U.S. patent application number 13/151935 was filed with the patent office on 2011-12-08 for methods and probe combinations for detecting melanoma.
This patent application is currently assigned to ABBOTT LABORATORIES. Invention is credited to Boris Bastian, Susan Jewell, Larry E. Morrison.
Application Number | 20110300540 13/151935 |
Document ID | / |
Family ID | 37809581 |
Filed Date | 2011-12-08 |
United States Patent
Application |
20110300540 |
Kind Code |
A1 |
Bastian; Boris ; et
al. |
December 8, 2011 |
METHODS AND PROBE COMBINATIONS FOR DETECTING MELANOMA
Abstract
The present invention is based on the discovery of methods and
combinations of probes to chromosomal regions that are gained or
lost or imbalanced in melanoma that provide highly specific and
sensitive assays for the detection of melanoma cells.
Inventors: |
Bastian; Boris; (Mill
Valley, CA) ; Morrison; Larry E.; (Glen Ellyn,
IL) ; Jewell; Susan; (Elmhurst, IL) |
Assignee: |
ABBOTT LABORATORIES
Abbott Park
IL
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Oakland
CA
|
Family ID: |
37809581 |
Appl. No.: |
13/151935 |
Filed: |
June 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11515505 |
Sep 1, 2006 |
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13151935 |
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60713799 |
Sep 2, 2005 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 2537/143 20130101;
C12Q 2600/158 20130101; C12Q 2600/112 20130101; C12Q 1/6841
20130101; C12Q 1/6841 20130101; C12Q 1/6886 20130101; C12Q 2537/143
20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-17. (canceled)
18. A combination of between two and ten probes comprising a probe
that targets chromosome region 6p and a probe that targets
chromosome region 6q, wherein the combination of probes has a
difference from ideal (DFI) value of less than about 0.29 for
melanoma.
19. The combination of probes of claim 18, wherein the combination
of probes has a DFI value of less than about 0.20.
20. The combination of probes of claim 18, wherein one of the
probes in the combination of between two and ten probes is a probe
that targets chromosome subregion 6p25.
21. The combination of probes of claim 18, wherein the combination
is a combination of at least three probes, wherein an additional
probe is selected from the group consisting of a chromosome 6
enumerator probe a chromosome 10 enumerator probe, a probe that
targets chromosome region 7q, a probe that targets chromosome
region 11q, a probe that targets chromosome region 17q, a probe
that targets chromosome region 1q, and a probe that targets
chromosome region 20q.
22. The combination of probes of claim 21, wherein the three probes
are selected from the group of probes consisting of: a probe that
targets chromosome subregion 6p25, a probe that targets chromosome
subregion 6q23 and a probe that targets chromosome subregion 11q13;
a probe that targets chromosome subregions 6p25, a probe that
targets chromosome subregion 6q23 and a chromosome 6 enumerator
probe; and a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 20q13 and a probe that targets
chromosome subregion 6q23.
23. The combination of probes of claim 18, wherein the combination
is a combination of at least four probes, wherein two additional
probes are selected from the group consisting of a chromosome 6
enumerator probe, a chromosome 10 enumerator probe, a probe that
targets chromosome region 7q, a probe that targets chromosome
region 11q, a probe that targets chromosome region 17q, a probe
that targets chromosome region 1q, and a probe that targets
chromosome region 20q.
24. The combination of probes of claim 23, wherein the four probes
are selected from the group consisting of: a probe that targets
chromosome subregion 6p25; a probe that targets chromosome
subregion 6q23, a chromosome 6 enumerator probe, and a chromosome
10 enumerator probe; a probe that targets chromosome subregion
6p25; a probe that targets chromosome subregion 6q23, a probe that
targets chromosome subregion 11q13, and a chromosome 6 enumerator
probe; a probe that targets chromosome subregion 6p25; a probe that
targets chromosome subregion 6q23, a probe that targets chromosome
subregion 11q13, and a chromosome 10 enumerator probe; a probe that
targets chromosome subregion 6p25; a probe that targets chromosome
subregion 6q23, a probe that targets chromosome subregion 1q31, and
a chromosome 10 enumerator probe; a probe that targets chromosome
subregion 6p25, a probe that targets chromosome subregion 17q25, a
probe that targets chromosome subregion 6q23, and a chromosome 6
enumerator probe; a probe that targets chromosome subregion 6p25, a
probe that targets chromosome subregion 20q13, a probe that targets
chromosome subregion 6q23, and a chromosome 6 enumerator probe; a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome subregion 7q34, a probe that targets chromosome
subregion 6q23, and a chromosome 6 enumerator probe; a probe that
targets chromosome subregion 6p25, a probe that targets chromosome
subregion 7q34, a probe that targets chromosome subregion 6q23, and
a probe that targets chromosome subregion 17q25; a probe that
targets chromosome subregion 6p25, a probe that targets chromosome
subregion 20q13, a probe that targets chromosome subregion 6q23,
and a probe that targets chromosome subregion 17q25; and a probe
that targets chromosome subregion 6p25, a probe that targets
chromosome subregion 6q23, a probe that targets chromosome
subregion 17q25, and a chromosome 10 enumerator probe.
25. The combination of probes of claim 20, wherein a second probe
in the combination of between two and ten probes targets chromosome
region 6q23.
26. The combination of probes of claim 21, wherein the combination
of three probes has at least two probes selected from the group of
two probes consisting of: a probe that targets chromosome subregion
6q23 and a chromosome 6 enumerator probe; a probe that targets
chromosome subregion 20q13 and a probe that targets chromosome
subregion 6q23.
27. The combination of probes of claim 23, wherein the combination
of four probes has at least three probes selected from the group of
three probes consisting of: a probe that targets chromosome
subregion 6q23, a chromosome 6 enumerator probe, and a chromosome
10 enumerator probe; a probe that targets chromosome subregion
6q23, a probe that targets chromosome subregion 11q13, and a
chromosome 6 enumerator probe; a probe that targets chromosome
subregion 6q23, a probe that targets chromosome subregion 11q13,
and a chromosome 10 enumerator probe; a probe that targets
chromosome subregion 6q23, a probe that targets chromosome
subregion 1q31, and a chromosome 10 enumerator probe; a probe that
targets chromosome subregion 17q25, a probe that targets chromosome
subregion 6q23, and a chromosome 6 enumerator probe; a probe that
targets chromosome subregion 20q13, a probe that targets chromosome
subregion 6q23, and a chromosome 6 enumerator probe; a probe that
targets chromosome subregion 7q34, a probe that targets chromosome
subregion 6q23, and a chromosome 6 enumerator probe; a probe that
targets chromosome subregion 7q34, a probe that targets chromosome
subregion 6q23, and a probe that targets chromosome subregion
17q25; a probe that targets chromosome subregion 20q13, a probe
that targets chromosome subregion 6q23, and a probe that targets
chromosome subregion 17q25; a probe that targets chromosome
subregion 6q23, and a probe that targets chromosome subregion
17q25, and a chromosome 10 enumerator probe.
28. A kit for diagnosing melanoma with a difference from ideal
(DFI) value of less than about 0.29, the kit comprising the
combination of probes of claim 18.
29. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 19.
30. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 20.
31. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 21.
32. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 22.
33. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 23.
34. The kit of claim 28, wherein kit comprises the combination of
probes of claim 24.
35. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 25.
36. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 26.
37. The kit of claim 28, wherein the kit comprises the combination
of probes of claim 27.
38. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome region 6q, and a chromosome 6 enumerator probe.
39. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of probes comprises a probe that targets
chromosome subregion 6p25, a probe that targets chromosome
subregion 6q23, and a chromosome 6 enumerator probe.
40. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome region 6q, a probe that targets chromosome region
11q.
41. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome subregion 6q23, and a probe that targets chromosome
region 11q.
42. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome region 6q, and a probe that targets chromosome subregion
11q13.
43. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome subregion 6q23, and a probe that targets chromosome
subregion 11q13.
44. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome region 6q, a chromosome 6 enumerator probe, and a probe
that targets chromosome region 11q.
45. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome subregion 6q23, a chromosome 6 enumerator probe, and a
probe that targets chromosome region 11q.
46. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome region 6q, a chromosome 6 enumerator probe, and a probe
that targets chromosome subregion 11q13.
47. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises a
probe that targets chromosome subregion 6p25, a probe that targets
chromosome subregion 6q23, a chromosome 6 enumerator probe, and a
probe that targets chromosome subregion 11q13.
48. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination of between two and ten probes comprises not
more than four probes.
49. The combination of probes of claim 18 or the kit of claim 28,
wherein the combination probes has a difference from ideal (DFI)
value of less than about 0.29 for distinguishing primary melanoma
from benign nevi.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This applications claims benefit of U.S. provisional
application No. 60/713,799, filed Sep. 2, 2005, which is herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Melanoma is an important clinical problem. The incidence and
mortality of melanoma has been increasing more rapidly than any
other malignancy except lung cancer in women. Pathology is the gold
standard for establishing the diagnosis of melanoma. Although many
cases can be classified reliably with current pathological
criteria, there is a significant subset of cases in which no
consensus can be reached even among expert pathologists. The effect
of the ambiguity on standard clinical practice is illustrated in a
recent study from The Netherlands. An expert panel reviewed 1069
consecutive melanocytic lesions that had been submitted for review
by clinical pathologists in order to identify the most common
diagnostic problems. In 14% (22/158) of the cases that had been
initially classified as invasive melanoma the panel considered the
lesions as benign, and in 16.6% (85/513) the panel considered
malignant what had been diagnosed as benign (Veenhuizen et al., J
Pathol. 182:266-72. 1997).
[0003] Diagnostic ambiguity has significant adverse consequences
for patients. Misclassifying a melanoma as benign may be fatal, and
diagnosing a benign lesion as malignant may result in significant
morbidity. Current medical practice with equivocal cases usually is
to consider them as malignant. However, the morbidity of the
therapeutic options--wide re-excision, sentinel lymph node biopsy,
and adjuvant alpha-interferon--coupled with the diagnostic
uncertainty frequently leads to pursuing a less aggressive
treatment regimen. Typically this includes a limited re-excision
and close clinical follow-up. Thus patients with benign lesions
suffer the side effects of a still significant surgery and the
emotional strain of the diagnosis, while those patients that in
fact have a melanoma may not receive the optimal treatment.
Currently there is no method to definitively resolve these
ambiguities. A diagnostic test that could reduce these
uncertainties would have a significant positive clinical impact.
This invention addresses this need.
[0004] Previous studies have shown that melanomas differ from nevi
by the presence of frequent gains or losses of particular
chromosomal regions. Comparative genomic hybridization (CGH) of
primary melanomas has identified losses at 6q, 8p, 9p, and 10q and
gains at 1q, 6p, chromosome 7, 8q, 17q, and 20q to be the most
common DNA copy number changes in melanoma (Bastian et al, Am J
Pathol. 163:1765-70, 2003). However, such studies do not provide
insight as to a combination of probes that will have a high level
of sensitivity and specificity to selectively detect melanoma. The
present invention is based on an assessment of the ability of
combinations of probes using a multi-color fluorescent in situ
hybridization (FISH) test to detect copy number changes of
chromosomal regions commonly found to be aberrant in melanoma.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention is based on the discovery of
combinations of probes that provide highly sensitive and specific
detection of melanoma and thus can distinguish malignant melanoma
from benign melanocytic lesions. Methods for the detection of
melanoma comprise evaluation of a biological sample from a
melanocytic lesion, typically by in situ hybridization, using a set
of at least two probes, often a set of at least three probes, and
in some embodiments, a set of four probes. Useful probes for
detecting melanoma can be selected from the following: a chromosome
6 enumerator probe, a chromosome 7 enumerator probe, a chromosome 8
enumerator probe, a chromosome 9 enumerator probe, a chromosome 10
enumerator probe, a probe that targets chromosome region 17q, a
probe that targets chromosome region 10q, a probe that targets
chromosome region 6p, a probe that targets chromosome region 6q, a
probe that targets chromosome region 9p, a probe that targets
chromosome region 8p, a probe that targets chromosome region 8q, a
probe that targets chromosome region 1q, a probe that targets
chromosome region 7q, a probe that targets chromosome region 20q,
and a probe that targets chromosome region 11q. Thus, a probe set
of the present invention comprises probes to chromosomal regions
selected from the group consisting of 1q, 6p, 6q, 7q, 8p, 8q, 9p,
10q, 11q, 17q, and 20q. Probe sets can also include chromosomal
enumerator probes to chromosomes 6, 7, 8, 9, or 10. Often, useful
probe set comprises at least one probe to a chromosomal subregion,
e.g., 1q23, 1q31, 6p25, 6q23, 7q34, 8p22, 8q24, 9p21, 10q23, 11q13,
17q25, or 20q13.
[0006] In one aspect, the invention provides a method of detecting
the presence of melanoma cells in a biological sample from a
patient, the method comprising:
a) contacting the sample with a combination of at least two probes,
wherein the probes are selected from group consisting of a
chromosome 6 enumerator probe, a chromosome 10 enumerator probe, a
probe that targets chromosome region 6p (e.g., 6p25), a probe that
targets chromosome region 6q (e.g., 6q23), a probe that targets
chromosome region 7q (e.g., 7q34), a probe that targets chromosome
region 11q (e.g., 11q13), a probe that targets chromosome region
17q (e.g., 17q25), a probe that targets chromosome region 1q (e.g.,
1q31), and a probe that targets chromosome region 20q (e.g.,
20q13); b) incubating each probe of the set with the sample under
conditions in which each probe binds selectively with a
polynucleotide sequence on its target chromosome or chromosomal
region to form a stable hybridization complex; c) detecting and
analyzing the hybridization pattern of the combination of probe for
the presence or absence of melanoma. A hybridization pattern
showing at least one gain or loss or imbalance at a chromosomal
region targeted by the probes is indicative of melanoma. The
combination of probes typically has a difference from ideal (DFI)
value, of about 0.29 or less. In some embodiments, the DFI value is
about 0.20 or less. Probe combinations for use in the methods of
the invention include 2-, 3-, and 4-probe combinations listed in
Table 6 that have a DFI value of about 0.29 or less.
[0007] Often, one of the probes in the combination of at least two
probes is a probe that targets chromosome subregion 6p25. In some
embodiments, a combination of two probes is one probe that targets
chromosome subregion 6p25 and a second probe selected from the
group consisting of a chromosome 10 enumerator probe, a probe that
targets subregion 11q13, and a probe that targets chromosome
subregion 6q23.
