Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature
Biotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958.
Arrays, particularly nucleic acid arrays can be produced according to a
wide variety of methods well known to those of skill in the art. For example,
in a simple
embodiment, "low density" arrays can simply be produced by spotting (e.g. by
hand using
a pipette) different nucleic acids at different locations on a solid support
(e.g. a glass
surface, a membrane, etc.).
This simple spotting approach has been automated to produce high density
spotted arrays (see, e.g., U.S. Patent No: 5,807,522). This patent describes
the use of an
automated systems that taps a microcapillary against a surface to deposit a
small volume
of a biological sample. The process is repeated to generate high density
arrays. Arrays
can also be produced using oligonucleotide synthesis technology. Thus, for
example,
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U.S. Patent No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and
92/10092
teach the use of light-directed combinatorial synthesis of high density
oligonucleotide
arrays.
In another embodiment the array., particularly a spotted array, can include
genomic DNA, e.g. overlapping clones that provide a high resolution scan of
the
amplicon corresponding to the region of interest. Amplicon nucleic acid can be
obtained
from, e.g., MACs, YACs, BACs, PACs, Pls, cosmids, plasmids, inter-Alu PCR
products
of genomic clones, restriction digests of genomic clone, cDNA clones,
amplification
(e.g., PCR) products, and the like.
In various embodiments, the array nucleic acids are derived from
previously mapped libraries of clones spanning or including the target
sequences of the
invention, as well as clones from other areas of the genome, as described
below. The
arrays can be hybridized with a single population of sample nucleic acid or
can be used
with two differentially labeled collections (as with an test sample and a
reference sample).
Many methods for immobilizing nucleic acids on a variety of solid
surfaces are known in the art. A wide variety of organic and inorganic
polymers, as well
as other materials, both natural and synthetic, can be employed as the
material for the
solid surface. Illustrative solid surfaces include, e.g., nitrocellulose,
nylon, glass, quartz,
diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose,
and
cellulose acetate. In addition, plastics such as polyethylene, polypropylene,
polystyrene,
and the like can be used. Other materials which may be employed include paper,
ceramics, metals, metalloids, semiconductive materials, cermets or the like.
In addition,
substances that form gels can be used. Such materials include, e.g., proteins
(e.g.,
gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides. Where
the solid
surface is porous, various pore sizes may be employed depending upon the
nature of the
system.
In preparing the surface, a plurality of different materials may be
employed, particularly as laminates, to obtain various properties. For
example, proteins
(e.g., bovine serum albumin) or mixtures of macromolecules (e.g., Denhardt's
solution)
can be employed to avoid non-specific binding, simplify covalent conjugation,
enhance
signal detection or the like. If covalent bonding between a compound and the
surface is
desired, the surface will usually be polyfunctional or be capable of being
polyfunctionalized. Functional groups which may be present on the surface and
used for
linking can include carboxylic acids, aldehydes, amino groups, cyano groups,
ethylenic
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groups, hydroxyl groups, mercapto groups and the like. The manner of linking a
wide
variety of compounds to various surfaces is well known and is amply
illustrated in the
literature.
For example, methods for immobilizing nucleic acids by introduction of
various functional groups to the molecules is known (see, e.g., Bischoff
(1987) Anal.
Biochem., 164: 336-344; Kremsky (1987) Nucl. Acids Res. 15: 2891-2910).
Modified
nucleotides can be placed on the target using PCR primers containing the
modified
nucleotide, or by enzymatic end labeling with modified nucleotides. Use of
glass or
membrane supports (e.g., nitrocellulose, nylon, polypropylene) for the nucleic
acid arrays
of the invention is advantageous because of well developed technology
employing
manual and robotic methods of arraying targets at relatively high element
densities. Such
membranes are generally available and protocols and equipment for
hybridization to
membranes is well known.
Target elements of various sizes, ranging from 1 mm diameter down to 1
pm can be used. Smaller target elements containing low amounts of
concentrated, fixed
probe DNA are used for high complexity comparative hybridizations since the
total
amount of sample available for binding to each target element will be limited.
Thus it is
advantageous to have small array target elements that contain a small amount
of
concentrated probe DNA so that the signal that is obtained is highly localized
and bright.
Such small array target elements are typically used in arrays with densities
greater than
104/cm2. Relatively simple approaches capable of quantitative fluorescent
imaging of 1
cm2 areas have been described that permit acquisition of data from a large
number of
target elements in a single image (see, e.g., Wittrup, Cytometry 16: 206-213,
1994).
Arrays on solid surface substrates with much lower fluorescence than
membranes, such as glass, quartz, or small beads, can achieve much better
sensitivity.
Substrates such as glass or fused silica are advantageous in that they provide
a very low
fluorescence substrate, and a highly efficient hybridization environment.
Covalent
attachment of the target nucleic acids to glass or synthetic fused silica can
be
accomplished according to a number of known techniques (described above).
Nucleic
acids can be conveniently coupled to glass using commercially available
reagents. For
instance, materials for preparation of silanized glass with a number of
functional groups
are commercially available or can be prepared using standard techniques (see,
e.g., Gait
(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Wash.,
D.C.).
CA 02368903 2001-10-05
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Quartz cover slips, which have at least 10-fold lower autofluorescence than
glass, can
also be silanized.
Alternatively, probes can also be immobilized on commercially available
coated beads or other surfaces. For instance, biotin end-labeled nucleic acids
can be
bound to commercially available avidin-coated beads. Streptavidin or anti-
digoxigenin
antibody can also be attached to silanized glass slides by protein-mediated
coupling using
e.g., protein A following standard protocols (see, e.g., Smith (1992) Science
258: 1122-
1126). Biotin or digoxigenin end-labeled nucleic acids can be prepared
according to
standard techniques. Hybridization to nucleic acids attached to beads is
accomplished by
suspending them in the hybridization mix, and then depositing them on the
glass substrate
for analysis after washing. Alternatively, paramagnetic particles, such as
ferric oxide
particles, with or without avidin coating, can be used.
In one particularly preferred embodiment, probe nucleic acid is spotted
onto a surface (e.g., a glass or quartz surface). The nucleic acid is
dissolved in a mixture
of dimethylsulfoxide (DMSO) and nitrocellulose and spotted onto amino-silane
coated
glass slides. Small capillaries tubes can be used to "spot" the probe mixture.
A variety of other nucleic acid hybridization formats are known to those
skilled in the art. For example, common formats include sandwich assays and
competition or displacement assays. Hybridization techniques are generally
described in
Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL
Press;
Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al.
(1969)
Nature 223: 582-587.
Sandwich assays are commercially useful hybridization assays for
detecting or isolating nucleic acid sequences. Such assays utilize a "capture"
nucleic acid
covalently immobilized to a solid support and a labeled "signal" nucleic acid
in solution.
The sample will provide the target nucleic acid. The "capture" nucleic acid
and "signal"
nucleic acid probe hybridize with the target nucleic acid to form a "sandwich"
hybridization complex. To be most effective, the signal nucleic acid should
not hybridize
with the capture nucleic acid.
Detection of a hybridization complex may require the binding of a signal
generating complex to a duplex of target and probe polynucleotides or nucleic
acids.
Typically, such binding occurs through ligand and anti-ligand interactions as
between a
ligand-conjugated probe and an anti-ligand conjugated with a signal.
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The sensitivity of the hybridization assays may be enhanced through use of
a nucleic acid amplification system that multiplies the target nucleic acid
being detected.
Examples of such systems include the polymerase chain reaction (PCR) system
and the
ligase chain reaction (LCR) system. Other methods recently described in the
art are the
nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga,
Ontario)
and Q Beta Replicase systems.
