U.S. patent application number 09/835181 was filed with the patent office on 2001-10-11 for detection of chromosome copy number changes to distinguish melanocytic nevi from malignant melanoma.
Invention is credited to Bastian, Boris, Pinkel, Daniel.
Application Number | 20010029021 09/835181 |
Document ID | / |
Family ID | 23109321 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010029021 |
Kind Code |
A1 |
Bastian, Boris ; et
al. |
October 11, 2001 |
Detection of chromosome copy number changes to distinguish
melanocytic nevi from malignant melanoma
Abstract
The present invention provides for methods of distinguishing
melanocytic nevi, such as Spitz nevi, from malignant melanoma. The
methods comprise contacting a nucleic acid sample from a patient
with a probe which binds selectively to a target polynucleotide
sequence on a chromosomal region such as 11p, which is usually
amplified in Spitz nevi. The nucleic acid sample is typically from
skin tumor cells located within a tumor lesion on the skin of the
patient. Using another probe which binds selectively to a
chromosomal region such as 1q, 6p, 7p, 9p, or 10q, which usually
show altered copy number in melanoma, the method can determine that
those tumor cells with no changes in copy number of 1q, 6p, 7p, 9p,
or 10q, are not melanoma cells but rather Spitz nevus cells. The
finding of amplifications of chromosome 11p would be an additional
indication of Spitz nevus.
Inventors: |
Bastian, Boris; (San
Francisco, CA) ; Pinkel, Daniel; (Walnut Creek,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
23109321 |
Appl. No.: |
09/835181 |
Filed: |
April 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09835181 |
Apr 13, 2001 |
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09288940 |
Apr 9, 1999 |
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6261775 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6886 20130101; C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method of distinguishing a Spitz nevus from malignant melanoma
in a skin tumor sample from a patient, the method comprising:
providing a skin tumor sample from a patient, detecting an absence
of change in copy number at 1q, 6p, 7p, or 10q in a nucleic acid
sample from the skin tumor sample, thereby distinguishing the Spitz
nevus from malignant melanoma.
2. The method of claim 1, further comprising detecting an absence
of change in copy number at 9p in the nucleic acid sample.
3. The method of claim 1, wherein the detecting step comprises:
contacting the nucleic acid sample with a probe which selectively
hybridizes to a target polynucleotide sequence on a chromosomal
region selected from the group consisting of 1q, 6p, 7p, or 10q,
wherein the probe is contacted with the sample under conditions in
which the probe selectively hybridizes with the target
polynucleotide sequence to form a stable hybridization complex;
detecting the formation of the hybridization complex; and detecting
an absence of change in copy number at 1q, 6p, 7p, or 10q, thereby
distinguishing the Spitz nevus from a malignant melanoma.
4. The method of claim 1, wherein the nucleic acid sample is an
interphase nucleus.
5. The method of claim 3, wherein the probe is labeled with a
detectable composition.
6. The method of claim 5, wherein the detectable composition is
selected from the group consisting of a direct label and an
indirect label.
7. The method of claim 6, wherein the direct label is Cy3.
8. The method of claim 6, wherein the indirect label is selected
from a group consisting of a digoxigenin and a biotin
9. The method of claim 6, wherein the indirect label is detected by
a fluorescent dye.
10. The method of claim 9, wherein the fluorescent dye is FITC.
11. The method of claim 3, further comprising contacting the sample
with a reference probe that selectively hybridizes to a
polynucleotide sequence at a chromosomal region different from 1q,
6p, 7p, or 10q.
12. The method of claim 11, wherein the reference probe is labeled
with a fluorescent label distinguishable from a label on the probe
that selectively hybridizes to the target polynucleotide
sequence.
13. The method of claim 3, wherein the step of detecting the
hybridization complex comprises determining the copy number of the
target polynucleotide sequence.
14. The method of claim 3, further comprising a step of blocking
the hybridization capacity of repetitive sequences in the
probe.
15. The method of claim 14, wherein the step of blocking the
hybridization capacity of repetitive sequence in the probe
comprises contacting unlabeled blocking nucleic acids comprising
repetitive sequences with the sample.