[0008] The methods of the invention can also employ a probe set
that has three probes selected from the group consisting of a
chromosome 6 enumerator probe, a chromosome 10 enumerator probe, a
probe that targets chromosome region 6p (e.g., 6p25), a probe that
targets chromosome region 6q (e.g., 6q23), a probe that targets
chromosome region 7q (e.g., 7q34), a probe that targets chromosome
region 11q (e.g., 11q13), a probe that targets chromosome region
17q (e.g., 17q25), a probe that targets chromosome region 1q (e.g.,
1q31), and a probe that targets chromosome region 20q (e.g.,
20q13). In some embodiments, one of the three probes in the
combination of three probes targets chromosome subregion 6p25.
Thus, a combination of three probes for detecting melanoma is often
selected from the group of:
[0009] a) a probe that targets chromosome subregions 6p25, a probe
that targets chromosome subregion 6q23 and a chromosome 6
enumerator probe;
[0010] b) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 1q31 and a probe that targets
chromosome subregion 17q25;
[0011] c) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 11q13 and a chromosome 10
enumerator probe;
[0012] d) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 11q13 and a probe that targets
chromosome subregion 17q25;
[0013] e) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 17q25 and a chromosome 10
enumerator probe;
[0014] f) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 1q31 and a chromosome 10
enumerator probe;
[0015] g) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 1q31 and a probe that targets
chromosome subregion 11q13;
[0016] h) a probe that targets chromosome subregion 1q31, a probe
that targets chromosome subregion 11q13 and a probe that targets
chromosome subregion 17q25;
[0017] i) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 7q34 and a probe that targets
chromosome subregion 17q25;
[0018] j) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 20q13 and a probe that targets
chromosome subregion 17q25;
[0019] k) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 20q13 and a probe that targets
chromosome subregion 11q13;
[0020] l) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 20q13 and a probe that targets
chromosome subregion 6q23;
[0021] m) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 11q13 and a chromosome 10
enumerator probe;
[0022] n) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 7q34 and a chromosome 10
enumerator probe; and
[0023] o) a probe that targets chromosome subregion 6p25, a probe
that targets chromosome subregion 20q13 and a chromosome 10
enumerator probe.
[0024] In some embodiments, a combination of at least four probes
can be used to detect melanoma, where the probes are selected from
the group consisting of a chromosome 6 enumerator probe, a
chromosome 10 enumerator probe, a probe that targets chromosome
region 6p (e.g., 6p25), a probe that targets chromosome region 6q
(e.g., 6q23), a probe that targets chromosome region 7q (e.g.,
7q34), a probe that targets chromosome region 11q (e.g., 11q13), a
probe that targets chromosome region 17q (e.g., 17q25), a probe
that targets chromosome region 1q (e.g., 1q31), and a probe that
targets chromosome region 20q (e.g., 20q13). In some embodiments,
one of the four probes in the combination targets chromosome
subregion 6p25. Thus, a combination of four probes for use in
detecting melanoma can be selected from the following group:
[0025] a) a probe to chromosome subregions 6p25; a probe to
chromosome subregion 6q23, a chromosome 6 enumerator probe, and a
chromosome 10 enumerator probe;
[0026] b) a probe to chromosome subregions 6p25; a probe to
chromosome subregion 6q23, a probe to chromosome subregion 11q13,
and a chromosome 6 enumerator probe;
[0027] c) a probe to chromosome subregions 6p25; a probe to
chromosome subregion 1q31, a probe to chromosome subregion 11q13,
and a probe to chromosome subregion 17q25;
[0028] d) a probe to chromosome subregions 6p25; a probe to
chromosome subregion 6q23, a probe to chromosome subregion 11q13,
and a chromosome 10 enumerator probe; and
[0029] e) a probe to chromosome subregions 6p25; a probe to
chromosome subregion 6q23, a probe to chromosome subregion 1q31,
and a chromosome 10 enumerator probe;
[0030] f) a probe to chromosome subregions 6p25, probe to
chromosome subregion 1q31, a probe to chromosome subregion 17q25,
and a chromosome 10 enumerator probe;
[0031] g) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 17q25, a probe to chromosome subregion 6q23,
and a chromosome 6 enumerator probe;
[0032] h) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 20q13, a probe to chromosome subregion 6q23,
and a chromosome 6 enumerator probe;
[0033] i) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 6q23,
and a chromosome 6 enumerator probe;
[0034] j) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 1q31,
and a probe to chromosome subregion 17q25;
[0035] k) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 20q13, a probe to chromosome subregion 1q31,
and a probe to chromosome subregion 17q25;
[0036] l) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 6q23,
and a probe to chromosome subregion 17q25;
[0037] m) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 20q13, a probe to chromosome subregion 6q23,
and a probe to chromosome subregion 17q25;
[0038] n) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 11q13,
and a chromosome 10 enumerator probe;
[0039] o) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 20q13, a probe to chromosome subregion 11q13,
and a chromosome 10 enumerator probe;
[0040] p) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 17q25,
and a probe to chromosome subregion 11q13;
[0041] q) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 20q13, a probe to chromosome subregion 17q25,
and a probe to chromosome subregion 11q13;
[0042] r) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 17q25,
and a chromosome 10 enumerator probe;
[0043] s) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 20q13, a probe to chromosome subregion 17q25,
and a chromosome 10 enumerator probe;
[0044] t) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 20q13,
and a probe to chromosome subregion 17q25;
[0045] u) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 6q23, a probe to chromosome subregion 17q25,
and a chromosome 10 enumerator probe;
[0046] v) a probe to chromosome subregions 6p25, a probe to
chromosome subregion 17q25, a probe to chromosome subregion 11q13,
and a chromosome 10 enumerator probe; and
[0047] x) a probe to chromosome subregion 17q25, a probe to
chromosome subregion 7q34, a probe to chromosome subregion 6q23,
and a chromosome 6 enumerator probe.
[0048] Probe combinations are not limited to those that comprise
probes that target the exemplary subregions, supra. Any probe that
targets the chromosome region of interest can be used.
[0049] In typical embodiments, the melanoma detection methods of
the invention employ a skin sample, such as a skin biopsy sample.
In some embodiments, the biological sample may be a formalin-fixed,
paraffin-embedded sample. The biological sample is hybridized under
conditions in which the members of the probe set selectively
hybridize to the target chromosome or chromosome region/subregion.
The probes are often labeled with fluorescent labels. In some
embodiments, hybridization of the probe set is performed
concurrently, i.e., the probes are hybridized at the same time to
the same sample. The hybridization pattern of the probe set is
evaluated to determine whether malignant melanoma cells are present
in the lesion.
[0050] The invention also provides combinations of probes (two-,
three-, or four-probe combinations) for diagnosing melanoma and
kits that contain such combinations of probes. The probes that are
members of the combinations are selected from the group consisting
of a chromosome 6 enumerator probe, a chromosome 10 enumerator
probe, a probe that targets chromosome region 6p (e.g., 6p25), a
probe that targets chromosome region 6q (e.g., 6q23), a probe that
targets chromosome region 11q (e.g., 11q13), a probe that targets
chromosome region 17q (e.g., 17q25), and a probe that targets
chromosome region 1q (e.g., 1q31). Typically, a combination of
probes of the invention has a DPI value of less than about 0.29 for
melanoma. Often, the combination of probes has a DFI of less than
about 0.2. A set of probes of the invention is thus any of the
two-, three-, or four-probe combinations specifically set forth
above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIGS. 1A, 1B, and 1C show analysis of the CGH melanoma data
for the sensitivity of individual loci to detect melanoma. The
plots show the entire genome from the p arm of chromosome 1 to the
q arm of chromosome 22 binned by chromosome band position along the
x-axis. The y-axis shows the specificity. FIG. 1A shows the
sensitivity for DNA copy number losses. FIG. 1B shows the
sensitivity for DNA copy number losses gains. FIG. 1C shows the
sensitivity for DNA copy number amplifications.
[0052] FIGS. 2A, 2B, and 2C provide exemplary ROC curves for
several combinations of probes.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The terms "melanoma" or "cutaneous melanoma" or "malignant
melanoma" refer to malignant neoplasms of melanocytes, which are
pigment cells present normally in the epidermis, in adnexal
structures including hair follicles, and sometimes in the dermis,
as well as extracutaneous sites such as the mucosa, meninx,
conjuctiva, and uvea. There are at least four types of cutaneous
melanoma: lentigo maligna melanoma, superficial spreading melanoma
(SSM), nodular melanoma, and acral lentiginous melanoma (AM).
Cutaneous melanoma typically starts as a proliferation of single
melanocytes, e.g., 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.
[0054] The term "melanocytic lesion" refers to an accumulation of
melanocytes that can undergo a benign, locally aggressive, or
malignant course. "Melanocytic lesion" encompasses both benign
melanocytic neoplasms, such as "nevi" and "lentigines" and
"melanocytomas"; malignant melanocytic neoplasms, and "melanoma"
and "malignant blue nevus".
[0055] 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 term "neoplastic" refers to both benign
and malignant atypical growth.
[0056] 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 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 and Russell (2001) Molecular Cloning: A Laboratory Manual
(3rd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring
Harbor Press, NY, and detailed discussion, below).
[0057] 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 encompassed 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.
[0058] The term "biological sample" or "specimen" is intended to
mean a sample obtained from a patient suspected of having, or
having melanoma. Typically, the sample comprises a formalin-fixed
paraffin-embedded skin biopsy of a body region suspected to contain
melanoma cells. In addition to patients suspected of having
melanoma, the biological sample may further be derived from a
subject that has been diagnosed with melanoma for confirmation of
diagnosis or establishing that all of the tumor was removed ("clear
margin"). In addition, the biological sample may be derived from
non-skin tissue such as lymph nodes to establish whether any
melanocytes present in this tissue represent melanoma or nevus. The
biological sample may be derived from a subject with an ambiguous
diagnosis in order to clarify the diagnosis. The sample may be
derived from a "punch", "shave", curettage, fine needle aspirate,
sentinel lymph node or excisional biopsy, or other excision of the
region including the suspected lesion or peripheral to the
suspected or known lesion in order to establish a clear margin.
Introduction
[0059] The current invention is based, in part, on the
identification of highly sensitive and specific chromosomal probe
sets that can be used to selectively detect melanoma. The probe
sets provide higher sensitivity and specificity than individual
probes. The invention thus provides methods and compositions for
the use of such probe sets. The probes encompass locus-specific
probes as well as chromosome enumeration probes, which typically
hybridize to centromeric regions.
Chromosomal Probes
[0060] Probes for use in the invention are used for hybridization
to nucleic acids that are present in biological samples from
patients that have a melanocytic tumor for which some degree of
suspicion exists that it could be melanoma. In situ hybridization
is usually employed in the methods of the invention. In typical
embodiments, the probes are labeled with fluorescent labels.
[0061] A "chromosomal probe" or "chromosomal probe composition"
refers to one or more polynucleotides that specifically hybridize
to a region of a chromosome. The target sequences to which the
probe can bind vary in length, typically from about 70,000
nucletoides to about 800,000 nucleotides. Smaller probes, e.g.,
that hybridize to a region of less than 100,000 nucleotides; or to
a region of less than 10,000 nucleotides, can also be employed.
[0062] A probe to a particular chromosomal region can comprise
multiple polynucleotide fragments, e.g., ranging in size from about
50 to about 1,000 nucleotides in length.
Chromosome Enumeration Probe
[0063] A chromosome enumeration probe is any probe able to
enumerate the number of specific chromosomes in a cell. A
chromosome enumeration probe typically recognizes and binds to a
region near to (referred to as "peri-centromeric") or at the
centromere of a specific chromosome, typically a repetitive DNA
sequence. The centromere of a chromosome is typically considered to
represent that chromosome entity since the centromere is required
for faithful segregation during cell division. Deletion or
amplification of a particular chromosomal region can be
differentiated from loss or gain of the whole chromosome
(aneusomy), within which it normally resides, by comparing the
number of FISH signals corresponding to the particular locus (copy
number) to the number of signals for the corresponding centromere.
One method for making this comparison is to divide the number of
signals representing the locus by the number of signals
representing the centromere. Ratios of less than one indicate
relative loss or deletion of the locus, and ratios greater than one
indicate relative gain or amplification of the locus. Similarly,
comparison can be made between two different loci on the same
chromosome, for example on two different arms of the chromosome, to
indicate imbalanced gains or losses within the chromosome.
[0064] In lieu of a centromeric probe for a chromosome, one of
skill in the art will recognize that a chromosomal arm probe may
alternately be used to approximate whole chromosomal loss or gain.
However, such probes are not as accurate at enumerating chromosomes
since the loss of signals for such probes may not always indicate a
loss of the entire chromosomes. Examples of chromosome enumeration
probes include CEP.RTM. probes (e.g., CEP.RTM. 12 and X/Y probes)
commercially available from Abbott Molecular, DesPlaines, Ill.
(formerly Vysis, Inc., Downers Grove, Ill.).
[0065] Chromosome enumerator probes and locus-specific probes that
target a chromosome region or subregion can readily be prepared by
those in the art or can be obtained commercially, e.g., from Abbott
Molecular, Molecular Probes, Inc. (Eugene, Oreg.), or Cytocell
(Oxfordshire, UK). Such probes are prepared using standard
techniques Chromosomal probes may be prepared, for example, from
protein nucleic acids, cloned human DNA such as plasmids, bacterial
artificial chromosomes (BACs), and P1 artificial chromosomes (PACs)
that contain inserts of human DNA sequences. A region of interest
may be obtained via PCR amplification or cloning. Alternatively,
chromosomal probes may be prepared synthetically.
Locus-Specific Probes
[0066] Probes that can be used in the invention include probes that
selectively hybridize to chromosome regions (e.g., 1q, 6p, 6q, 7q,
11q, 17q, and 20q) or subregions of the chromosome regions (e.g.,
1q23, 1q31, 6p25, 6q23, 7q34, 11q13, 17q25, or 20q13). Such probes
are also referred to as locus-specific probes. Locus-specific probe
targets preferably comprise at least 100,000 nucleotides. A
locus-specific probe selectively binds to a specific locus at a
chromosomal region that is known to undergo gain or loss in
melanoma. A probe can target coding or non-coding regions, or both,
including exons, introns, and/or regulatory sequences, such as
promoter sequences and the like.
[0067] When targeting of a particular gene locus is desired, probes
that hybridize along the entire length of the targeted gene are
preferred although not required. For cells of a given sample,
relative to those of a control, increases or decreases in the
number of signals for a probe indicate a gain or loss,
respectively, for the corresponding region. In some embodiments, a
locus-specific probe can be designed to hybridize to an oncogene or
tumor suppressor gene, the genetic aberration of which is
correlated with melanoma. Locus-specific probes can hybridize to
loci on chromosomal regions including, for example, 8q24, 9p21,
17q, and 20q13. Exemplary, locus-specific probes that target these
regions are probes to C-MYC, P16, HER2, and ZNF217,
respectively.
