U.S. patent application number 13/729225 was filed with the patent office on 2013-07-04 for automated analysis of circulating tumor cells.
This patent application is currently assigned to Ventana Medical Systems, Inc.. The applicant listed for this patent is Ryan Dittamore, Karl Garsha, Alexandra Dea Nagy, Michael Otter, Gary Pestano, Chol Steven Yun. Invention is credited to Ryan Dittamore, Karl Garsha, Alexandra Dea Nagy, Michael Otter, Gary Pestano, Chol Steven Yun.
Application Number | 20130171642 13/729225 |
Document ID | / |
Family ID | 47561845 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130171642 |
Kind Code |
A1 |
Pestano; Gary ; et
al. |
July 4, 2013 |
AUTOMATED ANALYSIS OF CIRCULATING TUMOR CELLS
Abstract
The disclosure provides methods for automated characterization
of circulating tumor cells (CTCs), for example using automated
tissue strainers. In specific examples, such methods permit
characterizing a prostate cancer sample by simultaneously or
contemporaneously detecting ERG rearrangements and PTEN deletions
in the same CTC. Also provided are kits that can be used with such
methods.
Inventors: |
Pestano; Gary; (Lafayette,
CO) ; Dittamore; Ryan; (San Diego, CA) ;
Garsha; Karl; (Sahuarita, AZ) ; Otter; Michael;
(Tucson, AZ) ; Yun; Chol Steven; (Tucson, AZ)
; Nagy; Alexandra Dea; (Oro Valley, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pestano; Gary
Dittamore; Ryan
Garsha; Karl
Otter; Michael
Yun; Chol Steven
Nagy; Alexandra Dea |
Lafayette
San Diego
Sahuarita
Tucson
Tucson
Oro Valley |
CO
CA
AZ
AZ
AZ
AZ |
US
US
US
US
US
US |
|
|
Assignee: |
Ventana Medical Systems,
Inc.
|
Family ID: |
47561845 |
Appl. No.: |
13/729225 |
Filed: |
December 28, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61581825 |
Dec 30, 2011 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/7.1; 530/389.7; 536/24.31 |
Current CPC
Class: |
G01N 33/57434 20130101;
G01N 2800/56 20130101; C12Q 1/6886 20130101; C12Q 2600/156
20130101; G01N 2800/52 20130101; G01N 2800/54 20130101 |
Class at
Publication: |
435/6.11 ;
435/7.1; 536/24.31; 530/389.7 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of analyzing a sample containing circulating tumor
cells (CTCs) using an automated instrument, comprising: obtaining
the sample from a subject, depositing the sample on a substrate
configured for use with the automated instrument, placing the
substrate within the automated instrument, retrieving targets on
the sample using the automated instrument, contacting the sample
with CTC identification reagents using the automated instrument,
contacting the sample with CTC characterization reagents using the
automated instrument, imaging the sample, locating the CTCs by
locating the CTC identification reagents, spectral imaging the CTCs
by location, and analyzing the sample by analyzing the spectral
imaging.
2. The method of claim 1, further comprising enriching the CTC
content of the sample using a capture antibody specific for an ETS
related gene (ERG) protein, a prostate specific membrane antigen
(PSMA) protein, or an epithelial cell adhesion molecule (EpCAM),
protein, wherein enriching the CTC content of the sample occurs
prior to depositing the sample on the substrate.
3. The method of claim 1, wherein the CTC characterization reagents
comprise nucleic acid probes directed to four genomic markers.
4. The method of claim 3, wherein the four genomic markers are
analyzed for gene expression and/or genetic
rearrangements/deletions.
5. The method of claim 3, wherein the four genomic markers comprise
a gene expression probe and a rearrangement/deletion probe
combination.
6. The method of claim 1, wherein the subject has prostate
cancer.
7. The method of claim 6, wherein the prostate cancer is a
castrate-resistant prostate cancer (CRPC).
8. The method of claim 7, wherein the CRPC is a metastatic CRPC
(mCRPC).
9. The method of claim 3, wherein the four genomic markers comprise
ETS related gene (ERG), phosphatase and tensin homolog (PTEN), and
centromere 10 (CEN-10).
10. The method of claim 3, wherein the nucleic acid probes are a 5'
ERG probe, a 3' ERG probe, a PTEN probe, and a CEN-10 probe.
11. The method of claim 1, wherein the CTC identification reagents
comprise immunohistochemical reagents directed to CTC protein
markers.
12. The method of claim 11, wherein the CTC protein markers
comprise a CD45 protein, a cytokeratin (CK) protein, an ERG
protein, an androgen receptor (AR), a PSMA protein, or combinations
thereof.
13. The method of claim 1, wherein the CTC characterization
reagents comprise nucleic acid probes directed to four genomic
markers and the CTC identification reagents comprise
immunohistochemical reagents directed to CTC protein markers.
14. The method of claim 13, wherein the four genomic markers
comprise ETS related gene (ERG), phosphatase and tensin homolog
(PTEN), and centromere 10 (CEN-10) and the CTC protein markers
comprise a CD45 protein, a CK protein, an ERG protein, an AR, a
PSMA protein, or combinations thereof.
15. The method of claim 1, wherein imaging the sample comprises
imaging immunofluorescence of the CTC identification reagents.
16. The method of claim 15, wherein imaging the sample comprises
using multi-spectral bandpass filters.
17. The method of claim 15, wherein the immunofluorescence emanates
from antibodies directly labeled with fluorophores.
18. The method of claim 17, wherein the immunofluorescence results
from exciting the fluorophores with spectrally filtered visible
light.
19. The method of claim 18, wherein the spectrally filtered visible
light includes a first selected range to excite a first fluorophore
and a second selected range to excite a second fluorophore, wherein
the first selected range does not significantly excite the second
fluorophore and the second selected range does not significantly
excite the first fluorophore.
20. The method of claim 18, wherein imaging the sample comprises
acquiring a first immunofluorescence image of the sample excited by
the first selected range and acquiring a second immunofluorescence
image of the sample excited by the second selected range and
locating the CTCs by locating the CTC identification reagents
comprises comparing or overlaying the first immunofluorescence
image and the second immunofluorescence image.
21. The method of claim 20, wherein imaging the first
immunofluorescence image identifies CK+ cells, the second
immunofluorescence image identifies CD45+ cells, wherein comparing
or overlaying comprises identifying cells that are CK+ and
CD45-.
22. The method of claim 20, wherein locating the CTCs by locating
the CTC identification reagents comprises algorithmically analyzing
the first immunofluorescence image and the second
immunofluorescence image using a computer.
23. The method of claim 22, wherein algorithmically analyzing
comprises digitally interrogating the images to measure cell size,
cell compartment localization of markers, and/or intensity of
marker expression.
24. The method of claim 1, wherein spectral imaging the CTCs
comprises spectral imaging luminescence of the CTC characterization
reagents, the luminescence emanating from specific binding moieties
labeled with quantum dots.
25. The method of claim 24, wherein the specific binding moieties
are directly labeled with the quantum dots.
26. The method of claim 24, wherein the specific binding moieties
are indirectly labeled with the quantum dots, the luminescence
emanating from the quantum dots labeling anti-hapten secondary
antibodies, the anti-hapten secondary antibodies being specific to
haptens labeling the specific binding moieties.
27. The method of claim 24, wherein the specific binding moieties
are nucleic acid probes.
28. The method of claim 24, wherein spectral imaging the CTCs
comprises exciting the quantum dots with radiation.
29. The method of claim 28, wherein the radiation is UV or near-UV
radiation.
30. The method of claim 28, wherein the radiation has a spectral
emission profile with a maximum between 300 and 400 nm.
31. The method of claim 24, wherein spectral imaging the CTCs
comprises multispectral imaging.
32. The method of claim 24, wherein spectral imaging the CTCs
comprises hyperspectral imaging.
33. The method of claim 1, wherein spectral imaging is guided by
the step of locating the CTCs to regions of interest to the
exclusion of regions devoid of interest.
34. The method of claim 33, wherein regions of interest comprise
CTCs.
35. The method of claim 18, wherein the spectrally filtered visible
light does not result in significant quantum dot luminescence.
36. The method of claim 1, wherein imaging the sample comprises
multi-spectral imaging immunofluorescence of the CTC identification
reagents and spectral imaging the CTCs comprises hyper-spectral
imaging the CTC characterization reagents.
37. The method of claim 36, wherein the multi-spectral and
hyper-spectral imaging differentiates at least about four CTC
identification reagents and/or CTC characterization reagents, at
least about five CTC identification reagents and/or CTC
characterization reagents, or at least about six CTC identification
reagents and/or CTC characterization reagents.
38. The method of claim 36, wherein the multi-spectral imaging
differentiates at least about two CTC identification reagents and
the hyper-spectral imaging differentiates at least about four CTC
characterization reagents.
39. The method of claim 1, wherein retrieving targets comprises
contacting the sample with a tris-based buffer having a slightly
basic pH and applying heat so that covalent bonds resulting from
fixation are broken.
40. The method of claim 1, further comprising contacting the sample
with a protease.
41. The method of claim 1, further comprising contacting the sample
with a citrate buffer having a slightly acidic pH and applying heat
so that covalent bonds resulting from fixation are broken.
42. The method of claim 1, wherein retrieving targets comprises
contacting the sample with a tris-based buffer having a slightly
basic pH and applying heat so that covalent bonds resulting from
fixation are broken and contacting the sample with a protease prior
to contacting the sample with CTC identification reagents, and the
method further comprises contacting the sample with a citrate
buffer having a slightly acidic pH and applying heat so that
covalent bonds resulting from fixation are broken and contacting
the sample with a protease prior to contacting the sample with CTC
characterization reagents.
43. The method of claim 1, wherein contacting the sample with the
CTC characterization reagent comprises a reagent directed towards
one or more housekeeping molecules in the CTCs, wherein analyzing
the sample includes comparing expression of the one or more
housekeeping molecules in the CTCs to a control representing the
one or more housekeeping molecules expected in a normal prostate
sample.
