U.S. patent application number 13/854764 was filed with the patent office on 2014-01-02 for method for detecting risk of progression of low grade cervical dysplasia.
This patent application is currently assigned to The U.S.A., as represented by the Secretary, Dept. of Health & Human Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health & Human Services, The United States of America, as represented by the Secretary, Department of Health & Human Services. Invention is credited to Sonia Andersson, Gert Auer, Kerstin Heselmeyer-Haddad, Catharina Larsson, Thomas Ried, Winfried Steinberg.
Application Number | 20140005063 13/854764 |
Document ID | / |
Family ID | 36778261 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140005063 |
Kind Code |
A1 |
Ried; Thomas ; et
al. |
January 2, 2014 |
METHOD FOR DETECTING RISK OF PROGRESSION OF LOW GRADE CERVICAL
DYSPLASIA
Abstract
The invention provides methods for identifying conditions of low
grade cervical dysplasia and assessing the progressive potential of
individual lesions to develop into high grade cervical dysplasia
and cervical squamous cell cancer as well as cervical
adenocarcinoma.
Inventors: |
Ried; Thomas; (Bethesda,
MD) ; Heselmeyer-Haddad; Kerstin; (Edewecht, DE)
; Steinberg; Winfried; (Soest, DE) ; Auer;
Gert; (Solna, SE) ; Andersson; Sonia;
(Stockholm, SE) ; Larsson; Catharina; (Stockholm,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Department of Health & Human Services; The United States of
America, as represented by the Secretary, |
|
|
US |
|
|
Assignee: |
The U.S.A., as represented by the
Secretary, Dept. of Health & Human Services
Rockville
MD
|
Family ID: |
36778261 |
Appl. No.: |
13/854764 |
Filed: |
April 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11884608 |
Sep 8, 2008 |
8409808 |
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PCT/US2006/006116 |
Feb 21, 2006 |
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13854764 |
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60654176 |
Feb 18, 2005 |
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Current U.S.
Class: |
506/9 ; 435/6.11;
435/7.4 |
Current CPC
Class: |
C12Q 2600/112 20130101;
G01N 2800/36 20130101; G01N 2800/56 20130101; C12Q 2600/118
20130101; G01N 33/57442 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
506/9 ; 435/6.11;
435/7.4 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT OF RIGHTS TO INVENTION MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0001] This work was supported by the National Institutes of
Health. The government may have certain rights to this invention.
Claims
1-16. (canceled)
17. A kit for assessing a change in a patient condition of low
grade cervical dysplasia to a condition of high grade cervical
dysplasia comprising a probe for detecting a genomic amplification
of chromosome 3q and instructions for using the probe to assess the
change in a patient condition of low grade cervical dysplasia to a
condition of high grade cervical dysplasia in accordance with a
method of any of claims 1-16.
18. A method for identifying a patient at risk of developing
invasive cervical carcinoma, comprising: making a first
identification of a genomic amplification of chromosome 3q in a
sample obtained from a patient known to have had a condition of
either i) low grade cervical dysplasia; or ii) no dysplasia thereby
identifying a patient at risk of developing invasive cervical
carcinoma.
19. The method of claim 18, wherein the genomic amplification is
within the 3q26 locus of chromosome 3q.
20. The method of claim 18, wherein the low grade cervical
dysplasia is a cervical intraepithelial neoplasm of grade 1.
21. The method of claim 18, wherein a cytologically normal pap
smear is obtained from a patient having no dysplasia.
22. The method of claim 19, wherein the genomic amplification is
detected by hybridizing the sample to a probe comprising a
detectable marker and a nucleic acid sequence that is complimentary
to a nucleic acid sequence of the 3q26 locus.
23. The method of claim 22, wherein the nucleic acid sequence of
the probe is complimentary to the telomerase gene, or a portion
thereof.
24. The method of claim 23, wherein the detectable marker emits a
fluorescent signal.
25. The method of claim 23 wherein the detectable marker is
chromogenic.
26. The method of claim 22, further comprising hybridizing the
sample to a centromere enumeration probe comprising a detectable
marker and a nucleic acid sequence that is complimentary to a
nucleic acid sequence proximate to the centromere of chromosome
3.
27. The method of claim 19, wherein the genomic amplification is
detected by Polymerase Chain Reaction.
28. The method of claim 19, wherein the genomic amplification is
detected by measuring the amount of the telomerase polypeptide.
29-63. (canceled)
64. The method of claim 22, wherein the nucleic acid sequence of
the probe is complimentary a nucleic acid sequence selected from
the group consisting of ectopic viral integration site 1,
myelodysplasia syndrome 1, myoneurin, G protein-coupled receptor
160, protein kinase C, SKI-like, claudin 11, eukaryotic translation
initiation factor 5A2, traf2 and nck interacting kinase,
phospholipase D1, growth hormone secretagogue receptor, tumor
necrosis factor 10, epithelial cell transforming sequence 2
oncogene, p53 target zinc finger protein,
phosphoinositide-3-kinase, mitofusin 1 and ubiquitin specific
protease 13.
65-67. (canceled)
68. A method of diagnosing adenocarcinoma in a subject comprising:
detecting genomic amplification of a telomerase gene or portion
thereof in a subject.
69. The method of claim 68, wherein the genomic amplification is
detected by hybridizing the sample to a probe comprising a
detectable marker and a nucleic acid sequence that is complimentary
to a nucleic acid sequence of the 3q26 locus.
70. The method of claim 69, wherein the probe comprises a contig of
four overlapping BAC clones.
71. The method of claim 68, wherein the nucleic acid sequence of
the probe is complimentary to the telomerase gene, or a portion
thereof.
72. The method of claim 68, wherein the sample is a preparation
from the cervix.
73. The method of claim 68, further comprising hybridizing the
sample to a centromere enumeration probe comprising a detectable
marker and a nucleic acid sequence that is complimentary to a
nucleic acid sequence proximate to the centromere of chromosome
3.
74. The method of claim 68, further comprising hybridizing the
sample to a centromere enumeration probe comprising a detectable
marker and a nucleic acid sequence that is complimentary to a
nucleic acid sequence proximate to the centromere of chromosome
7.
75-76. (canceled)
Description
RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The visualization of chromosomal aneuploidy and copy number
changes of specific cancer-associated genes has become an important
complement to routine morphological assessment of cytological
samples..sup.1 This approach is biologically valid and successful
because chromosomal aneuploidy and the resulting genomic imbalances
are specific for cancer cells, distinct for different carcinomas,
and occur early during disease progression..sup.2,3 Some genomic
imbalances are correlated with poor prognosis and treatment
failure,.sup.4-6 and others, such as amplification of the Her2/neu
oncogene in breast cancer, can guide therapeutic
decisions..sup.7
[0004] Like most other human carcinomas, cervical cancers are
defined by a conserved distribution of genomic imbalances. In
addition to infection with high-risk human papilloma
virus,.sup.9,10 the sequential transformation of cervical squamous
epithelium requires the acquisition of additional copies of
chromosome arm 3q,.sup.11 among other cytogenetic
abnormalities..sup.12 CGH analyses of cervical carcinomas have
shown that more than 85% of invasive cervical carcinomas carry
specific genomic imbalances that result in copy number increases of
chromosome arm 3q..sup.5,11,20-26 The region of minimal overlap
points to chromosome band 3q26, which contains the gene for the RNA
component of human telomerase (TERC)..sup.27
[0005] The implementation of cervical cancer screening programs has
greatly reduced disease incidence and mortality in industrialized
countries..sup.16,17 However, a single cytological evaluation
remains relatively insensitive, hence the need for frequent
follow-up investigations. This is attributable to sampling or
interpretation errors, and to the fact that some early lesions may
not have acquired recognizable phenotypic alterations..sup.17
Invasive cervical carcinomas develop through increasing stages of
cervical dysplasia and advance to CIN3, which is considered a
bonafide precancerous lesion that requires surgical intervention.
However, only about 10-15% of all low-grade dysplastic lesions
follow this path of linear progression. The identification of
markers of disease progression would therefore be of great clinical
interest.
[0006] A previous study demonstrated that visualization of
additional copies of TERC can serve as a specific and sensitive
test for 3q26 amplification in routinely collected cytological
samples, including samples of low grade cervical dysplasia..sup.8
This finding was consistent with, but not conclusive of, the
hypothesis that the 3q-imposed growth advantage reflects a point of
no return during the sequential malignant transformation of
cervical epithelial cells. For example, U.S. application Ser. No.
10/857,859, filed on Jun. 4, 2004 and published as U.S. Application
Publication No. 20050026190, indicates that the 3q26 amplification
can be used to selectively detect high grade cervical
intraepithelial neoplasias (CIN II and CIN III) and malignant
carcinomas in cervical biopsy and pap smear specimens without
detecting low grade cervical intraepithelial neoplasia. Thus, the
3q26 amplification was thought to be a distinguishing factor
between low grade and high grade cervical dysplasias and
adenocarcinomas.
SUMMARY OF THE INVENTION
[0007] It has now been that demonstrated that identification of a
3q amplification in low grade cervical dysplasia can provide
information regarding the progressive potential of individual
lesions to high grade cervical dysplasia and cancer.
[0008] In one aspect, the present invention provides a method for
assessing a change in a patient condition of low grade cervical
dysplasia to a condition of high grade cervical dysplasia or
cancer, comprising: detecting a genomic amplification of chromosome
3q in a sample from a patient having or previously having low grade
cervical dysplasia to thereby assess the change in the patient
condition to a high grade cervical dysplasia or cancer. Detecting
the genomic amplification of chromosome 3q indicates progression of
the patient condition to high grade cervical dysplasia.
[0009] In another aspect, the present invention provides a method
for monitoring a shift to a condition of high grade cervical
dysplasia from a condition of low grade cervical dysplasia in a
patient comprising: assaying for a genomic amplification of
chromosome 3q in a sample from a patient having or previously
having low grade cervical dysplasia to thereby monitor a shift to a
condition of high grade cervical dysplasia from a condition of low
grade cervical dysplasia. The method can further comprising
determining that the genomic amplification of chromosome 3q is
present in the sample or that the genomic amplification of
chromosome 3q is not present in the sample.
[0010] Low grade cervical dysplasias of the invention can be but
are not limited to cervical intraepithelial neoplasms of grade 1.
High grade cervical dysplasias of the invention include but are not
limited to cervical intraepithelial neoplasms of grade 2, cervical
intraepithelial neoplasms of grade 3 and carcinomas in situ.
[0011] In one embodiment, the sample obtained from the patient is a
preparation from the cervix, including but not limited to a pap
smear or a thin layer (e.g., mono-layer) suspension of cells.
[0012] According to specific embodiments of the invention, the
genomic amplification can be within the 3q26 locus of chromosome
3q. This locus includes the coding sequences of the telomerase
(TERC) gene. Thus, in one embodiment, the genomic amplification is
detected by hybridizing the sample to a probe comprising a
detectable marker and a nucleic acid sequence that is complimentary
to a nucleic acid sequence of the 3q26 locus. Methods of the
invention can further involve hybridizing the sample to a
centromere enumeration probe comprising a detectable marker and a
nucleic acid sequence that is complimentary to a nucleic acid
sequence proximate to the centromere of chromosome 3.
[0013] In one embodiment, the nucleic acid sequence of the probe is
complimentary to the telomerase gene, or a portion thereof.
[0014] In other embodiments, the nucleic acid sequence of the probe
is complimentary to a nucleic acid sequence of chromosome 3q, or a
portion thereof, including but not limited to EVI1 (ectopic viral
integration site 1, 3q24-3q28), MDS1 (myelodysplasia syndrome 1,
3q26), MYNN (myoneurin, 3q26.2), GPR160 (G protein-coupled receptor
160, 3q26.2-3q27), PRKCI (protein kinase C, 3q26.3), SKIL (SKI-like
3q26), CLDN11 (claudin 11, 3q26.2-3q26.3), EIF5A2 (eukaryotic
translation initiation factor 5A2, 3q26.2), TNIK (TRAF2 and NCK
interacting kinase, 3q26.2), PLD1 (phospholipase D1, 3q26), GHSR
(growth hormone secretagogue receptor, 3q26.31), TNSF10 (tumor
necrosis factor 10, 3q26), ECT2 (epithelial cell transforming
sequence 2 oncogene, 3q26.1-3q26.2), WIG1 (p53 target zinc finger
protein, 3q26.3-3q27), PIK3A (phosphoinositide-3-kinase, 3q26.3),
MFN1 (mitofusin 1, 3q26.32) and USP13 (ubiquitin specific protease
13, 3q26.2-3q26.3).