16. The method of claim 15, wherein the unlabeled blocking nucleic
acids are Cot-1 DNA.
17. The method of claim 3, wherein the probe is bound to a solid
substrate.
18. The method of claim 17, wherein the probe is a member of an
array.
Description
BACKGROUND OF THE INVENTION
[0001] The melanocyte can give rise to a plethora 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, dysplastic or
Clark's nevi, blue nevi, lentigo simplex, and deep penetrating
nevus. Pigmented spindle cell nevus is regarded as a subset of
Spitz nevi.
[0002] 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., J Am 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 differentiate Spitz nevi from melanoma.
The diagnostic difficulties are explained by overlapping
histological features. Both Spitz nevi and melanoma can be composed
of melanocytes with abundant cytoplasm and large nuclei. Nuclei can
be pleomorphic and contain macronucleoli. Mitotic figures,
sometimes numerous, occur in both neoplasms.
[0003] Melanoma refers to malignant neoplasms of melanocytes. Its
proper diagnosis and early treatment is of great importance because
advanced melanoma has a poor prognosis, but most melanomas are
curable if excised in their early stages. While clinicians make the
initial diagnosis of pigmented lesions of the skin, pathologists
make the final diagnosis. 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, which have many
variants that share some features of melanomas (LeBoit, P. E.
STIMULANTS 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 subset of cases remains, where an unambiguous
diagnosis cannot be reached (Farmer et al., DISCORDANCE IN THE
HISTOPATHOLOGIC DIAGNOSIS OF MELANOMA AND MELANOCYTIC NEVI BETWEEN
EXPERT PATHOLOGISTS, Human Pathol. 27: 528-31 (1996)). The most
frequent and important diagnostic dilemma is the differential
diagnosis between Spitz nevus, a neoplasm composed of large
epithelioid or spindled melanocytes, and melanoma.
[0004] Misdiagnosis of Spitz nevus as melanoma and vice versa has
been repeatedly reported in the literature (Goldes et al., Pediatr.
Dermatol., 1: 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 hazard of
mistaking a Spitz nevus for melanoma can be severe and traumatic:
The patients may be subjected to needless surgery, unable to plan
for the future, and psychologically traumatized. For obvious
reasons, the misdiagnosis of a melanoma as a benign nevus can have
even more dramatic consequences. 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 of Dermatology 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 al 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 S100, 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.
[0005] Thus, there exists a great need for improved and accurate
diagnostic methods to distinguish Spitz nevi from malignant
melanoma. The present invention addresses these and other
needs.
SUMMARY OF THE INVENTION
[0006] 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 on a
chromosomal region such as 11p, particularly 11p15.5, which is
usually 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., 1q, 6p, 7p, or 10q).
Usually, the copy number of the target region is measured.
[0007] 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.
[0008] 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.
[0009] The nucleic acid hybridization can be carried 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.
[0010] Definitions
[0011] To facilitate understanding the invention, a number of terms
are defined below.
[0012] 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.
[0013] The terms "Spitz nevi" or "Spitz nevus" refer to benign
melanocytic neoplasms that can have considerable histological
resemblance to melanoma. 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.
[0014] The terms "tumor" or "cancer" in an animal refers to the
presence of cells possessing characteristics typical of
cancer-causing cells, such as 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, or may be a non-tumorigenic cancer
cell, such as a leukemia cell. Cancers include, but are not limited
to melanomas, breast cancer, lung cancer, bronchus cancer,
colorectal cancer, prostate cancer, pancreas cancer, stomach
cancer, ovarian cancer, urinary bladder cancer, brain or central
nervous system cancer, peripheral nervous system cancer, esophageal
cancer, cervical cancer, uterine or endometrial cancer, cancer of
the oral cavity or pharynx, liver cancer, kidney cancer, testis
cancer, biliary tract cancer, small bowel or appendix cancer,
salivary gland cancer, thyroid gland cancer, adrenal gland cancer,
osteosarcoma, chondrosarcoma, and the like.