44. The method of claim 1, wherein analyzing the sample comprises
detecting one or more other prostate cancer-related molecules in
the CTCs and comparing expression of the one or more other prostate
cancer related molecules in the CTCs to a control the one or more
other prostate cancer-related molecules expected in a normal
prostate sample.
45. The method of claim 6, further comprising: predicting the
likelihood that the prostate cancer will respond to a poly-(ADP)
ribose polymerase (PARP) inhibitor, radiotherapy, or hormone
blocker; predicting the likelihood of disease recurrence,
predicting the likelihood of prostate cancer progression;
predicting the likelihood of prostate cancer metastasis; predicting
likelihood survival time; or combinations thereof.
46. The method of claim 6, further comprising: predicting that the
prostate cancer will respond to PARP inhibitor when the CTCs have
an ERG rearrangement and/or a PTEN deletion; predicting that the
prostate cancer will not respond to radiotherapy when the CTCs have
an ERG rearrangement and/or a PTEN deletion; predicting that the
prostate cancer will respond to a hormone blocker when the CTCs
have an ERG rearrangement and/or a PTEN deletion; predicting that
the prostate cancer has a higher likelihood of recurring when the
CTCs have an ERG rearrangement and/or a PTEN deletion; predicting
that the prostate cancer has a higher likelihood of progressing
when the CTCs have an ERG rearrangement and/or a PTEN deletion;
predicting that the prostate cancer is more likely to metastasize
when the CTCs have an ERG rearrangement and/or a PTEN deletion;
predicting a survival time of less than 5 years when the CTCs have
an ERG rearrangement and/or a PTEN deletion; or combinations
thereof.
47. A kit for characterizing prostate cancer, comprising: one or
more nucleic acid probes that can specifically detect an ERG
genomic rearrangement; one or more nucleic acid probes that can
specifically detect a PTEN genomic deletion; and one or more
nucleic acid probes that can specifically detect CEN-10; wherein
the nucleic acid probes comprise a quantum dot.
48. The kit of claim 47, further comprising: one or more antibodies
specific for an EpCAM protein; one or more antibodies specific for
a CD45 protein; one or more antibodies specific for a CK protein;
one or more antibodies specific for an ERG protein; one or more
antibodies specific for AR; one or more antibodies specific for a
PSMA protein; one or more microscope slides; or combinations
thereof.
49. The kit of claim 47, further comprising: one or more nucleic
acid probes or antibodies specific for one or more housekeeping
genes or proteins; one or more nucleic acid probes or antibodies
specific for one or more prostate cancer-related genes or proteins;
or combinations thereof.
50. A method of characterizing a prostate cancer, comprising:
isolating circulating tumor cells (CTCs) from a subject having
prostate cancer using anti-ETS related gene (ERG) antibodies;
spreading the isolated CTCs onto a glass slide to form a
homogeneous layer; contacting the CTCs on the glass slide with one
or more nucleic acid probes specific for ERG, PTEN, and CEN-10,
wherein each probe comprises one or more quantum dots; detecting
signals from the one or more quantum dots on the one or more
nucleic acid probes; determining whether one or more ERGs is
rearranged, whether one or more PTEN genes is deleted, and whether
CEN-10 is detected; and characterizing the prostate cancer based on
whether one or more ERGs is rearranged, whether one or more PTEN
genes is deleted, and whether CEN-10 is detected.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 61/581,825 filed Dec. 30, 2011, herein incorporated
by reference.
FIELD
[0002] The disclosure provides methods for automated
characterization of circulating tumor cells (CTCs), for example
using automated tissue stainers. Also provided are kits that can be
used with such methods.
BACKGROUND
[0003] Circulating tumor cells (CTCs) are primary tumor cells that
have shed into the vascular system and are potentially present
across the body, particularly in the bloodstream. CTCs may serve as
seeds for metastasis in locations divergent from the primary tumor,
posing a substantial health risk to the cancer patient. Research
indicates that CTCs derive from clones in the primary tumor and
that they serve an important role in the metastatic spread of
carcinomas. It has been demonstrated that CTCs reflect molecular
features of cells within the primary tumor, thus enabling
characterization of the tumor without a biopsy of the primary
tumor. CTCs also represent metastasis in action, and therefore,
monitoring and analyzing CTCs is indicative of the patient's
disease status. CTCs are not easily analyzed because they are
present in very small numbers in blood. For example, CTCs may be
found in frequencies of about 1-10 CTC per mL of whole blood in
patients with a metastatic disease. This small number contrasts
with the other cellular components within one mL of blood, (e.g. a
few million white blood cells, a billion red blood cells). Thus,
using CTCs for diagnostic purposes relies on the ability to isolate
or identify the CTCs within a vast matrix of other cells.
Furthermore, the rarity of the cells in a sample enhances the value
of the sample as analytical targets. CTCs are presently known to be
present in several epithelial cancers (e.g., breast, prostate,
lung, and colon) and clinical evidences indicate that patients with
metastatic lesions are more likely to have isolatable CTCs.
SUMMARY
[0004] Methods are provided for analyzing a sample known or
suspected of containing circulating tumor cells (CTCs) using an
automated instrument, and kits configured for the same.
Furthermore, provided herein are methods of characterizing cancer,
such as prostate cancer.
[0005] In some embodiments, the method includes one or more of the
following: obtaining the sample from a subject (such as a blood or
bone marrow sample), depositing or applying the sample on a
substrate configured for use with the automated instrument, placing
the substrate within the automated instrument, retrieving targets
on the sample using the automated instrument, contacting the sample
with CTC identification reagents using the automated instrument,
contacting the sample with CTC characterization reagents using the
automated instrument, imaging the sample, locating the CTC by
locating the CTC identification reagents, spectral imaging of the
CTC by location, and analyzing the sample by analyzing the spectral
imaging. In one example, the method includes enriching the CTC
content of the sample using a capture antibody specific for an ETS
related gene (ERG) protein, a prostate specific membrane antigen
(PSMA) protein, or an epithelial cell adhesion molecule (EpCAM)
protein, wherein enriching the CTC content of the sample occurs
prior to depositing or placing the sample on the substrate.
[0006] Furthermore, it is disclosed herein that ETS related gene
(ERG) rearrangements and phosphatase and tensin homolog (PTEN) gene
deletions, along with centromere 10 (CEN-10) can simultaneously or
contemporaneously be detected in CTCs, for example by using labeled
nucleic acid probes (such as those labeled with quantum dots). The
methods can be automated. Based on whether ERG rearrangements
and/or PTEN deletions are detected, a prostate cancer can be
characterized.
[0007] In some examples, the methods can include isolating CTCs
from a subject having prostate cancer, such as a castrate-resistant
prostate cancer (CRPC). Methods of isolating CTCs can include the
use of antibodies specific for EpCAM, ERG, PSMA, or combinations
thereof. The isolated CTCs are applied to a glass slide or other
substrate and fixed (for example using methods known in the art).
Novel spreading methods using prostate-specific antibodies as
discussed herein may also be used to isolate CTCs and apply them to
a substrate, such as a glass slide, before fixation. The mounted
and fixed CTCs are then contacted with one or more nucleic acid
probes specific for ERG, PTEN, and CEN-10, for example under
conditions sufficient for the nucleic acid probes to hybridize to
their complementary sequence in the CTCs. The nucleic acid probes
are labeled, for example with one or more quantum dots. For
example, the nucleic acid probe(s) specific for ERG, PTEN, and
CEN-10 can each labeled with a different quantum dot, to permit one
to distinguish the probes from one another. After allowing the
nucleic acid probes to hybridize to ERG, PTEN, and CEN-10, signals
from the one or more quantum dots on the one or more nucleic acid
probes are detected, for example by using spectral imaging. The
signals are then analyzed, to determine whether in the isolated
CTCs, one or more ERGs are rearranged, whether one or more PTEN
genes are deleted, and whether CEN-10 is detected. Based on whether
one or more ERGs is rearranged, whether one or more PTEN genes is
deleted, and whether CEN-10 is detected, the prostate cancer is
characterized.
[0008] In some examples, the method can also include contacting the
CTCs with one or more probes that permit detection of one or more
other prostate cancer-related molecules in the CTCs and/or one or
more housekeeping molecules in the CTCs.
[0009] Characterizing a prostate cancer can include predicting the
likelihood that the prostate cancer will respond to a particular
therapy, such as a poly-(ADP) ribose polymerase (PARP) inhibitor
(such as olaparib or MK4827), abiraterone or other hormone pathway
inhibitor, or radiotherapy, predicting the likelihood of disease
recurrence after treatment (such as a prostatectomy), predicting
the likelihood of prostate cancer progression, predicting the
likelihood of prostate cancer metastasis, predicting likelihood
survival time, or combinations thereof. For example, characterizing
a prostate cancer can include predicting that the prostate cancer
will respond to a PARP inhibitor or abiraterone, but not
radiotherapy, when the CTCs have an ERG rearrangement and/or a PTEN
deletion, predicting that the prostate cancer has a higher
likelihood of recurring (for example after prostatectomy) when the
CTCs have an ERG rearrangement and/or a PTEN deletion, predicting
that the prostate cancer has a higher likelihood of progressing
when the CTCs have an ERG rearrangement and/or a PTEN deletion,
predicting that the prostate cancer is more likely to metastasize
when the CTCs have an ERG rearrangement and/or a PTEN deletion,
predicting a survival time of less than 5 years when the CTCs have
an ERG rearrangement and/or a PTEN deletion, or combinations
thereof.
[0010] In addition, kits are provided that include one or more
nucleic acid probes that can specifically detect an ERG genomic
rearrangement, one or more nucleic acid probes that can
specifically detect a PTEN genomic deletion, and one or more
nucleic acid probes that can specifically detect CEN-10, wherein
the nucleic acid probes comprise a quantum dot. Such kits can
further include one or more antibodies specific for an EpCAM
protein, one or more antibodies specific for a CD45 protein, one or
more antibodies specific for a cytokeratin (CK) protein, one or
more antibodies specific for an ERG protein, one or more antibodies
specific for a PTEN protein, one or more antibodies specific for
androgen receptor (AR), one or more antibodies specific for a
prostate-specific membrane antigen (PSMA) protein, one or more
microscope slides, one or more nucleic acid probes specific for one
or more housekeeping genes, one or more nucleic acid probes
specific for one or more prostate cancer-related genes, or
combinations thereof.