[0015] In another embodiment, the detectable marker of the probe
emits a fluorescent signal.
[0016] In yet another embodiment, the detectable marker of the
probe is chromogenic.
[0017] In yet another embodiment, the genomic amplification is
detected by Polymerase Chain Reaction (PCR).
[0018] In yet another embodiment, the genomic amplification is
detected by measuring the amount of a polypeptide transcribed from
a gene having a locus within the genomic amplification.
[0019] In a specific embodiment, the genomic amplification is
detected by measuring the amount of the telomerase polypeptide.
[0020] In yet another aspect, the present invention provides a
method for identifying a patient at risk of developing invasive
cervical carcinoma, comprising: making a first identification of a
genomic amplification of chromosome 3q in a sample obtained from a
patient known to have had a condition of either i) low grade
cervical dysplasia or ii) no dysplasia thereby identifying a
patient at risk of developing invasive cervical carcinoma.
[0021] In a specific embodiment, a cytologically normal pap smear
can be obtained from a patient having no dysplasia but having a
genomic amplification of chromosome 3q.
[0022] In yet another aspect, the present invention provides a
method for assessing maintenance or regression of a patient
condition of low grade cervical dysplasia comprising: a) assaying
for a genomic amplification of chromosome 3q in a sample from a
patient having or previously having low grade cervical dysplasia
and b) determining that the genomic amplification of step a) is not
present, wherein the absence of the genomic amplification of step
a) indicates maintenance or regression of the patient condition of
low grade cervical dysplasia.
[0023] In one embodiment, regression is detected by obtaining a
cytologically normal pap smear from the patient.
[0024] In other aspects of the invention, kits are provided for
conducting methods of the invention.
[0025] In one embodiment, the present invention provides a kit for
assessing a change in a patient condition of low grade cervical
dysplasia to a condition of high grade cervical dysplasia
comprising a probe for detecting a genomic amplification of
chromosome 3q and instructions for using the probe to assess the
change in a patient condition of low grade cervical dysplasia to a
condition of high grade cervical dysplasia in accordance with
methods of the invention.
[0026] In another embodiment, the present invention provides a kit
for identifying a patient at risk of developing invasive cervical
carcinoma comprising a probe for detecting a genomic amplification
of chromosome 3q and instructions for using the probe to identify a
patient at risk of developing invasive cervical carcinoma in
accordance with methods of the invention.
[0027] In yet another embodiment, the present invention provides a
kit for monitoring a shift to a condition of high grade cervical
dysplasia from a condition of low grade cervical dysplasia in a
patient comprising a probe for detecting a genomic amplification of
chromosome 3q and instructions for using the probe to monitor the
shift to a condition of high grade cervical dysplasia from a
condition of low grade cervical dysplasia in accordance with
methods of the invention.
[0028] In yet another embodiment, the present invention provides a
kit for assessing maintenance or regression of a patient condition
of low grade cervical dysplasia comprising a probe for detecting a
genomic amplification of chromosome 3q and instructions for using
the probe to assess maintenance or regression of a patient
condition of low grade cervical dysplasia in accordance with
methods of the invention.
[0029] In one aspect, methods for assessing a change in a patient
condition of normal to a condition of low grade cervical dysplasia,
high grade cervical dysplasia or cancer and provided and comprise
detecting a genomic amplification of chromosome 3q in a sample from
a patient having or previously having a normal diagnosis to thereby
assess the change in the patient condition to a low grade cervical
dysplasia, high grade cervical dysplasia or cancer.
[0030] In one embodiment, detecting the genomic amplification of
chromosome 3q indicates progression of the patient condition to low
grad or high grade cervical dysplasia.
[0031] In another embodiment, the genomic amplification is within
the 3q26 locus of chromosome 3q.
[0032] In one aspect, methods of diagnosing adenocarcinoma in a
subject are provided and comprise detecting genomic amplification
of a telomerase gene or portion thereof in a subject.
[0033] In one embodiment, the genomic amplification is detected by
hybridizing the sample to a probe comprising a detectable marker
and a nucleic acid sequence that is complimentary to a nucleic acid
sequence of the 3q26 locus.
[0034] In another embodiment, the probe comprises a contig of four
overlapping BAC clones. In related embodiments, the probe comprises
five or six overlapping BAC clones.
[0035] In yet another embodiment, the nucleic acid sequence of the
probe is complimentary to the telomerase gene, or a portion
thereof.
[0036] In certain embodiments, the sample is a preparation from the
cervix.
[0037] In one embodiment the methods may further comprise
hybridizing the sample to a centromere enumeration probe comprising
a detectable marker and a nucleic acid sequence that is
complimentary to a nucleic acid sequence proximate to the
centromere of chromosome 3.
[0038] In one embodiment the methods may further comprise
hybridizing the sample to a centromere enumeration probe comprising
a detectable marker and a nucleic acid sequence that is
complimentary to a nucleic acid sequence proximate to the
centromere of chromosome 7.
[0039] In one embodiment the methods may further comprise
hybridizing the sample to a centromere enumeration probe comprising
a detectable marker and a nucleic acid sequence that is
complimentary to a nucleic acid sequence proximate to the
centromere of chromosomes 3 and 7.
[0040] In one aspect, kits for monitoring an adenocarcinoma in a
patient are provided and comprise a probe for detecting a genomic
amplification of chromosome 3q and instructions for using the probe
to monitor adenocarcinoma in accordance with the methods described
herein.
[0041] Other aspects of the invention are described in or are
obvious from the following disclosure, and are within the ambit of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
drawings, incorporated herein by reference. Various preferred
features and embodiments of the present invention will now be
described by way of non-limiting example and with reference to the
accompanying drawings in which:
[0043] FIG. 1 schematically depicts the triple-color FISH probe set
(Heselmeyer-Haddad et al., 2003).
[0044] FIG. 2A shows the results of the hybridization of the TERC
gene (red) to previously stained routine Pap smears from patient 9
(group 2, regressors, Table 3). Two distinct areas of the slide are
visualized. For simplicity, only the signals for the TERC probe are
shown. All nuclei exhibit two TERC signals indicating a normal
diploid status for these cells.
[0045] FIG. 2B shows the results of the hybridization of the TERC
gene (red) to previously stained routine Pap smears from patient 2
(group 1, progressors, Table 2). Multiple nuclei that appeared
aberrant during the cytological screening throughout the slide
reveal extra copies of TERC (shown as red signals). Note that both
larger nuclei and cells with small nuclei reveal increased copy
numbers for this gene (lower right area).
[0046] FIG. 2C shows the results of the hybridization of the TERC
gene (red) to previously stained routine Pap smears from patient 7
(group 1, Table 2). Note multiple 3q-positive cells in the Pap
smear (main pattern 2-3-3).
[0047] FIG. 2D shows the results of the hybridization of the TERC
gene (red) to previously stained routine Pap smears from patient 9
(group 3, Table 4). This case revealed four, occasionally five
copies of 3q on a diploid background (i.e., two signals for CEP7).
Interestingly, the subsequent CIN3 lesion showed the same main
pattern (2-3-4), supporting the hypothesis of clonal expansion.
[0048] FIG. 3A shows the ROC plot of sensitivity versus
1--specificity at thresholds ranging between 0 and 100% abnormal
cells for 3q gain, and FIG. 3b shows the plot of DFI versus
threshold for 3q gain. The white triangles in both 3A and 3B denote
the results when considering cells with >2 TERC signals/cell,
excluding tetraploidy as positive. The blue squares reflect the
results when only tetraploid cell were considered, i.e.,
hybridization patterns of four signals for each probe (4-4-4). The
red circles show the results when considering cells with any 3q
gain (>2 TERC signals/cell including cells with a tetraploid
hybridization pattern).
[0049] FIG. 4 depicts interphase FISH analysis of showing gain of
TERC in 3q26 in HPV infected and HPV negative cervical
adenocarcinomas. (A) Chromosomal location of the triple colour
probe set: CEP 7 (labeled with Spectrum Aqua, which is
pseudocloured in light-blue in panels B, C, E, and G), CEP 3
(Spectrum Green, pseudocoloured in green in panels B, C, E, and G),
and TERC (Spectrum Orange, pseudocoloured in red). (B) Normal
nuclei with the expected pattern of two signals per probe per
nucleus (2-2-2). (C and D) HPV negative case number 4. (E and F)
HPV infected case number 6, and (G and H) HPV positive case number
10. All cases exhibit varying extents of gain of TERC. The cell
clusters in C, E and G and in D, F and H show cells with identical
aberrant signal numbers with either the triple color probe set
shown, or with the TERC probe only, respectively.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0050] "Adenocarcinoma" refers at least to a cervical carcinoma as
known to one of skill in the art. Cervical adenocarcinoma develops
at least in part from the mucus-producing gland cells of the
endocervix.
[0051] "Low grade cervical dysplasia" refers to minor dysplastic
cervical lesions including cervical intraepithelial neoplasms of
grade 1.
[0052] "High grade cervical dysplasia" refers to carcinoma in situ
or high-grade squamous intraepithelial lesions including cervical
intraepithelial neoplasms of grade 2 or 3.
[0053] "Cervical intraepithelial neoplasms" or "CINs" are
categorized as grade 1, 2, or 3 depending on the extent of
aberration in cellular stratification. CIN 1 is characterized by
the presence of immature basal type cells in the lower third of the
epithelium. CIN 2 is characterized by the presence of immature
basal type cells in the lower two-thirds of the epithelium. In CIN
3, the full thickness of the epithelium contains undifferentiated
and nonstratified cells.
[0054] "Progression" of a patient condition of low grade cervical
dysplasia refers to malignant conversion of minor cervical
dysplastic lesions such as cervical intraepithelial neoplasms of
grade 1 or grade 2 to high grade cervical dysplasias.
[0055] "Maintenance" of a patient condition of low grade cervical
dysplasia refers to the continued presence of minor dysplastic
cervical lesions over time.
[0056] "Regression" of a patient condition of low grade cervical
dysplasia refers to a reduction or eradication of minor dysplastic
lesions over time. Eradication can be detected by a cytologically
normal pap smear in a patient previously diagnosed with low grade
cervical dysplasia.
[0057] "Genomic amplification" refers to a copy number increase in
a genomic sequence, such as a sequence encoding one or more
genes.
[0058] A "locus" refers to a chromosomal region defined according
to genetic maps of an organism. Typically, the genetic maps
designate the specific position (i.e., the "locus") occupied by a
given gene on a chromosome and at a particular locus, any one of
the variant genes may be present.
[0059] A "nucleic acid sequence" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise stated, would encompass known analogs of
natural nucleotides that can function in a similar manner as
naturally occurring nucleotides.
[0060] "Complimentary nucleic acid sequences" refer to contiguous
DNA or RNA sequences which have compatible nucleotides (e.g., A/T,
G/C) in corresponding positions, such that base pairing between the
sequences occurs. For example, the sense and anti-sense strands of
a double-stranded DNA helix are known in the art to be
complimentary.
[0061] A "probe" or a "nucleic acid probe", as used herein, is
defined to be a collection of one or more nucleic acid fragments
whose hybridization to a chromosomal nucleic acid sequence can be
detected.
[0062] "Hybridizing" refers to the binding of two single stranded
nucleic acids via complementary base pairing as well as to two or
more nucleic acids.
[0063] A "centromere enumeration probe" is a probe that hybridizes
to a chromosomal region proximate to the centromere, usually a
repeat sequence region, and can indicate the presence or absence of
an entire chromosome.
[0064] A "patient" is a female subject, preferably human, having or
previously having cancer or cervical dysplasia, e.g., low grade
cervical dysplasia, or a cytologically normal pap smear
[0065] Other definitions appear in context throughout the
disclosure.