[0015] The phrase "detecting a tumor or a cancer" refers to the
ascertainment of the presence or absence of cancer in an animal, in
this case, melanoma cells or premalignant melanocytes. "Detecting a
tumor or a cancer" can also refer to obtaining indirect evidence
regarding the likelihood of the presence of cancerous cells in the
animal or to the likelihood of predilection to development of a
cancer. Detecting a cancer 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
animal.
[0016] The term "animal" refers to a member of the kingdom
Animalia, characterized by multicellularity, the possession of a
nervous system, voluntary movement, internal digestion, etc. An
"animal" can be a human or other mammal. Preferred animals include
humans, non-human primates, and other mammals. Thus, it will be
recognized that the methods of this invention contemplate
veterinary applications as well as medical applications directed to
humans.
[0017] The terms "hybridizing specifically to" and "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 I, Ch. 2
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," Elsevier, N.Y. ("Tijssen"). Generally,
highly stringent hybridization and wash conditions are selected to
be about 5.degree. C. lower than the thermal melting point
(T.sub.m) for the specific sequence at a defined ionic strength and
pH. The T.sub.m 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 T.sub.m 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.degree. C. using standard hybridization solutions (see, e.g.,
Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.)
Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press,
NY, and detailed discussion, below), with the hybridization being
carried out overnight. An example of highly stringent wash
conditions is 0.15 M NaCl at 72.degree. C. for about 15 minutes. An
example of stringent wash conditions is a 0.2.times.SSC wash at
65.degree. 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 1.times.SSC at 45 .degree. C. for 15
minutes. An example of a low stringency wash for a duplex of, e.g.,
more than 100 nucleotides, is 4.times. to 6.times.SSC at 40.degree.
C. for 15 minutes.
[0018] The term "labeled with a detectable composition", 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 .sup.32P,
35S, .sup.3H, .sup.14C, .sup.125I, .sup.131I; 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), calorimetric labels (e.g.
colloidal gold), magnetic labels (e.g. Dynabeads.TM.), 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. A peptide can be
made detectable by incorporating predetermined polypeptide epitopes
recognized by a secondary reporter (e.g., leucine zipper pair
sequences, binding sites for secondary antibodies, transcriptional
activator polypeptide, metal binding domains, epitope tags). 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 sensitivity of the
detection can be increased by using chemical amplification
procedures using e.g. tyramide (Speel et al., J. Histochem.
Cytochem. 45:1439-46, (1997)).
[0019] 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 methylphosphonate 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.
[0020] 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 hybridized. The
nucleic acids of a target element can contain sequence(s) 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 .mu.m to about 3
mm, preferably between about 5 .mu.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 500 kb, and from about 50 kb to about 150 kb.
[0021] 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.
[0022] 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.
[0023] 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 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.
[0024] 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 polymerase
chain reaction (PCR) amplification products. The probes of the
present invention are produced from nucleic acids found in the
regions described herein. 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: R171-R174; Schummer (1997)
Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23: 120-124;
U.S. Pat. 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.
[0025] "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 isolated cells (e.g. isolated by another person), or by
performing the methods of the invention in vivo.
[0026] "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
[0027] 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).
[0028] 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 ideogramms. Thick lines indicate
amplifications.
[0029] 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.
[0030] FIG. 4 shows the frequency distribution of hybridization
signals after dual-target hybridization of probe RMC11B022 for
chromosome 11p (black bars) and RMC11P008 for chromosome 11q (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 11p 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
[0031] The present invention provides for unique and accurate
methods for distinguishing benign melanocytic nevi, such as Spitz
nevi, from malignant melanoma. This invention is based upon the
observation that chromosomal regions that have frequently altered
copy numbers in melanoma such as 1q, 6p, 7p, 9p, or 10q, are rarely
changed in Spitz nevi. In addition, Spitz nevi cells show a single
amplification of chromosomal region 11p, particularly 11p15.5, as
shown by the increase of its copy number, a phenomenon that is
exceedingly rare in melanoma. This difference in pattern of
chromosomal aberrations between Spitz nevi and melanoma can lead to
more accurate diagnostic distinction of Spitz nevi from
melanoma.