Nucleic acid hybridization simply involves providing a denatured probe
and target nucleic acid under conditions where the probe and its complementary
target
can form stable hybrid duplexes through complementary base pairing. The
nucleic acids
that do not form hybrid duplexes are then washed away leaving the hybridized
nucleic
acids to be detected, typically through detection of an attached detectable
label. It is
generally recognized that nucleic acids are denatured by increasing the
temperature or
decreasing the salt concentration of the buffer containing the nucleic acids,
or in the
addition of chemical agents, or the raising of the pH. Under low stringency
conditions
(e.g., low temperature and/or high salt and/or high target concentration)
hybrid duplexes
(e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed
sequences are not perfectly complementary. Thus specificity of hybridization
is reduced
at lower stringency. Conversely, at higher stringency (e.g., higher
temperature or lower
salt) successful hybridization requires fewer mismatches.
One of skill in the art will appreciate that hybridization conditions may be
selected to provide any degree of stringency. In a preferred embodiment,
hybridization is
performed at low stringency to ensure hybridization and then subsequent washes
are
performed at higher stringency to eliminate mismatched hybrid duplexes.
Successive
washes may be performed at increasingly higher stringency (e.g., down to as
low as 0.25
X SSPE-T at 37 °C to 70 °C) until a desired level of
hybridization specificity is obtained.
Stringency can also be increased by addition of agents such as formamide.
Hybridization
specificity may be evaluated by comparison of hybridization to the test probes
with
hybridization to the various controls that can be present.
In general, there is a tradeoff between hybridization specificity
(stringency) and signal intensity. Thus, in a preferred embodiment, the wash
is performed
at the highest stringency that produces consistent results and that provides a
signal
intensity greater than approximately 10% of the background intensity. Thus, in
a
preferred embodiment, the hybridized array may be washed at successively
higher
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stringency solutions and read between each wash. Analysis of the data sets
thus produced
will reveal a wash stringency above which the hybridization pattern is not
appreciably
altered and which provides adequate signal for the particular probes of
interest.
In a preferred embodiment, background signal is reduced by the use of a
detergent (e.g., C-TAB) or a blocking reagent (e.g., sperm DNA, cot-1 DNA,
etc.) during
the hybridization to reduce non-specific binding. In a particularly preferred
embodiment,
the hybridization is performed in the presence of about 0.1 to about 0.5 mg/ml
DNA (e.g.,
cot-1 DNA). The use of blocking agents in hybridization is well known to those
of skill
in the art (see, e.g., Chapter 8 in P. Tijssen, supra.)
Methods of optimizing hybridization conditions are well known to those of
skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques in
Biochemistry and
Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier,
N.Y.).
Optimal conditions are also a function of the sensitivity of label (e.g.,
fluorescence) detection for different combinations of substrate type,
fluorochrome,
excitation and emission bands, spot size and the like. Low fluorescence
background
membranes can be used (see, e.g., Chu (1992) Electrophoresis 13:105-114). The
sensitivity for detection of spots ("target elements") of various diameters on
the candidate
membranes can be readily determined by, e.g., spotting a dilution series of
fluorescently
end labeled DNA fragments. These spots are then imaged using conventional
fluorescence microscopy. The sensitivity, linearity, and dynamic range
achievable from
the various combinations of fluorochrome and solid surfaces (e.g., membranes,
glass,
fused silica) can thus be determined. Serial dilutions of pairs of
fluorochrome in known
relative proportions can also be analyzed. This determines the accuracy with
which
fluorescence ratio measurements reflect actual fluorochrome ratios over the
dynamic
range permitted by the detectors and fluorescence of the substrate upon which
the probe
has been fixed.
Probes useful in the methods described here are available from a number
of sources. For instance, Pl clones are available from the DuPont P1 library
(Shepard, et
al., Proc. Natl. Acad. Sci. USA, 92: 2629 (1994), and available commercially
from
Genome Systems. Various libraries spanning entire chromosomes are also
available
commercially (Clonetech, South San Francisco, CA), or from the Los Alamos
National
Laboratory.
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CA 02368903 2001-10-05
WO 00/61814 PCT/US00/09609
Labeling and Detection of Nucleic Acids.
In a preferred embodiment, the hybridized nucleic acids are detected by
detecting one or more labels attached to the sample or probe nucleic acids.
The labels
may be incorporated by any of a number of means well known to those of skill
in the art.
Means of attaching labels to nucleic acids include, for example nick
translation or end-
labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and
subsequent
attachment (ligation) of a nucleic acid linker joining the sample nucleic acid
to a label
(e.g., a fluorophore). A wide variety of linkers for the attachment of labels
to nucleic
acids are also known. In addition, intercalating dyes and fluorescent
nucleotides can also
be used.
Detectable labels suitable for use in the present invention include any
composition detectable by spectroscopic, photochemical, biochemical,
immunochemical,
electrical, optical or chemical means. Useful labels in the present invention
include biotin
for staining with labeled streptavidin conjugate, magnetic beads (e.g.,
DynabeadsTM),
fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent
protein, and
the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels
(e.g., 3H, ~ZSI,
3sS, '4C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others
commonly used in an ELISA), and colorimetric labels such as colloidal gold
(e.g., gold
particles in the 40 -80 nm diameter size range scatter green light with high
efficiency) or
colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Patents
teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752;
3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241.
A fluorescent label is preferred because it provides a very strong signal
with low background. It is also optically detectable at high resolution and
sensitivity
through a quick scanning procedure. The nucleic acid samples can all be
labeled with a
single label, e.g., a single fluorescent label. Alternatively, in another
embodiment,
different nucleic acid samples can be simultaneously hybridized where each
nucleic acid
sample has a different label. For instance, one target could have a green
fluorescent label
and a second target could have a red fluorescent label. The scanning step will
distinguish
cites of binding of the red label from those binding the green fluorescent
label. Each
nucleic acid sample (target nucleic acid) can be analyzed independently from
one another.
Suitable chromogens which can be employed include those molecules and
compounds which absorb light in a distinctive range of wavelengths so that a
color can be
24
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observed or, alternatively, which emit light when irradiated with radiation of
a particular
wave length or wave length range, e.g., fluorescers.
Desirably, fluorescers should absorb light above about 300 nm, preferably
about 350 nm, and more preferably above about 400 nm, usually emitting at
wavelengths
S greater than about 10 nm higher than the wavelength of the light absorbed.
It should be
noted that the absorption and emission characteristics of the bound dye can
differ from
the unbound dye. Therefore, when refernng to the various wavelength ranges and
characteristics of the dyes, it is intended to indicate the dyes as employed
and not the dye
which is unconjugated and characterized in an arbitrary solvent.
Fluorescers are generally preferred because by irradiating a fluorescer with
light, one can obtain a plurality of emissions. Thus, a single label can
provide for a
plurality of measurable events.
Detectable signal can also be provided by chemiluminescent and
bioluminescent sources. Chemiluminescent sources include a compound which
becomes
electronically excited by a chemical reaction and can then emit light which
serves as the
detectable signal or donates energy to a fluorescent acceptor. Alternatively,
luciferins can
be used in conjunction with luciferase or lucigenins to provide
bioluminescence.
Spin labels are provided by reporter molecules with an unpaired electron spin
which can
be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin
labels
include organic free radicals, transitional metal complexes, particularly
vanadium,
copper, iron, and manganese, and the like. Exemplary spin labels include
nitroxide free
radicals.
The label may be added to the target (sample) nucleic acids) prior to, or
after the hybridization. So called "direct labels" are detectable labels that
are directly
attached to or incorporated into the target (sample) nucleic acid prior to
hybridization. In
contrast, so called "indirect labels" are joined to the hybrid duplex after
hybridization.