[0068] Probes for use in the invention target region on chromosomal
arms (also referred to herein as a chromosomal region) that undergo
gain or loss in melanoma, e.g., 17q, 10q, 6p, 6q, 1q, 7q, 11q and
20q. Gain or loss can be determined using any probe that targets
the chromosome arm of interest. Often, probes that target
subregions, e.g., 17q25, 10q23, 6p25, 6q23, 1q23, 1q31, 7q34, 11q13
or 20q13, are employed. In the context of this invention, probes to
the chromosomal subregions noted herein are representative of the
chromosomal arm of interest. Further, the subregion designations as
used herein include the designated band and typically about 10
megabases of genomic sequence to either side.
Probe Selection Methods
[0069] Probe sets can be selected for their ability to simply
detect melanoma, but are typically selected for the ability to
discriminate between melanoma and other benign melanocytic lesions.
Thus, analyses of probe sets are typically performed to determine
the DFI values of different probe sets for discriminating between
melanoma and benign nevi.
[0070] Probe sets for use in the methods of the present invention
can be selected using the principles described in the examples.
Combinations of chromosomal probes within a probe set are chosen
for sensitivity, specificity, and detectability regarding melanoma.
Sensitivity refers to the ability of a test (e.g. FISH) to detect
disease (e.g. melanoma) when it is present. More precisely,
sensitivity is defined as True Positives/(True Positives+False
Negatives). A test with high sensitivity has few false negative
results, while a test with low sensitivity has many false negative
results. Specificity, on the other hand, refers to the ability of
test (e.g. FISH) to give a negative result when disease is not
present. More precisely, specificity is defined as True
Negatives/(True Negatives+False Positives). A test with high
specificity has few false positive results, while a test with a low
specificity has many false positive results.
[0071] In general, chromosomal probe sets with the highest combined
sensitivity and specificity for the detection of melanoma are to be
chosen. The combined sensitivity and specificity of a probe set can
be represented by the parameter distance from ideal (DFI), defined
as [(1-sensitivity).sup.2+(1-specificity).sup.2].sup.1/2. DFI
values range from 0 to 1.414, with 0 representing a probe set
having 100% sensitivity and 100% specificity and 1.414 representing
a probe set with 0% sensitivity and 0% specificity. In this
invention, probe sets chosen for the identification of melanoma
will have DFI values that are at most about 0.29. Probe sets that
have DFI values of less than about 0.20 usually provide better
results.
[0072] There is no limit to the number of probes that can be
employed in a set, however, in some embodiments, the number of
probes within a set that is to be viewed by a human observer (and
not with computer assisted imaging techniques) may be restricted
for practical reasons, e.g., by the number of unique fluorophores
that provide visually distinguishable signals upon hybridization.
For example, typically four or five unique fluorophores (e.g.,
which appear as red, green, aqua, and gold signals to the human
eye) can be conveniently employed in a single probe set. Generally,
the sensitivity of an assay increases as the number of probes
within a set increases. However, the increases in sensitivity
become smaller and smaller with the addition of more probes and at
some point the inclusion of additional probes to a probe set is not
associated with significant increases in the sensitivity of the
assay ("diminishing returns"). Increasing the number of probes in a
probe set may decrease the specificity of the assay. Accordingly, a
probe set of the present invention typically comprises two, three,
or four chromosomal probes, as necessary to provide optimal balance
between sensitivity and specificity.
[0073] Individual probes are chosen for inclusion in a probe set of
the present invention based on their ability to complement other
probes within the set. Specifically, they are targeted to
chromosomes or chromosomal subregions that are not frequently
altered simultaneously within a given melanoma. Thus, each probe in
a probe set complements the other(s), i.e., identifies melanoma
where the other probes in the set sometime fail to identify. One
method for determining which probes complement one another is to
identify single probes with the lowest DFI values for a group of
tumor specimens. Then additional probes can be tested on the tumor
samples that the initial probe failed to identify, and the probe
with the lowest DFI value measured in combination with the initial
probe(s) is added to the set. This may then be repeated until a
full set of chromosomal probes with the desired DFI value is
achieved.
[0074] Discrimination analysis is one method that can be used to
determine which probes are best able to detect melanoma. This
method assesses if individual probes are able to detect a
statistically different percentage of abnormal cells in test
specimens (e.g. melanoma) when compared to normal specimens. The
detection of cells with chromosomal (or locus) gains or chromosomal
(or locus) losses can both be used to identify neoplastic cells in
melanoma patients with melanocytic lesions. However, chromosomal
losses sometimes occur as an artifact in normal cells because of
random signal overlap and/or poor hybridization. In sections of
formalin-fixed paraffin-embedded material, commonly used to assess
skin biopsies, truncation of nuclei in the sectioning process can
also produce artifactual loss of chromosomal material.
Consequently, chromosomal gains are often a more reliable indicator
of the presence of neoplastic cells.
[0075] Cutoff values for individual chromosomal gains and losses
must be determined when choosing a probe set. The term "cutoff
value" is intended to mean the value of a parameter associated with
chromosomal aberration that divides a population of specimens into
two groups--those specimens above the cutoff value and those
specimens below the cutoff value. For example, the parameter may be
the absolute number or percentage of cells in a population that
have genetic aberrations (e.g., losses or gains for target
regions). If the number or percentage of cells in the specimen
harboring losses or gains for a particular probe is higher than the
cutoff value, the sample is determined to be positive for
melanoma.
[0076] A useful probe set often comprises a probe to a chromosomal
region selected from the group consisting of 1q23, 1q31, 6p25,
6q23, 7q34, 11q13, 17q25, and 20q13, and/or chromosome enumeration
probes for chromosomes 6 and 10, e.g., CEP.RTM. 6, and CEP.RTM. 10,
available from Abbott Molecular Inc. Such probe sets detect the
presence of melanoma and can discriminate melanoma from benign
specimens.
[0077] A probe set able to detect melanoma and/or discriminate
melanoma from benign specimens may comprise two or more probes
selected from the group of probes targeting 1q23, 1q31, 6p25, 6q23,
7q34, 11q13, 17q25, 20q13, or a chromosome enumerator probe, e.g.,
to the peri-centromeric regions of chromosomes 6 or 10. In some
embodiments, the probe set comprises probes to: a) 6p25 and a
chromosome 10 enumerator, b) 6p25 and 11q13, c) 6p25 and 6q23, d)
6p25 and 20q13, e) a chromosome 10 enumerator and 1q31, f) 6p25 and
1q31, g) 6q23 and 1q31, h) 7q34 and 6p25, i) 6q23 and 1q23, j)
6q23, 6p25 and a chromosome 6 enumerator, k) 6p25, 1q31 and 17q25,
l) 11q13, 6p25 and a chromosome 10 enumerator, m) 6p25, 11q13, and
17q25, n) 6p25, 1q31, and a chromosome 10 enumerator, o) 6p25,
1q31, and 11q13, p) 1q31, 11q13, and 17q25, q) 6q23, 6p25, and
11q13, r) 20q13, 7q34, and 6p25 s) 6p25, 6q23, a chromosome 6
enumerator, and a chromosome 10 enumerator, u) 6p25, 1q31, 11q13,
and 17q25, v) 6p25, 1q31, a chromosome 10 enumerator, and 17q25, w)
11q13, a chromosome 10 enumerator, 6q23, and 6p25, x) 6q23, 6p25,
1q31, and a chromosome 10 enumerator, or y) 6q23, a chromosome 6
enumerator, 6p25 and 11q13.
Probe Hybridization
[0078] Conditions for specifically hybridizing the probes to their
nucleic acid targets generally include the combinations of
conditions that are employable in a given hybridization procedure
to produce specific hybrids, the conditions of which may easily be
determined by one of skill in the art. Such conditions typically
involve controlled temperature, liquid phase, and contact between a
chromosomal probe and a target. Hybridization conditions vary
depending upon many factors including probe concentration, target
length, target and probe G-C content, solvent composition,
temperature, and duration of incubation. At least one denaturation
step must precede contact of the probes with the targets.
Alternatively, both the probe and nucleic acid target may be
subjected to denaturing conditions together while in contact with
one another, or with subsequent contact of the probe with the
biological sample. Hybridization may be achieved with subsequent
incubation of the probe/sample in, for example, a liquid phase of
about a 50:50 volume ratio mixture of 2-4.times.SSC and formamide,
at a temperature in the range of about 25 to about 55.degree. C.
for a time that is illustratively in the range of about 0.5 to
about 96 hours, or more preferably at a temperature of about 32 to
about 40.degree. C. for a time in the range of about 2 to about 16
hours. In order to increase specificity, use of a blocking agent
such as unlabeled blocking nucleic acid as described in U.S. Pat.
No. 5,756,696, the contents of which are herein incorporated by
reference, may be used in conjunction with the methods of the
present invention. Other conditions may be readily employed for
specifically hybridizing the probes to their nucleic acid targets
present in the sample, as would be readily apparent to one of skill
in the art.
[0079] Upon completion of a suitable incubation period,
non-specific binding of chromosomal probes to sample DNA may be
removed by a series of washes. Temperature and salt concentrations
are suitably chosen for a desired stringency. The level of
stringency required depends on the complexity of a specific probe
sequence in relation to the genomic sequence, and may be determined
by systematically hybridizing probes to samples of known genetic
composition. In general, high stringency washes may be carried out
at a temperature in the range of about 65 to about 80.degree. C.
with about 0.2.times. to about 2.times.SSC and about 0.1% to about
1% of a non-ionic detergent such as Nonidet P-40 (NP40). If lower
stringency washes are required, the washes may be carried out at a
lower temperature with an increased concentration of salt.
Detection of Probe Hybridization Patterns
[0080] The hybridization probes can be detected using any means
known in the art. Label-containing moieties can be associated
directly or indirectly with chromosomal probes. The term "label
containing moiety" or "detection moiety" generally refers to a
molecular group or groups associated with a chromosomal probe,
either directly or indirectly, that allows for detection of that
probe upon hybridization to its target. Different label containing
moieties are selected for each individual probe within a particular
set so that each hybridized probe is visually distinct from the
others upon detection. Preferably, fluorescence in situ
hybridization (FISH) is employed and the chromosomal probes are
labeled with distinct fluorescent label-containing moieties.
Fluorophores, organic molecules that fluoresce upon irradiation at
a particular wavelength, are typically directly attached to the
chromosomal probes. A large number of fluorophores are commercially
available in reactive forms suitable for DNA.
[0081] Attachment of fluorophores to nucleic acid probes is well
known in the art and may be accomplished by any available means.
Fluorophores can be covalently attached to a particular nucleotide,
for example, and the labeled nucleotide incorporated into the probe
using standard techniques such as nick translation, random priming,
PCR labeling, and the like. Alternatively, the fluorophore can be
covalently attached via a linker to the deoxycytidine nucleotides
of the probe that have been transaminated. Methods for labeling
probes are described in U.S. Pat. No. 5,491,224 and Molecular
Cytogenetics: Protocols and Applications (2002), Y.-S. Fan, Ed.,
Chapter 2, "Labeling Fluorescence In Situ Hybridization Probes for
Genomic Targets," L. Morrison et al., p. 21-40, Humana Press, both
references of which are herein incorporated by reference.
[0082] Exemplary fluorophores that can be used for labeling probes
include TEXAS RED (Molecular Probes, Inc., Eugene, Oreg.), CASCADE
blue aectylazide (Molecular Probes, Inc., Eugene, Oreg.),
SpectrumOrange.TM. (Abbott Molecular, Des Plaines, Ill.) and
SpectrumGold.TM. (Abbott Molecular).
[0083] One of skill in the art will recognize that other agents or
dyes can be used in lieu of fluorophores as label-containing
moieties. Luminescent agents include, for example,
radioluminescent, chemiluminescent, bioluminescent, and
phosphorescent label containing moieties. Alternatively, detection
moieties that are visualized by indirect means can be used. For
example, probes can be labeled with biotin or digoxygenin using
routine methods known in the art, and then further processed for
detection. Visualization of a biotin-containing probe can be
achieved via subsequent binding of avidin conjugated to a
detectable marker. The detectable marker may be a fluorophore, in
which case visualization and discrimination of probes may be
achieved as described above for FISH.
[0084] Chromosomal probes hybridized to target regions may
alternatively be visualized by enzymatic reactions of label
moieties with suitable substrates for the production of insoluble
color products. Each probe may be discriminated from other probes
within the set by choice of a distinct label moiety. A
biotin-containing probe within a set may be detected via subsequent
incubation with avidin conjugated to alkaline phosphatase (AP) or
horseradish peroxidase (HRP) and a suitable substrate.
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium
(NBT) serve as substrates for alkaline phosphatase, while
diaminobenzidine serves as a substrate for HRP.
[0085] In embodiments where fluorophore-labeled probes or probe
compositions are used, the detection method can involve
fluorescence microscopy, flow cytometry, or other means for
determining probe hybridization. Any suitable microscopic imaging
method may be used in conjunction with the methods of the present
invention for observing multiple fluorophores. In the case where
fluorescence microscopy is employed, hybridized samples may be
viewed under light suitable for excitation of each fluorophore and
with the use of an appropriate filter or filters. Automated digital
imaging systems such as the MetaSystems, BioView or Applied Imaging
systems may alternatively be used.
Screening and Diagnosis of Patients for Melanoma
[0086] The detection methods of the invention comprise obtaining a
biological sample from a subject having melanoma or suspected of
having melanoma. The biological sample is typically a tissue sample
that comprises the melanocytic lesion. Often, the biological sample
is formalin-fixed and paraffin embedded. The sample is contacted
with chromosomal probes to selectively detect melanoma in the
sample, if any, under conditions for specifically hybridizing the
probes to their nucleic acid targets present in the sample. The
probes of the set can be hybridized concurrently or sequentially
with the results of each hybridization imaged digitally, the probe
or probes stripped, and the sample thereafter hybridized with the
remaining probe or probes. Multiple probe sets can also be
hybridized to the sample in this manner.
[0087] The biological sample can be from a patient suspected of
having melanoma or from a patient diagnosed with melanoma, e.g.,
for confirmation of diagnosis or establishing a clear margin, or
for the detection of melanoma cells in other tissues such as lymph
nodes. The biological sample can also be from a subject with an
ambiguous diagnosis in order to clarify the diagnosis. The
biological sample can also be from a subject with a
histopathologically benign lesion to confirm the diagnosis.