[0032] 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.
[0033] 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.
General Methods for Measuring Chromosomal Abnormality
[0034] 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.
[0035] 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., J
Invest Dermatol 88, 753-7 (1987); Otsuka et al., Clin Exp Dermatol
18, 421-4 (1993); Vogt et al., Am J Dermatopathol 18, 142-50
(1996)). However, routine application of these techniques has been
hampered by the complexity of the procedure and more importantly by
its lack of sensitivity.
[0036] 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, 818-21 (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.
[0037] 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., J Pathol. 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 number of signals between 15 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.
[0038] Detection of Copy Number
[0039] 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
1q, 6p, 7p, 9p, 10q, and 11p.
[0040] Hybridization-based Assays
[0041] 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.
[0042] 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.
[0043] 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.
[0044] The probes are typically labeled, e.g., with radioisotopes
or fluorescent reporters. The preferred size range is from about
200 bp to about 1000 bases, more preferably between about 400 to
about 800 bp for double stranded, nick translated nucleic
acids.
[0045] 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.
[0046] 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.
[0047] 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, Vol. 33: In
Situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J.
(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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.).
[0052] This simple spotting approach has been automated to produce
high density spotted arrays (see, e.g., U.S. Pat. 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, U.S. Pat. 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.
[0053] 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, P1s, cosmids, plasmids, inter-Alu PCR
products of genomic clones, restriction digests of genomic clone,
cDNA clones, amplification (e.g., PCR) products, and the like.
[0054] 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).
[0055] 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.
[0056] 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 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.
[0057] 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.
[0058] Target elements of various sizes, ranging from 1 mm diameter
down to 1 .mu.m 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 10.sup.4/cm.sup.2. Relatively simple
approaches capable of quantitative fluorescent imaging of 1
cm.sup.2 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).
[0059] 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.). Quartz cover slips, which have at least 10-fold lower
autofluorescence than glass, can also be silanized.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.times.SSPE-T at 37.degree.
C. to 70.degree. 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.
[0068] 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 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.
[0069] 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.)
[0070] 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.).
[0071] 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.
[0072] Probes useful in the methods described here are available
from a number of sources. For instance, P1 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, Calif.), or
from the Los Alamos National Laboratory.
[0073] Labeling and Detection of Nucleic Acids
[0074] 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.
[0075] 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.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, Texas red,
rhodamine, green fluorescent protein, and the like, see, e.g.,
Molecular Probes, Eugene, Oreg., USA), radiolabels (e.g., .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse
radish peroxidase, alkaline phosphatase and others commonly used in
an ELISA), and calorimetric 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. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
[0076] 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.
[0077] 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 observed or, alternatively,
which emit light when irradiated with radiation of a particular
wave length or wave length range, e.g., fluorescers.
[0078] Desirably, fluorescers should absorb light above about 300
nm, preferably about 350 nm, and more preferably above about 400
nm, usually emitting at wavelengths 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 referring 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.
[0079] 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.
[0080] 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.
[0081] The label may be added to the target (sample) nucleic
acid(s) 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 Biochemistry and
Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes,
P. Tijssen, ed. Elsevier, N.Y., (1993)).
[0082] 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.
[0083] 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, Calif.) 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.
[0084] 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).
[0085] Amplification-based Assays
[0086] 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 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.).
[0087] Other suitable amplification methods include, but are not
limited to ligase chain reaction (LCR) (see Wu and Wallace (1989)
Genomics 4: 560, Landegren et al. (1988) Science 241: 1077, and
Barringer et al. (1990) Gene 89: 117, transcription amplification
(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), and
self-sustained sequence replication (Guatelli et al. (1990) Proc.
Nat. Acad. Sci. USA 87: 1874).
[0088] Detection of Gene Expression
[0089] As indicated below, a number of oncogenes 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.
[0090] Detection of Gene Transcripts
[0091] 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.
[0092] 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.
[0093] Detection of Expressed Protein
[0094] The "activity" of the target oncogene can also be detected
and/or quantified by detecting or 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), immunoelectrophoresis, radioimmunoassay (RIA),
enzyme-linked immunosorbent assays (ELISAs), immunofluorescent
assays, western blotting, and the like.