II. Methods of the Invention
[0066] Preparation of Samples
[0067] Biological samples obtained from patients are typically
cervical preparations, including a pap smear or a thin layer
suspension of cells. A biological sample is a sample that contains
cells or cellular material, e.g., cells or material derived from
the uterine cervix of the uterus. Examples of cervical specimens
include cervical biopsies, smears, scrapes and the like. Typically,
cells are harvested from a biological sample and prepared using
techniques well known in the art. Samples can comprise any number
of cells that is sufficient for a clinical diagnosis, and typically
contain at least about 100 cells. In a typical assay, the
hybridization pattern is assessed in about 25-5,000 cells. Numerous
methods are available for collecting cervical cells for evaluation.
For example, cells from the ectocervix and
endocervix/transformation zone are collected using well-known
devices such as endocervical brushes (or "brooms") or wooden and
plastic spatulas. Conventional smears are prepared by spreading
cells evenly and thinly onto a glass slide. The slide is then fixed
rapidly by immersion into 95% ethanol or spraying with a commercial
fixative according to manufacturer instructions.
[0068] For the ThinPrep.TM. collection method (Cytyc Corp.,
Boxborough, Mass.), cells are transferred from the cervix into the
fixative PreservCyt.TM.. This allows cells to be preserved until
ready for further processing. Cells are then gently dispersed,
randomized and collected onto a TransCyt.TM. membrane filter by
drawing the sample across the filter with a vacuum until an optimal
number of cells is deposited into the filter. The cells can be
further processed as desirable. In another method, the cells
collected into PreservCyt.TM. or other fixative solution can be
further washed by centrifuging, removing the supernatant and
resuspending in Carnoys solution (3:1 Methanol:Acetic acid),
repeating (e.g., three times) as desired. Cells are then
transferred to a glass slide by dropping a small aliquot of cell
suspension directly onto the slide. Slides are typically dried
overnight.
[0069] Where hybridization methods are desired, such biological
samples comprise DNA in a form suitable for hybridization to one of
the probes of the invention. The nucleic acid can be total genomic
DNA, total mRNA, genomic DNA or mRNA from particular chromosomes,
or selected sequences (e.g. 3q sequences).
[0070] The biological sample can optionally be prepared such that
the nuclei in the biological sample remain substantially intact and
comprise interphase nuclei prepared according to standard
techniques. The biological sample can also comprise substantially
intact condensed chromosome (e.g. a metaphase chromosome). Such
condensed chromosomes or interphase nuclei are suitable for use as
hybridization targets in situ hybridization techniques (e.g.
FISH).
[0071] Detection of Chromosomal Abnormalities
[0072] An overabundance of mRNA and protein can result from
transcription of duplicated genes within the amplified region of
chromosome 3q. Accordingly, methods for detecting the genomic
amplification can include methods known in the art for measuring
mRNA levels (e.g., RT-PCR) and protein levels (e.g., western
blotting, enzyme linked immunosorbent assays (ELISAs),
immunoprecipitations and immunofluorescence). Protein expression
can be evaluated using an immunoassay. An immunoassay is an assay
that utilizes an antibody to specifically bind to the analyte
(e.g., telomerase protein). The immunoassay is characterized by
detection of specific binding of the protein to an antibody.
Western blot (immunoblot) analysis can also be used to quantify the
presence of the proteins in the sample. This technique generally
comprises separating sample proteins by gel electrophoresis on the
basis of molecular weight, transferring the separated proteins to a
suitable solid support, (such as a nitrocellulose filter, a nylon
filter, or derivatized nylon filter), and incubating the sample
with the antibodies that specifically bind the desired protein.
[0073] Genomic amplifications can also be identified through
hybridization methods in which a probe is bound to a nucleotide
sequence within a sample or obtained from a sample. Essentially,
the genomic amplification is detected by hybridizing a probe that
is complimentary to a nucleic acid sequence of chromosome 3q (e.g.,
3q26).
[0074] Suitable hybridization formats are well known to those of
skill in the art and include, but are not limited to, variations of
southern blots, northern blots, CGH, in situ hybridization and
quantitative amplification methods such as quantitative PCR (see,
e.g. Sambrook et al., Kallioniemi et al., Proc. Natl. Acad Sci USA,
89: 5321-5325 (1992), and PCR Protocols, A Guide to Methods and
Applications, Innis et al., Academic Press, Inc. N.Y., (1990)). The
sample can also comprise isolated nucleic acids immobilized on a
solid surface (e.g., nitrocellulose) for use in southern or dot
blot hybridizations and the like. In various blot formats (e.g.,
dot blots, Southern blots, and Northern blots) nucleic acids (e.g.,
genomic DNA, cDNA or RNA) are hybridized to a probe specific for
the target region. Either the probe or the target can be
immobilized on the solid surface. Procedures for carrying out
Southern hybridizations are well known to those of skill in the
art. see, e.g., Sambrook et al. Gain or loss of chromosomes or
chromosomal regions can be assessed by methods of in situ
hybridization in which the hybridization pattern of the chromosomal
probe or set of chromosomal probes (e.g., the number of signals for
each probe) is examined, and the number of signals is recorded. In
situ hybridization comprises the following major steps: (1)
fixation of tissue or biological structure to analyzed; (2)
prehybridization treatment of the biological structure to increase
accessibility of target DNA, and to reduce nonspecific binding; (3)
hybridization of the mixture of nucleic acids to the nucleic acid
in the biological structure or tissue; (4) posthybridization washes
to remove nucleic acid fragments not bound in the hybridization and
(5) detection of the hybridized nucleic acid fragments. The reagent
used in each of these steps and their conditions for use vary
depending on the particular application.
[0075] In some applications it is necessary to block the
hybridization capacity of repetitive sequences. In this case, human
genomic DNA or Cot1 DNA is used as an agent to block such
hybridization. The preferred size range is from about 200 bp to
about 1000 bases, more preferably between about 400 to about 800 bp
for double stranded, nick translated nucleic acids.
[0076] Hybridization protocols for the particular applications
disclosed here are described in Pinkel et al. Proc. Natl. Acad.
Sci. USA, 85: 9138-9142 (1988) and in EPO Pub. No. 430,402.
Suitable hybridization protocols can also be found in Methods in
Molecular Biology Vol. 33: In Situ Hybridization Protocols, K. H.
A. Choo, ed., Humana Press, Totowa, N.J., (1994) and Kallioniemi et
al., Proc. Natl. Acad Sci USA, 89: 5321-5325 (1992).
[0077] Typically, it is desirable to use dual color FISH, in which
two probes are utilized, each labeled by a different fluorescent
dye. A test probe that hybridizes to the region of interest is
labeled with one dye, and a control probe that hybridizes to a
different region is labeled with a second dye (e.g., a centromere
enumeration probe). A nucleic acid that hybridizes to a stable
portion of the chromosome of interest, such as the centromere
region, is often most useful as the control probe. In this way,
differences between efficiency of hybridization from sample to
sample can be accounted for.
[0078] The FISH methods for detecting chromosomal abnormalities can
be performed on single cells from the sample. Paraffin embedded
tumor sections can be used, as can fresh or frozen material.
Because FISH can be applied to the limited material, touch
preparations prepared from uncultured primary tumors can also be
used (see, e.g., Kallioniemi, A. et al., Cytogenet. Cell Genet. 60:
190-193 (1992)). For instance, small biopsy tissue samples from
tumors can be used for touch preparations (see, e.g., Kallioniemi,
A. et al., Cytogenet. Cell Genet. 60: 190-193 (1992)). Small
numbers of cells obtained from aspiration biopsy or cells in bodily
fluids (e.g., blood, urine, sputum and the like) can also be
analyzed. Comparison of the intensity of the hybridization signal
from the probe for the target region with the signal from a probe
directed to a control (non amplified or deleted) such as
centromeric DNA, provides an estimate of the relative copy number
of the target nucleic acid.
[0079] Chromosomal Probes
[0080] Using the results provided here, one of skill can prepare
nucleic acid probes that are complimentary to the sequences of 3q
known to have genetic alterations. In particular, such nucleic acid
sequences include but are not limited to TERC (telomerase, 3q26),
EVI1 (ectopic viral integration site 1, 3q24-3q28), MDS1
(myelodysplasia syndrome 1, 3q26), MYNN (myoneurin, 3q26.2), GPR160
(G protein-coupled receptor 160, 3q26.2-3q27), PRKCI (protein
kinase C, 3q26.3), SKIL (SKI-like 3q26), CLDN11 (claudin 11,
3q26.2-3q26.3), EIF5A2 (eukaryotic translation initiation factor
5A2, 3q26.2), TNIK (TRAF2 and NCK interacting kinase, 3q26.2), PLD1
(phospholipase D1, 3q26), GHSR (growth hormone secretagogue
receptor, 3q26.31), TNSF10 (tumor necrosis factor 10, 3q26), ECT2
(epithelial cell transforming sequence 2 oncogene, 3q26.1-3q26.2),
WIG1 (p53 target zinc finger protein, 3q26.3-3q27), PIK3A
(phosphoinositide-3-kinase, 3q26.3), MFN1 (mitofusin 1, 3q26.32)
and USP13 (ubiquitin specific protease 13, 3q26.2-3q26.3).
[0081] In a preferred embodiment, a genomic probe for the
telomerase (TERC) gene on chromosome band 3q26 is applied in
combination with two control probes (CEP3 and CEP7).
[0082] The probe is often labeled with a detectable marker so that
its binding to the target nucleic acid sequence can be identified.
In some embodiments the probes are attached to a solid surface as
an array of nucleic acid molecules. The probe is produced from a
source of nucleic acids from one or more particular (preselected)
portions of the genome, for example one or more clones, an isolated
whole chromosome or chromosome fragment, or a collection of
polymerase chain reaction (PCR) amplification products. The probes
of the present invention are produced from nucleic acids found in
the regions of genetic alteration as described herein. The probe
can be processed in some manner, for example, by blocking or
removal of repetitive nucleic acids or enrichment with unique
nucleic acids.
[0083] The sample or the probes used in the invention can be
isolated nucleic acids immobilized on a solid surface (e.g.,
nitrocellulose) for use in Southern or dot blot hybridizations and
the like. In some embodiments, the sample or probes may comprise an
array of nucleic acids as described for instance in WO 96/17958.
The techniques capable of producing high density arrays for various
applications are also known in the art (see, e.g., Fodor et al.
Science 767-773 (1991) and U.S. Pat. No. 5,143,854). In some cases,
the nucleic acids can be amplified using standard techniques such
as PCR, prior to the hybridization.
[0084] A number of methods can be used to identify probes which
hybridize specifically to the 3q26 region other than those
exemplified here. For instance, probes can be generated by the
random selection of clones from a chromosome specific library, and
then mapped by digital imaging microscopy. This procedure is
described in U.S. Pat. No. 5,472,842. Briefly, a genomic or
chromosome specific DNA is digested with restriction enzymes or
mechanically sheared to give DNA sequences of at least about 20 kb
and more preferably about 40 kb to 300 kb. Techniques of partial
sequence digestion are well known in the art. See, for example
Perbal, A Practical Guide to Molecular Cloning 2nd Ed., Wiley N.Y.
(1988). The resulting sequences are ligated with a vector and
introduced into the appropriate host. Exemplary vectors suitable
for this purpose include cosinids, yeast artificial chromosomes
(YACs), bacterial artificial chromosomes (BACs) and P1 phage.
Various libraries spanning entire chromosomes are also available
commercially from for instance Genome Systems. Alternatively,
libraries can be obtained from the BAC/PAC clone registry or from
for the Caltech clones.