[0011] The foregoing and other objects and features of the
disclosure will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0013] FIG. 1 is a digital image at 40.times. magnification of
LNCaP cells applied onto a glass slide using a cytocentrifuge,
fixed, and stained with antibodies specific for pan-cytokeratin
(labeled with quantum dot that emits at 605 nm), and CD45 (labeled
with quantum dot that emits at 705 nm). The cells were also stained
with DAPI to show the nuclei. LNCaP cells were positive for CK, but
negative for CD45.
[0014] FIGS. 2 and 3 are digital images of CTCs applied onto a
glass slide using a cyto centrifuge, fixed, and labeled with probes
for 5'-end ERG, 3'-end ERG, PTEN, and CEN 10. The cells were also
stained with DAPI to show the nuclei.
[0015] FIG. 4 is a schematic showing a method starting with a
sample showing illustrative steps to reach an evaluation of
CTCs.
[0016] FIG. 5 is a schematic showing a method for the automated
analysis of CTCs.
SEQUENCE LISTING
[0017] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
All sequence database accession numbers referenced herein are
understood to refer to the version of the sequence identified by
that accession number as it was available on the filing date of
this application. In the accompanying sequence listing:
[0018] SEQ ID NOS: 1 and 2 are a human Ets related gene (ERG)
nucleic acid coding sequence and corresponding protein sequence,
respectively.
[0019] SEQ ID NOS: 3 and 4 are a human phosphatase and tensin
homolog (PTEN) nucleic acid coding sequence and corresponding
protein sequence, respectively.
[0020] SEQ ID NO: 5 is an exemplary nucleic acid probe specific for
centromere 10. This clone is from American Type Culture Collection
(ATCC Cat#61397; pA10RP8). The size of the insert is 1.36 kb. The
sequence shown in nucleotides 160 to 498 are repeated in the
clone.
DETAILED DESCRIPTION
I. Terms
[0021] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which a disclosed invention
belongs. Definitions of common terms in molecular biology may be
found in Benjamin Lewin, Genes V, published by Oxford University
Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). The singular terms "a," "an," and "the" include
plural referents unless context clearly indicates otherwise.
Similarly, the word "or" is intended to include "and" unless the
context clearly indicates otherwise. "Comprising" means
"including"; hence, "comprising A or B" means "including A" or
"including B" or "including A and B." Suitable methods and
materials for the practice and/or testing of embodiments of the
disclosed methods are described below. Such methods and materials
are illustrative only and are not intended to be limiting. Other
methods and materials similar or equivalent to those described
herein also can be used. For example, conventional methods well
known in the art to which a disclosed invention pertains are
described in various general and more specific references,
including, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press,
1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d
ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates, 1992
(and Supplements to 2000); Ausubel et al., Short Protocols in
Molecular Biology: A Compendium of Methods from Current Protocols
in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.
[0022] All sequences associated with the GenBank.RTM. accession
numbers referenced herein are incorporated by reference (e.g., the
sequence present on Dec. 30, 2011 is incorporated by reference).
All references cited herein are incorporated by reference.
[0023] In order to facilitate review of the various disclosed
embodiments, the following explanations of specific terms are
provided:
[0024] Androgen Receptor (AR): (OMIM 313700): A type of nuclear
receptor that is activated by binding of either of the androgenic
hormones testosterone or dihydrotestosterone in the cytoplasm and
then translocating into the nucleus. The main function of the
androgen receptor is as a DNA-binding transcription factor that
regulates gene expression. The AR gene is located on chromosome X.
AR sequences are publically available, for example from
GenBank.RTM. (e.g., accession numbers NP.sub.--000035, AAA51772.1,
and P10275.2 (proteins) and NM.sub.--000044.3, and
NM.sub.--001011645.2 (nucleic acids)).
[0025] Antibodies specific for AR proteins are publicly available,
for example from abcam (catalog numbers ab47569 and ab9474), Cell
Signaling Technology (catalog numbers 7395, and 5153), and Santa
Cruz Biotechnology, Inc. (catalog numbers sc-7305, sc-52309, and
sc-52984.
[0026] Antibody: A polypeptide ligand including at least a light
chain or heavy chain immunoglobulin variable region which
specifically recognizes and binds an epitope of an antigen, such as
an endothelial marker or a fragment thereof. Antibodies are
composed of a heavy and a light chain, each of which has a variable
region, termed the variable heavy (V.sub.H) region and the variable
light (V.sub.L) region. Together, the V.sub.H region and the
V.sub.L region are responsible for binding the antigen recognized
by the antibody. In one example, an antibody specifically binds to
an EpCAM, CK, or CD45 protein, but not to other proteins.
[0027] This includes intact immunoglobulins and the variants and
portions of them well known in the art, such as Fab' fragments,
F(ab)'.sub.2 fragments, single chain Fv proteins ("scFv"), and
disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a
fusion protein in which a light chain variable region of an
immunoglobulin and a heavy chain variable region of an
immunoglobulin are bound by a linker, while in dsFvs, the chains
have been mutated to introduce a disulfide bond to stabilize the
association of the chains. The term also includes genetically
engineered forms such as chimeric antibodies (for example,
humanized murine antibodies), heteroconjugate antibodies (such as,
bispecific antibodies). See also, Pierce Catalog and Handbook,
1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,
Immunology, 3.sup.rd Ed., W. H. Freeman & Co., New York,
1997.
[0028] Typically, a naturally occurring immunoglobulin has heavy
(H) chains and light (L) chains interconnected by disulfide bonds.
There are two types of light chain, lambda (.lamda.) and kappa (k).
There are five main heavy chain classes (or isotypes) which
determine the functional activity of an antibody molecule: IgM,
IgD, IgG, IgA and IgE.
[0029] Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs". The extent of the framework region and CDRs has
been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services, 1991, which is hereby incorporated by reference). The
Kabat database is now maintained online. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in
three-dimensional space.
[0030] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found. For
example, an antibody that binds CK will have a specific V.sub.H
region and the V.sub.L region sequence, and thus specific CDR
sequences. Antibodies with different specificities (i.e. different
combining sites for different antigens) have different CDRs.
Although it is the CDRs that vary from antibody to antibody, only a
limited number of amino acid positions within the CDRs are directly
involved in antigen binding. These positions within the CDRs are
called specificity determining residues (SDRs).
[0031] References to "V.sub.H" or "VH" refer to the variable region
of an immunoglobulin heavy chain, including that of an Fv, scFv,
dsFv or Fab. References to "V.sub.L" or "VL" refer to the variable
region of an immunoglobulin light chain, including that of an Fv,
scFv, dsFv or Fab.
[0032] A "monoclonal antibody" is an antibody produced by a single
clone of B-lymphocytes or by a cell into which the light and heavy
chain genes of a single antibody have been transfected. Monoclonal
antibodies are produced by methods known to those of skill in the
art, for instance by making hybrid antibody-forming cells from a
fusion of myeloma cells with immune spleen cells. Monoclonal
antibodies include humanized monoclonal antibodies.
[0033] A "polyclonal antibody" is an antibody that is derived from
different B-cell lines. Polyclonal antibodies are a mixture of
immunoglobulin molecules secreted against a specific antigen, each
recognizing a different epitope. These antibodies are produced by
methods known to those of skill in the art, for instance, by
injection of an antigen into a suitable mammal (such as a mouse,
rabbit or goat) that induces the B-lymphocytes to produce IgG
immunoglobulins specific for the antigen which are then purified
from the mammal's serum.
[0034] A "chimeric antibody" has framework residues from one
species, such as human, and CDRs (which generally confer antigen
binding) from another species, such as a murine antibody that
specifically binds an endothelial marker.
[0035] A "humanized" immunoglobulin is an immunoglobulin including
a human framework region and one or more CDRs from a non-human (for
example a mouse, rat, or synthetic) immunoglobulin. The non-human
immunoglobulin providing the CDRs is termed a "donor," and the
human immunoglobulin providing the framework is termed an
"acceptor." In one embodiment, all the CDRs are from the donor
immunoglobulin in a humanized immunoglobulin. Constant regions need
not be present, but if they are, they are substantially identical
to human immunoglobulin constant regions, e.g., at least about
85-90%, such as about 95% or more identical. Hence, all parts of a
humanized immunoglobulin, except possibly the CDRs, are
substantially identical to corresponding parts of natural human
immunoglobulin sequences. Humanized immunoglobulins can be
constructed by means of genetic engineering (see for example, U.S.
Pat. No. 5,585,089).
[0036] Binding affinity: Affinity of one molecule for another, such
as an antibody for an antigen (for example, an EpCAM, CK, ERG,
PTEN, PSMA, AR, or CD45 protein). In one example, affinity is
calculated by a modification of the Scatchard method described by
Frankel et al., Mol. Immunol., 16:101-106, 1979. In another
example, binding affinity is measured by an antigen/antibody
dissociation rate. In yet another example, a high binding affinity
is measured by a competition radioimmunoassay. In several examples,
a high binding affinity is at least about 1.times.10.sup.-8 M. In
other examples, a high binding affinity is at least about
1.5.times.10.sup.-8, at least about 2.0.times.10.sup.-8, at least
about 2.5.times.10.sup.-8, at least about 3.0.times.10.sup.-8, at
least about 3.5.times.10.sup.-8, at least about
4.0.times.10.sup.-8, at least about 4.5.times.10.sup.-8, or at
least about 5.0.times.10.sup.-8 M.
[0037] Cancer: Malignant neoplasm, for example one that has
undergone characteristic anaplasia with loss of differentiation,
increased rate of growth, invasion of surrounding tissue, and is
capable of metastasis.