Often, the indirect label is attached to a binding moiety that has been
attached to the
target nucleic acid prior to the hybridization. Thus, for example, the target
nucleic acid
may be biotinylated before the hybridization. After hybridization, an avidin-
conjugated
fluorophore will bind the biotin bearing hybrid duplexes providing a label
that is easily
detected. The nucleic acid probe may also be labeled with digoxigenin and then
detected
with an antibody that is labeled with a fluorochrom, or an enzyme such as
horseradish
peroxidase or alkaline phosphatase. For a detailed review of methods of
labeling nucleic
acids and detecting labeled hybridized nucleic acids see Laboratory Techniques
in
CA 02368903 2001-10-05
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Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid
Probes,
P. Tijssen, ed. Elsevier, N.Y., (1993)).
Fluorescent labels are easily added during an in vitro transcription
reaction. Thus, for example, fluorescein labeled UTP and CTP can be
incorporated into
the RNA produced in an in vitro transcription.
The labels can be attached directly or through a linker moiety. In general,
the site of label or linker-label attachment is not limited to any specific
position. For
example, a label may be attached to a nucleoside, nucleotide, or analogue
thereof at any
position that does not interfere with detection or hybridization as desired.
For example,
certain Label-ON Reagents from Clontech (Palo Alto, CA) provide for labeling
interspersed throughout the phosphate backbone of an oligonucleotide and for
terminal
labeling at the 3' and 5' ends. As shown for example herein, labels can be
attached at
positions on the ribose ring or the ribose can be modified and even eliminated
as desired.
The base moieties of useful labeling reagents can include those that are
naturally
occurring or modified in a manner that does not interfere with the purpose to
which they
are put. Modified bases include but are not limited to 7-deaza A and G, 7-
deaza-8-aza A
and G, and other heterocyclic moieties.
It will be recognized that fluorescent labels are not to be limited to single
species organic molecules, but include inorganic molecules, multi-molecular
mixtures of
organic and/or inorganic molecules, crystals, heteropolymers, and the like.
Thus, for
example, CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be
easily
derivatized for coupling to a biological molecule (Bruchez et al. (1998)
Science, 281:
2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped
cadmium
selenide) have been covalently coupled to biomolecules for use in
ultrasensitive
biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
Amplification-based Assay
In another embodiment, amplification-based assays can be used to measure
copy number. In such amplification-based assays, the nucleic acid sequences
act as a
template in an amplification reaction (e.g. Polymerase Chain Reaction (PCR).
In a
quantitative amplification, the amount of amplification product will be
proportional to the
amount of template in the original sample. Comparison to appropriate (e.g.
healthy
tissue) controls provides a measure of the copy number of the desired target
nucleic acid
sequence. Methods of "quantitative" amplification are well known to those of
skill in the
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WO 00/61814 PCT/US00/09609
art. For example, quantitative PCR involves simultaneously co-amplifying a
known
quantity of a control sequence using the same primers. This provides an
internal standard
that may be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR
are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications,
Academic Press, Inc. N.Y.).
Other suitable amplification methods include, but are not limited to ligase
chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et
al.
(1988) Science 241: 1077, and Barnnger et al. (1990) Gene 89: 117,
transcription
amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), and
self
sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci.
USA 87:
1874).
Detection of Gene Expression
As indicated below, a number of oncogenes, particularly H-RAS, are found
in the regions of amplification disclosed here. Thus, oncogene activity can be
detected
by, for instance, measuring levels of the gene transcript (e.g. mRNA), or by
measuring
the quantity of translated protein.
Detection of Gene Transcripts.
Methods of detecting and/or quantifying t gene transcripts using nucleic
acid hybridization techniques are known to those of skill in the art (see
Sambrook et al.
supra). For example , a Northern transfer may be used for the detection of the
desired
mRNA directly. In brief, the mRNA is isolated from a given cell sample using,
for
example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is
then
electrophoresed to separate the mRNA species and the mRNA is transferred from
the gel
to a nitrocellulose membrane. As with the Southern blots, labeled probes are
used to
identify and/or quantify the target mRNA.
In another preferred embodiment, the gene transcript can be measured
using amplification (e.g. PCR) based methods as described above for directly
assessing
copy number of the target sequences.
Detection of Expressed Protein
The presence of polypeptides encoded by regions of the chromosome that
are amplified, e.g, H-RAS can also be detected and/or quantified by detecting
or
27
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WO 00/61814 PCT/LTS00/09609
quantifying the expressed polypeptide. The polypeptide can be detected and
quantified
by any of a number of means well known to those of skill in the art. These may
include
analytic biochemical methods such as electrophoresis, capillary
electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography (TLC),
hyperdiffusion chromatography, and the like, or various immunological methods
such as
fluid or gel precipitin reactions, immunodiffusion (single or double),
immunoelectro-
phoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),
immunofluorescent assays, western blotting, and the like. For a review of the
general
immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology,
volume 37
(Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.
1991).
Immunoassays can be performed in any of several configurations, which
are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow
&
Lane, supra. Immunoassays also often use a labeling agent to specifically bind
to and
label the complex formed by the antibody and antigen. The labeling agent may
itself be
one of the moieties comprising the antibody/antigen complex. For example, in
detecting
H-RAS, the labeling agent may be a labeled H-RAS polypeptide or a labeled anti-
H-RAS
antibody. Alternatively, the labeling agent may be a third moiety, such a
secondary
antibody, that specifically binds to the antibody/H-RAS complex (a secondary
antibody is
typically specific to antibodies of the species from which the first antibody
is derived).
Other proteins capable of specifically binding immunoglobulin constant
regions, such as protein A or protein G, may also be used as the label agent.
These
proteins exhibit a strong nonimmunogenic reactivity with immunoglobulin
constant
regions from a variety of species (see, e.g., Kronval et al., J. Immunol.
111:1401-1406
(1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)). The labeling
agent can be
modified with a detectable moiety, such as biotin, to which another molecule
can
specifically bind, such as streptavidin. A variety of detectable moieties are
well known to
those skilled in the art.
Throughout the assays, incubation and/or washing steps may be required
after each combination of reagents. Incubation steps can vary from about 5
seconds to
several hours, optionally from about 5 minutes to about 24 hours. However, the
incubation time will depend upon the assay format, antigen, volume of
solution,
concentrations, and the like. Usually, the assays will be carned out at
ambient
28
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WO 00/61814 PCT/US00/09609
temperature, although they can be conducted over a range of temperatures, such
as 10°C
to 40°C.
Immunoassays for detecting polypeptides in a sample may be either
competitive or noncompetitive. Noncompetitive immunoassays are assays in which
the
amount of antigen is directly measured. In one preferred "sandwich" assay for
detecting,
for example, H-RAS, the anti-H-RAS antibodies can be bound directly to a solid
substrate
on which they are immobilized. These immobilized antibodies then capture the H-
RAS
protein present in the test sample. The H-RAS thus immobilized is then bound
by a
labeling agent, such as a second antibody bearing a label. Alternatively, the
second
antibody may lack a label, but it may, in turn, be bound by a labeled third
antibody
specific to antibodies of the species from which the second antibody is
derived. The
second or third antibody is typically modified with a detectable moiety, such
as biotin, to
which another molecule specifically binds, e.g., streptavidin, to provide a
detectable
moiety.
In competitive assays, the amount of a polypeptide present in the sample is
measured indirectly by measuring the amount of a known, added (exogenous)
protein
displaced (competed away) from an anti-polypeptide antibody by the unknown
polypeptide present in a sample. In one competitive assay, for example, a
known amount
of H-RAS protein is added to a sample and the sample is then contacted with an
antibody
that specifically binds to the H-RAS protein. The amount of exogenous H-RAS
protein
bound to the antibody is inversely proportional to the concentration of H-RAS
protein
present in the sample. In a particularly preferred embodiment, the antibody is
immobilized on a solid substrate. The amount of H-RAS bound to the antibody
may be
determined either by measuring the amount of H-RAS present in a H-RAS/antibody
complex, or alternatively by measuring the amount of remaining uncomplexed
protein.