[0085] Once a probe library is constructed, a subset of the probes
is physically mapped on chromosome 3q. FISH and digital image
analysis can be used to localize clones along the desired
chromosome. Briefly, the clones are mapped by FISH to metaphase
spreads from normal cells using e.g., FITC as the fluorophore. The
chromosomes can be counterstained by a stain which stains DNA
irrespective of base composition (e.g., propidium iodide), to
define the outlining of the chromosome. The stained metaphases are
imaged in a fluorescence microscope with a polychromatic
beam-splitter to avoid color-dependent image shifts. The different
color images are acquired with a CCD camera and the digitized
images are stored in a computer. A computer program is then used to
calculate the chromosome axis, project the two (for single copy
sequences) FITC signals perpendicularly onto this axis, and
calculate the average fractional length from a defined position,
typically the p-telomere. This approach is described, for instance,
in U.S. Pat. No. 5,472,842.
[0086] Probes that hybridize specific chromosomal loci are
available commercially from Vysis, Inc. (Downers Grove, Ill.) and
Cancer Genetics, Inc., River Vale, N.J. Alternatively, probes can
be made non-commercially using well known techniques. Sources of
DNA for use in constructing DNA probes include genomic DNA, cloned
DNA sequences such as bacterial artificial chromosomes (BAC),
somatic cell hybrids that contain one or a part of a human
chromosome along with the normal chromosome complement of the host,
and chromosomes purified by flow cytometry or microdissection. The
region of interest can be isolated through cloning or by
site-specific amplification via the polymerase chain reaction
(PCR). See, for example, Nath, et al., Biotechnic Histochem, 1998,
73 (1): 6-22; Wheeless, et al., Cytometry, 1994, 17:319-327; and
U.S. Pat. No. 5,491,224. Synthesized oligomeric DNA or PNA probes
can also be used.
[0087] Sequence information of the genes identified herein permits
the design of highly specific hybridization probes or amplification
primers suitable for detection of target sequences from these
genes. As noted above, the complete sequence of these genes are
known. Means for detecting specific DNA sequences within genes are
well known to those of skill in the art. For instance,
oligonucleotide probes chosen to be complementary to a selected
subsequence within the gene can be used. Alternatively, sequences
or subsequences may be amplified by a variety of DNA amplification
techniques (for example via polymerase chain reaction, ligase chain
reaction, transcription amplification, etc.) prior to detection
using a probe. Amplification of DNA increases sensitivity of the
assay by providing more copies of possible target subsequences. In
addition, by using labeled primers in the amplification process,
the DNA sequences may be labeled as they are amplified.
[0088] The size of the chromosomal region detected by the probes
used in the invention can vary, for example, from a several hundred
base pair probe sequence to a large segment of 150,000 bases. For
locus-specific probes, that are directly labeled, it is preferred
to use probes of at least 100,000 bases in complexity, and to use
unlabeled blocking nucleic acid, as disclosed in U.S. Pat. No.
5,756,696, herein incorporated by reference, to avoid non-specific
binding of the probe. It is also possible to use unlabeled,
synthesized oligomeric nucleic acid or protein nucleic acid as the
blocking nucleic acid. For targeting a particular gene locus, it is
preferred that the probes span the entire genomic coding locus of
the gene.
[0089] Probe of the invention can be labeled with a detectable
marker. Methods of labeling nucleic acids are well known to those
of skill in the art. Preferred labels are those that are suitable
for use in in situ hybridization. The nucleic acid probes can be
detectably labeled prior to the hybridization reaction.
Alternatively, a detectable label which binds to the hybridization
product may be used. Such detectable labels include any material
having a detectable physical or chemical property and have been
well-developed in the field of immunoassays.
[0090] The labels can be coupled to the probes in a variety of
means known to those of skill in the art. In some embodiments the
nucleic acid probes are labeled using nick translation or random
primer extension (Rigby, et al. J. Mol. Biol., 113: 237 (1977) or
Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)). Particularly
preferred methods for labeling probes are described in U.S. Pat.
No. 5,491,224. These methods involve direct labeling the probes by
chemical modification of cytosine residues.
[0091] As used herein, a detectable marker is any composition
detectable by spectroscopic, photochemical, biochemical, enzymatic,
immunochemical, or chemical means. Useful labels in the present
invention include radioactive labels (e.g. .sup.32P, .sup.125I,
.sup.14C, .sup.3H, and .sup.35S), fluorophores (e.g. fluorescein,
rhodamine, Texas Red), electron-dense reagents (e.g. gold), enzymes
(e.g. enzymes that metabolize substrates to produce a chromogenic
signal), colorimetric labels (e.g. colloidal gold), magnetic labels
(e.g. Dynabeads.TM.) and the like. Examples of labels which are not
directly detected but are detected through the use of directly
detectable label include biotin and dioxigenin as well as haptens
and proteins for which labeled antisera or monoclonal antibodies
are available.
[0092] Chromosomal probes can contain any detectable marker that
facilitates the detection of the probe when hybridized to a
chromosome. Effective markers include both direct and indirect
labels as described below.
[0093] Chromosomal probes can be directly labeled with a detectable
marker. Examples of detectable markers include fluorophores, i.e.,
organic molecules that fluoresce after absorbing light, and
radioactive isotopes, e.g., (e.g. .sup.32P, .sup.125I, .sup.14C,
.sup.3H, and .sup.35S). Fluorophores can be directly labeled
following covalent attachment to a nucleotide by incorporating the
labeled nucleotide into the probe with standard techniques such as
nick translation, random priming, and PCR labeling. Alternatively,
deoxycytidine nucleotides within the probe can be transaminated
with a linker. The fluoropore can then be covalently attached to
the transaminated deoxycytidine nucleotides. See, e.g., U.S. Pat.
No. 5,491,224 to Bittner, et al., which is incorporated herein by
reference. Useful probe labeling techniques are described in
Molecular Cytogenetics Protocols and Applications, Y.-S. Fan, Ed.,
Chap. 2, "Labeling Fluorescence In Situ Hybridization Probes for
Genomic Targets", L. Morrison et. al., p. 21-40, Humana Press,
(2002), incorporated herein by reference.
[0094] Examples of fluorophores that can be used in the methods
described herein are: 7-amino-4-methylcoumarin-3-acetic acid
(AMCA), Texas Red.TM. (Molecular Probes, Inc., Eugene, Oreg.);
5-(and -6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and
-6)-carboxyfluorescein; fluorescein-5-isothiocyanate (FITC);
7-diethylaminocoumarin-3-carboxylic acid,
tetramethyl-rhodamine-5-(and -6)-isothiocyanate; 5-(and
-6)-carboxytetramethylrhodamine; 7-hydroxy-coumarin-3-carboxylic
acid; 6-[fluorescein 5-(and -6)-carboxamido]hexanoic acid;
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic
acid; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 5-(and
-6)-carboxyrhodamine 6G; Cascade.TM. blue aectylazide (Molecular
Probes, Inc., Eugene, Oreg.), SpectrumOrange.TM., SpectrumAqua.TM.,
SpectrumGreen.TM., SpectrumGold.TM., and SpectrumRed.TM. (Vysis,
Inc., Downers Grove, Ill.).
[0095] When multiple probes are used, fluorophores of different
colors can be chosen such that each chromosomal probe in the set
can be distinctly visualized. Preferably the probe panel of the
invention will comprise four separate probes, each labeled with a
separate fluorophore. It is also within the scope of the invention
to use multiple panels sequentially on the same sample: in this
embodiment, after the first panel is hybridized, the results are
imaged digitally, the sample is destained and then is hybridized
with a second panel. Probes can be viewed with a fluorescence
microscope and an appropriate filter for each fluorophore, or by
using dual or triple band-pass filter sets to observe multiple
fluorophores. See, e.g., U.S. Pat. No. 5,776,688 to Bittner, et
al., which is incorporated herein by reference. Any suitable
microscopic imaging method can be used to visualize the hybridized
probes, including automated digital imaging systems, such as those
available from MetaSystems or Applied Imaging. Alternatively,
techniques such as flow cytometry can be used to examine the
hybridization pattern of the chromosomal probes.
[0096] Probes can also be labeled indirectly, e.g., with biotin or
digoxygenin by means well known in the art. However, secondary
detectable markers or further processing are then required to
visualize the labeled probes. For example, a probe labeled with
biotin can be detected by avidin conjugated to a detectable marker,
e.g., a fluorophore. Additionally, avidin can be conjugated to an
enzymatic marker such as alkaline phosphatase or horseradish
peroxidase. Such enzymatic markers can be detected in standard
colorimetric reactions using a substrate for the enzyme. Substrates
for alkaline phosphatase include
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
Diaminobenzoate can be used as a substrate for horseradish
peroxidase. Fluorescence detection of a hybridized biotin or other
indirect labeled probe can be achieved by use of the commercially
available tyramide amplification system.
[0097] The present invention is additionally described by way of
the following illustrative, non-limiting Examples that provide a
better understanding of the present invention and of its many
advantages.
EXAMPLES
Example 1
Collection of Patient Samples and Cytological Screening
[0098] 59 previously stained and routinely diagnosed Pap smear
samples from 34 patients were collected from the archive of the
Laboratory of Cytopathology at the Klink Kloster Paradiese in
Soest, Germany, with informed consent, and assigned to three
groups. The Pap smears were evaluated according to established
routine diagnostic procedures, i.e., initial screening by a
cytotechnologist, and--when aberrant cells were found--a consensus
diagnosis by two cytopathologists.
[0099] In brief, all samples were stained according to standard
procedures and were embedded in a permanent mounting medium under
coverslips. Cytological images were acquired from the samples. From
cytologically normal lesions, 15-30 brightfield images were taken
from areas on the slides that contained epithelial cells with
reasonable cell density. If, during the screening process of normal
Pap smears, cells that appeared suspicious were encountered, images
were taken of these, as well. From the CIN lesions and the
carcinomas, between 15 and 30 images were acquired from areas that
contained phenotypically suspicious cells using a 20.times. Leica
Phase contrast dry objective (NA 0.5). The xy coordinates of these
areas were recorded.
[0100] Cytological grading was performed according to a custom
classification system in Germany. Table 1 presents the conversion
of the German classification system (based on the Munich
nomenclature).sup.13 to the Bethesda nomenclature.sup.14 and the
nomenclature using cervical intraepithelial neoplasias.
TABLE-US-00001 TABLE 1 Comparison of terminology: Cytological
classification systems Cervical Bethesda intraepithelial Pap I-V
(used In classification neoplasia (CIN) Germany) Normal Normal Pap
I, Pap II ASCUS ASCUS Pap IIw, Pap III LSIL (low-grade CIN 1 Pap
IIID squamous intraepithelial lesion) HSIL (high-grade CIN 2
squamous CIN 3 Pap IVa, Pap IVb intraepithelial lesions) Carcinoma
Carcinoma Pap V
[0101] The patient groups were as follows: the first patient group
(n=22 samples) consisted of 12 cases for which the initial
diagnosis was CIN1 and CIN2 (PapIIId). Matched Pap smears screened
two months to two years after the initial diagnosis revealed
progression to CIN3 (PapIV). This group was selected to evaluate
whether CIN1 and CIN2 lesions that progress to CIN3 already carry
extra copies of chromosome arm 3q.
[0102] The second group (n=19 samples) consisted of 10 cases
wherein Pap smears were assessed as CIN1 or CIN2, and whose
subsequent Pap smears, several months to two years later, were
cytologically normal. It was hypothesized that the CIN1 and CIN2
lesions in this group would not have acquired gains of 3q.
[0103] The third group (n=23 samples) consisted of 12 cases for
whom the Pap smears were initially diagnosed as normal. However, in
all instances, these women developed CIN3 (PapIVa/b) (n=11) or
cervical carcinomas (PapV) (n=1) after a follow-up period of only
one to three years. In this group, it would be of interest to learn
whether some of the normal Pap smears were already initially
positive for 3q gain and to assess why cytological screening had
not identified aberrant cells.
Example 2
FISH and Signal Enumeration
[0104] Prior to in situ hybridization, the coverslips were removed
by incubating the slides in xylene for 2-4 days. After removal of
the coverslips, the slides were washed twice in xylene, rehydrated,
and de-stained in 0.5% HCl/70% EtOH for 1-2 hours. Slides were
pre-treated with 0.05% pepsin for 10-30 minutes and fixed in 100%
EtOH.