[0095] Kits for Use in Diagnostic and/or Prognostic
Applications
[0096] 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.
[0097] In addition, the kits may include instructional materials
containing directions (i.e., protocols) for the practice of the
methods of this invention. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
EXAMPLES
Example One: CGH and FISH Studies of Spitz Nevi Cells
[0098] 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.
[0099] Material and Methods
[0100] Tumor Material
[0101] 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 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.
[0102] DNA Preparation
[0103] Paraffin material: 30 .mu.m sections were cut, with a 5
.mu.m section for H & E every 5 sections. The unstained 30
.mu.m sections were placed on glass slides and an area of interest
was microdissected without de-paraffinizing.
[0104] 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, 1 mM EDTA, 0.5% Tween 20
buffer. DNA was extracted with phenol-chloroform-isoamylalc- ohol
(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 12 .mu.g.
[0105] Comparative Genomic Hybridization (CGH) and Digital
Image
[0106] Analysis
[0107] All tumors were measured both with the tumor DNA labeled
with fluorescein-12-dUTP (DuPont, Inc., Boston, Mass.), 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 .mu.g human Cot-1 DNA
(Life Technologies, Inc., Gaithersburg, Md.) dissolved in 10 .mu.l
hybridization buffer (50% formamide, 10% dextrane sulfate, and
2.times.SSC, pH 7.0). Hybridization was carried out for 2-3 days at
37.degree. 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 (50% formamide in
2.times.SSC, pH) at 45.degree. C., once in PN buffer (0.1 M
NaH.sub.2PO.sub.4, 0.1 M Na.sub.2HPO.sub.4, 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.
[0108] Controls and Threshold Definitions
[0109] 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.
[0110] 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 11p of Spitz nevi
cells is not seen among melanoma cells, as shown in FIG. 1.
[0111] Fluorescence In-situ Hybridization (FISH)
[0112] Dual-color FISH was carried out on tissue sections of the
cases in which tissue was left after CGH (14/17). Probes mapping to
the short arm (RMC11B022 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, Ill.) or Digoxigenin (Boehringer
Mannheim, Indianapolis, Ind.). 6 .mu.m 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.degree. C., in 4 mg/ml Pepsin in 0.2 N HCl at 37.degree. C. for
4-8 min, dehydrated by increasing strength ethanol and air-dried.
Slides were denatured in 70% formamide, 2.times.SSC pH 7.0 for 5
min at 72.degree. C., and dehydrated again in a graded ethanol
series. 2.5 to 25 ng of each of the labeled probes together with 20
.mu.g Cot-1 DNA (Life Technologies, Inc., Gaithersburg, Md.) were
dissolved in 10 .mu.l hybridization buffer (50% formamide, 10%
dextrane sulfate, and 2.times.SSC, pH 7.0) and denatured for 10 min
at 72.degree. C. Hybridization was carried out for 48-72 hours at
37.degree. C. Slides were washed three times in washing solution
(50% formamide in 2.times.SSC, pH 7.0) at 45.degree. C., once in
2.times.SSC at 45.degree. C., once in 2.times.SSC at room
temperature (RT), and once in 0.1% Triton X100 in 4.times.SSC/ at
RT. Subsequently, sections were incubated with 10% BSA in
4.times.SSC in a moist chamber at 37.degree. C., and then with a
FITC labeled anti-digoxigenin antibody (Boehringer Mannheim,
Indianapolis, Ind.) diluted in 4.times.SSC with 10% BSA. Sections
were counterstained with 4,6-diamino-2-phenylindole (Sigma, St.
Louis, Mo.) 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.
[0113] Results
[0114] 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. 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).
[0115] FISH measurements were performed to tissue sections in order
to study the histopathologic distribution of the recurrent gain on
chromosome 11p 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 11p
(11p15.5, clone RMC11B022) and a reference probe mapping to
chromosome 11q23 (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 6 .mu.m 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.