Biological samples can be obtained using any of a number of methods
in the art. Examples of biological samples comprising potential
melanocytic lesions include those obtained from excised skin
biopsies, such as punch biopsies, shave biopsies, fine needle
aspirates, or surgical excisions; or biopsy from non-cutaneous
tissues such as lymph node tissue, mucosa, meninx, conjuctiva, or
uvea. In other embodiments, the biological sample can be obtained
by shaving, waxing, or stripping the region of interest on the
skin.
[0088] As noted, a biological sample can be treated with a fixative
such as formaldehyde and embedded in paraffin and sectioned for use
in the methods of the invention. Alternatively, fresh or frozen
tissue can be pressed against glass slides to form monolayers of
cells known as touch preparations, which contain intact nuclei and
do not suffer from the truncation artifact of sectioning. These
cells may be fixed, e.g., in alcoholic solutions such as 100%
ethanol or 3:1 methanol:acetic acid. Nuclei can also be extracted
from thick sections of paraffin-embedded specimens to reduce
truncation artifacts and eliminate extraneous embedded material.
Typically, biological samples, once obtained, are harvested and
processed prior to hybridization using standard methods known in
the art. Such processing typically includes protease treatment and
additional fixation in an aldehyde solution such as
formaldehyde.
Prescreening of Samples
[0089] Prior to detection, cell samples may be optionally
pre-selected based on apparent cytologic abnormalities as disclosed
in U.S. Pat. No. 6,174,681, the contents of which are herein
incorporated by reference. Pre-selection identifies suspicious
cells, thereby allowing the screening to be focused on those cells.
Pre-selection allows for faster screening and increases the
likelihood that a positive result will not be missed. During
pre-selection, cells from a biological sample are placed on a
microscope slide and visually scanned for cytologic abnormalities
commonly associated with dysplastic and neoplastic cells. Such
abnormalities include abnormalities in nuclear size, nuclear shape,
and nuclear staining, as assessed by counterstaining nuclei with
nucleic acid stains or dyes such as propidium iodide or
4,6-diamidino-2-phenylindole dihydrochloride (DAPI) usually
following hybridization of probes to their target DNAs. Typically,
neoplastic cells harbor nuclei that are enlarged, irregular in
shape, and/or show a mottled staining pattern. Propidium iodide,
typically used at a concentration of about 0.4 .mu.g/ml to about 5
.mu.g/ml, is a red-fluorescing DNA-specific dye that can be
observed at an emission peak wavelength of 614 nm. DAPI, typically
used at a concentration of about 125 ng/ml to about 1000 ng/ml, is
a blue fluorescing DNA-specific stain that can be observed at an
emission peak wavelength of 452 nm. In this case, only those cells
pre-selected for detection are subjected to counting for
chromosomal losses and/or gains. Preferably, pre-selected cells on
the order of at least 20, and more preferably at least 30-40, in
number are chosen for assessing chromosomal losses and/or gains.
Preselection of a suspicious region on a tissue section may be
performed on a serial section stained by conventional means, such
as H&E or PAP staining, and the suspect region marked by a
pathologist or otherwise trained technician. The same region is
then located on the serial section stained by FISH and nuclei
enumerated within that region. Within the marked region,
enumeration may be limited to nuclei exhibiting abnormal
characteristics as described above.
[0090] Alternatively, cells for detection may be chosen independent
of cytologic or histologic features. For example, all
non-overlapping cells in a given area or areas on a microscope
slide may be assessed for chromosomal losses and/or gains. As a
further example, cells on the slide, e.g., cells that show altered
morphology, on the order of at least about 50, and more preferably
at least about 100, in number that appear in consecutive order on a
microscope slide may be chosen for assessing chromosomal losses
and/or gains.
Hybridization Pattern
[0091] The term "target region" or "nucleic acid target" refers to
a nucleotide sequence that resides at a specific chromosomal
location whose loss and/or gain is indicative of the presence of
melanoma. The "target region" or "nucleic acid target" is to be
specifically recognized by a probe of the present invention and
hybridize to the same in the method of the present invention.
[0092] The hybridization pattern for the set of chromosomal probes
to the target regions is detected and recorded for cells chosen for
assessment of chromosomal losses and/or gains. Hybridization is
detected by the presence or absence of the particular signals
generated by each of the chromosomal probes. The term
"hybridization pattern" is intended to refer to the quantification
of chromosomal losses/gains for those cells chosen for such
assessment, relative to the number of the same in an evenly matched
control sample, for each probe throughout a chosen cell sample. The
quantification of losses/gains can include determinations that
evaluate the ratio of one locus to another on the same or a
different chromosome. Once the number of target regions within each
cell is determined, as assessed by the number of regions showing
hybridization to each probe, relative chromosomal gains and/or
losses may be quantified. A relative gain or loss for each probe is
determined by comparing the number of distinct probe signals in
each cell to the number expected in a normal cell, i.e., where the
copy number should be two. Non-neoplastic cells in the sample, such
as keratinocytes, fibroblasts, and lymphocytes, can be used as
reference normal cells. More than the normal number of probe
signals is considered a gain, and fewer than the normal number is
considered a loss. Alternatively, a minimum number of signals per
probe per cell can be required to consider the cell abnormal (e.g.,
5 or more signals). Likewise for loss, a maximum number of signals
per probe can be required to consider the cell abnormal (e.g., 0
signals, or one or fewer signals).
[0093] The percentages of cells with at least one gain and/or loss
are to be recorded for each locus. A cell is considered abnormal if
at least one of the identified genetic aberrations identified by a
probe set of the present invention is found in that cell. A sample
may be considered positive for a gain or loss if the percentage of
cells with the respective gain or loss exceeds the cutoff value for
any probes used in an assay. Alternatively, two or more genetic
aberrations can be required in order to consider the cell abnormal
with the effect of increasing specificity. For example, wherein
gains are indicative of a melanoma malignancy or precursor lesion,
a sample is considered positive if it contains, for example, at
least four cells showing gains of at least two or more
probe-containing regions.
Probe Combinations and Kits for Use in Diagnostic and/or Prognostic
Applications
[0094] The invention includes highly specific and sensitive
combinations of probes that can be used to detect melanoma and kits
for use in diagnostic, research, and prognostic applications. These
combinations of probes typically have a DFI of less than about 0.29
and often less than about 0.20. Kits include probe sets and can
also include reagents such as buffers and the like. 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
Probe Selection
[0095] CGH Database, The CGH data on which probe selection was
based had been acquired at the University of California, San
Francisco and has been published previously (Bastian et al, Am J
Pathol. 163:1765-70, 2003). The data were from 136 primary
cutaneous melanoma specimens (63 superficial spreading melanomas
(SSM), 30 lentigo maligna melanomas, (LMM), 23 acral-lentiginous
(ALM), 4 nodular melanomas (NM), 10 not classifiable (NC), and 6
melanomas arising within a nevus) and 53 benign nevi specimens (19
blue nevi, 7 congenital nevi, 27 Spitz nevi). The genome from the
1p telomere to the 22q telomere (chromosomes X and Y omitted) was
divided into 571 `bins` according to the Giemsa banding pattern of
chromosomes and the CGH ratio corresponding to each bin was
interpreted as reflecting chromosomal gain (tumor to reference
fluorescence intensity ratio) (ratio>1.2), and loss
(ratio<0.8). These thresholds were based on hybridizations of
normal DNA versus normal DNA as published previously (Bastian et
al, Cancer Res.; 58:2170-5, 1998). The sensitivities,
specificities, and DFI values to discriminate between nevi and
melanomas were calculated at each bin for identifying melanoma by
losses or gains. To determine the best combination of bins to
discriminate nevi from melanoma, bins were examined in combinations
of up to 4, calculating sensitivity, specificity, and DFI value for
each combination.
FISH Probe Sets
[0096] FISH was performed with eight unique probe sets. Each probe
set contained three or four chromosome enumeration probes or locus
specific identifiers to centromeres or specific loci of chromosomes
(Table 1) that were prevalent in the better performing probe
combinations (low DFI values) as determined from the CGH data
analysis (see above). The chromosome enumerator probes were
included to determine allelic gain or loss of the corresponding
locus specific identifiers on those chromosomes (e.g., 9p21 on
chromosome 9) or aneusomy of those chromosomes.
TABLE-US-00001 TABLE 1 FISH Probes and Gene Target Locations Used
for Probe Selection Probe Set Aqua Green Orange Gold Red I CEP
.RTM. 10 17q25 (TK1) 6p25 9p21 (RREB1) (p16) II CEP .RTM. 9 8q24
(MYC) 8p22 (LPL) III CEP .RTM. 10 10q23 1q23 (PTEN) (NTRK1) IV CEP
.RTM. 6 CEP .RTM. 7 7q34 (BRAF) V CEP .RTM. 8 CEP .RTM. 9 20q13 VI
7q34 6p25 20q13 9p21 (BRAF) (RREB1) (p16) VII 6q23 6p25 CEP .RTM. 6
(MYB) (RREB1) VIII CEP .RTM. 10 17q25 (TK1) 1q31 (COX2) 11q13
(CCND1)
[0097] With the exception of the LSI.RTM. 17q25 (TK1), LSI.RTM.
6p25 (RREB1), LSI.RTM. 7q34 (BRAF), LSI.RTM. LPL (8p22), LSI.RTM.
1q31 (COX2), and LSI.RTM. 1q23 (NTRKI) probes, the LSI.RTM. and
CEP.RTM. probes are commercially available from Abbott Molecular
Inc. (www.abbottmolecular.com) although LSI.RTM. p16 (9p21) and
LSI.RTM. 20q13 are commercially available only labeled with
SpectrumOrange.TM.. Instead of the SpectrumOrange.TM. label, the
nucleic acid starting material was transaminated and then
chemically labeled using Texas Red.RTM. 44 (trademark of Molecular
Probes, Eugene, Oreg.), and 5-(and 6-)-carboxyrhodamine 6G (gold),
respectively, The transamination and labeling process is described
in Bittner, et al. U.S. Pat. No. 5,491,224, incorporated herein by
reference.
[0098] The LSI.RTM. 1q31 (COX2) probe was made from five BAC clones
(Identification Nos. RPCI-11-70N10, RPCI-11-809F11, RPCI-11-104B23,
RPCI-11-457L10, RPCI-11-339I2). The LSI.RTM. 17q25 (TK1) probe was
made from two BAC clones (Identification Nos. RPCI11-219g17,
RPCI11-153a23), LSI.RTM. 6p25 (RREB1) was made from two BAC clones
(Identification Nos. RPCI11-405o10, RPCI11-61o1). LSI.RTM. 7q34
(BRAF) was made from two BAC clones (Identification Nos.
RPCI11-837g3, CITD-2516j12), LSI.RTM. LPL (8p22) was made from two
clones (Identification Nos. 67-o21, CTD-2286-L9). LSI.RTM. 1q23
(NTRK1) was made from two BAC clones (Identification Nos.
RPCI11-107d16, RPCI11-356j7). Clones were obtained from BAC Pac
Resources or Invitrogen. The probes were transaminated and labeled
using Texas Red (red), 6-[fluorescein 5-(and
-6)-carboxamido]hexanoic acid, succinimidyl ester (green), 5-(and
6-)-carboxyrhodamine 6G, succinimidyl ester (gold) or 5-(and
-6)-carboxytetramethylrhodamine, succinimidyl ester.
Study Population and Preparation of Samples for Analysis
[0099] The study included specimens of 86 melanomas and 31 benign
nevi assembled into microtissue arrays as well as sections from 94
melanoma and 95 benign nevi FFPE skin biopsy specimens.
[0100] Paraffin blocks containing tissue biopsy specimens were
sectioned at 5 .mu.M thickness and mounted onto SuperFrost
Plus.RTM. positively charged slides (ThermoShandon, Pittsburgh,
Pa.). All slides were baked at 56.degree. C. overnight to fix the
tissue onto the slides, and were then stored at room temperature.
In preparation for in situ hybridization, array specimen slides
were de-paraffinized by soaking in 3 changes of Hemo-De.TM. Solvent
and Clearing Agent (Scientific Safety Solvents, Keller, Tex.) for 5
minutes each, followed by two 1-minute rinses in absolute ethanol.
After drying, the specimens were further prepared for in situ
hybridization by treatment in 45% formic acid/0.3% H.sub.2O.sub.2
for 15 minutes followed by a 3 minute rinse in water. Samples were
then immersed in Vysis Pretreatment Solution (Abbott Molecular Inc)
at 80.degree. C. for 10 minutes, and rinsed in water for 3 minutes.
The slides were then immersed in a solution of Proteinase K (35
.mu.g proteinase K/ml buffer; Abbott Molecular Inc.) at 37.degree.
C. for 10 minutes, immersed in 0.01 N HCl for 3 minutes, rinsed in
water for 3 minutes, dehydrated in 70%, 85%, and 100% ethanol for 1
minute each, and allowed to dry. In preparation for in situ
hybridization, tissue sections were de-paraffinized by soaking in 3
changes of Hemo-De.TM. Solvent and Clearing Agent (Scientific
Safety Solvents, Keller, Tex.) for 5 minutes each, followed by two
1-minute rinses in absolute ethanol. After drying, the specimens
were further prepared for in situ hybridization by treatment in
1.times.SSC pH 6.3 at 80.degree. C. for 35 minutes, and rinsed in
water for 3 minutes. The slides were then immersed in a solution of
Protease (4 mg protease/ml 0.2 N HCl) at 37.degree. C. for 15
minutes, rinsed in water for 3 minutes, dehydrated in 70%, 85%, and
100% ethanol for 1 minute each, and allowed to dry.
FISH Hybridization
[0101] The prepared specimen slides were hybridized with FISH probe
solutions in a HYBrite.TM. automated co-denaturation oven (Abbott
Molecular Inc). The slides were placed on the oven surface, probe
solution was placed over the tissue section (typically 10 .mu.l), a
coverslip was applied over the probe solution, and the edges of the
coverslip were sealed to the slide with rubber cement. The oven
co-denaturation/hybridization cycle was set for denaturation at
73.degree. C. for 5 minutes, and hybridization at 37.degree. C. for
16-18 h. After hybridization, the slides were removed from the
HYBrite, and the rubber cement was removed. The slides were placed
in room-temperature 2.times.SSC(SSC=0.3 M NaCl, 15 mM sodium
citrate)/0.3% Nonidet P40 (NP40; Abbott Molecular Inc.) for 2 to 10
minutes to remove the coverslips. The slides were then immersed in
73.degree. C. 2.times.SSC/0.3% NP40 for 2 minutes for removal of
nonspecifically bound probe, and allowed to dry in the dark. DAPI I
antifade solution (Abbott Molecular Inc.) was applied to the
specimen to allow visualization of the nuclei.