The amount of H-RAS may be detected by providing a labeled H-RAS molecule.
A hapten inhibition assay is another preferred competitive assay. In this
assay, the known protein is immobilized on a solid substrate. A known amount
of
antibody to the protein is added to the sample, and the sample is then
contacted with the
immobilized protein. The amount of antibody bound to the known immobilized
protein is
inversely proportional to the amount of protein present in the sample. Again,
the amount
of immobilized antibody may be detected by detecting either the immobilized
fraction of
antibody or the fraction of the antibody that remains in solution. Detection
may be direct
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where the antibody is labeled or indirect by the subsequent addition of a
labeled moiety
that specifically binds to the antibody as described above.
Western blot (immunoblot) analysis is used to detect and quantify the
presence of a polypeptide in a sample. The technique generally comprises
separating
sample proteins by gel electrophoresis on the basis of molecular weight,
transfernng the
separated proteins to a suitable solid support, (such as a nitrocellulose
filter, a nylon filter,
or derivatized nylon filter), and incubating the sample with the antibodies
that specifically
bind the polypeptide, e.g., H-RAS, and/or antibodies that specifically bind to
mutated
versions of the polypeptide. The polypeptide antibodies specifically bind to
the
polypeptide on the solid support. These antibodies may be directly labeled or
alternatively may be subsequently detected using labeled antibodies (e.g.,
labeled sheep
anti-mouse antibodies) that specifically bind to the antibodies.
Other assay formats include liposome immunoassays (LIA), which use
liposomes designed to bind specific molecules (e.g., antibodies) and release
encapsulated
reagents or markers. The released chemicals are then detected according to
standard
techniques (see Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 (1986)).
Kits for Use in Diagnostic and/or Prognostic Applications
For use in diagnostic, research ,and therapeutic applications suggested
above, kits are also provided by the invention. In the diagnostic and research
applications
such kits may include any or all of the following: assay reagents, buffers,
nucleic acids
for detecting the target sequences and other hybridization probes and/or
primers. A
therapeutic product may include sterile saline or another pharmaceutically
acceptable
emulsion and suspension base.
In addition, the kits may include instructional materials containing
directions (i. e., protocols) for the practice of the methods of this
invention. While the
instructional materials typically comprise written or printed materials they
are not limited
to such. Any medium capable of storing such instructions and communicating
them to an
end user is contemplated by this invention. Such media include, but are not
limited to
electronic storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media
(e.g., CD ROM), and the like. Such media may include addresses to Internet
sites that
provide such instructional materials.
CA 02368903 2001-10-05
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EXAMPLES
Example One' CGH and FISH studies of Spitz nevi cells
This example demonstrates that using CGH studies, Spitz nevi cells are
shown to have a gain in chromosomal region 11. Seventeen (17) cases of Spitz
nevi were
studied using CGH. The procedures of CGH were performed following standard
protocols as described as follows.
Material and Methods
Tumor Material
Formalin-fixed, paraffin-embedded tissue from Spitz nevi from 17 patients
were retrieved from the archives of the Department of Dermatology (University
Hospital,
Wurzburg, Germany) and the Dermatopathology Section, Departments of Pathology
and
Dermatology (University of California, San Francisco). We selected lesions
that had an
1 S extensive and densely cellular dermal component that allowed the
collection of mostly
melanocytes and had at most a sparse lymphocytic infiltrate, so that
lymphocyte DNA
would not obscure aberrations in the neoplastic cells.
DNA Preparation
Paraffin material: 30~m sections were cut, with a S~m section for H & E
every 5 sections. The unstained 30 pm sections were placed on glass slides and
an area of
interest was microdissected without de-paraffinizing.
Microdissection was carried out manually under a dissecting microscope.
Depending on the size of the tumor 20-60 unstained sections were used and
regions with
a high density of tumor cells were separated from normal cells. The dissected
tumor parts
were collected in tubes and de-paraffinized by washing with xylene and
ethanol. DNA
extraction and labeling was performed as published by Isola et al. (8).
Briefly, tissue was
incubated until complete digestion (3 days) with proteinase K (Life
Technologies, Inc.,
Gaithersburg, MD) in a 50 mM Tris pH8.5, 1mM EDTA, 0.5% Tween 20 buffer. DNA
was extracted with phenol-chloroform-isoamylalcohol (25:24:1, v/v),
precipitated with
7.5 M ammonium acetate and 100% ethanol, and resuspended in water. The amount
of
DNA obtained ranged from 2 to l2pg.
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Comparative Genomic Hybridization (CGH) and Digital Image
Analysis
All tumors were measured both with the tumor DNA labeled with
fluorescein-12-dUTP (DuPont, Inc., Boston, MA), and reference DNA with Texas
red-5-
dUTP ("standard" labeling), and with the labeling reversed. Labeling was
performed by
Nick translation. Nick translation conditions were adjusted so that the mean
probe
fragment size after labeling ranged between 800 and 1500 bp. The hybridization
mixture
consisted of 200-1000 ng of labeled tumor DNA, 200 ng inversely labeled sex-
matched
normal human reference DNA from peripheral blood lymphocytes, and 25 ug human
Cot-
1 DNA (Life Technologies, Inc., Gaithersburg, MD) dissolved in 10 pl
hybridization
buffer (SO% formamide, 10% dextrane sulfate, and 2 X SSC, pH 7.0).
Hybridization was
carried out for 2-3 days at 37°C to normal metaphases (9). All samples
were investigated
with a single batch of metaphase slides. Slides were washed three times in a
washing
solution (SO% formamide in 2 X SSC, pH) at 45°C, once in PN buffer (0.1
M NaH2P04,
0.1 M Na2HP04, and 0.1 % Nonidet P40, pH 8.0), and once in distilled water
(both 10
minutes at room temperature). Slides were counterstained with 4,6-diamino-2-
phenylindole in an anti-fade solution. Hybridization quality was evaluated as
published
previously (7). Digital images were collected from five metaphases with a
Photometrics
CCD camera (Microimager 1400, Xillix Technologies, Vancouver, British
Columbia,
Canada) on a standard fluorescence microscope. The average tumor/reference
fluorescence ratios along each chromosome were calculated with custom CGH
analysis
software. Measurements were made on at least 4 copies of each autosome.
Controls and Threshold Definitions
Normal DNA and DNA from tumor cell lines with known aberrations
were used as controls. We regarded a region as aberrant when 1 ) either the
standard
labeling or the reverse labeling resulted in a tumor:reference fluorescent
ratios <0.80 or >
1.2 or 2) both the standard and the reverse labeling resulted in a
tumor:reference
fluorescent ratios <0.85 or >1.15.
Results of this experiment showed that 13 tumors did not show any
chromosomal aberrations. One case had an isolated gain of the distal part of
chromosome
7, 7q21-qter. Three cases showed a single high level gain of the entire short
arm of
chromosome 11 (Fig. 2). This phenomenon of a gain in chromosome l lp of Spitz
nevi
cells is not seen among melanoma cells, as shown in Fig. 1.
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Fluorescence in-situ hybridization (FISH)
Dual-color FISH was carned out on tissue sections of the cases in which
tissue was left after CGH (14/17). Probes mapping to the short arm (RMCl 1B022
and
RMC11P014) and the long arm (RMC11P008) of chromosome 11 were obtained from
the
resource of the laboratory. Probes were labeled by nick translation with Cy3
(Amersham,
Arlington Heights, IL) or Digoxigenin (Boehringer Mannheim, Indianapolis IN).