[0116] 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 11p 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 11p ranged from 1.8-3.0. 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
11p 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 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.
[0117] 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 11p14
(RMC11P014). The number of signals in the nuclei of the tumor cells
with this probe was similar to that found with the probe for
11p15.
[0118] One probe mapped to the distal part of the p-arm of
chromosome 11 and the second probe mapped to 11q. Of the three
cases that showed a gain of chromosome 11p, 6-10 signals of the 11p
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 11p gain in CGH, only one additional case
showed an amplification of lip. 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 11p 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.
[0119] 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).
1TABLE 1 Aberra- Case Sex Age tions Clinical Information Site
Histology Follow-up S1 f 17.9 none Since one year slowly enlarging
Shin compound with FOD, 5.4y 1.1 .times. 1.4 cm reddish papule with
desmoplasia brown rim S2 m 31.0 none Since one unchanged cherry
pit- Lower arm dermal with desmoplasia FOD, 3.4y sized dome-shaped
reddish nodule S3 f 21.5 none NA Upper back compound FOD, 1.2y S4 f
7.2 none Since one year cherry-pit-sized Knee dermal with
desmoplasia FOD, 4.0y tumor S5 f 23.8 Amp. Since one year
discretely enlarging Knee compound FOD, 7.5y 11p (FISH) S6 m 7.4
none Since 3 month slowly growing Ear helix compound with FOD, 9.3y
lentil-sized, slightly keratotic, desmoplasia elevated S7 m 13.7
none Since years 1.5 cm, raised, firm Lateral compound FOD, 2.6y
nodule abdomen S8 f 3.2 none Since 1.5 years growth of a Cheek PSCT
FOD, 6.9y pigmented tumor S9 f 31.3 none NA Thigh compound FOD,
2.3y S11 f 3.0 Gain of NA Inner thigh PSCT FOD, 8.1y 7q31- qter S12
f 23.0 none NA Epigastrium PSCT FOD, 4.2y S13 f 45.8 Amp. NA Thorax
dermal with desmoplasia FOD, 5.3y of 11p S14 f 10.8 none since 7
months itching, reddish Cheek compound NA papule S15 f 26.7 none NA
Shin dermal FOD, 1.2y S16 f 23.2 Amp. 1.5 years ago curettage of a
lesion Tip of nose compound with scar tissue FOD, 7.3y of 11p that
arose 4 months earlier. Lesion recurred one month later and was
again curetted. Recurred again after 2 months and was excised. Now,
after 2 months, again recurring. Reddish, sharply circumscribed
lentil-sized papule. S17 m 12.5 none NA Mid back compound NA S18 m
11.3 Amp. enlarging skin lesion for three Calf compound with NA of
11p month desmoplasia
[0120] Discussion
[0121] 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 11p 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.
[0122] The finding of an increased copy number of chromosome 11p 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 11p 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 11p gain, those genes, or the pathways
they belong to, are activated by a different mechanism than
increase of gene copy number.
[0123] A gain of chromosome 11p was not seen in any of the 102
primary melanomas we have analyzed by CGH. Gains including 11p 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).
[0124] 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 50% 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 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 11p,
9, and 10. Gains of chromosome 11p could be interpreted as in favor
of Spitz nevus, and losses of chromosomes 9 and/or 10 as in favor
of melanoma.
[0125] 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
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 1q, 6p, 7p, and 10q, 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.
[0126] 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 11q 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 11p, because the increased copy number of this chromosomal
arm was present in all cells of the lesions
[0127] 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 of chromosome 11p 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
[0128] This example demonstrates FISH experiments using chromosome
9 probes in detecting primary melanoma cells.
[0129] 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.
[0130] 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. 1q, 6p, 7p, 9p, 10q, and 11p), (b) probes should give
strong and reproducible hybridization signals.
Example Three: Tissue Hybridization Protocols
[0131] 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.
[0132] 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.degree. 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.
Alternative labeling approaches may be employed so as to be able to
detect three differentially labeled probes in one
hybridization.
[0133] 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.
[0134] 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., J Pathol
173, 227-33 (1994)).
[0135] 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.
* * * * *