[0038] CD45: (OMIM 151460): A member of the protein tyrosine
phosphatase (PTP) family, that is specifically expressed in
hematopoietic cells. The human CD45 gene (also known as protein
tyrosine phosphatase, receptor type, C (PTPRC)) is located on
chromosome 1 (1q31-q32). CD45 sequences are publically available,
for example from GenBank.RTM. (e.g., accession numbers
NP.sub.--002829.2, and NP.sub.--035340 (proteins) and
NM.sub.--002838.3, and NM.sub.--011210 (nucleic acids)).
[0039] Antibodies specific for CD45 proteins are publicly
available, for example from abcam (catalog number ab10558),
Millipore (catalog numbers FCMAB126F, 05-1410, and FCMAB118P), and
Santa Cruz Biotechnology, Inc. (catalog numbers sc-25590, sc-70686,
sc-66201, and sc-20056).
[0040] Centromere 10 (CEN-10): The region of chromosome 10 (e.g.,
10p11.1-q11.1) where the centromere is located. Its presence can be
detected using centromere 10-specific nucleic acid probes.
[0041] Circulating tumor cells (CTCs): Tumor cells produced during
tumorigenesis found in peripheral blood or bone marrow (when in
bone marrow referred to as disseminated tumor cells, DTCs), which
can result in metastatic disease. CTC are found in frequencies of
about 1 to 10 CTC per mL of whole blood in patients with metastatic
disease. CTC cells are EpCAM positive, CD45 negative, and CK
positive (such as positive when using a pan-keratin antibody).
[0042] Complementary: A nucleic acid molecule is said to be
"complementary" with another nucleic acid molecule if the two
molecules share a sufficient number of complementary nucleotides to
form a stable duplex or triplex when the strands bind (hybridize)
to each other, for example by forming Watson-Crick, Hoogsteen or
reverse Hoogsteen base pairs. Stable binding occurs when a nucleic
acid molecule (e.g., nucleic acid probe or primer) remains
detectably bound to a target nucleic acid sequence (e.g., ERG,
PTEN, or CEN-10 target nucleic acid sequence) under the required
conditions.
[0043] Complementarity is the degree to which bases in one nucleic
acid molecule (e.g., nucleic acid probe or primer) base pair with
the bases in a second nucleic acid molecule (e.g., target nucleic
acid sequence). Complementarity is conveniently described by
percentage, that is, the proportion of nucleotides that form base
pairs between two molecules or within a specific region or domain
of two molecules. For example, if 10 nucleotides of a 15 contiguous
nucleotide region of a nucleic acid probe or primer form base pairs
with a target nucleic acid molecule, that region of the probe or
primer is said to have 66.67% complementarity to the target nucleic
acid molecule.
[0044] In the present disclosure, "sufficient complementarity"
means that a sufficient number of base pairs exist between one
nucleic acid molecule or region thereof (such as a region of a
probe or primer) and a target nucleic acid sequence (e.g., a ERG or
PTEN nucleic acid sequence) to achieve detectable binding. A
thorough treatment of the qualitative and quantitative
considerations involved in establishing binding conditions is
provided by Beltz et al. Methods Enzymol. 100:266-285, 1983, and by
Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd
ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0045] Contact: Placement in direct physical association including
both in solid or liquid form, under conditions that allow the
agents to interact. For example, a CTC (or population of isolated
CTCs on a slide) can be incubated with a specific binding agent
(e.g., nucleic acid probe or antibodies), such as one or more ERG
probes, PTEN probes, and CEN-10 probes, thereby permitting
detection of nucleic acid molecules in the CTCs that have
sufficient complementarity to the probe.
[0046] Control: A sample or standard used for comparison with a
test sample, such as a biological sample, e.g., a biological sample
obtained from a patient (or plurality of patients) or a cell
culture. In some embodiments, the control is a sample obtained from
a healthy patient (or plurality of patients) (also referred to
herein as a "normal" control), such as a normal sample (e.g., one
that does not have prostate cancer, such as a normal prostate
sample). In some embodiments, the control is a CTC or plurality of
CTCs obtained from a patient (or plurality of patients) with CRPC
or mCRPC known to have ERG rearrangements and/or PTEN deletions. In
some embodiments, the control is a CTC or plurality of CTCs
obtained from a patient (or plurality of patients) with CRPC or
mCRPC known to not have ERG rearrangements and/or PTEN deletions.
In some embodiments, the control is a historical control or
standard value (i.e., a previously tested control sample or group
of samples that represent baseline or normal values).
[0047] Cytokeratins (CK): Proteins of keratin-containing
intermediate filaments found in the intracytoplasmic cytoskeleton
of epithelial tissue. There are about 20 different epithelial CK
genes. The subsets of cytokeratins expressed by a particular
epithelial cell depends mainly on the type of epithelium, the
moment in the course of terminal differentiation and the stage of
development. Exemplary CK include keratins 4, 5, 6, 7, 8, 10, 13,
14, and 18. Antibodies specific for CK proteins are publicly
available. For example a pan-keratin antibody can detect multiple
CKs, such as CKs 4, 5, 6, 8, 10, 13 and 18 or the 56.5 kD, 50 kD,
50' kD, 48 kD, and 40 kD cytokeratins of the acidic subfamily and
65-67 kD, 64 kD, 59 kD, 58 kD, 56 kD, and 52 kD cytokeratins of the
basic subfamily. Exemplary CK antibodies are available from Ventana
(catalog numbers 760-2595 and 760-2135), Cell Signaling Technology
(catalog numbers 4528 and 4545), Abcam (catalog number ab8068), and
Thermo Scientific (catalog number MS-744-A).
[0048] Detect: To determine if an agent (e.g., a protein or nucleic
acid molecule) is present or absent. For example, the use of a
probe specific for a particular gene (e.g., ERG or PTEN) permits
detection of the desired nucleic acid molecule in a CTC, such as an
ERG rearrangement or PTEN deletion. For example, the use of an
antibody specific for a particular protein (e.g., CK or EpCAM)
permits detection of the desired protein in a CTC, such as CK or
EpCAM. In some examples this can further include quantification. In
particular examples, an emission signal from a label (such as a
quantum dot) is detected. Detection can be in bulk, so that a
macroscopic number of molecules can be observed simultaneously.
Detection can also include identification of signals from single
molecules using microscopy and such techniques as total internal
reflection to reduce background noise.
[0049] Diagnose: The process of identifying a medical condition or
disease, for example from the results of one or more diagnostic
procedures. In one example, the disclosed methods allow for
diagnosis of a more aggressive form of a prostate cancer if an ERG
rearrangement and/or and PTEN deletion is detected in CTC cells
from the patient with prostate cancer.
[0050] Epithelial cell adhesion molecule (EpCAM) (OMIM 185535): A
pan-epithelial differentiation antigen expressed on most cancer
cells. The human EpCAM gene (also known as tumor-associated calcium
signal transducer 1 (TACSTD1) and CD326) is located on chromosome 2
(2p21). EpCAM sequences are publically available, for example from
GenBank.RTM. (e.g., accession numbers NP.sub.--002345.2,
AAH14785.1, and NP.sub.--032558.2 (proteins) and NM.sub.--002354,
and NM.sub.--008532.2 (nucleic acids)) and UniProt (e.g., accession
numbers P16422 (protein)).
[0051] Antibodies specific for EpCAM proteins are publicly
available, for example from abcam (catalog number ab20160),
Millipore (catalog numbers CP63-100UG, OP187-100UG, MAB4444, and
CBL251), and Santa Cruz Biotechnology, Inc. (catalog numbers
sc-71059, sc-73491, sc-23788, and sc-73942).
[0052] Gene: A nucleic acid (e.g., genomic DNA, cDNA, or RNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., mRNA). The
polypeptide can be encoded by a full-length coding sequence or by
any portion of the coding sequence so long as the desired activity
or functional properties (e.g., enzymatic activity, ligand binding,
signal transduction, immunogenicity, etc.) of the full-length or
fragment is/are retained. The term also encompasses the coding
region of a structural gene and the sequences located adjacent to
the coding region on both the 5'- and 3'-ends for a distance of
about 1 kb or more on either end such that the gene corresponds to
the full-length mRNA. Sequences located 5' of the coding region and
present on the mRNA are referred to as 5' untranslated sequences.
Sequences located 3' or downstream of the coding region and present
on the mRNA are referred to as 3' untranslated sequences. The gene
as present in (or isolated from) a genome contains the coding
regions ("exons") interrupted with non-coding sequences termed
"introns." Introns are absent in the processed RNA (e.g., mRNA)
transcript. Provided herein are methods that permit detection of
ERG rearrangements and PTEN gene deletions.
[0053] Hybridization: Oligonucleotides and their analogs hybridize
by hydrogen bonding, which includes Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary bases.
Generally, nucleic acid molecules are composed of nitrogenous bases
that are either pyrimidines (cytosine (C), uracil (U), and thymine
(T)) or purines (adenine (A) and guanine (G)). These nitrogenous
bases form hydrogen bonds between a pyrimidine and a purine, and
the bonding of the pyrimidine to the purine is referred to as "base
pairing." More specifically, A will hydrogen bond to T or U, and G
will bond to C. "Complementary" refers to the base pairing that
occurs between two distinct nucleic acid sequences. For example, an
oligonucleotide probe can be complementary to an ERG or PTEN gene
sequence (or portion thereof), or a CEN-10 sequence (or portion
thereof).
[0054] "Specifically hybridizable" and "specifically complementary"
are terms that indicate a sufficient degree of complementarity such
that stable and specific binding occurs between the oligonucleotide
and the nucleic acid target. The oligonucleotide probe need not be
100% complementary to its target sequence to be specifically
hybridizable.
[0055] Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method of choice and the composition and length of the hybridizing
nucleic acid sequences. Generally, the temperature of hybridization
and the ionic strength (especially the Na.sup.+ concentration) of
the hybridization buffer will determine the stringency of
hybridization, though waste times also influence stringency.
Hybridization of an oligonucleotide sequence can be modified by
incorporating un-natural bases into the sequence, such as
incorporating locked nucleic acids or peptide nucleic acids.