6pm
sections were mounted on positively charged glass slides (Fisher Scientific,
Pittsburgh,
PA), deparaffinized, and hydrated by decreasing strength ethanol. Sections
were
incubated for 2-4 min in 1M sodium thiocyanate at 80°C , in 4 mg/ml
Pepsin in 0.2 N
HCI at 37°C for 4-8 min, dehydrated by increasing strength ethanol and
air-dried. Slides
were denatured in 70% formamide, 2x SSC pH 7.0 for 5 min at 72°C, and
dehydrated
again in a graded ethanol series. 2.5 to 25 ng of each of the labeled probes
together with
20pg Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD) were dissolved in
101
hybridization buffer (50% formamide, 10% dextrane sulfate, and 2 x SSC, pH
7.0) and
denatured for 10 min at 72°C. Hybridization was carried out for 48-72
hours at 37°C.
Slides were washed three times in washing solution (50% formamide in 2 x SSC,
pH 7.0)
at 45°C, once in 2x SSC at 45°C, once in 2x SSC at room
temperature (RT), and once in
0.1% Triton X100 in 4 x SSC/ at RT. Subsequently, sections were incubated with
10%
BSA in 4 x SSC in a moist chamber at 37 °C, and then with a FITC
labeled anti-
digoxigenin antibody (Boehringer Mannheim, Indianapolis IN) diluted in 4 x SSC
with
10% BSA. Sections were counterstained with 4,6-diamino-2-phenylindole (Sigma,
St.
Louis, MS) in an anti-fade solution. The two-tailed student's t-test was used
for the
comparison of FISH signals for the locus of interest and the reference probe.
Results
Table 1 shows the clinical information of the Spitz nevi patients, and
aberrations found by CGH and FISH. Patient age ranged from 3-45 years (mean 18
years). Follow-up was available from most patients. The follow-up time was 1.2-
9 years
(mean 4.9 years). All patients with available follow-up were free of disease
by the end of
the follow-up interval. In one case (case 16) 2 recurrences prior to the final
excision of
the lesion that entered the study occurred, possibly because the tumor was
curetted twice.
Recut sections of all cases represented typical Spitz nevi by
histopathological
examination. 13 of the 17 tumors (76%) showed no DNA copy number changes by
CGH.
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Three cases (18%) showed gain of the entire short arm of chromosome 11 as the
sole
abnormality. (Fig. 3). One case showed gain of chromosome 7q21-qter as the
only
abnormality (Fig. 3).
FISH measurements were performed to tissue sections in order to study the
histopathologic distribution of the recurrent gain on chromosome 1 lp and to
find
potential minor populations of cells with this aberration in the cases with
normal CGH
profiles. A test probe was selected that mapped to the distal part of
chromosome l lp
( 1 lp 15.5, clone RMC 11 B022) and a reference probe mapping to chromosome 11
q23
(clone RMC11P008). In all experiments keratinocytes of the epidermis adjacent
to the
lesion were used as internal controls. As the hybridization was carried out on
sections of
6pm thickness, many nuclei were not fully represented in the slide. For
counting
hybridization signals, we selected nuclei that appeared minimally truncated
when the
focus of the microscope was slightly altered. The nuclear signal counts in
keratinocytes
for the q-arm and the p-arm probe ranged from mean values of 1.6-1.9 and 1.7-
1.9,
respectively (Fig. 4b, 4d, 4f). A mean of 2.0 is expected if all counted
nuclei are intact
and the hybridization efficiency is 100%. The numbers of p-arm signals tended
to be
slightly higher than that of the q-arm, which can be explained by the larger
size of the p-
arm probe, resulting in slightly higher hybridization efficiencies.
However, this difference was not statistically significant. As the variance
of the signal number was low (0.16-0.24) in this control population of
supposedly normal
cells, counting 20 cells per tumor was sufficient to establish the success of
the
hybridization. When analyzing the neoplastic cells, 20 cells of each
morphologically
distinct subpopulation were counted. The three cases that had a gain of
chromosome l lp
by CGH showed a mean of 3.5-5.3 signals with the p-arm probe compared to a
mean of
1.5-2.1 counts for the q-arm probe (Fig. 4c,). This difference was highly
significant
(p<0.00001). The counts for the q-arm (control) probes were not statistically
different
from signal counts in keratinocytes (normal cells) of the respective lesions.
The ratio of
p-arm signals to q-arm signals in the cases with increased copies of
chromosome 1 lp
ranged from 1.8-3Ø The increased signal number of the p-arm probe was
present in
virtually every cell of each the nevi. From the 14 tumors that had no gain of
chromosome
1 lp by CGH twelve could be studied by FISH. In the other two cases the
paraffin blocks
were exhausted. Of these twelve cases, eleven had no significant differences
in signal
distribution of the probes for p-arm and the q-arm of chromosome 11 (Fig. 4a,
4b). One
case (case 5) had 2.4 p-arm signals vs. 1.9 q-arm signals, a difference which
was
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statistically significant (p=0.01). In two cases (cases 3 and 15) a
subpopulation of cells
was present that had increased numbers of both the q-arm and the p-arm signal
(Fig. 4e).
These cells mostly had considerably larger nuclei than the tumor cells with 1-
2 signals,
and are thus likely to be polyploid.
As illustrated in Fig. 3, the area of chromosome 11 that was found by CGH
to be gained in three cases seems to be identical. The profiles of case 13 and
case 16
suggest the highest increase of DNA copy number towards the p-telomere.
However, the
profiles of the CGH measurement in which the labeling was reversed showed a
decrease
of red:green fluorescence ratio toward the telomere, indicating that the p-
telomeric ratio
increase is artifactual. To confirm this, FISH experiments were performed with
a
different probe for the p-arm that mapped more proximally to l 1p14
(RMC11P014). The
number of signals in the nuclei of the tumor cells with this probe was similar
to that found
with the probe for 11 p 15.
One probe mapped to the distal part of the p-arm of chromosome 11 and
the second probe mapped to l lq. Of the three cases that showed a gain of
chromosome
1 lp, 6-10 signals of the 1 lp probe per nucleus were detected, whereas the
probe that
mapped to the q-arm only gave two signals. Interestingly, the signal number
was
virtually constant over the entire lesion, suggesting a clonal nature of the
neoplasms.
Among the other Spitz nevi studied which showed no indication of l lp gain in
CGH,
only one additional case showed an amplification of l lp. All other cases had
two signals
of both markers. Exceptions were cells with large nuclei that occurred in
three tumors.
Those cells had up to 10 signals of both markers. These findings suggest that
gain of
chromosome 1 lp is a recurrent aberration in Spitz nevi. The cells of Spitz
nevi are
diploid, with the exception of cells with large nuclei that can be polyploid.
Spitz nevi are
clonal neoplasms.
Table 1: Clinical information of the Spitz nevi and aberrations found by CGH
and FISH.
(PSCT = pigmented spindle cell tumor, FOD = free of disease, NA = not
available).
CA 02368903 2001-10-05
WO 00/61814 PCT/US00/09609
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36
CA 02368903 2001-10-05
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37
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WO 00/61814 PCT/US00/09609
Discussion
These results show that the majority of Spitz nevi have a normal
chromosomal complement, but that a subset may have abnormalities. We detected
gains
of chromosome l lp in 4/17 cases. Thus Spitz nevi is one of the many benign
lesions that
contain genetic abnormalities at the chromosomal level. The pattern of
chromosomal
aberrations in Spitz nevi shows clear differences from that observed in
primary cutaneous
melanoma. In the latter only a small minority of cases does show no
aberrations when
analyzed by CGH. In this example, CGH measurements were performed on 102
melanomas and only 5 cases did not show changes. The 5 cases without
detectable
aberrations had considerable contamination of normal cells in the tumor that
may account
for at least a part for these findings. In the Spitz nevi of the present study
cases with an
inflammatory component were excluded, so that contamination of normal cells
could not
have accounted for the high frequency of negative findings.