Enumeration of FISH Signals
[0102] Slides were analyzed with an epi-fluorescence microscope
equipped with single band-pass filters for the DAPI counterstain,
SpectrumAqua.TM., and SpectrumGold.TM., SpectrumGreen.TM.,
SpectrumOrange.TM., and SpectrumRed.TM.. FISH signal enumeration
was performed without knowledge of the patient's clinical or
histologic findings. Probe sets I-V were hybridized to tissue
microarrays and 40 cells analyzed per specimen. Probe sets VI-VIII
were hybridized to specimen sections and 30 cells analyzed per
specimen. For specimens with low cellularity, a minimum of 20
nuclei was required for inclusion in the data analysis.
Analysis of Enumeration Data
[0103] The FISH number of signals recorded for each cell nucleus
within a specimen can be used to classify the corresponding loci as
normal (2 signals for loci on autosomes), abnormal due to loss (1
or 0 signals), or abnormal due to gain (greater than 2 signals).
Several parameters were defined as measures of abnormal locus gains
and losses within a specimen:
Parameters Based on Percentages of Cells Enumerated within a
Specimen:
[0104] % Gain: 1) for a single locus, % Gain is the percentage of
cells with >2 signals corresponding to that locus. [0105] 2) for
gain of one locus (locus 1) relative to another locus (locus 2),
Gain is the percentage of cells with more locus 1 signals than
locus 2 signals.
[0106] % Loss: 1) for a single locus, % Loss is the percentage of
cells with <2 signals corresponding to that locus. [0107] 2) for
loss of one locus (locus 1) relative to another locus (locus 2),
e.g., MYB/CEP 6, Loss is the percentage of cells with fewer locus 1
(e.g., MYB) signals than locus 2 (e.g., CEP 6) signals.
[0108] % Abnormal: 1) for a single locus, % Abnormal is the
percentage of cells with >2 signals or <2 signals
corresponding to that locus. [0109] 2) for abnormal balance between
two loci (locus 1 and locus 2),
[0110] Abnormal is the percentage of cells for which the number of
locus 1 signals does not equal the number of locus 2 signals.
Parameters Based on Averages Over all Cells Enumerated within a
Single Specimen:
[0111] average locus copy number: For a single locus, average copy
number is the number of signals corresponding to that locus summed
over all cells enumerated, divided by number of cells
enumerated.
[0112] average locus ratio: For a ratio of two loci (locus 1/locus
2), e.g., MYB/CEP 6 shown in Table 6, average locus ratio is the
number of locus 1 (e.g., MYB) signals summed over all cells
enumerated, divided by the number of locus 2, (e.g., CEP 6) signals
summed over all cells enumerated.
[0113] The values of the above five parameters were tabulated for
each locus and relevant locus pairs (e.g., loci on the same
chromosome such as 6q23 (MYB) and CEP 6) for each specimen, and the
means (x) and standard deviations (s) of each parameter were
calculated for the nevi and melanoma specimen groups, excluding
specimens of insufficient signal quality for enumeration.
[0114] The discriminate value (DV), defined as
(x.sub.1-x.sup.2).sup.2/(s.sub.1.sup.2+s.sub.2.sup.2) was used as a
measure of the ability of a specific locus aberration, measured by
one of the five parameters above, to distinguish between a sample
from the group of patients having melanoma and a sample from the
group of patients not exhibiting melanoma. In this formula, x1 and
s1 refer to the mean (x) and standard deviation (s) of a specific
locus parameter for the melanoma specimen group, and x2 and s2
refer to the mean and standard deviation of that parameter for the
benign nevi specimen group. Larger DV values are indicative of a
greater ability to distinguish between the two groups of patients.
As another measure of discrimination, the Student's t-test was used
to compare the values measured for each parameter between the nevi
and melanoma specimen groups in order to determine if the
differences between the two groups were statistically significant
(probabilities <0.05 were considered significant).
[0115] Sensitivities and specificities were calculated by applying
cutoffs to the various locus parameters for each of the 17 loci.
For the % Gain, % Loss, and % Abnormal parameters, a specimen was
considered positive for a particular locus if the value of that
parameter exceeded the cutoff value. For the average locus copy
number and average locus ratio parameters, cutoff values could be
applied to distinguish: 1) aberrantly high copy number or ratio,
(denoted as r-gain in Table 6), for which parameter values greater
than the cutoff value indicated a specimen was positive, 2)
aberrantly low copy number or ratio, (denoted by r-loss in Table
6), or 3), for which parameter values less than the cutoff value
indicated a specimen was positive, or 3) or both conditions 1) and
2). Note that cutoffs values for 1) and 2) may be different.
[0116] The sensitivity for detecting specimens with a particular
diagnosis was equal to the fraction of specimens in that group that
were positive. Specimens that did not provide at least 20 cells
with FISH signals of sufficient quality for counting were excluded
from the calculation. Specificity, relative to a control group, was
calculated as one minus the fraction of the control group specimens
that were positive using the same criteria (false positives). For
single probes, sensitivity, specificity, and DFI were calculated
for cut-off values between 0 and 100% abnormal cells, at 1%
increments. The parameter `distance from ideal` (DFI), which
incorporates both sensitivity and specificity, was used to assess
the relative performance of each probe or combination of
probes.
[0117] For combinations of probes cutoffs were applied by two
different methods that allowed each locus, as measured by the
parameter selected for that locus, to have a different cutoff
value. If any of the loci targeted by the probe combination were
positive for the respective cutoff, then the specimen was
considered positive. Permutation analysis of individual cut-off
values between 0 and 100% abnormal cells at 1% increments was not
practical for combinations of three or more probes (due to the
excessive computation time required), so cut-offs based on the
means and standard deviations of the locus parameters for the
benign nevi specimen group were calculated first. Cut-offs were
generated as x+n*s, where x and s are the mean and standard
deviation for a particular locus-specific parameter in the nevi
specimen group and n is a multiplier typically ranging from -1 to 3
in increments of 0.1. For probe combinations the cut-off values
were calculated using the respective x and s for each probe and
parameter in the combination, but for the same value of n. In order
to identify the best cutoff values for discriminating between
malignancy and benign specimens, cutoff values were calculated for
the lowest value of n (e.g., -1), sensitivity, specificity, and DFI
values were then calculated based on those cutoffs, n was
incremented (e.g., by 0.1), and the calculations were repeated
until a maximum value of n (e.g., 3) was reached This procedure
provided cut-off values adjusted to each probe based on the level
of abnormality and extent of variation in the nevi group. To a
first approximation, basing cut-offs on x and common multiples of s
establishes a similar specificity relative to the nevi group for
each probe and parameter in the combination for each value of n
(assuming a normal distribution of parameter values). Probes and
probe combinations at each cut-off or set of cut-off values were
sorted from lowest to highest DFI in order to identify the better
performing probe combinations. Optimal cut-off values for top
performing probe combinations (lowest DFI values) were further
refined by a second method. By this method cutoffs were
independently varied in small increments over a reduced range of
cutoffs flanking the optimal cutoffs established by the first
method.
[0118] Probe complementation was evaluated by calculating
sensitivity, specificity, and DFI values for all possible probe
combinations up to combinations of four probes, over a range of
cutoffs as described above. Each probe was also examined using the
five different parameters for measuring aberrations. The relevant
parameters examined are included in Tables 2 through 6. Two probes
complement one another if a lower DFI value can be achieved for the
two probes collectively, than for either probe individually.
Typically, only probes providing p-values less than 0.05 in the
discrimination analysis (Table 3) were utilized in these
calculations in order to reduce the likelihood that low DFI values
would result from the combination of random events, and to reduce
the computation time. Receiver Operator Characteristics (ROC)
graphs were generated by plotting sensitivity versus 1--specificity
for a particular probe and parameter or probe combination over the
range of cutoff values examined (see above). Since independently
varied cut-off values in probe combinations generates multiple
sensitivity values for each specificity value, only the highest
sensitivity value at each specificity value was plotted,
representing the optimal combination of cut-off values for each
specificity. Relative performance of a probe or combination of
probes can be assessed from these curves by the areas under the
curves (better performance indicated by larger areas) or by the
distance of closest approach to the point (0, 1) on the graph (100%
specificity, 100% sensitivity). Notice that the distance of any
point on the curves to the point (0, 1) is equal to the DFI value,
and probe combinations with lower DFI values perform better than
those with higher DFI values. The cut-offs associated with the
lowest DFI value for a particular probe combination are the optimal
cutoffs for that combination. However, depending upon the
application, points on the ROC curves with somewhat lower DFI
values may be selected, after considering the relative clinical
importance of sensitivity and specificity. For example, a point on
the curve with a slightly higher sensitivity but lower specificity
and higher DFI may be chosen over another point on the curve that
has a lower sensitivity and higher specificity and lower DFI if it
is more important to identify as many cancers as possible, at the
expense of a higher false positive rate.
Results
[0119] CGH Database Analysis and Selection of Probes for FISH. CGH
data were used to identify genetic loci for FISH probe development.
The first step in identifying loci was to calculate the
sensitivity, specificity, and DFI value at each of the 571 bins
across the genome from 1p through 22q with respect to the 136
melanoma specimens and 56 benign nevi specimens. Specificities were
100% at nearly all loci, so only the sensitivities are plotted
versus bin number in FIG. 1 parts A (losses), B (gains), and C
(amplifications). Loci with highest sensitivities for detecting
melanoma include 1p21, 3q29, 6q25.3, 8p23, 9p21, 9q21.1, 10p15,
10q23.3, 11q25, 13q34, 16q24, and 17pter for loss; 1q22, 3p21.3,
6p25.3, 7p21, 7q33, 8q24.2, 15q26, 17q25, and 20q13.3 for gain; and
5p15.3, 11q13.2, and 22q13.1 for amplification. Regions of gain and
loss were often broad, at times including whole chromosomes or
chromosome arms, indicating FISH probe placement would not be
critical, i.e., FISH probes in neighboring bands should usually
yield similar efficiency for detecting abnormality.
[0120] Centromere status was assessed from the status of
non-repetitive sequences flanking the centromere. Centromeres
predicted to have high sensitivities for detecting melanoma based
on the CGH data included centromeres 3, 6, 9, and 10 for losses,
and centromeres 1, 6, 7, 8, and 20 for gains.
[0121] The second step in identifying loci for FISH probe
development was to examine the ability of different loci to
complement one another for detecting malignancy (i.e. sensitivity
of combined loci is greater than individual sensitivities of the
loci). To this end, sensitivities, specificities and DFI values
were calculated for all possible combinations of two, three, and
four of the loci listed above. Table 7 shows sensitivities and DFI
values for loci and locus combinations with lowest DFI values for
the 136 melanoma specimens in the CGH database. For single loci,
sensitivities and DFI values are listed for imbalance (gain, loss,
or amplification), gain, loss, or amplification. For locus
combinations, only the sensitivities and DFI values for imbalance
are listed. The individual loci and combinations of loci with the
lowest DFI values (FISH) are listed in Table 6 ordered from lowest
to highest DFI values. The 14 loci appearing most commonly in the
combinations with the lowest DFI values were selected for
evaluation by FISH on TMA and include the probe sets I-V in Table
1. Upon analysis of the 14 loci, a subset of the FISH probes was
assessed on tissue sections of individual melanocytic tumors (probe
set VD. Additional combinations off new probes were also tested on
such sections (Probe set VII-VIII; Table 1) to identify loci that
complemented one another for detecting malignancy.
Discrimination Analysis
[0122] The ability of each FISH probe to discriminate between the
group of patients having melanoma and patients having a benign
nevus was initially examined by comparing the means and standard
deviations of the different parameters for each locus tested
between the benign nevi specimen group and the melanoma specimen
group. For the tissue microarray hybridizations and for each locus
or locus ratio, Table 2 lists the relevant data for the benign nevi
specimen group, including the number of specimens evaluated (N),
the means of the percent of cells with gain (% Gain) or loss (%
Loss), the average locus copy number (single loci) or average locus
ratio (ratios of 2 loci), and the corresponding standard
deviations. Means and standard deviations were calculated for the
melanoma specimen group and are listed in Table 3 for the tissue
microarray. Table 3 also lists DVs and p-values, quantities that
reflect the ability of particular probes or probe ratios to
differentiate between melanoma and normal specimens. The DVs and
p-values were consistent in that lower p-values were accompanied by
higher DVs. Entries of NA in Table 3 for DV and p-values indicate
that the mean of the melanoma group was lower than that of the
benign nevi group.
[0123] The p-values listed in Table 3 indicate that gains of
centromere 8, centromere 9, 1q23, 6p25, 7q34, 17q25, and 20q13
loci, and gain of 7q34 relative to centromere 7 occur in a
significantly higher percentage of cells in melanoma specimens than
in normal specimens. In addition, loss of the 6p25, 8p22, 8q24,
9p21, 17q25, centromere 6, and centromere 10 loci, and loss of
10q23 (identified using PTEN probe) relative to centromere 10,
occur in a significantly higher percentage of cells for melanoma
specimens than for normal specimens. Based on DV, the gain of 20q13
and centromere 6 loci showed the greatest level of discrimination
between melanoma and nevi, both having DV>1. Likewise, the loss
of 9p21, centromere 6, and 17q25 loci showed DV>1.
[0124] Based on the data in Table 3, a subset of probes was
hybridized to entire tissue sections and analyzed in the same
manner using DVs and p-values to confirm the data collected on
tissue microarrays. As seen in Table 5, probes selected based upon
cells with gain from the tissue microarray data have a similar
performance. Several new probes were analyzed on tissue sections
(probe sets VII and VIII; Table 1). Means and standard deviations
were calculated for melanoma and are listed in Table 5 for the
sections. Table 5 also lists DVs and p-values, quantities that
reflect the ability of particular probes or probe ratios to
differentiate between melanoma and normal specimens. The DVs and
p-values were consistent in that lower p-values were accompanied by
higher DVs.
[0125] The p-values listed in Table 5 indicate that gains of 1q31,
6p25, 6q23, 7q34, 9p21, 11q13, 17q25, 20q13, and relative gain of
6q23 to centromere 6 occur in a significantly higher percentage of
cells for melanoma specimens than for normal specimens. Based on
the DV, gain of 6p25 showed the greatest level of discrimination
between melanoma and nevi, having a DV=0.7.
Single Probe Sensitivities, Specificities, and DFI Values
[0126] Sensitivities, specificities, and DFI values were calculated
for individual probes hybridized to sections over a range of
cut-off values and are listed at the top of Table 6. For melanoma
versus nevi, the best DFI values (i.e. lowest DFI values) were
obtained for 6p25, 1q31, 7q34, and 20q13 in order from lowest to
highest DFI (Table 6). Of these probes, gain of 6p25 was
consistently identified as highest performing by the different
methods of assessing single probe performance including DV values,
p-values from Student's t-test comparisons and 2-tail Fisher's
exact test, and DFI values.