[0056] Immunohistochemistry (IHC): A method of determining the
presence, amount or distribution of an antigen (such as a protein)
in a sample (for example, a CTC) by detecting interaction of the
antigen with a specific binding agent, such as an antibody. A
sample suspected of containing CTCs, and thus an EpCAM antigen, is
incubated with an antibody under conditions permitting
antibody-antigen binding. Antibody-antigen binding can be detected
by means of a detectable label conjugated to the antibody (direct
detection) or by means of a detectable label conjugated to a
secondary antibody, which is raised against the primary antibody
(e.g., indirect detection). Exemplary detectable labels that can be
used for IHC include, but are not limited to, radioactive isotopes,
fluorochromes (such as fluorescein, fluorescein isothiocyanate, and
rhodamine), and enzymes (such as horseradish peroxidase or alkaline
phosphatase).
[0057] Isolated: An "isolated" biological component (e.g., a
nucleic acid molecule or protein) or cell (such as a CTC) has been
substantially separated or purified away from other biological
components or other cells in which the component naturally occurs.
For example, a biological component can be substantially separated
or purified away from other chromosomal and extra-chromosomal DNA
and RNA, proteins and/or organelles. For isolated CTCs, such CTCs
are substantially separated or purified away from other cells in
the blood or bone marrow, such as lymphocytes and RBC. Nucleic
acids and proteins that have been "isolated" include nucleic acids
and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acids, such as probes for the detection of ERG rearrangements, PTEN
deletions, and CEN-10.
[0058] Label: An agent capable of detection, for example by
spectrophotometry, spectral imaging, flow cytometry, or microscopy.
For example, one or more labels can be attached to an antibody,
thereby permitting detection of a target protein (such as EpCAM,
CD45, or CK). Furthermore, one or more labels can be attached to a
nucleic acid molecule, thereby permitting detection of a target
nucleic acid molecule (such as ERG, PTEN, or CEN-10). Exemplary
labels include radioactive isotopes, fluorophores, quantum dots,
chromophores, ligands, chemiluminescent agents, enzymes, and
combinations thereof.
[0059] Normal cells or tissue: Non-tumor, non-malignant cells and
tissue.
[0060] Probe: An isolated nucleic acid capable of hybridizing to a
target nucleic acid (such as an ERG, PTEN, or CEN-10 nucleic acid
sequence), which can include a detectable label or reporter
molecule. Exemplary labels include quantum dots, radioactive
isotopes, enzyme substrates, co-factors, ligands, chemiluminescent
or fluorescent agents, haptens, and enzymes. In a particular
example, a probe includes at least one quantum dot. Methods for
labeling and guidance in the choice of labels appropriate for
various purposes are discussed, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989) and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and
Wiley-Intersciences (1987).
[0061] Probes are generally at least 50 nucleotides in length, such
as at least 51, at least 52, at least 53, at least 54, at least 55,
at least 56, at least 57, at least 58, at least 59, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 120, at
least 140, at least 160, at least 180, at least 200, at least 250,
at least 300, at least 350, at least 400, at least 450, at least
500, or more contiguous nucleotides complementary to the target
nucleic acid molecule (such as ERG, PTEN, or CEN-10), such as
20-500 nucleotides, 100-500 nucleotides, 100-250 nucleotides, or
20-250 nucleotides. In some examples, a probe comprises a plurality
of different probe sequences which can each bind to the target
nucleic acid molecule.
[0062] Prognose: The process of determining the likely outcome of a
subject having a disease (e.g., prostate cancer) in the absence of
additional therapy. In one example, the disclosed methods allow for
prognosis of a more aggressive form of a prostate cancer if ERG
rearrangements and/or PTEN deletions are detected in CTCs from the
patient. For example, the prognosis can relate to predicting future
events, such as life expectancy (e.g., likelihood of survival in 1
year, 3 years or 5 years), predicting the likely recurrence of
prostate cancer after prostatectomy, and/or predicting the likely
metastasis of a prostate cancer (e.g., after prostatectomy).
[0063] Prostate-specific membrane antigen (PSMA): (OMIM 600934): A
type 2 integral membrane glycoprotein found in prostate and other
tissues. Also known as folate hydrolase 1. PSMA has two enzymatic
activities, one as a prostate-specific integral membrane folate
hydrolase and the other as a carboxypeptidase. The human PSMA gene
is located on chromosome 11 (11p11.12). PSMA sequences are
publically available, for example from GenBank.RTM. (e.g.,
accession numbers AAA60209.1, AAM34479.1, and AAC83972.1 (proteins)
and NM.sub.--004476.1, and NG.sub.--029170.1 (nucleic acids)).
[0064] Antibodies specific for PSMA proteins are publicly
available, for example from Abcam (catalog number ab403, ab53774,
ab53690), Leica Microsystems (clone number PSMA-L-A), and Santa
Cruz Biotechnology, Inc. (catalog numbers sc-10269, sc-69665,
sc-130546, and sc-59674).
[0065] Quantitating or quantifying: Determining or measuring a
quantity (such as an absolute or relative quantity) of a molecule,
such as the quantity of an ERG or PTEN gene, such as a number of
ERG rearrangements, PTEN deletions, or CEN-10 present in isolated
CTCs.
[0066] Quantum dots: Inorganic semiconductor crystalline
nanoparticles that fluoresce stably and possess a uniform surface
area that can be attached to a nucleic acid probe or antibody,
thereby permitting detection of the target of the nucleic acid
probe or antibody. Although generally spherical, quantum dots
attached to nucleic acid probes or antibodies can be of any shape
(such a spherical, tubular, pyramidal, conical or cubical), but
particularly suitable nanoparticles are spherical.
[0067] Generally, quantum dots can be prepared with relative
monodispersity (for example, with the diameter of the core varying
approximately less than 10% between quantum dots in the
preparation), as has been described previously (Bawendi et al., J.
Am. Chem. Soc. 115:8706, 1993). Quantum dots known in the art have,
for example, a core selected from the group consisting of CdSe,
CdS, and CdTe (collectively referred to as "CdX").
[0068] Signal: A detectable change or impulse in a physical
property that provides information. In the context of the disclosed
methods, examples include electromagnetic signals such as light,
for example light of a particular quantity or wavelength. In
certain examples the signal is the disappearance of a physical
event, such as quenching of light. A characteristic signal is the
particular signal expected when a particular nucleic acid probe
specifically hybridizes to its complementary nucleic acid sequence
in the CTCs. For example, a characteristic signal can be the
resulting signal emitted from a quantum dot present on the nucleic
acid probe, which can be predicted by the particular quantum dot
attached to or associated with the nucleic acid probe.
[0069] Specific binding (or derivations of such phrase, such as
specifically binds, specific for, etc.): The particular interaction
between one binding partner (such as a gene-specific probe or
protein-specific antibody) and another binding partner (such as a
target of a gene-specific probe or protein-specific antibody). Such
interaction is mediated by one or, typically, more non-covalent
bonds between the binding partners (or, often, between a specific
region or portion of each binding partner). Thus, an
oligonucleotide stably binds to a target nucleic acid (e.g., PTEN,
ERG, CEN-10) if a sufficient amount of the oligonucleotide forms
base pairs or is hybridized to its target nucleic acid.
[0070] In contrast to non-specific binding sites, specific binding
sites are saturable. Accordingly, one exemplary way to characterize
specific binding is by a specific binding curve. A specific binding
curve shows, for example, the amount of one binding partner (the
first binding partner) bound to a fixed amount of the other binding
partner as a function of the first binding partner concentration.
As the first binding partner concentration increases under these
conditions, the amount of the first binding partner bound will
saturate. In another contrast to non-specific binding sites,
specific binding partners involved in a direct association with
each other (e.g., a probe-mRNA or antibody-protein interaction) can
be competitively removed (or displaced) from such association by
excess amounts of either specific binding partner. Such competition
assays (or displacement assays) are very well known in the art.
[0071] Subject: Includes any multi-cellular vertebrate organism,
such as human and non-human mammals (e.g., veterinary subjects such
as cats or dogs). In some examples, a subject or patient is one who
has cancer, or is suspected of having cancer, such as prostate
cancer, such as a castration-resistant prostate cancer (CRPC) or a
metastatic prostate cancer such as metastatic castration-resistant
prostate cancer (mCRPC).
[0072] Substrate: A material or surface to which other molecules
(such as isolated CTCs) can be attached. In particular examples,
the substrate is made of biocompatible material that is transparent
to light, including glass and quartz. For example, the substrate
can be a glass microscope slide (such as one that is 3 cm long by 1
cm wide by 0.25 cm thick). Glass microscope slides are commercially
available and are sold pretreated, resulting in positively charged
slides. In one example, positively charged glass surfaces are
prepared by chemical reaction of slides with
3-aminopropyltriethoxysilane (APES).
[0073] Under conditions sufficient for: Any environment that
permits the desired activity, for example, that permits an antibody
to bind an antigen, such as EpCAM, CD45, ERG, AR, PSMA, or CK, or
that permits a nucleic acid probe to bind its complementary target
sequence, such as ERG, PTEN, or CEN-10, and the interaction to be
detected.
[0074] An example includes contacting a nucleic acid probe with
CTCs under conditions sufficient to allow hybridization of the
nucleic acid probe it is complementary nucleic acid molecule in the
CTCs (if present), for example to determine whether the
complementary nucleic acid molecule is present in the CTCs, such as
ERG rearrangements or PTEN deletions.
[0075] Unique Emission Signal: An emission signal that conveys
information about a specific event, such as the emission spectrum
for a particular label (such as a quantum dot), which can be
distinguished from other signals (such as emission spectrum signals
from other labels). Examples in association with the disclosed
methods include associating one or more individual labels (such as
a quantum dot) with each different type of nucleic acid probe (such
as a nucleic acid probe specific for PTEN vs. a nucleic acid probe
specific for ERG), such that hybridization of the a nucleic acid
probe with its complementary sequence in CTCs results in a unique
signal or a combination of signals (such as quantum dots that emit
at different unique wavelengths). Each label has a unique emission
signal corresponding to a particular nucleic acid probe specific.