The finding of an increased copy number of chromosome 1 lp in four out
of 17 lesions indicates that this aberration represents a recurrent change in
Spitz nevi. It
suggests that increased dosage of genes of chromosome 1 lp has relevance in
the
pathogenesis of this tumor. As the gained genomic fragment is large,
additional studies
are warranted to refine the extent of the region as a first step toward
identifying the
critical gene(s). It may well be that in the Spitz nevi without l lp gain,
those genes, or the
pathways they belong to, are activated by a different mechanism than increase
of gene
copy number.
A gain of chromosome l lp was not seen in any of the 102 primary
melanomas we have analyzed by CGH. Gains including l lp were found in only two
cases of the 239 published karyotypes, mostly from metastatic melanomas (11,
12).
However, in these two cases the gain extended far on to the q-arm ( 12).
Furthermore, none of the most frequent findings in primary melanomas,
losses of chromosomes 9 and 10, was found in any of the Spitz nevi. However,
chromosome 7 was gained in SO% of our melanoma cases (7), and one of the Spitz
nevi
showed a gain of chromosome 7q23-pter. Thus, even though future studies will
be
required to determine the full spectrum and frequency of aberrations in Spitz
nevi, the
current data clearly shows that there is a clear difference in the pattern of
chromosomal
gains and losses in melanoma and Spitz nevi. These differences raise the
possibility of
defining genetic markers that can be used for diagnostic purposes. Cytogenetic
studies
38
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have been of great help in the classification of soft tissue tumors and can
provide pivotal
diagnostic information (13). A diagnostic test for spitzoid melanocytic
neoplasms might
include copy number detection of chromosomes l lp, 9, and 10. Gains of
chromosome
1 lp could be interpreted as in favor of Spitz nevus, and losses of
chromosomes 9 and/or
as in favor of melanoma.
It is indeed remarkable that the chromosomal alterations most frequently
found in primary melanomas are absent in Spitz nevi. And this absence of
aberrations
frequently found in melanoma may be a difference that may indeed offer
diagnostic
opportunities. Previous CGH studies on 32 primary melanomas showed losses of
10 chromosome 9p in 82%, chromosome 10 in 63%, and 6q in 28% of the cases
(Bastian et
al. 1998). Frequent gains in melanoma involved chromosome 7p (50%), 8q (34%),
and
6p (28%). None of these changes was found in our series of Spitz nevi. Note
that one
study found interstitial deletions of chromosome 9p in two out of 27 Spitz
nevi indicating
that losses of 9p are not exclusive to melanoma (Healy E, et al., ALLELOTYPES
OF
PRIMARY CUTANEOUS MELANOMA AND BENIGN MELANOCYTIC NEVI, Cancer Res 56: 589,
1996). It may thus be that the determination of copy number of other
chromosomal
regions such as lq, 6p, 7p, and lOq, may prove to be more helpful in the
differential
diagnosis. The efficacy of such a test needs to be evaluated through the
analysis of a
larger set of tumors with the inclusion of cases that have conflicting
histopathologic
criteria but have known follow-up. This will permit determination of the
sensitivity and
specificity under clinically relevant conditions.
FISH measurements not only confirmed the CGH findings but also
allowed some interesting insight into the ploidy and clonality of Spitz nevi.
Since almost
all cells in the nevi had 2 copies of the control locus on 1 lq by FISH and
CGH showed no
aberrant copy numbers for that locus, the large majority of the cells in these
nevi are
diploid, which is consistent with previous flow cytometry studies ( 10). Two
cases had a
subpopulation of cells with large nuclei. Those cells elevated copy number had
elevated
FISH signals for the two loci tested, indicating that the increased nuclear
size is most
likely due to polyploidy. These data also show that Spitz nevi are probably
comprised of
a monoclonal population of melanocytes. This can be concluded from the three
cases
with a gained 1 lp, because the increased copy number of this chromosomal arm
was
present in all cells of the lesions
In summary, this example shows that in Spitz nevi, (I) the majority of
cases have a normal chromosomal complement at the level of CGH resolution,
(II) gains
39
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of chromosome 1 lp represent a recurrent aberration in a subset of lesions,
(III) Spitz nevi
are probably clonal neoplasms, (IV) the majority of the melanocytes of a Spitz
nevus are
diploid with the exception of cells with large nuclei which can be polyploid,
and (V) the
clear differences in the location and frequencies of the cytogenetically
detectable
aberrations in primary cutaneous melanoma and Spitz nevi make CGH and FISH
promising techniques for refining diagnostic accuracy of this difficult
differential
diagnosis.
Example Two: FISH study of melanocytic tumor using chromosome 9 probes
This example demonstrates FISH experiments using chromosome 9 probes
in detecting primary melanoma cells.
PI-clones for chromosome 9 were similarly used for FISH studies of
sections of primary melanomas. Loss of chromosome 9 was the most frequent
finding in
the CGH-study of melanoma. The FISH experiments showed that in most cases of
melanoma 0-1 signals per nucleus with a probe for chromosome 9p was detected,
whereas
a simultaneously hybridized reference locus revealed more than 2 signals per
nucleus.
This indicates that FISH is capable of detecting homozygous and heterozygous
deletions
in tissue sections.
The selection of hybridization probes will thus be based on the following
criteria: (a) the corresponding chromosomal regions should show frequent
aberration in
one neoplasm and not in the other (e.g. lq, 6p, 7p, 9p, lOq, and l lp), (b)
probes should
give strong and reproducible hybridization signals.
Example Three: Tissue Hvbridization Protocols
This example demonstrates the use of tissue hybridization protocols in
studying the difference in signal ratios per chromosome locus between melanoma
cells
and Spitz nevi cells.
A hybridization protocol is adapted from Thompson et al., Cancer Genet
Cytogenet 83, 93-104 (1995). Briefly, tissue sections are mounted on
positively charged
slides. The slides are heated at 55°C for about 30 minutes and
deparaffinized with
xylene, and ethanol dehydrated. They are then sequentially incubated in NaSCN,
followed by Pepsin. After being denatured in formamide, they are hybridized
using
standard techniques. Probes will be labeled directly with Cy-3 and indirectly
with
digoxigenin that will later be detected with FITC-labeled anti-digoxigenin
antibodies.
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Alternative labeling approaches may be employed so as to be able to detect
three
differentially labeled probes in one hybridization.
Based on previous studies, it is expected that counting signals of each
hybridization probe in a total of 25 tumor cell nuclei and 25 nuclei of normal
tissue cells
will suffice. One parameter for decision making will be the ratio of average
number of
signals per locus per tumor cell compared to the average number of signals per
locus in
normal cells within the tissue (e.g. keratinocytes of the epidermis or
epidermal
appendages). According to the preliminary studies, the ratio is expected to be
less than
one for loci frequently lost in melanoma and more than one for loci gained in
Spitz nevi.
The second parameter will be the variance of the signal number per tumor
cell. Based on previous studies and experience of others, the variance is
expected to be
significantly higher in malignant tumors than in benign tumors (De Wit et al.,
JPathol
173, 227-33 ( 1994)).
Example Four: Identification of H-RAS mutations
This example demonstrates that a mutated H-RAS gene is associated with
Spitz nevus. The procedures to identify H-RAS mutations were performed
following
standard protocols as described as follows.