Complementation Analysis
[0127] In order to determine which probes work best in combination,
complementation analysis was performed. Sensitivities,
specificities, and DFI values for discriminating melanoma from
benign nevi were calculated for all possible probe combinations of
1 to 4 probes. Combinations of as many as 4 probes were analyzed,
as four probes are easily combined into a multicolor probe set
suitable for viewing through the microscope (visible light emitting
labels). Resulting data for top performing probe combinations are
listed in Table 6.
Receiver Operator Curves
[0128] ROC plots were generated using sensitivities and
specificities calculated over the range of cutoff value tested. The
ROC curves for a few examples of better performing probe
combinations, as judged by lower DFI values in Table 6, are plotted
in FIGS. 2A, 2B, and 2C. ROC curves in FIGS. 2A, 2B, and 2C
illustrate the relationships between sensitivity and specificity
for detecting melanoma specimens relative to the collective group
of nevi specimens using probes that detect both loss and gain of
loci. ROC curves for the 4 best performing single probes are
plotted in order to show improvements (complementation) afforded by
combinations of probes (FIG. 2C). The entries in Table 6 correspond
to the point on each curve that lies closest to the top left corner
of the plot, (0,1). From these plots, or the data in Table 6, it
can be seen that gain of 6p25 is complemented by CEP.RTM. 10
deletion (0 signals/cell), gain of 11q13, or gain of 6q23 for
discriminating between melanoma and benign nevi. Since a single
probe could provide a DFI as low as 0.2905 (6p25 gain), probe
combinations should desirably provide DFI <0.2905 to be worth
the added expense of including additional probes in the assay, and
the added time required to enumerate the probes. The ROC curves in
FIG. 2B representing two-probe combinations can each provide DFI
<0.2905, while the three-probe combinations can provide a DFI
<0.2622 (FIG. 2B), and the four-probe combination can provide a
DFI <0.1937 (FIG. 2C).
[0129] The best overall probe combination is seen in FIG. 2C. The
ROC curves show that 6p25 gain is complemented by the ratio for
6q23 to centromere 6 (compare ROC curve for the 6p25 gain alone in
FIG. 2A and the ROC curve for 6p25 in combination with the ratio
for 6q23 to centromere 6 in FIG. 2B), and 6p25 gain combined with
the ratio for 6q23 to centromere 6 are further complemented by
17q25 gain (FIG. 2C). The corresponding ROC curve has a DFI
value=0.1127 with a sensitivity of 91% and a specificity of 93%.
The next best performing probe set in FIG. 2C is the set of 6p25,
the ratio of 6q23 to centromere 6, and 11q13 gain. The
corresponding ROC curve has a DFI=0.1257 with a sensitivity of 88%
and specificity of 97% (Table 6).
[0130] As measurements of locus loss can be complicated by
truncation artifacts (paraffin sections) and signal overlaps (any
specimen preparation), probe combinations that only rely on the
measurement of locus gain were also evaluated. The data for the
four probe set of all gains including probes 6p25, 6q23, ratio for
6q23 to centromore 6, and 17q25 are included in Table 6 and an
example ROC curve is plotted in FIG. 2C. The corresponding ROC
curve has a DFI=0.1257 with a sensitivity of 88% and specificity of
97% (Table 6).
Melanoma Detection
[0131] The four-color probe sets 6p25, 17q25, 6q23, and CEP.RTM. 6;
and 6p25, 6q23, CEP.RTM. 6 and 11q13 described in this probe
selection study can be used to assess formalin fixed paraffin
embedded skin biopsy samples for the presence of cells that have
chromosomal abnormalities consistent with a diagnosis of melanoma.
Samples are prepared for FISH hybridization and subject to
hybridization with the probe set as described in the probe
selection study. Cells from each sample are evaluated by
enumerating 20 to 200 sequential cells, as described in the probe
selection study. Samples demonstrating percentages of cells with
r-gain of 6p25, 6p25 abnormal, gain of 17q25 or a ratio of total
number of 6q23 to centromere 6 signals .gtoreq.1.1 that are greater
than cutoffs of 2.0%, 66%, 14% or 26%, respectively, are considered
positive for melanoma. Samples demonstrating percentages of cells
with a r-gain of 6p25, 6p25 abnormal, gain of 11q13 or a ratio of
total number of 6q23 to centromere 6 signals .gtoreq.1.1 that are
greater than cutoffs of 2.0, 66%, 13% and 26%, respectively, are
considered positive for melanoma. Hybridization patterns for other
probe sets useful for detecting melanoma are shown in Table 6.
Additional Probe Sets
[0132] Probe sets that can be useful in detecting melanoma can also
be selected based on the DFI values, e.g., less than 0.15, from CGH
studies, supra.
[0133] Thus, other probe combinations that have been found to be
useful have two probes where the two probes are:
a) a chromosome 9 enumerator probe and a probe to chromosome
subregion 9p21; b) probes to chromosome subregions 9p21 and 20q13;
c) a chromosome 8 enumerator probe and a probe that target
chromosome subregion 9p21; d) a chromosome 6 enumerator probe and a
probe that targets chromosome subregion 20q13; e) a chromosome 6
enumerator probe and a chromosome 8 enumerator probe; f) probes to
chromosome subregions 20q13 and 6p25; g) probes to chromosome
subregions 9p21 and 6p25; h) a chromosome 8 enumerator probe and a
probe that targets chromosome subregion 6p25; i) a chromosome 6
enumerator probe and a probe that targets chromosome subregion
9p21; j) probes that target chromosome subregions 9p21 and 17q25;
k) a chromosome 8 enumerator probe and a probe that target
chromosome subregion 20q13; l) probes that target chromosome
subregions 9p21 and 7q34; m) probes that target chromosome
subregions 9p21 and 1q23 n) probes that target chromosome
subregions 20q13 and 1q23; and o) a chromosome 9 enumerator probe
and a probe that target chromosome subregion 20q13.
[0134] Useful probe combinations having three probes are:
a) probes to the chromosome subregions 20q13, 9p21, and 7q34; b) a
chromosome 9 enumerator probe, a probe that targets chromosomal
subregion 20q13, and a probe that targets chromosome subregion
9p21; c) a chromosome 9 enumerator probe, a chromosome 8 enumerator
probe, and a probe that targets chromosome subregion 9p21; d) a
chromosome 9 enumerator probe, a probe that targets chromosomal
subregion 7q34, and a probe that targets chromosome subregion 9p21;
e) a chromosome 8 enumerator probe, a probe that targets
chromosomal subregion 20q13, and a probe that targets chromosome
subregion 6p25; f) a chromosome 6 enumerator probe, a probe that
targets chromosomal subregion 20q13, and a probe that targets
chromosome subregion 9p21; g) a chromosome 6 enumerator probe, a
probe that targets chromosomal subregion 20q13, and a probe that
targets chromosome subregion 9p21; h) a chromosome 8 enumerator
probe, a chromosome 10 enumerator probe, and a probe that targets
chromosome subregion 9p21; i) a chromosome 8 enumerator probe, a
probe that targets chromosomal subregion 6p25, and a probe that
targets chromosome subregion 9p21; and j) three probes target
chromosome subregions 9p21, 6p25, and 7q34.
[0135] Useful probe combinations having four probes are:
a) probes to chromosome subregions 9p21, 6p25, 7q34, and 20q13; b)
probes to chromosome subregions 1q23, 6p25, 7q34, and 20q13; c) a
chromosome 6 enumerator probe, a probe that targets chromosome
subregion 9p21, a probes that targets chromosome subregion 20q13,
and a probe that targets chromosome subregions 7q34; and d) a
chromosome 10 enumerator probe, a probe that targets chromosome
subregion 9p21, a probe that targets chromosome subregion 20q13,
and a probe that targets chromosome subregions 7q34.
[0136] Thus, probe combinations of at least two probes include:
a) a chromosome 9 enumerator probe and a probe to chromosome region
9p; b) a probe that targets chromosome region 9p and a probe that
targets chromosome region 20q; c) a chromosome 8 enumerator probe
and a probe that targets chromosome region 9p; d) a chromosome 6
enumerator probe and a probe that targets chromosome region 20q; e)
a probe that targets chromosome region 20q and a probe that targets
chromosome region 6p; f) a probe that targets chromosome region 9p
and a probe that targets chromosome region 6p; g) a chromosome 8
enumerator probe and a probe that targets chromosome region 6p; h)
a chromosome 6 enumerator probe and a probe that targets chromosome
region 9p; i) a probe that targets chromosome region 9p and a probe
that targets chromosome region 17q; j) a chromosome 8 enumerator
probe and a probe that target chromosome region 20q; k) a probe
that targets chromosome region 9p and a probe that targets
chromosome region 7q; l) a probe that targets chromosome region 9p
and a probe that targets chromosome region 1q; m) a probe that
targets chromosome region 20q and a probe that targets chromosome
region 1q; and n) a chromosome 9 enumerator probe and a probe that
target chromosome region 20q.
[0137] Probe combinations of at least three probes include:
a) a probe that targets chromosome region 20q, a probe that targets
chromosome region 9p, and a probe that targets chromosome region
7q; b) a chromosome 9 enumerator probe, a probe that targets
chromosomal bregion 20q, and a probe that targets chromosome region
9p; c) a chromosome 9 enumerator probe, a chromosome 8 enumerator
probe, and a probe that targets chromosome region 9p; d) a
chromosome 9 enumerator probe, a probe that targets chromosomal
region 7q, and a probe that targets chromosome region 9p; e) a
chromosome 8 enumerator probe, a probe that targets chromosomal
region 20q, and a probe that targets chromosome region 6p; f) a
chromosome 6 enumerator probe, a probe that targets chromosomal
region 20q, and a probe that targets chromosome region 9p; g) a
chromosome 6 enumerator probe, a probe that targets chromosomal
region 20q, and a probe that targets chromosome region 9p; h) a
chromosome 8 enumerator probe, a chromosome 10 enumerator probe,
and a probe that targets chromosome region 9p; i) a chromosome 8
enumerator probe, a probe that targets chromosomal region 6p, and a
probe that targets chromosome region 9p; and j) a probe that
targets chromosome region 9p, a probe that targets chromosome
region 6p, and a probe that targets chromosome region 7q.
[0138] Probe combinations of at least four probes include:
a) a probe that targets chromosome region 9p, a probe that targets
chromosome region 6p, a probe that targets chromosome region 7q,
and a probe that targets chromosome region 20q; b) a probe that
targets chromosome region 1q, a probe that targets chromosome
region 6p, a probe that targets chromosome region 7q, and a probe
that targets chromosome region 20q; c) a chromosome 6 enumerator
probe, a probe that targets chromosome region 9p, a probe that
targets chromosome region 20q, and a probe that targets chromosome
region 7q; and d) a chromosome 10 enumerator probe, a probe that
targets chromosome region 9p, a probe that targets chromosome
region 20q, and a probe that targets chromosome region 7q.
Other Embodiments
[0139] It is to be understood that, while the invention has been
described in conjunction with the detailed description, thereof,
the foregoing description is intended to illustrate and not limit
the scope of the invention. Other aspects, advantages, and
modifications of the invention are within the scope of the claims
set forth below.
[0140] All publications, patents, and patent applications cited in
this specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
TABLE-US-00002 TABLE 2 Nevus specimen set on tissue microarray
Probe signals/cell Cells with gain Cells with loss or Probe 1/Probe
2 Ave. % sd % Ave. % sd % Ave. locus sd copy cells cells cells
cells copy number number PROBE N with gain gain with loss with loss
or ratio of ratio NTRK1 (1q23) 29 4.80 6.85 36.95 20.81 1.69 0.25
RREB1 (6p25) 31 3.20 5.34 30.65 11.73 1.74 0.16 CEP 6 22 8.60 7.52
49.57 19.51 1.61 0.28 BRAF (7q34) 22 6.16 5.42 49.34 18.18 1.59
0.22 CEP 7 22 11.48 8.11 35.88 19.16 1.79 0.26 LPL (8p22) 23 27.93
14.73 16.92 10.99 2.26 0.36 MYC (8q24) 23 28.62 9.47 17.79 9.75
2.24 0.27 CEP 8 22 0.81 2.36 50.50 19.56 1.50 0.20 p16 (9p21) 31
3.84 4.99 33.82 10.07 1.73 0.21 CEP 9 22 0.84 1.84 45.53 16.69 1.55
0.17 PTEN (10q23) 29 5.97 6.71 43.42 17.96 1.64 0.24 CEP 10(probe
set 2) 31 3.51 2.89 42.52 10.90 1.60 0.15 CEP 10(probe set 5) 29
5.95 8.05 47.58 18.23 1.61 0.28 CEP 10(avg. for set 2 and 5) 33
4.67 4.26 44.92 10.04 1.60 0.15 TK1 (17q25) 31 3.40 6.14 29.22
10.50 1.75 0.16 20q13 22 1.14 3.43 47.02 24.64 1.54 0.26 LPL/MYC 23
20.29 10.77 19.17 7.40 1.01 0.09 PTEN/CEP10 29 19.13 9.72 16.05
7.16 1.03 0.11 BRAF/CEP7 22 7.05 6.46 25.63 7.66 0.89 0.07
RREB1/CEP6* 21 1.12 0.22 LPL/CEP8* 18 1.48 0.31 MYC/CEP8* 18 1.49
0.24 p16/CEP9* 22 1.13 0.18 (sd = standard deviation, DV =
discriminate value) *ratios are estimates based on average signals
per cell for each probe measured in different hybridizations.