This signal can be used to determine which nucleic acid probe has
successfully hybridized to its complementary sequence in the
CTCs.
II. Automated Analysis of CTCs
[0076] One aspect of blood tests is that they can be safely
performed at many points during cancer treatment or diagnosis,
whereas solid tumor biopsies are often invasive and can only be
performed intermittently. The ability to monitor disease
progression over time allows appropriate therapy modifications, for
example to improve a patient's quality of life. To this end, the
disclosed methods were developed to improve the lives of those
afflicted with cancer. The automation and technologies implemented
through the disclosed methods provide the requisite sensitivity and
reproducibility to detect CTCs in patients with metastatic disease.
In particular, methods that permit simultaneous or contemporaneous
detection of multiple genetic and protein markers in a single
sample have been developed. For example, contemporaneous detection
of ERG, PTEN, and CEN-10, in the same CTC is demonstrated
herein.
[0077] One or more steps of the method can be automated. In
illustrative embodiments, the methods include automated chemical
treatment steps to decrease the variability between assays, to
achieve consistency of detection, or both. For example, one or more
of the steps can be automated, such as a hybridization and
detection steps. Manual hybridizations can have a variable rate of
stripping the CTC from the slide, whereas automation is more
reliable. Automation provides faster results, turnaround time and
reduces site-to-site variability. In one example, automation allows
at least 30 samples (such as at least 50, at least 100, or even at
least 500 samples, such as 10, 20, 30, 40, 50, 100, 200, 250, 500,
or 1000 samples) to be tested simultaneously or
contemporaneously.
[0078] Provided herein are methods of analyzing a sample, such as
one known or suspected of containing CTCs, using an automated
instrument. Such methods can include obtaining a sample from a
subject (such as a subject known to have cancer), depositing or
applying the sample on a substrate configured for use with the
automated instrument, placing the substrate within the automated
instrument, retrieving targets on the sample using the automated
instrument, contacting the sample with CTC identification reagents
using the automated instrument, contacting the sample with CTC
characterization reagents using the automated instrument, imaging
the sample, locating the CTCs by locating the CTC identification
reagents, spectral imaging f the CTCs by location, analyzing the
sample by analyzing the spectral imaging, or combinations
thereof.
[0079] In one example, the method includes enriching the CTC
content of the sample using a capture antibody specific for an ERG
protein, a prostate specific membrane antigen (PSMA) protein, or an
epithelial cell adhesion molecule (EpCAM) protein, wherein
enriching the CTC content of the sample occurs prior to depositing
or placing the sample onto the substrate. Methods of obtaining
samples that may contain CTCs are routine, and include obtaining a
blood or bone marrow sample. In some examples, the sample is used
directly, while in other examples the sample is manipulated (e.g.,
concentrated, diluted, treated to enrich or separate CTC cells,
treated to remove undesired cells (such WBC or RBC, for example by
lysing RBC), or combinations thereof). In some examples, a blood
sample is obtained and fractionated and the serum fraction is used
for further analysis.
[0080] In one example, the CTC identification reagents and the CTC
characterization reagents are selected so that the method provides
medical value, for example, the method provides information that is
medically actionable.
[0081] In one embodiment, the CTC identification reagents include
immunohistochemical (IHC) reagents directed to (or specific for)
CTC protein markers, such as one or more of CD45 protein,
cytokeratin (CK) protein, ERG protein, androgen receptor (AR), or
PSMA protein. Such CTC identification reagents permit determination
that a particular cell is a CTC cell. In one example, CTC
identification reagents can include antibodies or other binding
agents specific for CTC protein markers, such as one or more of
CD45, CK, ERG, AR, or PSMA. For example, CTC identification
reagents include antibodies specific for 1, 2, 3, 4, or all of
CD45, CK, ERG, AR, PSMA, or combinations thereof. Such
identification reagents can include a detectable label, such as a
quantum dot. In some examples, for example if several antibodies
are used on the same sample, each antibody specific for a
particular protein includes a different detectable label. For
example, FIG. 1 shows a prostate cancer cell line (LNCaP)
exhibiting CK+ staining while being negative for CD45, as detected
by CTC identification reagents (CK and CD45 antibodies).
[0082] In one example, the CTC characterization reagents include
nucleic acid probes directed to genomic markers, such as 1, 2, 3,
4, or 5 different genomic markers, such as genomic markers for
cancer diagnosis or prognosis. Such CTC characterization reagents
permit characterization of the CTC cell, for example to permit
diagnosis or prognosis of a patient with cancer. Such probes can
include a detectable label, such as a quantum dot. In some
examples, for example if several probes are used on the same
sample, each probe specific for a particular target includes a
different detectable label. In one embodiment, the genomic markers
are analyzed for gene expression and/or genetic
rearrangements/deletions. For example, the genomic markers can be
detected using a gene expression probe and a rearrangement/deletion
probe combination. In a particular example, the CTC
characterization reagents include nucleic acid probes specific for
(e.g., can selectively hybridize to) ETS related gene (ERG),
phosphatase and tensin homolog (PTEN), and centromere 10 (CEN-10).
In some examples, the CTC characterization reagents include nucleic
acid probes specific for different regions of the same gene (such
as one probe specific for the 5'-end and a second probe specific
for the 3'-end of a gene).
[0083] In a particular embodiment, the CTC characterization
reagents include nucleic acid probes directed to genomic markers
(such as at least 2, at least 3 or at least at 4 genomic markers)
and the CTC identification reagents include IHC reagents directed
to CTC protein markers. In one embodiment, the genomic markers
include ERG, PTEN, and CEN-10 and the CTC protein markers include
one or more of CD45 protein, CK protein, ERG protein, AR, and PSMA
protein. In some examples, the CTCs are also stained with DAPI. As
shown in FIGS. 2 and 3, a multiplexed quantum dot FISH assay can be
achieved using probes for 5'-end ERG, 3'-end ERG, PTEN, and CEN-10
(labeled with quantum dots that emit at 655 nm, 565 nm, 605 nm, and
585 nm, respectively) as described herein (Example 1).
[0084] The method of analyzing a sample known or suspected of
containing circulating CTCs can include an imaging step. In one
example, imaging includes imaging immunofluorescence of the CTC
identification reagents (for example by detecting the label
associated with each antibody used). In another example, imaging
includes using multi-spectral bandpass filters. The
immunofluorescence can emanate from antibodies labeled directly or
indirectly with fluorophores or the immunofluorescence can result
from exciting the fluorophores with spectrally filtered visible
light. In one embodiment, the spectrally filtered visible light
includes a first selected range to excite a first fluorophore and a
second selected range to excite a second fluorophore, wherein the
first selected range does not significantly excite the second
fluorophore and the second selected range does not significantly
excite the first fluorophore. Imaging the sample can include
acquiring a first immunofluorescence image of the sample excited by
the first selected range and acquiring a second immunofluorescence
image of the sample excited by the second selected range (and
acquiring additional immunofluorescence images for each label if
more than two CTC identification reagents were used) and locating
or identifying the CTCs by locating or visualizing the CTC
identification reagents, which can include comparing or overlaying
the first immunofluorescence image and the second
immunofluorescence image (and additional images if so obtained).
For example, imaging the first immunofluorescence image can
identify CK+ cells, and the second immunofluorescence image can
identify CD45+ cells, wherein comparing or overlaying includes
identifying cells that are CK+ and CD45-. In another embodiment,
locating the CTCs by locating the CTC identification reagents
includes algorithmically analyzing the first immunofluorescence
image and the second immunofluorescence image (and additional
immunofluorescence image s if obtained) using a computer. In one
embodiment, algorithmically analyzing includes digitally
interrogating the images to measure cell size, cell compartment
localization of markers, and/or intensity of marker expression.
[0085] In one example, spectral imaging of the CTCs includes
spectral imaging luminescence of the CTC characterization reagents,
the luminescence emanating from labeled specific binding moieties
(such as nucleic acid probes), such as nucleic acid probes labeled
with quantum dots. The specific binding moieties can be directly
labeled (e.g., with the quantum dots), or can be indirectly labeled
(e.g., with the quantum dots) (the luminescence emanating from the
quantum dots labeling anti-hapten secondary antibodies, the
anti-hapten secondary antibodies being specific to haptens labeling
the specific binding moieties). Spectral imaging the CTCs can
include exciting the label (e.g., quantum dots) with radiation,
such as UV or near-UV radiation. In one embodiment, the radiation
has a spectral emission profile with a maximum between 300 nm and
400 nm. Spectral imaging the CTCs can alternatively include
multispectral imaging or hyperspectral imaging. Spectral imaging
can be guided by the step of locating the CTCs to regions of
interest to the exclusion of regions devoid of interest. Exemplary
regions of interest include CTCs. In another embodiment, the
spectrally filtered visible light does not result in significant
quantum dot luminescence.
[0086] In one embodiment, imaging the sample includes
multi-spectral imaging immunofluorescence of the CTC identification
reagents and spectral imaging the CTC includes hyper-spectral
imaging the CTC characterization reagents. In one embodiment, the
multi-spectral and hyper-spectral imaging differentiates at least
four CTC identification reagents and/or CTC characterization
reagents, at least five CTC identification reagents and/or CTC
characterization reagents, or at least six CTC identification
reagents and/or CTC characterization reagents. In another
embodiment, the multi-spectral imaging differentiates at least two
CTC identification reagents (such as 2, 3, 4, or 5 CTC
identification reagents) and the hyper-spectral imaging
differentiates at least four CTC characterization reagents (such as
4, 5, 6, 7 or 8 CTC characterization reagents).
[0087] FIG. 4 is a schematic showing an illustrative method 100 of
analyzing a sample 101 containing circulating tumor cells (CTCs)
using an automated instrument. In this particular embodiment, an
optional first step is enriching the CTCs 102. The CTCs are
deposited on a substrate 103, resulting in CTCs being present on a
substrate 104. Subsequently, CTCs are identified 105 and
characterized 106.