Selection of cases: Paraffin blocks of Spitz nevi were retrieved randomly from
the
archives of the Dermatopathology section of the Departments of Dermatology and
Pathology at the University of California, San Francisco. We performed a
computer
search of the database of the Dermatopathology Section with the following
criteria: select
all cases from 1/1/98 to 12/31/98 that were assigned a main diagnosis of one
30 different
descriptive variants of Spitz nevus that are used in our laboratory. Cases
sent in as a slide
in consultation were excluded in order to avoid a bias towards unusual Spitz
nevi. The
request yielded 144 cases from which blocks were available.
In addition to these cases, 22 cases of the Department of Dermatology
University of Wiirzburg, Germany were included in the study. These cases had
originally
been retrieved for comparative genomic hybridization and only included Spitz
nevi with
at least 1 mm thickness.
Assembly of tissue arrays: Tissue arrays were constructed according to Kononen
et. al.,
(Nat. Med. 4:844-847, 1998). In brief, a tissue arraying instrument (Beecher
Instruments,
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Silver Spring, MD) was used to punch 0.8 mm biopsy cores of the most cellular
areas of
the nevi. The biopsy cores were arrayed in recipient paraffin blocks,
according to the
manufacturer's instructions. Multiple sections of 6 pm thickness were cut with
a
microtome using an adhesive-coated tape sectioning system (Instrumedics,
Hackensack,
NJ). H&E sections were used for the histological examination of the biopsy
cores. Only
cases with at least one area with a cohesive population of neoplastic
melanocytes were
included in the analysis.
FISH to formalin-fixed tissue microarray sections: Dual-color FISH was carried
out on
tissue sections of the array as described previously (Bastian et al., J.
Invest. Dermatol.
113:1065-1069, 1999). We used a BAC clone (RMC11B022) that contained H-RAS for
the detection of amplifications of chromosome l lp, and a reference P1 clone
(RMC 11 P008) for the q-arm of chromosome 11. Probes were labeled with Cy3
(Amersham, Arlington Heights, IL) or with digoxigenin (Boehringer Mannheim,
Indianapolis IN) by nick-translation. Tissue sections were deparaffinized,
hydrated, and
pre-treated for 2-4 min in 1M sodium thiocyanate at 80°C, in 4 mg/ml
Pepsin in 0.2 N
HCl at 37°C for 4-8 min. After dehydration, sections were denatured in
70% formamide,
2x SSC pH 7.0 for 5 min at 72°C, and hybridized over 48-72 h at
37°C in lOpl
hybridization buffer (SO% formamide, 10% dextran sulfate, and 2 x SSC, pH 7.0,
20pg
Cot-1 DNA (Life Technologies, Inc., Gaithersburg, MD)). Slides were washed
three
times in washing solution (50% formamide in 2 x SSC, pH 7.0) at 45°C,
once in 2x SSC
at 45°C, once in 2x SSC at room temperature (RT), and once in 0.1%
TritonX100 in 4 x
SSC/ at RT. Subsequently, sections were incubated with 10% BSA in 4 x SSC in a
moist
chamber at 37 °C, and then with a FITC labeled anti-digoxigenin
antibody (Boehringer
Mannheim, Indianapolis IN) diluted in 4 x SSC with 10% BSA. Sections were
counterstained with 4,6-diamino-2-phenylindole (Sigma, St. Louis, MS) in an
anti-fade
solution. FISH signals were scored with a fluorescence microscope Zeiss (Jena,
Germany) using a 63X objective. Criteria for amplification were: at least
three times
more test probe signals than reference signals in at least 30% of the tumor
cells.
DNA Sequence Analysis: DNA was extracted from 30 ~m sections from which the
tumor-
bearing areas were dissected manually with a scalpel under a dissecting
microscope. Two
to three sections were collected in a 0.5 ml tube and after washing with
xylene and
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ethanol were incubated at 55 °C with 0.4mg/ml proteinase K (Life
Technologies, Inc.,
Gaithersburg, MD) in PCR buffer (Perkin Elmer) containing 0.5% Tween 20 for
three
days. Fresh proteinase K was added every 24h to a final concentration of
0.4mg/ml.
HRAS codon 12 primers were 5'-AGGAGACCCTGTAGGAGGA-3' (SEQ ID NO:1)
(forward) and 5'-CGCTAGGCTCACCTCTATAGTG-3' (SEQ ID N0:2) (reverse) and
codon 61 primers were 5'-CTGCAGGATTCCTACCGGA-3' (SEQ ID N0:3) and 5'-
ACTTGGTGTTGTTGATGGCA-3' (SEQ ID N0:4). PCR was carned out in a Gene
Amp PCR System 9700 Thermal Cycler (Perkin Elmer) in 25 ~l reaction volumes.
Each
PCR reaction contained 3.5 mM MgCl2, 0.2 mM dNTP, 0.625 U Taq Gold Polymerise
(Perkin Elmer), 1X PCR Buffer II, 0.5 ~M each of forward and reverse primer,
and 50-
300 ng of genomic DNA. PCR cycling conditions were as follows: 95 °C
for 15 min
followed by 35 cycles of 95 °C for 15 sec, 55 °C for 30 seconds,
and 72 °C for 60
seconds, and a final hold at 72 °C for 10 minutes.
Prior to sequencing, PCR products were purified using the PCR product
Pre-sequencing kit (Amersham, Arlington Heights, IL) to remove excess primers
and
nucleotides. Fluorescent DNA sequencing was carried out using Big Dye
terminator
sequencing chemistry (PE Applied Biosystems). Briefly, 30-SO ng of purified
PCR
product and 3.2 pmol of sequencing primer were used for sequencing in a 15 ~1
reaction
according to the manufacturer's instructions. The sequencing products were
purified
using a Sephadex G50 column, dried in a vacuum concentrator and resuspended in
3 pl of
gel loading buffer (83% deionized formamide, 17% gel loading dye) (PE Applied
Biosystems). 0.5 pl of the sample was then loaded on a denaturing sequence gel
on an
ABI automated DNA sequencer. All samples were sequenced in both forward and
reverse directions to confirm the presence/absence of mutations. The data were
analyzed
using the Sequencher software (Gene Codes, Ann Arbor, MI).
FISH analysis of chromosome l lp copy number using tissue arra,~
High-quality hybridizations of cases in which tumor cells could be
definitively identified were obtained from 102 cases. This yield of 61.4% is
relatively
low compared to arrays that we have constructed from melanomas. Most of the
cases that
could not be analyzed were very small Spitz nevi that consisted only of single
cells or
small nests of functional melanocytes so that the neoplastic melanocytes could
not be
reliably recognized in the array. Thirty nine cases (38.2%) were from male and
61
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(59.8%) from female patients, in two cases the gender was not known. The mean
age was
30.0 years. 52 (51 %) of the cases had features of the pigmented spindle cell
nevus variant
of Spitz (PSCI~.
The hybridization efficiency could be assessed by counting the
hybridization signals in normal epidermis that was present in many of the
biopsies. The
average copy numbers for test and reference probes in normal keratinocytes
were 1.7 and
1.6, respectively. Hybridizations were analyzed of three separate sections of
the array,
and counts from two or more sections were available for 47 (46.1 %) cases. In
45 (95.7%)
of these, the result of the separate counts were identical, in one case a
definitive
amplification was seen in one analysis, and was not found in the cells present
in the other
section. Amplifications were only scored if more than 30% of the tumor cells
had at least
3-fold increased signals of l lp when compared to the reference probe on l lq.
According
to these criteria, amplification of 1 lp was found in 12 (11.8%) cases. The
average
thickness of cases with amplifications was significantly greater than the
thickness of cases
with normal copy number of l lp (1.1 mm vs. 0.6 mm, p=0.01). The amplification
frequency within the randomly retrieved set of cases was 6/84 (7.1 %), whereas
of the 18
cases that had been selected for thickness, 6 (33.3%) showed amplifications of
chromosome l lp.