[0105] The three color fluorescent probe panel has been previously
described in detail..sup.8 It consists of a BAC contig that
contains the human telomerase gene (TERC, labeled with
SpectrumOrange.TM.), a centromere enumeration probe for chromosome
3 (CEP3, labeled with SpectrumGreen.TM.), and a control probe for
the centromere of chromosome 7 (CEP7, labeled with
SpectrumAqua.TM.). The probe set is depicted schematically in FIG.
1.
[0106] Details of the hybridization conditions and
post-hybridization washes were previously described..sup.8 The
probes were provided by Vysis, Inc./Abbott Laboratories (Downers
Grove, Ill.). About one third of the samples were hybridized using
a probe cocktail provided by Cancer Genetics, Inc. (Milford,
Mass.). In this probe set, the Spectrum Orange labeled TERC probe
was replaced with a BAC contig specific for TERC that was directly
labeled with rhodamine using the protocol developed by Kreatech
(http://www.kreatech.com). In brief, this protocol relies on the
use of a molecule consisting of a platinum complex, a detectable
molecule, and a leaving group, which is displaced upon reaction
with the target. The molecule forms a co-ordinative bond, firmly
coupling it to the target. The performance of the probe sets was
comparable (data not shown).
[0107] After hybridization, the cell nuclei were counterstained
with DAPI and embedded in an antifade solution. Details of the FISH
procedure, which can also be retrieved at
http://www.riedlab.nci.nih.gov, are as follows: the hybridized
slides were washed in 50% formamide/2.times.SSC (pH 7-7.5) for
3.times.5 min at 45.degree. C., shaking. The slides were washed in
0.1.times.SSC at 45.degree. C. for 3.times.5 min, shaking. The
slides were dipped in 4.times.SSC/0.1% Tween 20. 120 .mu.L Blocking
Solution (3% BSA/4.times.SSC/0.1% Tween 20) were added to the
slides, which were then covered with a 24 mm.times.60 mm coverslip
in a moist hybridization chamber at 37.degree. C. for 30 min.
[0108] The slides were dipped in 4.times.SSC/0/15 Tween 20 to wash
off the blocking solution. The slides were stained for 5 min in
DAPI staining solution in a light-protected coplin jar. The slides
were washed for 5 min in 2.times.SSC, shaking. The slides were then
dehydrated by dipping through an ethanol series of: 70%, 90%, and
100% and air-dried. Finally, 35 .mu.L of antifade solution
(1,4-phenylene-diamine-based) were applied, the slides were covered
with 24 mm.times.60 mm coverslips and stored in a light-protected
container at 4.degree. C. until imaging of the slide.
[0109] After relocation, FISH images were acquired using a
40.times. Leica oil immersion objective (NA 1.25). Representative
images of both cytology and FISH results are displayed in FIG. 2.
In order to acquire images for the identical area on the slides,
several fluorescent images were taken. Different numbers of cells
were evaluated per case: this number was dependent upon cell
density and on the number of morphologically aberrant cells (as
identified by prior Pap staining). Signal enumeration was primarily
focused on those cells that appeared suspicious by routine
cytological screening (see examples in FIG. 2). The signal
enumeration procedure therefore differed from the one previously
described..sup.8 Cases were considered positive for the 3q assay
when more than 20% of the cells exhibited a TERC signal number
greater than 2.
[0110] Successful analysis of sequential samples was possible in 12
of 22 cases in group 1, in 10 of 19 cases in group 2, and in 12 of
23 cases in group 3. The morphological images that were acquired
before FISH were reviewed and evaluated independently by two
cytopathologists to ensure a direct correlation of the genetic with
the cytological diagnosis. In the few cases of minor discordance
between the two pathologists, a consensus diagnosis was
achieved.
[0111] The results are summarized in Tables 2-4.
TABLE-US-00002 TABLE 2 Group 1: Progressors with 3q positive
PAPIIID. Patient case number, date of birth, Pap smear dates,
hybridization patterns, review diagnosis, and 3q status for the Pap
IIID and the subsequent Pap IVa in patient. Review Diagnosis
(consensus diagnosis of two pathologists evaluating the Pap images
Hybridization patterns observed in corresponding to the Pap IIID
and the number of nuclei the areas that counted for each pattern
hybridized with Patterns are described in the Case No. Date of
Birth Date of Pap IIID 3q) following order: CEP7-CEP3-3q 3q status
1 Jan. 30, 1964 October 2000 Pap IVa, CIN3 1x 2-2-4, 1x 2-2-3, 3x
?-?-3, 1x 3-3-3, 1x 5-5-5, Gain 1x ?-?-5 2 Dec. 12, 1947 August
2000 Pap IIID, CIN 2 14x 2-2-3, 1x 2-3-3, 13x 3-3-3 Gain 3 Mar. 27,
1968 June 2001 Pap IIID, CIN 2 3x 2-3-3, 2x 2-3-3or4, 2x 2-3-4, 3x
2-4-4, 2x 3-3-3 Gain 4 Jan. 21, 1962 January 2001 Pap IIID, CIN 2
6x 2-2-5, 10x 2-2-6, 4x 2-?-4or5 Gain 5 Sep. 21, 1959 June 2000 Pap
IIID, CIN 2 6x ?-2-2, 18x ?-3-4, 5x ?-2-4, 6x ?-?-4 Gain 6 Oct. 25,
1954 July 2000 Pap IIID, CIN 2 20x 2-2-2, 2x 2-2-3, 2x 2-3-4, 5x
3-4-4, 4x 3-4-5 Gain 7 Jul. 5, 1960 October 2000 Pap IIID, CIN 2
10x 2-2-2, 12x 2-3-3, 2x 2-4-4 Gain 8 Jan. 7, 1961 December 1997
Pap IIID, CIN 2 11x 2-2-2, 1x 2-2-3, 1x 2-4-4, 8x 4-4-4, 1x 4-4-5
Tetraploid 9 74 1997 Pap IIw, ASCUS 2x 2-2-2, 1x 2-2-4, 4x 4-4-4
Tetraploid 10 Jun. 11, 1962 October 1998 Pap IIID, CIN 2 42x 4-4-4,
1x 3-3-3? Tetraploid 11 Dec. 28, 1957 March 1997 Pap IIID, CIN 2 5x
2-2-2, 5x 4-4-4 Tetraploid 12 Sep. 24, 1961 1999 Pap IIID, CIN 2 2x
2-2-2, 29x 4-4-4, 1x 4-4-4or5 Tetraploid Review Diagnosis
(consensus diagnosis of two pathologists evaluating the Pap images
Hybridization patterns observed in corresponding to the Pap IVa and
the number of the areas that nuclei counted for each pattern
hybridized with Patterns are described in the Case No. Date of Pap
IVa 3q) following order: CEP7-CEP3-3q 3q status 1 December 2000 Pap
IVa, CIN3 5x 2-3-3, 1x 3-3-3, 1x 3-4-4, 1x 4-5-5 Gain 2 October
2000 Pap IVa, CIN3 9x 2-2-3, 3x 2-3-3, 14x 3-3-3 Gain 3 September
2001 Pap IVa, CIN3 53x 2-2-3, 4x 2-3-3, 4x 2-3-4, 1x 2-2-5, 5x
3-3-3 Gain 4 March 2001 Pap IVa, CIN3 5x ?-?-6, 4x ?-?-5or6 Gain 5
July 2001 Pap IVa, CIN3 9x 2-2-2, 1x 2-2-3, 3x 2-3-3, 2x 2-2-4, 5x
2-3-4 Gain 6 September 2000 Pap IVa, CIN3 1x ?-?-2, 2x ?-?-4, 2x
2-?-5, 11x 3-?-5, 2x ?-?- Gain 5, 1x 3-?-6 7 2003 Pap IVa, CIN3
n.d. n.d. 8 February 1998 Pap IVa, CIN3 1x 3-3-3, 1x 3-4-4, 7x
4-4-4, 1x 4-4-5, 4x 4-5-5, Gain 2x 5-5-5 9 January 1999 Pap IVa,
CIN3 2x 2-2-2, 2x 3-4-4, 15x 4-4-4, 1x 4-4-5, 2x 4-4- Gain 4or5, 2x
4-5-5, 1x 4-4-6, 1x 5-5-5 10 December 1999 Pap IVa, CIN3 17x 4-4-4,
5x 4-5-5, 1x 5-5-5 Gain 11 July 1997 Pap IVa, CIN3 13x 2-2-2, 6x
4-4-4 Tetraploid 12 July 2001 Pap IVa, CIN3 4x 2-2-2, 17x 4-4-4, 1x
4-4-5? Tetraploid "Main patterns" are marked in bold "n.d." not
determined Table 2. Shown are the hybridization patterns and number
of cells with a specific hybridization pattern observed in
individual Pap smears. The column "3q status" reflects the
interpretation of the hybridization patterns as it pertains to 3q
copy numbers
[0112] Nine of the 12 CIN3 lesions in group 1 (Table 2:
progressors) revealed varying degrees of cells with extra copies of
chromosome arm 3q, and two were tetraploid (hybridization pattern
of four signals for all three probes, 4-4-4); one case was not
determined. Seven of the preceding matched CIN1 and CIN2 lesions
were positive for 3q gain, as well, indicating that those CIN1/CIN2
lesions with a high likelihood for progression frequently carry
extra copies of this genetic marker. The remaining five lesions
were tetraploid, including the precursors of the two tetraploid
CIN3 lesions.
TABLE-US-00003 TABLE 3 Group 2: Regressors. Patient case number,
date of birth, Pap smear dates, hybridization patterns, review
diagnosis, and 3q status for the Pap IIID and the subsequent normal
Pap smear of patient. Review Diagnosis (consensus diagnosis of two
pathologists evaluating the Pap images Hybridization patterns
observed in corresponding to the Pap IIID and the number of the
areas that nuclei counted for each pattern hybridized with Patterns
are described in the Case No. Date of Birth Date of Pap IIID 3q)
following order: CEP7-CEP3-3q 3q status 1 ######## October 1999 Pap
IIID, CIN 2 29x 2-2-2 Diploid 2 Sep. 3, 1959 January 2001 Pap IIID,
CIN 2 25x 2-2-2, 1x 2-3-3? Diploid 3 Mar. 28, 1969 2000 Pap IIID,
CIN1/2 40x 2-2-2 Diploid 4 Apr. 6, 1969 2000 Pap IIID, CIN 2 46x
2-2-2, 3x 4-4-4, 1x 4-4-4? Diploid 5 Jun. 5, 1977 July 1999 Pap
IIID, CIN 2 49x 2-2-2, 1x 2-2-3?, 1x 2-2-4, 2x 4-4-4 Diploid 6 Feb.
28, 1970 February 1999 Pap IIID, CIN1/2 57x 2-2-2, 1x 2-2-3?, 3x
2-2-4 Diploid 7 Apr. 15, 1947 August 1999 Pap IIID, CIN 2 19x
2-2-2, 2x 4-?-4, 1x ?-?-4or5 Diploid 8 ######## 1999 Pap IIID, CIN
1/2 18x 2-2-2, 1x 2-2-4, 4x 4-4-4, 1x 4-?-4 Tetraploid 9 Aug. 1,
1954 2000 Pap IIID, CIN 1 50x 2-2-2, 1x 2-2-4?, 13x 4-4-4
Tetraploid 10 Sep. 14, 1973 February 2001 Pap IIID, CIN 2 10x
2-2-2, 7x 4-4-4, 1x 4-?-4 Tetraploid Review Diagnosis (consensus
Hybridization patterns diagnosis of two observed in the Pap II
pathologists and the number of evaluating the nuclei counted for
Pap images each pattern corresponding to Patterns are the areas
that described in the hybridized with following order: CEP7- Case
No. Date of Pap II 3q) CEP3-3q 3q status 1 August 2000 Pap I,
normal 34x 2-2-2 Diploid 2 2001 n.d. not evaluable n.d. 3 December
2000 n.d. n.d. n.d. 4 2001 n.d. not evaluable n.d. 5 December 1999
n.d. not evaluable n.d. 6 2000 n.d. n.d. n.d. 7 July 2001 Pap II,
normal 20x 2-2-2 Diploid 8 April 2000 n.d. not evaluable n.d. 9
2001 n.d. n.d. n.d. 10 2001 n.d. not evaluable n.d. "Main patterns"
are marked in bold "n.d." not determined Table 3 Shown are the
hybridization patterns and number of cells with a specific
hybridization pattern observed in individual Pap smears. The column
"3q status" reflects the interpretation of the hybridization
patterns as it pertains to 3q copy numbers
[0113] In group 2 (Table 3: regressors), seven of the
non-progressing CIN1/CIN2 lesion were diploid (hybridization
pattern of two signals each for all probes, 2-2-2); three cases
were tetraploid (4-4-4), and none of the cases showed a gain of 3q.