[0088] FIG. 5 shows one embodiment of a method of treating CTCs 200
subsequent to the CTCs being deposited on a substrate 104. In this
embodiment, the method includes permeabilizing the CTCs 201,
retrieving targets 202, contacting the sample with CTC
identification reagents 203 (e.g., those that can be used for
immunofluorescence, IF, or immunohistochemistry, IHC). According to
various embodiments, the method may optionally include a second
target retrieval step 204. The method proceeds with contacting the
sample with CTC characterization reagents 205 (e.g., those that can
be used for in situ hybridization, FISH, CISH, SISH, IF, or IHC),
locating the CTCs by locating the CTC identification reagents 206
(e.g. IF or IHC guidance) and spectral imaging the CTCs by location
207. Subsequent to the imaging, the data obtained through the
imaging process can be analyzed, thus informing the user or
pathologist about the nature of the CTCs analyzed.
[0089] One or more steps of methods described herein can be
automated. Exemplary automated systems available through Ventana
Medical Systems, Inc., Tucson, Ariz. include SYMPHONY.RTM. Staining
System (catalog #: 900-SYM3), VENTANA.RTM.BenchMark Automated Slide
Preparation Systems (catalog #s: N750-BMKXT-FS, N750-BMKU-FS), and
VENTANA.RTM. BenchMark Special Stains automated slide stainer.
These systems employ a microprocessor controlled system including a
revolving carousel supporting radially positioned slides. A stepper
motor rotates the carousel placing each slide under one of a series
of reagent dispensers positioned above the slides. Bar codes on the
slides and reagent dispensers permits the computer controlled
positioning of the dispensers and slides so that different reagent
treatments can be performed for each of the various tissue samples
by appropriate programming of the computer.
[0090] Illustrative instrumentation systems are designed to
sequentially apply reagents to tissue sections or other samples
(such as a blood sample) mounted on one by three inch glass
microscope slides under controlled environmental conditions. The
instrument performs several basic functions such as reagent
application, washing (to remove a previously applied reagent), jet
draining (to reduce the residual buffer volume on a slide
subsequent to washing), application of a light oil (to contain
reagents and prevent evaporation), and other instrument functions.
Exemplary staining instruments process slides on a rotating
carousel. The slides maintain a stationary position and a dispenser
carousel rotates the reagents above the fixed slides. The processes
described herein can be performed using various physical
configurations. The process of clarifying and staining tissue or
other biological sample on a slide consists of the sequential
repetition of basic instrument functions described above.
Essentially a reagent is applied to the sample then incubated for a
specified time at a specific temperature. When the incubation time
is completed the reagent is washed off the slide and the next
reagent is applied, incubated, and washed off, etc., until all of
the reagents have been applied and the staining process is
complete.
[0091] For related disclosure, reference is made to Richards et al.
U.S. Pat. No. 6,296,809, which describes an apparatus and methods
for automatically staining or treating multiple tissue samples
mounted on microscope slides so that each sample can receive an
individualized staining or treatment protocol even when such
protocols require different temperature parameters. More
specifically, described is an apparatus comprising a computer
controlled, bar code driven, staining instrument that automatically
applies chemical and biological reagents to tissue or cells mounted
or affixed to standard glass microscope slides. A plurality of
slides are mounted in a circular array on a carousel which rotates,
as directed by the computer, to a dispensing location placing each
slide under one of a series of reagent dispensers on a second
rotating carousel positioned above the slides. Each slide receives
the selected reagents (e.g. DNA probes and/or antibodies) and is
washed, mixed, and/or heated in an optimum sequence and for the
required period of time. In one embodiment, the sample is mounted
on a glass microscope slide. In one embodiment, the glass
microscope slide is configured to be compatible with an automated
slide staining instrument.
[0092] A. Prostate Cancer CTC Evaluation
[0093] In 2008, it was estimated that prostate cancer alone will
account for 25% of all cancers in men and will account for 10% of
all cancer deaths in men (Jemal et al., CA Cancer J. Clin.
58:71-96, 2008). Prostate cancer typically is diagnosed with a
digital rectal exam ("DRE") and/or prostate specific antigen (PSA)
screening. An abnormal finding on DRE and/or an elevated serum PSA
level (e.g., >4 ng/ml) can indicate the presence of prostate
cancer. When a PSA or a DRE test is abnormal, a transrectal
ultrasound may be used to map the prostate and show any suspicious
areas. Biopsies of various sectors of the prostate are used to
determine if prostate cancer is present.
[0094] Oncologists have a number of treatment options available,
including different combinations of chemotherapeutic drugs that are
characterized as "standard of care," and a number of drugs that do
not carry a label claim for particular cancer, but for which there
is evidence of efficacy in that cancer. The best chance for a good
treatment outcome requires that patients promptly receive optimal
available cancer treatment(s) and that such treatment(s) be
initiated as quickly as possible following diagnosis. On the other
hand, some cancer treatments have significant adverse effects on
quality of life; thus, it is equally important that cancer patients
do not unnecessarily receive potentially harmful and/or ineffective
treatment(s).
[0095] Currently, the following therapeutics are available for the
treatment of castrate resistant prostate cancer (CRPC):
immunotherapy with Sipuleucel-T, chemotherapy with Docetaxel,
Cabazitaxel, radio-bone therapy with Radium 223,
anti-androgen-MDV-3100, and hormonal therapy with abiraterone.
Understanding the best therapy and sequence of therapies is not
well understood given each therapy has both non-responders and
super responders. Given the metastatic setting of mCRPC, CTCs can
be monitored in these patients using enumeration. Additionally,
patients with a positive response to cytotoxic (Docetaxel &
Cabitaxel) and immunotherapy (Sipuleucel-T) treatments, have
heterogeneous bone scan and PSA measurements. As such, PSA and bone
scans are not always a reliable surrogate marker of progression in
CRPC patients. Many patients also harbor de novo resistance to
cytotoxic or immunotherapy treatments, with no good progression
marker.
[0096] Given that some prostate cancers need not be treated while
others almost always are fatal, and further given that the disease
treatment can be unpleasant at best, there is a strong need for
methods that allow care givers to predict the expected course of
disease, including the likelihood of cancer recurrence, long term
survival of the patient, expected response to a particular
treatment, and the like, and to select the most appropriate
treatment option accordingly. An automated method of analyzing CTCs
and kits for the same would provide a valuable benefit to cancer
patients. Since blood is accessible and easy to collect, an
automated analysis of CTCs is of great need for early stage
detection of cancer as well as for neoplastic progression and
recurrence monitoring.
[0097] Thus provided are methods of characterizing a prostate
cancer, such as castrate-resistant prostate cancer (CRPC) or
metastatic CRPC (mCRPC). Such methods can include obtaining or
isolating CTCs from a subject having prostate cancer. The method
can further include detecting one or more prostate cancer related
molecules in the CTCs (or in a separate prostate cancer sample),
for example using labeled nucleic acid probes or antibodies
specific for such prostate cancer-related molecules. In some
examples, the detected prostate cancer-related molecule is compared
to expression of the one or more other prostate cancer related
molecules in the prostate cancer sample to a control representing
expression of the one or more other prostate cancer related
molecules expected in a normal prostate sample. Prostate cancer
related molecules include those whose expression is known to be
altered (such as increased or decreased) in a prostate cancer
sample, relative to a normal prostate cancer sample. Examples
include but are not limited to: growth arrest specific 1 (GAS1;
OMIM 139185), wingless-type MMTV integration site family member 5
(WNT5A; OMIM 164975), thymidine kinase 1 (TK1; OMIM 188300), V-raf
murine sarcoma viral oncogene homolog B1 (BRAF; OMIM 164757), ETS
translocation variant 4 (ETV4; OMIM 600711), tumor protein p63
(OMIM 603273), BCL-2 (OMIM 151430), Ki67 (OMIM 176741), ERK5 (OMIM
602521), androgen receptor (AR; OMIM 313700), prostate specific
antigen (PSA; OMIM 176820), ETS translocation variant 1 (ETV1; OMIM
600541), prostate-specific membrane antigen (PSMA; OMIM 600934),
serine protease inhibitor Kazal-type 1 (SPINK1; OMIM 167790),
measures of nuclear morphology (including nuclear size and shape
variation characteristics), or combinations thereof.
[0098] The use of both PTEN and CEN-10 probes increases the
accuracy of determining that a PTEN deletion is present. For
example, in previous assays, only PTEN probes were used. If there
was no detectable signal from the PTEN probe, it was concluded that
the sample had a PTEN deletion. However, such methods fail to
account for other reasons why no signal was detected, thus
increasing the number of false PTEN deletion determinations. To
increase the accuracy, the disclosed methods also use a probe
specific for centromere 10. Deletion of PTEN does not result in
deletion of centromere 10. Thus, only if a positive signal is
obtained from the CEN-10 probe, is the signal from the PTEN probe
considered. For example, if no signal is detected in the
CTC-containing sample for the CEN-10 probe, the assay is
disregarded as faulty. However, if a signal is detected in the
CTC-containing sample for the CEN-10 probe (indicating the presence
of CEN-10), then the signal or absence of signal from the PTEN
probe is considered, wherein a positive CEN-10 and negative PTEN
signal indicates a PTEN deletion, while a positive CEN-10 and
positive PTEN signal indicates the absence of PTEN deletion. In
some examples, the method further includes comparing the signals
detected from the ERG, PTEN, and CEN-10 nucleic acid probes to a
CTC cell having (1) no ERG rearrangements, no PTEN deletions, and a
detectable CEN-10, or (2) one or more ERG rearrangements, one or
more PTEN deletions, and a detectable CEN-10.
[0099] In illustrative embodiments, a method of analyzing a sample
containing CTCs using an automated instrument includes relates is
used for a subject has prostate cancer. In one embodiment, the
prostate cancer is a castrate-resistant prostate cancer (CRPC). In
another embodiment, the CRPC is a metastatic CRPC (mCRPC). In one
embodiment, four genomic markers are characterized, the four
markers including ERG, PTEN, and CEN-10. In a specific embodiment,
the nucleic acid probes are a 5' ERG probe, a 3' ERG probe, a PTEN
probe, and a CEN-10 probe.