H RAS mutations
Oncogenic mutations of H-RAS typically involve codons 12, 13 in exon 1
and codon 61 in exon 2 (Barbacid, M., Annu. Rev. Biochem: 56:779-827, 1987) .
We
sequenced exons l and 2 of H-RAS of 9 cases in which FISH detected an
amplification of
chromosome 1 lp, and in 13 cases in which FISH showed normal copy numbers of
chromosome 1 lp. Five of nine cases (56%) with 1 lp amplification had HRAS
mutations,
significantly more (p=0.002) than in the cases with normal l lp copy numbers,
in which
only one (8%) had a mutation. Three mutations were 61 Gln->Leu (fig. 1 E), two
61 Gln-
>Arg, and one 12 Gly->Arg.
Additionally, we sequenced H-RAS in 11 Spitz nevi used for our previous
CGH analysis (Bastian et al., J. Invest. Dermatol. 113:1065-1069, 1999). H-RAS
mutations were identified in all three cases (100%) in which CGH detected
increased
copies of chromosome l lp. All of these involved codon 61; two cases had a
transition of
glutamine to arginine, and the other to leucine. The seven cases in which CGH
found
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normal copy numbers of chromosome 11 p had wild-type sequences of both exons
of H-
RAS.
Of the total of 33 Spitz nevi in which H-RAS was sequenced, 8/12 cases
(67%) with amplified 1 lp had H-RAS mutations, significantly more (p<0.0001)
than in
the cases with normal copies of chromosome 1 lp (1/21 or 5%).
Histologic features of cases with amplified chromosome l In
Amplifications were most common in compound or predominantly
intradermal Spitz nevi (11/47 or 23.4%), and only rarely occurred in the
pigmented
spindle cell variant of Spitz nevus (1/52 or 1.9%; p=0.0007). The cases with
amplification of 1 lp frequently showed several histological features that
occurred
infrequently in the cases with normal copy number of chromosome l lp (Table
2). The
tumors commonly showed single cells splayed between collagen bundles at the
base
resulting in a pattern of haphazardly arranged collagen and marked desmoplasia
(8/12,
p=0.0005). Cells typically had vesicular nuclei with delicate nuclear
membranes, and
ample amphophilic cytoplasm. Cells at the base frequently (5/12) seemed to be
surrounded by thin, eosinophilic membranes), that were only seen in 3 of the
87 cases that
had normal copy numbers of l lp (p<0.0001). These membranes stained positive
with a
reticulin stain (not shown). Cases with l lp amplifications were also notably
more
pleomorphic (p<0.0001) with nuclei varying in sizes and shapes and staining
intensity
and in some cases intranuclear inclusions. These features were also present in
the three
cases that had shown amplification of chromosome 1 lp by comparative genomic
hybridization in a previous study (Bastian et al., J. Invest. Dermatol.
113:1065-1069,
1999). No association of 1 lp amplification with patient age or sex was found.
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Table 2. Histological features associated with amplification of chromosome 1
lp in Spitz
nevi.
Histology n llp amplifiedllp normal p-Value
Desmoplasia 26 (25.5%) 8 (30.8%) 18 (69.2%) 0.000552
Eosinophilic 8 (7.8%) 5 (62.5%) 3 (37.5%) 0.000004
membranes
Single cells 23 (22.5%) 5 (21.7%) 12 (52.2%) 0.014305
between collagen
bundles
Marked nuclear30 (29.4%) 10 (33.3%) 20 (66.7%) 0.000015
pleomorphism
The percentages m the n column retere to the total number of 102 cases. The
other
percentages refer to the number of cases that have the respective biological
feature.
Cell proliferation using a Ki-67 antibody was assessed in all 12 cases with
amplifications of l lp and 24 cases with normal copy numbers of chromosome l
lp.
Immunostains were performed on sections of the original blocks to allow
inspection of
sufficient numbers of cells. In 11/12 (92%) of the cases with l lp
amplification and 20/24
(83%) with normal 1 lp copy numbers the number of labeled nuclei ranged from 0
to
maximal 1 %. A total of five cases had higher labeling rates: Four had normal
1 lp copy
numbers and a labeling index of at most 5%, mostly affecting melanocytes at
the demo-
epidermal junction. One case that had an average of 6 copies of chromosome 1
lp by
FISH analysis and a 61 G->A mutation had a labeling index of 10%. It was a
superficial
biopsy, so that the proliferation rate in deeper areas of the nevus could not
be assessed.
Overexpression of a,,~i3 inte_grin in ~pitz nevi with l lp amplification
The expression of the cellular adhesion molecule a,,(33 integrin is
correlated with tumor progression and invasion in melanoma (Albelda et al.,
Cancer Res.
1990, 50:6757-6764) and has recently been reported to be expressed in Spitz
nevus (Van
Belle et al., Hum. Pathol. 1999, 30:562-567). The pattern of single cells
between
collagen bundles leading to a considerable remodelling of collagen frequently
found in
cases with 1 lp amplification was indicative of a marked invasive capacity of
the cells.
Immunohistochemistry to detect (33 integrin was performed to determine the
level of a,,(33
integrin expression. This analysis showed that of a total of 80 informative
cases, 29
(41.4%) showed expression (1+ and above) of a,,~i3 integrin. a,,(33 integrin
expression was
significantly (p<0.0001 ) associated with amplification of HRAS. Of 29 cases
which
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CA 02368903 2001-10-05
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expressed a"(33 integrin, 9 had amplifications of HRAS. Of 41 cases without
expression
of a,,(33 integrin, only two cases had an amplification of chromosome l lp. In
all cases
with l lp amplification the expression pattern of a,,(33 integrin was
membranous.
In summary, this example shows that H-RAS mutations are present in a
S subset of Spitz nevus and can be used as a target in typing tumor samples to
assist in the
differential diagnosis of a Spitz nevus.
Example Five: Identification of 1 lp isochromosomes
Ten Spitz nevi samples that showed amplification of H-RAS (see, e.g.,
Example 1) were analyzed for the presence of an 1 lp isochromosome using FISH.
Two probes were employed for the analysis. The first probe RPCI-
1156c13, which was labeled with FITC and detected as a green fluorescent
signal, maps
to chromosome l lp to a region adjacent to the centromere, l lpl 1.2. The
second probe
RPCI-11135h08, which was labeled with Cy3 and detected as a red fluorescent
label,
hybridizes to sequences on the q arm of chromosome 11 adjacent to the
centromere at
l lql 1. Normal control tissue showed only the presence of paired
hybridization signals
containing two colors, i.e., the pair of signals included one red signal and
one green
signal. The results from all of the 10 samples from Spitz nevi indicated the
additional
presence of paired hybridization signals of the same color (green). These
results
demonstrate the presence of an 1 lp isochromosome.
It is understood that the examples and embodiments described herein are
for illustrative purposes only and that various modifications or changes in
light thereof
will be suggested to persons skilled in the art and are to be included within
the spirit and
purview of this application and scope of the appended claims. All
publications, patents,
and patent applications cited herein are hereby incorporated by reference in
their entirety
for all purposes.
47
CA 02368903 2001-10-05
WO 00/61814 PCT/US00/09609
Sequence Listing:
SEQ ID NO: 1 HRAS codon 12 forward primer
S'-AGGAGACCCTGTAGGAGGA-3'
SEQ ID NO: 2 HRAS codon 12 reverse primer
5'-CGCTAGGCTCACCTCTATAGTG-3'
SEQ ID N0:3 HRAS codon 61 forward primer
5'-CTGCAGGATTCCTACCGGA-3'
SEQ ID N0:4 HRAS codon 61 reverse primer
5'-ACTTGGTGTTGTTGATGGCA-3'
48