These findings demonstrate that CIN1/CIN2 lesions that
spontaneously regress do not carry a gain of TERC.
TABLE-US-00004 TABLE 4 Shown are the hybridization patterns and
number of cells with a specific hybridization pattern observed in
individual Pap smears. The column "3q status" reflects the
interpretation of the hybridization patterns as it pertains to 3q
copy numbers Review Diagnosis (consensus diagnosis of two
pathologists evaluating the Pap images Hybridization patterns
observed corresponding to in the Pap I/II and the number of the
areas that nuclei counted for each pattern hybridized with Patterns
are described in the Case No. Date of Birth Date of Pap I/II 3q)
following order: CEP7-CEP3-3q 3q status 1 Sep. 22, 1941 Pap I: 1999
Pap I/II, normal 17x 2-2-2, 1x 2-2-3?, 1x 2-3-3? Diploid 2 ########
Pap II: 1999 Pap II, normal 14x 2-2-2, 1x 2-2-3?, 1x 2-3-3? Diploid
3 Jul. 8, 1958 Pap I: October 1996 Pap II, normal 15x 2-2-2, 2x
2-2-3? Diploid 4 Apr. 16, 1951 Pap I: Sept 97 Pap II, normal 16x
2-2-2, 1x 2-4-4?, 1x 3-3-3? Diploid 5 Jun. 17, 1973 Pap II:
February 1998 Pap II, normal 34x 2-2-2, 1x 2-3-3? Diploid 6
######## Pap II: March 2000 Pap IIID, CIN1 22x 2-2-2, 1x 5-5-5?
Diploid 7 ######## Pap I: July 2001 Pap I, normal 22x 2-2-2, 2x
?-?-3? Diploid 8 Feb. 28, 1967 Pap II: 1998 Pap II, normal 18x
2-2-2 Diploid 9 Sep. 23, 1961 Pap II: July 97 Pap II, normal 20x
2-2-2, 1x 2-3-3?, 16x 2-3-4, 2x 2-3-5 Gain 10 Jul. 13, 1950 Pap I:
1996 Pap IIID, CIN 2 3x 2-2-2, 4x 2-?-3, 1x 2-2-3, 3x 2-?-4, 1x 2-
Gain 2-4, 3x 2-?-5, 1x 2-2-5, 2x 2-5-5, 1x2-2-6, 1x 2-?-7, 1x
3-?-4, 2x 4-4-4 11 ######## Pap II: 1997 Pap IVa, CIN 3 8x 2-2-2,
46x 2-5-5 Gain 12 May 18, 2015 Pap II: August 1998 Pap II, normal
52x 2-?-2, 15x 2-?-3, 3x 2-2-3, 2x 4-?-4, 1x Gain 4-?-8 Review
Diagnosis (consensus diagnosis of two pathologists evaluating the
Pap images Hybridization patterns observed in the corresponding to
Pap IVa/b and the number of nuclei the areas that counted for each
pattern Case hybridized with Patterns are described in the
following No. Date of Pap IVa/IVb 3q) order: CEP7-CEP3-3q 3q status
1 Pap IVb: February 2001 Pap IVb, CIN3 4x 2-2-3, 2x 2-3-3, 1x
3-3-3, 1x 2-3-4, 1x 2-5-5, 3x 3 Gain 5-5, 4x 4-5-5 2 Pap IVa: May
2000 Pap IVa, CIN3 5x 2-2-3, 5x 2-3-3, 3x 2-4-4, 3x 4-4-4, 1x 5-4-4
Gain 3 Pap IVa: January 1999 Pap IVa, CIN3 1x 2-2-2, 6x 2-2-3, 3x
2-3-3, 1x 2-2-4, 1x 2-3-4 Gain 4 Pap IVa: June 1998 Pap IVa, CIN3
4x 2-2-2, 2x 2-2-3, 6x 2-3-3, 1x 2-3-4, 2x 3-3-3, 2x 3 Gain 3-4, 1x
3-3-5, 1x 4-4-4, 4x 3-3-? 5 Pap IVa: February 1999 Pap IVa, CIN3
18x 2-2-2, 6x 2-2-3, 4x 2-3-3, 1x 3-3-3 Gain 6 Pap IVa: August 2001
Pap IVa, CIN3 1x 2-2-2, 2x 3-4-4, 7x 4-4-4, 4x 4-5-5, 9x 5-5-5 Gain
7 Pap IVb: April 2002 Pap IVb, CIN3 9x ?-?-3, 4x ?-3-3, 2x 2-3-3
Gain 8 Pap IVa: June 2000 Pap IVa, CIN3 61x 4-4-4, 1x 2-2-3?, 3x
4-4-5?, 1x 3-4-4? Tetraploid 9 Pap IVa: March 2000 Pap IVa, CIN 3
2x 2-2-2, 3x 2-3-3, 98x 2-3-4, 1x 2-3-5, 2x 2-4-5, Gain 4x 4-6-6 10
Pap V: April 1999 Pap IVa, CIN 3 4x 2-2-2, 1x 2-2-3, 3x 2-3-3, 6x
2-2-4, 8x 2-2-5, 8x Gain 2-2-6, 1x 2-2-7, 3x 2-2-8, 26x 2-4-4, 1x
2-4-5, 1x 2- 4-6, 1x 2-4-5, 13x 2-5-5, 1x 2-4-6, 1x 2-6-9, 1x 3-3-
3, 10x 3-4-4, 1x 3-3-5, 1x 3-3-6, 1x 3-3-7, 1x 3-4-5, 1x 3-5-7, 1x
3-6-6, 3x 4-4-4 11 Pap IVa: August 1999 Pap IVa, CIN 3 6x 2-2-2, 5x
2-4-4, 128x 2-5-5 Gain 12 Pap V: February 1999 Pap V, Carcinoma 6x
2-2-2, 10x 2-2-3, 1x 3-2-3, 3x 2-3-5, 1x 2-2-8, 1x Gain 2-3-8, 1x
3-3-8, 87x 4-3-8, 1x 4-4-5, 1x 4-4-8, 1x 5- 4-8 "Main patterns" are
marked in bold
[0114] Eleven of the 12 CIN3 lesions and carcinomas in group 3
(Table 4: normal Pap smear followed by CIN3 or carcinoma) revealed
a 3q gain. One lesion (#8, Table 4) was tetraploid. Of note, four
of the 12 cytologically normal Pap smears already exhibited a gain
of 3q. This indicates that the visualization of additional copies
of the TERC gene could serve as an early and specific marker in
cytologically normal Pap smears obtained from women who are prone
to develop CIN3 lesions or invasive disease.
[0115] Of note, a certain number of cases in the groups of
progressors and regressors showed signal patterns that are
compatible with a tetraploidization of the genome (i.e., four
copies of CEP7, CEP3, and 3q, referred to as 4-4-4 in Tables 2-4).
The hybridization patterns observed in the 3q-positive CIN3 lesions
and their matched CIN1/2 precursors suggest that a certain
chromosomal aneuploidy, once established, is maintained during
tumor progression, which again suggests clonal expansion (Tables
2-4). It is striking that none of the 3q-positive lesions in which
the gain of TERC occurred on a diploid background (as assessed with
the copy numbers for CEP3 and CEP7) showed a tetraploid
hybridization pattern at lower-grade lesions.
[0116] All CIN3 lesions for which the corresponding premalignant
lesions were tetraploid, maintained tetraploidy or developed 3q
gain on a tetraploid background. This observation indicates that
the gain of 3q can occur on the basis of either a diploid or
tetraploid genome. Thus, quantitative DNA content measurement alone
may not suffice as a diagnostic method in cervical
cytology..sup.30
[0117] While none of the samples in group 2 (regressors) were
positive for 3q gain, three lesions were tetraploid. This implies
that tetraploidization per se does not modify the genome such that
progression is unavoidable. This is consistent with previous
observations indicating that genome duplication can occur as a
physiological response to certain environmental
challenges..sup.30,31
[0118] In the review diagnosis of the morphological images by two
experienced cytopathologists, one of the four 3q positive lesions
was upgraded from normal to CIN2 (Pap IIID), and another one was
upgraded to CIN3 (Pap IVa), whereas the diagnosis for two of them
remained as previously determined, i.e., normal.
[0119] The comparison of the cytological phenotype of the cells
with the genetic makeup indicates that, in two cases, the
dysplastic cells were present on the slide, yet were indeed
overlooked (a known problem in cervical cytology); however, in two
other cases, the cellular phenotype appeared normal on review
despite the presence of chromosomal aneuploidy and gain of 3q. This
demonstrates that the acquisition of specific genomic gains can
precede phenotypic alterations appreciable by morphological
inspection. Examples of images of these cases are displayed in FIG.
2.
[0120] FIG. 2A corresponds to a Pap smear assessed as Pap IIID
(CIN1). Of note, the morphologically suspicious cells do not carry
extra copies of the TERC genes (two copies per cell only). FIG. 2B
corresponds to a Pap smear assessed as Pap IIID (CIN2). FIG. 2C
corresponds to the Pap smear of a patient initially diagnosed as
Pap IIID (October 2000) and considered a regressor because
subsequent Pap smears were normal (2001), but assessed via a 2002
Pap smear as CIN2 and via a 2003 Pap smear as CIN3 (and,
accordingly, assigned to group 1). FIG. 2D corresponds to a Pap
smear repeatedly judged as morphologically normal, though the
patient presented with a CIN3 lesion after 28 months.
[0121] Of note, in many instances, the cells that were positive for
additional copies of 3q were located next to each other on the
diagnostic slides. This indicates a clonal evolution event, where
extra copies of 3q render a growth advantage to cervical epithelial
cells, which eventually results in a cell population in cervical
carcinomas in which the majority of the cells are positive for 3q.
Accordingly, visualization of additional copies of the TERC gene in
premalignant dysplastic lesions is not only informative for the
diagnosis of dysplasia, but can also provide information regarding
the progressive potential of individual lesions.
[0122] The review diagnosis of the morphological images of the
areas evaluable with the 3q marker was in concordance with the
initial diagnosis for 53 out of the 59 specimens (90%). Six
specimens were up- or downgraded from the original diagnosis,
including the cases discussed above (Group 3, Table 4, case 10 from
Pap I to Pap IIID, and case 11 from Pap II to Pap IVa). The
original Pap V diagnosis of case 10 (Table 4) was downgraded to Pap
IVa. In group 3, Table 4, case 6 was upgraded from Pap II to Pap
IIID, and in group 1, Table 2, case 1 was upgraded from Pap IIID to
Pap IVa, and case 9 was downgraded from Pap IIID to Pap IIw,
ASCUS.
[0123] As hypothesized, some of the Pap smears that had previously
been assessed as normal revealed the presence of 3q, the detection
of which would have resulted in an earlier diagnosis. Indeed, seven
of 12 CIN1/CIN2 lesions that progressed to CIN3 were found positive
for 3q (all matched CIN3 lesions carried amplified TERC). In strong
contrast, none of the spontaneously regressing lesions showed a 3q
gain.
Example 3
Statistical Evaluation of Data
[0124] A. Fisher's Exact Test
[0125] The statistical evaluation was based on the Fisher's exact
test and the exact binomial parameter estimation, which is suited
for small sample numbers. The Fisher's exact test was used for
2.times.2 contingency table analysis of the categorical data. The
two categorical variables used are the pathological assessment
(progression and regression) and the detection of genomic
aberrations, which is either positive or negative. Each cell in
Table 5 reflects the observed outcomes from patient samples.