TABLE-US-00003 TABLE 3 Melanoma cases on tissue microarray Cells
with Cells with Probe signals/cell gain loss or Probe 1/Probe 2
Ave. % sd % Ave. % sd % Ave. locus sd copy cells cells cells cells
with copy number number PROBE N with gain with gain DV p with loss
loss DV p or ratio of ratio DV p NTRK1 (1q23) 75 19.15 17.15 0.60
0.00 30.58 14.55 0 NA 1.97 0.40 0.34 0.00 RREB1 (6p25) 66 16.06
17.48 0.50 0.00 42.36 17.88 0 0.00 1.88 0.91 0.02 0.24 CEP 6 86
3.15 5.38 0.35 NA 78.14 16.60 1 0.00 1.26 0.23 0.93 NA BRAF (7q34)
86 12.98 14.78 0.19 0.00 48.14 16.83 0 NA 1.70 0.51 0.04 0.12 CEP 7
86 15.72 13.61 0.07 0.07 39.36 14.95 0 0.44 1.81 0.32 0.00 0.82 LPL
(8p22) 39 23.09 17.05 0.05 NA 31.71 15.32 1 0.00 2.16 1.19 0.01 NA
MYC (8q24) 39 19.00 15.31 0.29 NA 34.86 15.68 1 0.00 1.93 0.39 0.43
NA CEP 8 84 14.52 12.62 1.14 0.00 44.07 15.80 0 NA 1.76 0.33 0.43
0.00 p16 (9p21) 66 5.38 7.67 0.03 0.24 60.47 17.98 2 0.00 1.43 0.27
0.76 NA CEP 9 84 10.62 11.30 0.73 0.00 50.65 17.78 0 0.21 1.63 0.32
0.05 0.12 PTEN (10q23) 75 7.87 9.26 0.03 0.25 42.94 16.57 0 NA 1.67
0.26 0.01 0.57 CEP 10 93 6.71 7.42 0.06 0.06 52.04 14.63 0 0.00
1.51 0.25 0.08 NA TK1 (17q25) 66 8.16 9.52 0.18 0.00 53.74 16.88 2
0.00 1.52 0.33 0.41 NA 20q13 84 21.91 19.32 1.12 0.00 34.19 14.80 0
NA 2.02 0.60 0.54 0.00 LPL/MYC 39 28.43 13.86 0.21 0.01 21.05 10.28
0 0.41 1.11 0.42 0.06 0.15 PTEN/CEP10 75 16.95 8.48 0.03 NA 20.21
11.14 0 0.03 0.99 0.12 0.06 NA BRAF/CEP7 86 16.15 12.36 0.43 0.00
26.59 11.74 0 0.65 0.95 0.30 0.04 0.07 RREB1/CEP6* 51 1.55 0.81
0.26 0.00 LPL/CEP8* 34 1.14 0.20 0.82 NA MYC/CEP8* 34 1.11 0.24
1.25 NA p16/CEP9* 55 0.87 0.18 1.02 NA (sd = standard deviation, DV
= discriminate value)
TABLE-US-00004 TABLE 4 Nevus specimen set on sections Nevus
Specimen Set Number Ave. % S.D. % Ave. % S.D. % Ave. % S.D. % of
speci- cells with cells with cells with cells with cells with cells
with Ave. S.D. PROBE mens gain gain loss loss imbalance imbalance
ratio ratio RREB1 (6p25) 81 8.2922 10.4349 44.1975 13.7617 52.4897
12.9371 1.6226 0.2536 BRAF (7q34) 81 4.5268 6.5682 62.8395 15.4592
67.3663 13.6673 1.3554 0.2285 20q13 81 10.7819 11.5413 43.9506
12.1548 54.7325 11.2776 1.6416 0.2811 p16 (9p21) any del 81 5.4733
7.9461 52.9424 15.0422 58.4156 14.1321 1.4572 0.2653 p16 (9p21) del
(1 signal/cell) 81 5.4733 7.9461 44.1358 13.0679 49.6090 12.7755
1.4572 0.2653 p16 (9p21) del (0 signals/cell) 81 5.4733 7.9461
8.8066 6.3532 14.2798 9.1929 1.4572 0.2653 RREB1/p16 any del 81
34.8560 12.4058 23.5597 10.7230 58.4156 11.9777 1.1388 0.2304
BRAF/p16 any del 80 24.1511 10.1229 33.0258 10.8556 57.1769 11.6528
0.9455 0.1836 20q13/p16 any del 80 24.1667 10.1258 33.0208 10.8600
57.1875 11.6458 1.1457 0.2547 RREB1/BRAF 81 39.6296 11.0649 18.5597
8.9704 58.1893 10.5548 1.2140 0.1848 RREB1/20q 81 27.9424 10.1320
29.2387 9.4577 57.1811 11.3855 0.9997 0.1343 BRAF/20q 79 18.8819
8.8301 39.1561 10.7048 58.0380 12.1063 0.8357 0.1200 TK1 (17q25) 30
5.5412 5.0317 46.9534 9.7086 52.4946 10.0194 1.5283 0.1508 CCND
(11q13) 30 4.3226 5.5357 43.2903 11.8843 47.6129 12.5514 1.5704
0.1952 COX2 (1q23) 30 2.8817 2.8669 60.5771 10.0991 63.4588 9.3219
1.2926 0.1724 CEP10 any del 30 1.4444 2.5795 62.6165 12.7613
64.0609 12.1295 1.3353 0.1365 CEP10 del (1 signal/cell) 30 1.4444
2.5795 57.3154 14.4824 58.7599 13.6419 1.3353 0.1365 CEP10 del (0
signals/cell) 30 1.4444 2.5795 5.3011 4.9629 6.7455 6.2607 1.3353
0.1365 TK1/CEP10 any del 30 36.1219 8.8908 21.2473 8.9356 57.3692
8.3119 1.1549 0.1575 CCND/CEP10 any del 30 37.9068 11.4420 18.2652
10.4954 56.1720 10.2684 1.1874 0.1838 COX2/CEP10 any del 30 28.9283
7.5494 31.0860 10.5999 60.0143 9.2432 0.9743 0.1356 TK1/CCND 30
26.4588 9.5039 29.6846 8.5423 56.1434 9.4157 0.9863 0.1454 TK1/COX2
30 39.6416 7.8529 22.2616 7.5379 61.9032 7.4944 1.1995 0.1755
CCND/COX2 30 39.1792 9.6529 20.3728 9.7491 59.5520 10.0786 1.2392
0.2647 MYB (6q23) 13 10.8974 22.8989 26.0256 12.5377 36.9231
19.3631 1.8795 0.3941 CEP 6 13 12.9487 20.0018 27.1795 11.8514
40.1282 14.5370 1.8795 0.3655 MYB/CEP 6 13 18.3333 5.0000 17.5641
9.7329 35.8974 9.4432 0.9493 0.0561
TABLE-US-00005 TABLE 5 Melanoma specimen set on sections All
Malignancies No. Ave. % S.D. % Discri- Ave. % S.D. % Discri- Ave. %
S.D. % Discrimate Ave. locus S.D. Discrimi- of cells cells mate p
cells cells mate p cells cells Value- p copy copy nate Value speci-
with with Value- % with with Value- % Ab- Ab- % Ab- % Ab- number
number copy number p PROBE mens gain gain % gain gain loss loss %
loss loss normal normal normal normal or ratio or ratio or ratio
ratio RREB1 94 27.3759 19.4419 0.7480 0.0000 30.0000 13.3959 0.5465
0.0000 57.3759 13.9263 0.0661 0.0173 2.1238 0.5225 0.7444 0.0000
BRAF (1q34) 93 14.4086 14.8353 0.3710 0.0000 49.3190 18.9428 0.3058
0.0000 63.7276 12.1370 0.0396 0.669 1.6756 0.4600 0.3888 0.0000
20q13 94 22.1631 17.8049 0.2877 0.0000 35.1064 15.7543 0.1976
0.0000 57.2695 12.4910 0.0227 0.1599 1.9702 0.5375 0.2936 0.0000
p16 (9p21) 94 8.7234 10.8768 0.0582 0.0241 56.3120 19.6776 0.0185
0.2017 65.0355 14.2327 0.1089 0.0024 1.4220 0.4224 0.0050 0.5042
any del p16 (9p21) del 94 8.7234 10.8768 0.0582 0.0241 42.6596
15.4392 0.0053 0.4943 51.3830 12.6000 0.0098 0.3580 1.4220 0.4224
0.0050 0.5042 (1 signal/cell) p16 (9p21) del 94 8.7234 10.8768
0.0582 0.0241 13.6525 13.6001 0.1042 0.0025 22.3759 14.0797 0.2318
0.0000 1.4220 0.4224 0.0050 0.5042 (0 signals/cell) RREB1/p16 94
52.4468 17.9417 0.6503 0.0000 15.9574 10.0283 0.2681 0.0000 68.4042
14.0891 0.2918 0.0000 1.7599 1.6497 0.1390 0.0005 any del BRAF/p16
88 37.7652 19.1567 0.3948 0.0000 27.1212 12.7375 0.1245 0.0014
64.8864 11.7330 0.2174 0.0000 1.4277 1.8232 0.0692 0.0155 any del
20q13/p16 93 37.9570 19.2243 0.4028 0.0000 26.9693 12.7600 0.1296
0.0010 64.9462 11.5847 0.2231 0.0000 1.7361 2.8371 0.0430 0.0487
any del RREB1/BRAF 91 45.1648 15.5576 0.0841 0.0074 19.4872 11.2098
0.0042 0.5480 64.6520 12.2915 0.1591 0.0003 1.2979 0.2949 0.0582
0.0251 RREB1/20q 93 37.3118 13.7811 0.3000 0.0000 27.4552 14.3826
0.0107 0.3298 64.7670 11.5198 0.2194 0.0000 1.1116 0.2500 0.1553
0.0003 BRAF/20q 90 24.4815 11.9942 0.1413 0.0006 41.4444 13.5382
0.0176 0.2222 65.9259 11.2711 0.2274 0.0000 0.8771 0.2166 0.0279
0.1212 TK1 (17q25) 28 17.5509 17.1183 0.4531 0.0012 36.0326 13.6046
0.4269 0.0010 53.5835 16.4147 0.0032 0.7638 1.8624 0.4326 0.5317
0.0005 CCND (11q13) 28 20.0029 26.4299 0.3372 0.0045 32.3322
15.6338 0.3114 0.0043 52.3351 19.7416 0.0407 0.2865 2.5904 2.9406
0.1198 0.0780 COX2 (1q31) 28 10.1183 12.5577 0.3156 0.0057 48.2324
13.7921 0.5215 0.0003 58.3506 10.7742 0.1286 0.0596 1.5906 0.3159
0.6854 0.0001 CEP10 any del 28 3.8433 5.6027 0.1513 0.0454 57.1083
12.8267 0.0927 0.1070 60.9516 12.3963 0.0321 0.3390 1.4004 0.2094
0.0679 0.1705 CEP10 del 28 3.8433 5.6027 0.1513 0.0454 49.8174
11.7569 0.1616 0.0343 53.6607 10.7429 0.0862 0.1183 1.4004 0.2094
0.0679 0.1705 (1 signal/cell) CEP10 del 28 3.6433 5.6027 0.1513
0.0454 7.2909 8.3930 0.0416 0.2821 11.1342 10.6674 0.1259 0.0650
1.4004 0.2094 0.0679 0.1705 (0 signals/cell) TK1/CEP10 28 43.5521
14.4407 0.1920 0.0239 17.8182 8.7643 0.0841 0.1242 61.1703 13.8627
0.0553 0.2160 1.3550 0.3886 0.2279 0.0157 any del CCND/CEP10 28
49.7590 18.2212 0.3034 0.0051 14.8285 7.6359 0.0701 0.1578 64.5874
13.5475 0.2451 0.0107 1.7550 1.5584 0.1308 0.0659 any del
COX2/CEP10 28 35.7275 12.3092 0.2217 0.0156 24.5204 7.9564 0.2454
0.0098 60.2479 12.2438 0.0002 0.9353 1.1439 0.1974 0.5009 0.0004
any del TK1/CCND 28 26.4201 15.8924 0.0000 0.9912 35.5192 22.2996
0.0597 0.2028 61.9393 18.2061 0.0800 0.1397 0.9729 0.3676 0.0012
0.8576 TK1/COX2 28 36.8700 15.5970 0.0252 0.4029 23.6260 12.4005
0.0088 0.6181 60.4961 15.3093 0.0068 0.6626 1.2017 0.3441 0.0000
0.9768 CCND/COX2 28 42.9226 21.8386 0.0246 0.4096 20.9330 12.9700
0.0012 0.8540 63.8556 15.8538 0.0525 0.2273 1.5917 1.4590 0.0565
0.2181 MYB (6q23) 13 31.5608 35.3830 0.2404 0.0920 15 14.5688
0.3214 0.0523 47 29.2514 0.0775 0.3270 2.3313 0.6907 0.3229 0.0545
CEP 6 13 25.2285 33.9744 0.0970 0.2751 22.8986 14.4728 0.0524
0.4177 48.1271 25.3944 0.0747 0.3366 2.1713 0.7598 0.1198 0.2286
MYB/CEP 6 13 28.1828 13.6087 0.4615 0.0269 16.8785 12.6174 0.0019
0.8781 45.0613 19.2473 0.1827 0.1412 0.9252 0.1435 0.0245
0.5804
TABLE-US-00006 TABLE 6 # Probe 4 # # nevi melanoma SPEC DFI vs
PROBE 1 Pobe 1 Parameter PROBE 2 Probe 2 parameter PROBE 3 Probe 3
Parameter PROBE 4 Parameter Probes specimens specimens SENS vs norm
norm RREB1 (6p25) r-gain or % gain 1 30 33 0.7273 0.9000 0.2905
RREB1 (6p25) r-gain or 1 30 33 0.7879 0.8000 0.2915 % abnormal
RREB1 (6p25) r-gain 1 30 33 0.7273 0.8667 0.3036 COX2 (1q31) r-gain
1 30 33 0.7300 0.8333 0.3145 BRAF (7q34) % gain 1 81 93 0.6452
0.7160 0.4545 20q13 % gain 1 81 94 0.6809 0.6667 0.4615 RREB1
(6p25) r-gain or CCND % gain 2 30 33 0.8485 0.8700 0.1966 %
abnormal (11q13) MYB(6q23) r-gain RREB1 r-gain or 2 30 33 0.8485
0.8700 0.1996 (6p25) % abnormal CEP10 % loss RREB1 r-gain or 2 30
33 0.8182 0.8700 0.2235 (no signals) (6p25) % abnormal RREB1 (6p25)
r-gain or CEP10 % abnormal 2 30 33 0.7576 0.9000 0.2622 % abnormal
(0 or >2 signals) CEP10 % loss RREB1 r-gain 2 30 33 0.7879
0.8000 0.2915 (no signals) (6p25) MYB(6q23) r-gain RREB1 r-gain 2