[0126] The null hypothesis (H.sub.0) postulates that the presence
of genomic aberrations (either the gain of 3q or tetraploidy) and
the progression status are independent from one another. The
progression rate is defined as the number of cases that progress
over the total number of cases tested. The upper and lower
endpoints of the exact confidence intervals for estimation of this
binomial parameter were denoted as P.sub.L(.alpha.) by
P.sup..alpha..sub.L(n,B) and P.sub.U(.alpha.) by
P.sup..alpha..sub.u (n, B) and were determined based on the
equations below..sup.15
P L .alpha. ( n . , B ) B B + ( n - B + 1 ) f .alpha. / 2 , 2 ( n -
B + 1 ) , 2 B ##EQU00001## and ##EQU00001.2## P u .alpha. ( n , B )
= 1 - P L .alpha. ( n , n - B ) ##EQU00001.3##
B refers to the number of progressions (successes) in the n
Bernoulli trials, and f.sub..gamma.,n1,n2 is the upper
.gamma..sup.th percentile for the F distribution with n.sub.1
degree of freedom in the numerator and n.sub.2 degrees of freedom
in the denominator.
[0127] The results are summarized in Table 5.
TABLE-US-00005 TABLE 5 Statistical evaluation p-value 95%
confidence from interval of Aberration Pathological assessment
Fisher's Progression progression rate profile Progression
Regression test rate lower upper 4-4-4 5 3 0.6749 0.625 0.2449
0.9148 2-2-2 and 7 7 0.5000 0.2304 0.7696 3q gain 3q gain 7 0
0.0053 1.0000 0.5904 1.0000 2-2-2 and 5 10 0.3333 0.1182 0.6162
4-4-4 3q gain and 12 3 0.0007 0.8000 0.5191 0.9567 4-4-4 2-2-2 0 7
0.0000 0.0000 0.4096 Table 5. Statistical analysis for contingency
tables and confidence intervals of the progression rate. From the
left, the column classifier for all three 2.times.2 contingency
tables is the pathological assessment (progression and regression).
The row classifiers are the detection of tetraploidy (top 2.times.2
table), gain of the chromosome 3q (center 2.times.2 table) and gain
of the 3q including tetraploidy (bottom 2.times.2 table),
respectively. The two-tailed p-value for each table is derived from
the Fisher's exact test. On the right, the progression rate for the
different hybridization patterns was calculated based on the cell
counts in the contingency tables. Its exact 95% confidence interval
was obtained using the method described in the Materials and
Methods section.
[0128] For the patients whose Pap smears contained tetraploid
cells, the odds (5 to 3) are in favor of progression, yet the
p-value is 0.6749. Therefore, H.sub.0 cannot be rejected (i.e.,
there is no strong statistical evidence that tetraploidy is
associated with progression).
[0129] For cases with a 3q gain versus diploid and tetraploid
cases, the p value is 0.0053. Thus, there is strong evidence to
reject H.sub.0, indicating that additional copies of 3q and
progression are associated. The 95% confidence interval ranges from
0.5904 to 1.0000. Thus, with 95% confidence, the probability of
progression is expected to be 59% to 100%.
[0130] For patients that have either a gain of 3q or tetraploidy,
the p value for the test is 0.0007. Therefore, H.sub.0 can be
rejected with confidence. A significant correlation exists between
additional copies of 3q and carcinoma development. A 52% to 96%
progression rate is expected in patients with either 3q-positive or
tetraploid samples.
[0131] If the afore-mentioned cases in the groups of progressors
and regressors that showed signal patterns that are compatible with
a tetraploidization of the genome (see Example 2, above) are
included using a conservative threshold of 20%, the test achieves a
sensitivity of 100% (i.e., the association of progression with
either tetraploidy or TERC gain) with a specificity of 70%, which
is defined as the association of regression with the absence of 3q
positive patterns, i.e. 3q gain including tetraploidy (seven of 10
cases, as per Table 5).
[0132] B. ROC Analysis
[0133] Receiver Operator Characteristic (ROC) analysis was used to
further establish optimal thresholds and identify FISH parameters
that best predicted progression. ROC curves were generated by
plotting the sensitivity for predicting progression versus 1 minus
the specificity for predicting regression, calculated at percent
cell thresholds ranging from 0% to 100% (1% increments). Curves
were generated based on the percentage of tetraploid cells (4-4-4
hybridization pattern), the percentage of cells with 3q gain (>2
TERC signals per cell, excluding tetraploidy), and the percentage
of cells with either tetraploidy or 3q gain (i.e., >2 TERC
signals per cell, including tetraploidy).
[0134] In FIG. 3A, comparison of the ROC curves shows that
tetraploidy alone (blue squares) is not a good indicator of
progression, while 3q gain (defined for this purpose as >2 TERC
signals/cell, exclusive of tetraploidy, white triangles) is a
better indicator, and 3q gain including tetraploidy (any gain of
3q, red circles) shows very good ROC characteristics.
[0135] In the ROC plot, curves that come closest to the ideal
values of 100% sensitivity and 100% specificity (top left corner of
ROC graph, see FIG. 3A) provide the best combination of sensitivity
and specificity (assuming equal importance of each), and optimal
thresholds are typically selected from points near the `breaks` in
the curves (region closest to top left corner; curve slope near
45.degree.).
[0136] The point on the latter ROC curve lying closest to the top
left corner of the graph represents sensitivity and specificity
values of 91.7% (11/12) and 100% (10/10), respectively, which are
obtained for cell percentage thresholds of 45 to 49%. However, for
identifying women likely to progress, higher sensitivity is
preferred and 100% sensitivity (12/12) with 90% specificity ( 9/10)
are achieved with thresholds ranging between 25 to 39%. To further
ensure identification of likely progressors, a more conservative
threshold of 20% was used in the present study.
[0137] C. DFI Analysis
[0138] A better view of the dependence of sensitivity and
specificity on threshold can be obtained by plotting the Distance
From Ideal (DFI) versus threshold (FIG. 3B, which uses the same
data used to construct FIG. 3A). DFI is defined as the distance
from the ideal point (0, 1) on the ROC plot (100% sensitivity, 100%
specificity) and is calculated as
[(1-sensitivity).sup.2+(1-specificity).sup.2].sup.1/2. DFI is
smallest for the best combined sensitivity and specificity (giving
equal weight to each) and varies from a value of 0 for thresholds
providing 100% sensitivity and 100% specificity, i.e., the ideal
point to a maximum value of 2.sup.1/2.
[0139] The curves identify the threshold ranges providing the
lowest combined sensitivities and specificities, and emphasize that
3q gain inclusive of tetraploidy (red circles) is better suited to
predict progression (lowest and broadest minimum of the three
curves) than tetraploidy alone (blue squares) or 3q gain alone
(white triangles). Minima on a DFI curve indicate the best values
for thresholds, and broad minima are indicative of more robust
assays, since placement of thresholds is less critical.
[0140] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding, it will be apparent to those skilled in the art
that certain changes and modifications can be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
numbered claims.
Example 4
Frequent Gain of TERC in Cervical Adenocarcinomas
[0141] Tumor Material
[0142] The study includes formalin-fixed paraffin embedded tumour
tissue specimens from 12 primary cervical adenocarcinomas diagnosed
and surgically treated at the Karolinska University
Hospital-Huddinge during 1992-2000 (Table 6). All tumour cases were
identified from the Swedish Central Cancer Registry organized by
the National Board of Health and Welfare. This registry includes
all cases of malignant tumours diagnosed histopathologically after
1959, whereby each tumour is identified by a topographical and
histopathological code. All tumour samples were collected with
informed consent and approval from the local ethics committee.
TABLE-US-00006 TABLE 6 Clinical details and HPV status of the 12
adenocarcinomas in the study. Cancer Last Pap smear Tumor Tumor
Lymph node HPV infection Follow-up Case no Lab id. diagnosis
year/result Age at diagnosis stage differentiation involvement
status time outcome 1 230-3 1992 1990/normal 30 yrs 1B Poor No HPV
16 13 yrs AwoD 2 T15-2 1992 1989/normal 46 yrs 1B Poor No HPV 18 13
yrs AwoD 3 186-5 1995 1992/normal 37 yrs 1B Well No HPV 18 10 yrs
AwoD 4 120-7 1997 1987/normal 39 yrs 1B Well No HPV negative 8 yrs
AwoD 5 549-3 1992 1978/normal 50 yrs 2B Well Yes HPV negative 13
yrs AwoD 6 149-4 1994 1992/normal 32 yrs 1B Poor No HPV 18 11 yrs
AwoD 7 162-0 2000 1997/normal 62 yrs 1B Well No HPV 45 4 yrs DoD 8
164-0 2000 1998/normal 64 yrs 1B Poor No HPV negative 5 yrs AwoD 9
179-9 1999 1996/normal 63 yrs 2B Moderate Yes HPV negative 6 yrs
AwoD 10 235-9 2000 1999/inflammation 53 yrs 1A Well No HPV 16 5 yrs
AwoD 11 062-7 1997 1996/normal 45 yrs 1B Well No HPV 18 7 yrs DoD
12 132-3 1993 1992/normal 49 yrs 1A Well No HPV 18 11 yrs AwoD AwoD
= Alive without disease; DoD = Dead of disease
[0143] The clinical information for each case is detailed in Table
6 and has been previously published (Andersson et al, 2003a). The
histopathological diagnosis was based on WHO criteria. The 12
tumours were classified as cervical adenocarcinomas and all were
without any squamous cell component. Seven tumours were well
differentiated (58%), one was moderately differentiated (8%) and
four tumours were poorly differentiated (33%). Clinical staging was
according to the FIGO classification for cervical cancer (Benedet
et al, 2000). At initial diagnosis, 10 tumors were classified as
stage I (83%), and two tumors as stage II (17%). Two patients
initially exhibited lymph node involvement (17%). All patients were
previously sampled for Pap smear analysis. All but one Pap smear
(which was evaluated as showing signs of cellular inflammation)
were assessed as normal at the time of the most recent cytological
evaluation (Table 6). All patients were retrospectively followed-up
from the time of diagnosis until June 2005, and disease recurrence
and survival data recorded.
[0144] HPV Status
[0145] Results from HPV screening analyses have been previously
published for all cases (Andersson et al, 2003a). Briefly, the
analyses were performed on extracted DNA obtained from sections of
paraffin blocks of which the preceding section had been used for
morphological diagnosis. A fragment of 139-148 bp was amplified
from the L1 region with GP5+/GP6+ primers, HPV-typed by direct DNA
sequencing and comparison to known HPV sequence databases using the
BLAST algorithm (www.ncbi.nlm.nih.gov/BLAST). Eight of the 12
tumours were thus found to be HPV-positive; five tumours were
infected with HPV 18, two with HPV 16, and one with HPV 45 (Table
6). The remaining four tumour specimens were HPV-negative. The mean
age at cancer diagnosis was significantly lower for the
HPV-positive cases as compared to the HPV negative women (44.2
years, SD=10.4 vs. 54 years, SD=10.8; p=0.001).
[0146] Preparation of Nuclei Suspensions for FISH Analysis
[0147] Single layer nuclei preparations for interphase FISH
hybridizations were prepared using the Hedley method with
modifications (Castro et al, 1993). A 50 .mu.m section was cut from
each of the 12 formalin fixed paraffin-embedded tumour tissue
samples. After deparaffinization in xylene, the section was
rehydrated in an ethanol series and in distilled water, and the
section was disintegrated in 500 .mu.l of 0.1% Protease/1.times.PBS
(Protease:Type XXIV, Bacterial, P 8038, Sigma St. Luis, Mo. and
Dulbecco's 1.times.PBS, Life Technologies, Rockville, Md.) at
45.degree. C. for 45-60 minutes. The reaction was stopped by adding
500 .mu.l 1.times.PBS at room temperature. The sample was then
filtered through a nylon membrane (CN 051, DAKO, Glostrup, Denmark)
centrifuged and resuspended in 1.times.PBS. Cytospin slides were
prepared by Shandon Cytospin.RTM., and fixed in an ethanol
series.
[0148] Fluorescence In Situ Hybridization (FISH)
[0149] Triple-color FISH analysis was performed on each case using
the following probe set: a centromere specific probe for chromosome
7 (CEP.RTM.7); a centromere specific probe for chromosome 3 (CEP3);
and a contig consisting of four overlapping BAC clones containing
the TERC gene at chromosomal location 3q26. All probes were
obtained from Vysis/Abbott Molecula, Inc. (Des Plaines, Ill.). The
details for this probe set, its sensitivity and specificity, as
well as experimental conditions were published previously
(Heselmeyer-Haddad et al, 2003). In short, CEP7 was labeled with
Spectrum Aqua.TM. (SA), CEP3 with Spectrum Green.TM. (SG) and the
TERC contig with Spectrum Orange.TM. (SO), using chemical labeling
as described (Bittner et al, 1996). Before hybridization, the
cytospin slides were pretreated with a pepsin digestion, and fixed
in an ethanol series. Slides were denatured in 70% formamide,
2.times.SSC for 3.5 minutes at 80.degree. C. The probes were
denatured according to the manufacturer's recommendations. After
overnight hybridization at 37.degree. C., the slides were first
washed four times in 50% formamide/2.times.SSC at 45.degree. C.
(once for 3 minutes and three times for 7 minutes), followed by
washes in 2.times.SSC at 45.degree. C. for 5 minutes and in
2.times.SSC/0.1% NP40 at 45.degree. C. for 5 minutes. The slides
were counterstained with 4,6-diamidino-2-phenylindole (DAPI), and
subsequently embedded in an antifade solution.
[0150] Scoring of FISH Results
[0151] FISH and image analyses were carried out using a Leica
DM-RXA fluorescence microscope (Leica, Wetzlar, Germany) equipped
with custom optical filters for DAPI, SA, SG and SO (Chroma
Technologies, Brattleboro, Vt.) and 40.times. Plan Apo (NA 1.25)
objective. Images were taken in areas of optimal cell density with
minimal cellular clumps using an ORCA ER (IEEE1394 I/F) digital
camera (Hamamatsu, Bridgewater, N.J.). Leica Q-Fluoro was used to
acquire multifocus images for each of the DAPI, SA, SG and SO
optical filters. Ten to 16 images were acquired and signal
enumeration was performed on these digital images for 208-641
nuclei for each case. The counted signals were listed and evaluated
in Excel based customized software.
[0152] Nuclei that could not be evaluated (e.g., because of
insufficient hybridization or overlapping nuclei) were excluded
from further analysis. The results for all "countable" nuclei were
registered in relocation charts in form of patterns for the entire
probe panel. For example, the pattern (2-3-3), refers to two
signals for CEP7, three signals for CEP3 and three signals for TERC
in a given nuclei. Nuclei with normal signal numbers for the three
probes (i.e., 2-2-2) were recorded as "diploid", and nuclei with
four signals for each probe (pattern 4-4-4) were considered
"tetraploid". The background level for the CEP7-CEP3-TERC probe
panel was previously evaluated on cytological slides that contained
nuclei from normal cervical cells (Heselmeyer-Haddad et al, 2003).
This confirmed that deviation from the expected (2-2-2) pattern was
seen in less than 2% of normal cells.
[0153] Twelve cervical adenocarcinomas (Table 6) were evaluated for
copy number changes of the TERC locus at chromosomal band 3q26
using interphase FISH. The three-colour probe panel consisting of
CEP7-CEP3-TERC was simultaneously hybridized to cytospin slides
with interphase nuclei prepared from formalin fixed tissue
sections. All cases were successfully hybridized and analyzed,
whereby 208 to 641 nuclei per case were scored. FIG. 1 shows
representative hybridizations. Both, normal "diploid" patterns
(2-2-2), as well as nuclei with non-diploid patterns were observed.
The results are summarized in Table 7. In all 12 tumours a
significant proportion of "non-diploid" nuclei was detected, and
nine of the tumours exhibited >50% "non-diploid" nuclei. In the
individual tumours, nuclei with >2 TERC signals were found in
similar frequencies as cells with "non-diploid" pattern, and almost
all nuclei with "non-diploid" pattern exhibited >2 TERC signals.
In the "non-diploid" nuclei a "tetraploid" pattern (4-4-4) was not
commonly observed. In ten of the tumors less than 1% of the nuclei
exhibited (4-4-4) and in two cases (4-4-4) was seen in 4% and 12%
respectively (Table 7). Thus, gain of TERC was present in all
tumours studied and was not an effect of "tetraploid" status.
TABLE-US-00007 TABLE 7 Results from interphase FISH analysis of the
12 cervical adenocarcinomas. Nuclei "Non- with diploid" Relative
Case Nuclei "Diploid" "Non- "Tetraploid" >2 with >2 Signals
per cell: mean (range) TERC vs. Most common "non" no counted
[2-2-2] diploid"* [4-4-4] TERC TERC CEP7 CEP3 TERC CEP7 CEP3
diploid" patterns 1 376 33% 67% 1% 66% 99% 2.2 (2-5) 2.2 (2-5) 3.0
(2-8) 1.4 1.4 [2-2-3] [2-2-4] 2 391 31% 69% 0% 64% 92% 2.1 (2-4)
2.1 (2-4) 3.2 (2-7) 1.5 1.6 [2-2-4] [2-2-3] [2-2-5] 3 215 27% 73%
12% 68% 94% 3.0 (2-6) 3.1 (2-8) 3.3 (2-9) 1.1 1.1 [4-4-4] [4-3-4]
[4-3-3] [3-3-3] [3-4-4] 4 466 40% 60% 0% 59% 99% 2.0 (1-4) 3.2
(2-5) 4.4 (2-12) 2.1 1.4 [2-4-6] [2-3-5] [2-4-5] [2-4-7] 5 336 68%
32% 1% 27% 86% 2.1 (2-4) 2.1 (2-4) 2.4 (2-6) 1.1 1.1 [2-2-3]
[2-2-4] [2-3-2] 6 641 69% 31% 1% 28% 92% 2.0 (0-4) 2.3 (2-8) 2.6
(2-8) 1.3 1.1 [2-3-4] [2-3-5] [2-2-3] [2-3-3] 7 412 28% 72% 0% 71%
99% 2.1 (2-4) 2.1 (2-8) 4.2 (2-15) 2.0 2.0 [2-2-5] [2-2-4] [2-2-6]
[2-2-3] 8 626 46% 54% 1% 54% 98% 2.0 (2-4) 2.2 (2-5) 2.9 (2-12) 1.4
1.3 [2-2-4] [2-2-3] [2-3-3] [2-2-5] 9 253 29% 71% 0% 64% 91% 2.6
(2-5) 3.0 (2-7) 3.4 (2-11) 1.3 1.1 [2-2-3] [2-3-3] [2-2-4] [3-2-2]
[3-4-4] 10 286 30% 70% 0% 66% 95% 2.7 (2-7) 2.9 (2-7) 5.2 (2-22)
2.0 1.8 [2-2-4] [2-2-3] [2-2-5] [2-2-6] 11 393 79% 21% 4% 18% 83%
2.2 (2-6) 2.2 (2-6) 2.3 (2-6) 1.04 1.04 [2-2-3] [4-4-4] [2-3-2] 12
208 20% 80% 0% 79% 99% 1.9 (0-4) 2.0 (0-5) 3.0 (2-7) 1.6 1.5
[2-2-3] [2-2-4] *Background level of deviation from (2-2-2) in
normal nuclei is less than 2% (Heselmayer-Hadad et al., 2003)
[0154] Intra-Tumour Heterogeneity for Gain and Amplification of the
TERC Locus
[0155] It was observed that different FISH patterns in the
individual cases. For several of the tumors one or a few
predominating patterns were seen. In addition all cases exhibited
varying proportions of different but recurrent "non-diploid"
patterns. Comparison of signal numbers recorded for TERC versus
CEP7 and CEP3 showed increased relative TERC copies in the 12 cases
studied (range 1.04-2.0; Table 7). The absolute mean number of TERC
signals per nuclei ranged between 2.3 (case 11) and 5.2 (case 10),
and in five of the tumours nuclei with more than 10 signals were
repeatedly encountered (Table 7). FIG. 4 illustrates nuclei of case
10 with amplification of more than 20 TERC signals, together with 6
signals for CEP3 and CEP7 (FIGS. 1G and H). On the level of
individual nuclei the distribution of FISH signals for TERC was of
similar type, in that the signals were randomly distributed in the
nuclei without obvious clustering. These findings demonstrate that
the gain of TERC is not only and always a result of increased
chromosome 3 copy numbers, but instead a subchromosomal
gain/amplification of the TERC gene locus.
Gain of TERC is Independent of HPV Infection Status
[0156] The clinical and follow-up information as well as HPV status
of the 12 cervical adenocarcinomas are provided in Table 6. Eight
cases were HPV positive and four were HPV negative. Very high
proportions of "non-diploid" nuclei with >2 TERC signals were
demonstrated in HPV positive (83-99%) as well as HPV negative
(86-99%) tumours. The absolute numbers of TERC signals per nucleus
were similarly increased in HPV positive (mean 3.4; range 2-22) and
HPV negative (mean 3.3; range 2-12) tumours. No apparent
associations between TERC copy numbers and clinical
characteristics, such as age at diagnosis, stage, differentiation,
lymph node involvement or outcome at follow-up, were noted. Thus,
gain of TERC is characteristic of cervical adenocarcinomas per se,
and independent of HPV infection status.
[0157] A series of 12 primary cervical adenocarcinomas were
analyzed for gain or amplification of the human telomerase gene
TERC, which maps to chromosome band 3q26. Eight of the 12
carcinomas (67%) were HPV-positive (five tumors were positive for
HPV 18, two for HPV 16, and one for HPV 45) (Andersson et al,
2003a; Skyldberg et al, 1999). The remaining four tumour specimens
were HPV-negative. FISH was then used with a custom designed probe
panel that includes the human telomerase gene (TERC) to assess the
potential of this genetic marker to ameliorate the morphological
diagnosis of cervical adenocarcinomas.
[0158] Detection of a trisomy by CGH requires this numerical
aberration to be present in at least 40% of the cells. FISH, of
course, detects changes on a single cell basis and is therefore not
sensitive to dilution. Additionally, small regional low copy number
amplicons might escape detection by CGH.
[0159] HPV-DNA has not been identified in the same proportion in
adenocarcinomas as in squamous cervical carcinomas (Andersson et
al, 2003b; Skyldberg et al, 1999; Tenti et al, 1996). Perhaps
oncogenic factors other than HPV are more likely to play a role in
the malignant transformation of cervical adenocarcinomas (Skyldberg
et al, 1999; Pirog et al, 2000).
[0160] This data shows that 10 of the 12 women had a normal Pap
smear within 3 years prior to the diagnosis of invasive disease,
and without wishing to be bound by any particular scientific
theory, may support the hypothesis of a rapid-onset carcinoma. On
the other hand, since the sensitivity of a Pap smear for the
detection of cervical adenocarcinomas is very low (Krane et al,
2001), the rapid-onset hypothesis may not apply to these cases.
[0161] Cervical squamous carcinomas are defined by a non-random and
recurrent distribution of genomic imbalances. In addition to HPV,
the sequential transformation of cervical squamous epithelium
requires the acquisition of additional copies of chromosome arm 3q.
Using a genomic probe for the TERC gene on chromosome band 3q26 in
combination with two centromere specific probes (CEP3 and CEP7), a
high copy number of this locus was shown in cervical
adenocarcinomas. Gain or amplification of 3q was found in all
cervical adenocarcinomas investigated. Application of this probe
set provides an objective genetic test for the diagnosis of
cervical adenocarcinomas and might assist in the interpretation of
smears with a high degree of glandular cells.
[0162] An objective molecular marker in this patient group is
needed in the art because of the cytomorphologically difficult
identification of these cells and the limited numbers of
representative cells due to technical problems in sampling these
lesions. As shown herein, many women with cervical adenocarcinoma
presented with morphologically normal Pap smears, often only 1-2
years prior to diagnosis.
[0163] The following specific references, also incorporated by
reference, are indicated above by corresponding reference
number.
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