30 33 0.7273 0.8667 0.3036 (6p25) 20q13 % gain RREB1 % gain 2 81 94
0.7766 0.7778 0.3151 (6p25) RREB1 (6p25) r-gain CCND % gain 2 30 33
0.7273 0.8333 0.3196 (11q13) CEP10 % loss COX2 r-gain 2 30 33
0.8182 0.7333 0.3228 (no signals) (1q31) RREB1 (6p25) r-gain COX2
r-gain 2 30 33 0.8788 0.7000 0.3236 (1q31) COX2 (1q31) r-gain CEP10
% abnormal 2 30 33 0.8182 0.7000 0.3508 (0 or >2 signals)
MYB(6q23) r-gain COX2 r-gain 2 30 33 0.7273 0.7667 0.3589 (1q31)
BRAF (7q34) % gain RREB1 % gain 2 81 93 0.7312 0.7407 0.3735 (6p25)
MYB(6q23) % gain COX2 r-gain 2 30 33 0.6970 0.7667 0.3825 (1q31)
MYB(6q23)/CEP 6 % gain RREB1 r-gain or 3 30 33 0.8500 0.9333 0.1669
(6p25) % abnormal MYB(6q23)/CEP 6 % gain RREB1 % abnormal 3 30 33
0.8182 0.9333 0.1937 (6p25) or % gain BRAF (7q34) r-gain RREB1
r-gain or TK1 (17q25) % gain 3 30 33 0.8182 0.9333 0.1937 (6p25) %
abnormal 20q13 r-gain RREB1 r-gain or TK1 (17q25) % gain 3 30 33
0.8182 0.9333 0.1937 (6p25) % abnormal RREB1 (6p25) % gain COX2 %
gain TK1 (17q25) % gain 3 30 30 0.8333 0.9000 0.1944 (1q31)
MYB(6q23)/CEP 6 % gain MYB r-gain RREB1 (6p25) % gain 3 30 33
0.8200 0.9000 0.2075 (6q23) 20q13 r-gain RREB1 r-gain or CCND
(11q13) % gain 3 30 33 0.7879 0.9000 0.2345 (6p25) % abnormal CCND
(11q13)/ % gain RREB1 % gain 3 30 33 0.7900 0.8700 0.2488 CEP10
(6p25) CEP10 % loss RREB1 % gain TK1 (17q25) % gain 3 30 33 0.7879
0.8667 0.2505 (no signals) or (6p25) % gain 20q13 r-gain MYB r-gain
RREB1 (6p25) r-gain or 3 30 33 0.7576 0.9333 0.2514 (6q23) %
abnormal BRAF (7q34) r-gain or % gain RREB1 r-gain TK1 (17q25) %
gain 3 30 33 0.7576 0.9333 0.2514 (6p25) 20q13 r-gain or % gain
RREB1 r-gain TK1 (17q25) % gain 3 30 33 0.7576 0.9333 0.2514 (6p25)
RREB1 (6p25) r-gain COX2 r-gain or % gain TK1 (17q25) % gain 3 30
33 0.7576 0.9333 0.2514 (1q31) RREB1 (6p25) % gain CCND % gain TK1
(17q25) % gain 3 30 30 0.8333 0.8000 0.2603 (11q13) RREB1 (6p25) %
gain CEP10 % gain TK1 (17q25) % gain 3 30 30 0.8333 0.8000 0.2603
CCND/CEP10 r-gain or CEP10 % loss RREB1 (6p25) r-gain 4 30 33
0.7576 0.9000 0.2622 any del % abnormal (no signals) BRAF (7q34)
r-gain CEP10 % loss RREB1 (6p25) r-gain or 4 30 33 0.7576 0.9000
0.2622 homo del (no signals) % abnormal 20q13 r-gain CEP10 % loss
RREB1 (6p25) r-gain or 4 30 33 0.7576 0.9000 0.2622 homo del (no
signals) % abnormal CEP10 % loss COX2 r-gain RREB1 (6p25) % gain 3
30 33 0.7576 0.9000 0.2622 (no signals) (1q31) COX2 (1q31) r-gain
CEP10 % abnormal RREB1 (6p25) % gain 3 30 33 0.7576 0.9000 0.2622
(0 or >2 signals) CEP10 % loss RREB1 r-gain CCND (11q13) % gain
3 30 33 0.7576 0.9000 0.2622 (no signals) (6p25) RREB1 (6p25)
r-gain CEP10 % abnormal CCND (11q13) % gain 3 30 33 0.7576 0.9000
0.2622 (0 or >2 signals) RREB1 (6p25) r-gain COX2 r-gain RREB1
(6p25) % abnormal CCND (11q13) % gain 3 30 33 0.7576 0.9000 0.2622
(1q31) COX2/CEP10 % gain RREB1 % gain 3 30 33 0.7879 0.8333 0.2698
(6p25) CCND/CEP10 % gain CEP10 % loss RREB1 (6p25) r-gain 3 30 33
0.7576 0.9000 0.2622 (no signals) COX2 (1q31) % gain CCND % gain
TK1 (17q25) % gain 3 30 30 0.8000 0.8000 0.2828 (11q13) RREB1
(6p25) % gain COX2 % gain CCND (11q13) % gain 3 30 30 0.7667 0.8333
0.2867 (1q31) CEP10 % loss RREB1 % gain COX2 (1q31) % gain 3 30 33
0.7273 0.8667 0.3036 (no signals) (6p25) MYB(6q23) r-gain RREB1
r-gain CCND (11q13) % gain 3 30 33 0.7273 0.8667 0.3036 (6p25) COX2
(1q23) r-gain RREB1 % gain CCND (11q13) % gain 3 30 33 0.7273
0.8667 0.3036 (6p25) CEP10 % abnormal RREB1 % gain CCND(11q13) %
gain 3 30 33 0.7273 0.8667 0.3036 (0 or >2 signals) (6p25) 20q13
% gain BRAF % gain RREB1 (6p25) % gain 3 81 93 0.6989 0.8148 0.3535
(7q34) MYB(6q23)/ % gain RREB1 r-gain or TK1 (17q25) % gain 4 30 33
0.9091 0.9333 0.1127 CEP 6 (6p25) % abnormal MYB(6q23)/ % gain
RREB1 r-gain or CCND (11q13) % gain 4 30 33 0.8788 0.9667 0.1257
CEP 6 (6p25) % abnormal MYB(6q23)/ % gain MYB r-gain RREB1 (6p25) %
gain TK1 (17q25) % gain 4 30 33 0.8788 0.9667 0.1257 CEP 6 (6q23)
MYB(6q23)/ % gain CEP10 signals) RREB1 (6p25) % gain 4 30 33 0.8788
0.9000 0.1571 CEP 6 MYB(6q23)/ % gain RREB1 r-gain or CEP10 %
abnormal 4 30 33 0.8788 0.9000 0.1571 CEP 6 (6p25) % abnormal (0 or
>2 signals) 20q13 r-gain MYB(6q23)/ % gain RREB1 (6p25) r-gain
or 4 30 33 0.8485 0.9333 0.1655 CEP 6 % abnormal BRAF (7q34) r-gain
MYB(6q23)/ % gain RREB1 (6p25) r-gain 4 30 33 0.8485 0.9000 0.1815
CEP 6 BRAF (7q34) r-gain COX2 (1q31) r-gain RREB1 (6p25) % gain TK1
(17q25) % gain 4 30 33 0.8182 0.9333 0.1937 20q13 r-gain COX2
(1q31) r-gain RREB1 (6p25) % gain TK1 (17q25) % gain 4 30 33 0.8182
0.9333 0.1937 RREB1 (6p25) % gain COX2 (1q31) % gain CCND (11q13) %
gain TK1 (17q25) % gain 4 30 30 0.8333 0.9000 0.1944 20q13 r-gain
MYB(6q23)/ % gain MYB(6q23) r-gain RREB1 (6p25) % gain 4 30 33
0.8182 0.9000 0.2075 CEP 6 RREB1 (6p25) % gain COX2 (1q31) % gain
CEP10 any del % gain TK1 (17q25) % gain 4 30 30 0.8333 0.8667
0.2134 BRAF (7q34) r-gain MYB(6q23) r-gain RREB1 (6p25) r-gain TK1
(17q25) % gain 4 30 33 0.7879 0.9667 0.2147 20q13 r-gain MYB(6q23)
r-gain RREB1 (6p25) r-gain TK1 (17q25) % gain 4 30 33 0.7879 0.9667
0.2147 CCND/CEP10 r-gain BRAF r-gain RREB1 (6p25) r-gain or 4 30 33
0.7879 0.9333 0.2224 (7q34) % abnormal CCND/CEP10 r-gain 20q13
r-gain RREB1 (6p25) r-gain or 4 30 33 0.7879 0.9333 0.2224 %
abnormal BRAF (7q34) r-gain MYB(6q23)/ % gain MYB(6q23) r-gain
RREB1 (6p25) % gain 4 30 33 0.7879 0.9333 0.2224 CEP 6 BRAF (7q34)
r-gain or MYB(6q23)/ % gain RREB1 (6p25) % gain 4 30 33 0.7879
0.9000 0.2345 % gain CEP 6 20q13 r-gain or MYB(6q23)/ % gain RREB1
(6p25) % gain 4 30 33 0.7879 0.9000 0.2345 % gain CEP 6 BRAF (7q34)
r-gain RREB1 r-gain TK1 (17q25) % gain CCND (11q13) % gain 4 30 33
0.7879 0.9000 0.2345 (6p25) 20q13 r-gain RREB1 r-gain TK1 (17q25) %
gain CCND (11q13) % gain 4 30 33 0.7879 0.9000 0.2345 (6p25)
TK1/CEP10 r-gain BRAF r-gain RREB1 (6p25) r-gain or 4 30 33 0.7576
0.9667 0.2447 (7q34) % abnormal TK1/CEP10 r-gain 20q13 r-gain RREB1
(6p25) r-gain or 4 30 33 0.7576 0.9667 0.2447 % abnormal BRAF
(7q34) r-gain 20q13 r-gain RREB1 (6p25) r-gain TK1 (17q25) % gain 4
30 33 0.7576 0.9667 0.2447 BRAF (7q34) r-gain RREB1 % gain TK1
(17q25) % gain CEP10 % gain 4 30 33 0.7879 0.8667 0.2505 (6p25)
20q13 r-gain RREB1 % gain TK1 (17q25) % gain CEP10 % gain 4 30 33
0.7879 0.8667 0.2505 (6p25) CCND/CEP10 % gain MYB(6q23) r-gain
RREB1 (6p25) % gain 4 30 33 0.7879 0.8667 0.2505 TK1 (17q25) r-gain
or % gain BRAF r-gain MYB(6q23)/CEP % gain 4 30 33 0.7576 0.9333
0.2514 (7q34) BRAF (7q34) r-gain CEP10 % loss RREB1 (6p25) r-gain
TK1 (17q25) % gain 4 30 33 0.7576 0.9333 0.2514 (no signals) 20q13
r-gain CEP10 % loss RREB1 (6p25) r-gain TK1 (17q25) % gain 4 30 33
0.7576 0.9333 0.2514 (no signals) CEP10 % loss RREB1 r-gain COX2
(1q31) r-gain TK1 (17q25) % gain 4 30 33 0.7576 0.9333 0.2514 (no
signal) (6p25) BRAF (7q34) r-gain RREB1 r-gain CEP10 % abnormal TK1
(17q25) % gain 4 30 33 0.7576 0.9333 0.2514 (6p25) 20q13 r-gain
RREB1 r-gain CEP10 % abnormal TK1 (17q25) % gain 4 30 33 0.7576
0.9333 0.2514 (6p25) MYB(6q23) % gain RREB1 r-gain CEP10 % abnormal
TK1 (17q25) % gain 4 30 33 0.7576 0.9333 0.2514 (6p25) CCND/CEP10
r-gain RREB1 % abnormal TK1 (17q25) % gain 4 30 33 0.7576 0.9333
0.2514 any del (6p25) or % gain MYB(6q23) r-gain CEP10 % abnormal
RREB1 (6p25) % gain COX2 (1q31) % gain 4 30 33 0.7576 0.9333 0.2514
MYB(6q23)/ % gain MYB r-gain RREB1 (6p25) r-gain CCND (11q13) %
gain 4 30 33 0.7576 0.9333 0.2514 CEP 6 (6q23) MYB(6q23)/ % gain
RREB1 r-gain or CCND (11q13) % gain 4 30 33 0.7576 0.9333 0.2514
CEP 6 (6p25) % abnormal MYB(6q23) r-gain RREB1 r-gain COX2 (1q31)
r-gain CEP10 % abnormal 4 30 33 0.7576 0.9000 0.2622 (6p25) (0 or
>2 signals) COX2/CEP10 r-gain CCND/ r-gain CEP10 % loss RREB1
(6p25) r-gain 4 30 33 0.7576 0.9000 0.2622 CEP10 (no signals)
TABLE-US-00007 TABLE 7 IMBALANCE LOSS GAIN AMPL PROBE 1 PROBE 2
PROBE 3 PROBE 4 SENS DFI SENS DFI SENS DFI SENS DFI 09p22 0.59 0.41
0.59 0.41 0.00 1.00 0.00 1.00 10q26.3 0.36 0.64 0.35 0.65 0.01 0.99
0.00 1.00 06p25 0.35 0.65 0.00 1.00 0.33 0.67 0.02 0.98 09q21.2
0.35 0.65 0.34 0.66 0.01 0.99 0.00 1.00 01q22 0.32 0.68 0.00 1.00
0.32 0.68 0.00 1.00 07p21 0.32 0.68 0.01 0.99 0.31 0.69 0.01 0.99
07q36 0.32 0.68 0.01 0.99 0.29 0.71 0.01 0.99 10p15 0.28 0.72 0.28
0.72 0.00 1.00 0.00 1.00 08p23.2 0.27 0.73 0.18 0.82 0.10 0.90 0.00
1.00 06q25.3 0.26 0.74 0.25 0.75 0.01 0.99 0.00 1.00 17q25 09p22
0.72 0.28 09p22 06p25 0.70 0.30 09p22 06q25.3 0.69 0.31 09p22 01q22
0.69 0.31 09p22 08p23.2 0.68 0.32 09p22 07p21 0.68 0.32 10q26.3
09p22 0.68 0.32 09p22 07q36 0.68 0.32 09p22 08q24.2 0.68 0.32
20q13.3 09p22 0.66 0.34 17q25 09p22 06p25 0.79 0.21 17q25 09p22
08p23.2 0.78 0.22 17q25 10q26.3 09p22 0.77 0.23 17q25 09p22 08q24.2
0.77 0.23 20q13.3 17q25 09p22 0.77 0.23 09p22 08p23.2 06q25.3 0.76
0.24 09p22 08p23.2 06p25 0.76 0.24 09p22 07p21 06p25 0.76 0.24
09p22 06p25 01q22 0.76 0.24 17q25 09p22 06q25.3 0.76 0.24 10cen
17q25 09p22 06p25 0.82 0.18 17q25 10q26.3 09p22 06p25 0.82 0.18
17q25 09p22 08p23.2 06p25 0.82 0.18 20q13.3 17q25 09p22 08p23.2
0.82 0.18 01cen 09p22 06q25.3 06p25 0.82 0.18 06cen 17q25 10q26.3
09p22 0.82 0.18 06cen 17q25 09p22 08p23.2 0.82 0.18 06cen 09p22
08p23.2 01q22 0.82 0.18 08cen 17q25 09p22 06p25 0.82 0.18 10cen
06cen 17q25 09p22 0.82 0.18
* * * * *