[0100] B. Isolating Circulating Tumor Cells
[0101] Methods for isolating or enriching CTCs, for example from
blood, fractions thereof (such as serum), or bone marrow, are
known. However, those methods have not, prior to the present
disclosure, been capable of providing a sample which is
translatable to an automated tissue stainer. In one example, CTCs
are isolated from a sample using antibodies or other specific
binding agents against one or more cell surface antigens present on
the CTCs (but not present on blood cells, such as leukocytes). One
exemplary antigen is the epithelial cell adhesion molecule (EpCAM).
For example, the CellSearch system (Veridex) can be used to isolate
CTCs, which uses ferrofluids loaded with an EpCAM antibody to
capture CTCs. In some examples, the CTCs are collected and isolated
from a patient after cancer diagnosis, and in some examples after
prostatectomy or other therapy. In illustrative embodiments, the
isolation of CTCs generates about 900 .mu.L of a cellular
suspension containing the CTCs and other cells (e.g. white blood
cells). In one embodiment, the enriched cells are bound to magnetic
beads.
[0102] Methods of isolating CTCs include approaches relying on the
physical properties, expression of biomarkers, or functional
characteristics of CTCs. In one example, a blood sample or bone
marrow is obtained from the patient suspected of having, known to
have or had cancer (such as prostate cancer, for example a CRPC or
mCRPC), and used to isolate CTCs. In some examples, the CTCs are
collected and isolated after cancer diagnosis, for example after
prostatectomy or other therapies. In some examples, the patient has
biochemically or histologically confirmed prostate cancer, elevated
PSA, or both. The subject can be a human or other mammal (such as a
dog or cat) with cancer. A typical subject with prostate cancer is
a human male; however, any mammal that has a prostate cancer can
serve as a source of CTCs. In one example 1 to 10 mls of blood is
obtained, such as 7.5 ml. In some examples the blood is
fractionated, and CTC isolated from the serum fraction. In some
examples, CTCs are isolated by subjecting the blood sample to
differential lysis to remove red blood cells. In some examples CTCs
are isolated from bone marrow.
[0103] In one example, CTCs are isolated using antibodies against
one or more cell surface antigens present on the CTCs (but not
present on blood cells, such as leukocytes). CTCs express
epithelial cell surface markers that are absent from normal
leukocytes. For example, the epithelial cell adhesion molecule
(EpCAM) is expressed in cells of epithelial origin, but it is
absent in blood cells. Therefore, antibodies specific for EpCAM can
be used to isolate CTCs, such as a polyclonal or monoclonal
antibody, or fragment thereof. Such antibodies are commercially
available. In one example, EpCAM-specific antibodies are conjugated
to a solid substrate, such as a bead, microscope slide, or
microtiter plate, for example magnetic beads. This enables easy
purification of the CTCs bound to the EpCAM antibodies, such that
cells not bound to the EpCAM antibodies can be easily separated
away from the CTCs. CTCs bound to the EpCAM antibodies and thus the
solid support can be purified, for example by washing away unbound
cells or capturing the bound cells through a magnetic field. In one
specific example, the CellSearch system (Veridex) is used to
isolate CTCs. This method uses ferrofluids loaded with an EpCAM
antibody to capture CTCs. In some examples, the isolated CTCs are
also stained with DAPI.
[0104] The isolated CTCs can be further analyzed to ensure that the
cells isolated are CTCs. For example, the isolated cells can be
analyzed using CTC identification reagents, such as determining
whether cytoplasmic epithelial cytokeratins (CK) and CD45 are
present or absent. The leukocyte-specific marker CD45 is used as a
control to exclude contaminating leukocytes. CTCs are those
EpCAM-captured cells that are both positive for cytokeratins and
negative for CD45. For example, nucleic acid probes, antibodies
(e.g., monoclonal and/or polyclonal antibodies) or other specific
binding agents (such as aptamers) specific for CKs or CD45 can be
used. The antibodies can be detected by direct labeling of the
antibodies themselves, for example, with radioactive labels,
fluorescent labels, hapten labels such as, biotin, or an enzyme
such as horseradish peroxidase or alkaline phosphatase.
Alternatively, an unlabeled primary antibody is used in conjunction
with a labeled secondary antibody, comprising antisera, polyclonal
antisera or a monoclonal antibody specific for the primary
antibody.
[0105] In one example, CTCs are isolated based on their physical
properties. Several physical properties distinguish CTCs from most
normal blood cells. These include the larger size of most
epithelial cells and differences in density, charge, migratory
properties, and some properties of specific cell types (e.g.,
melanocytic granules in melanoma cells). Differences in buoyant
density can be used to separate mononucleated cells, including
CTCs, from red blood cells through gradient centrifugation.
Isolation of CTCs by virtue of their increased size, compared with
leukocytes, can be accomplished using filtration-based approaches,
such as isolation by size of epithelial tumor cells and
microelectromechanical systems. Although most CTCs derived from
epithelial cancers are larger than leukocytes, there is a
significant variation in their size range. In one example CTCs are
isolated using filter-based methods.
[0106] In one example, a microfluidic platform for single-step
isolation of CTCs from unprocessed blood specimens is used (e.g.,
see Nagrath et al., Nature. 450:1235-1239, 2007; Stott et al., Sci.
Trans'. Med. 2:25ra23, 2010). For example, the CTC-chip is a
silicon chamber etched with 78,000 microposts that are coated with
an anti-EpCAM antibody. Whole blood is flowed through the chip,
permitting capture of CTCs to microposts. In another example, a
herringbone-chip is used, which makes use of a microvortex mixing
device (Stott et al., Proc. Natl. Acad. Sci. USA. 107:18392-18397,
2010). Instead of using three-dimensional microposts to break up
flow streamlines and enhance cell collisions with antibody-coated
posts, the HB-chip uses calibrated microfluidic flow patterns to
drive cells into contact with the antibody-coated walls of the
device.
[0107] C. Application of CTCs to Solid Substrates
[0108] CTCs, such as isolated CTCs or those present in a sample
(such as a blood or fraction thereof, or bone marrow), can be
applied onto a substrate (e.g., glass slide). For example, after
isolating the CTCs they can be adhered to a substrate. In another
example, after obtaining the sample (and in some examples lysing
the red blood cells), the remaining white blood cells and CTCs can
be adhered to a substrate.
[0109] In illustrative embodiments, the CTCs can be cytospun,
smeared (i.e. roller method), or immunocoated (e.g. adhered to PSMA
antibody coating) onto the substrate. Exemplary solid substrates
include but are not limited to a polycarbonate substrate, a glass
substrate (such as a glass slide that is positively charged), or
other suitable substrate. The substrate surface may be coated or
modified by chemical or physical means, such as a protein or amino
acid coating.
[0110] In one example, CTCs can be applied to a substrate by
cytocentrifuging the cells onto a glass slide generally following
published protocols. For example, cells can be centrifuged onto
commercially available precoated glass slides (e.g., VWR,
Superfrost Plus slides) at 400 rpm for 4 minutes using a
cytocentrifuge (e.g., Cytospin 4, Shandon Thermo Scientific).
Because cell integrity may be lost due to centrifugal forces, a
cell spreading deposition method can be used as an alternative
cytocentrifugation. The spreading method is gentler than that of
cytocentrifugation, thereby better preserving cellular shape and
volume prior to fixation. This method also tends to apply CTCs to a
substrate in a generally homogeneous manner. In illustrative
embodiments, a roller method is used for depositing CTCs onto the
substrate.
[0111] Cell components suspended in a liquid on a surface can be
homogeneously distributed onto a substrate as follows: positioning
a liquid carrier having an upper surface on a table, positioning a
distributing bar above the liquid carrier upper surface a distance
from about 50 .mu.m to about 1000 .mu.m (such as 350 .mu.m to about
1000 .mu.m), applying a liquid comprising cell components suspended
therein onto the liquid carrier upper surface, and moving one or
more of the distributing bar, the table, and liquid carrier to
distribute the cell components suspended in the liquid on the
liquid carrier upper surface uniformly. In one embodiment, the
liquid sufficiently adheres to the distributing bar to enable the
movement and distribution of the liquid uniformly on the liquid
carrier upper surface. In another embodiment, the cell components
within the liquid are separated. In another embodiment, the cell
components within the liquid comprise CTCs. The liquid carrier
upper surface can include a coating or other cell retaining
property. In one embodiment, the surface of the liquid carrier
positioned on the table has an at least partly electrostatically
charged surface or a coating of one or more antibodies and/or
lipophilic molecules thereon.
[0112] In yet other embodiments, the method further includes at
least one of these steps: a. removing the liquid after the liquid
has been homogeneously distributed on the liquid carrier surface,
wherein a uniform layer of cell components are retained on the
carrier surface; b. drying the liquid carrier to retain the cell
components uniformly positioned on the carrier surface; and c.
application of additional fluid or fluids to the liquid carrier
upper surface after drying the liquid carrier, such as for fixation
or staining purposes of the cell components retained thereon. In
one embodiment, the cell components suspended in the liquid include
at least one subgroup which is to be detected. In another
embodiment, the liquid carrier is a microscope slide. Reference is
made to co-pending U.S. Application Ser. No. 61/621,107 for
disclosure related to the roller method; incorporated by reference
herein in its entirety.
[0113] D. Fixation and Permeabilization
[0114] In some examples, after the CTCs are attached to a
substrate, they are fixed, for example with a crosslinking agent
such as NBF. For example, CTCs can be fixed after the attachment in
10% NBF (for example at room temperature for at least 10 minutes,
at least 15 minutes, or at least 20 minutes, such as 5 to 30, 10 to
30 or 15 to 30 minutes, such as 20 minutes), followed by rinsing in
phosphate buffered saline (PBS) (such as 3.times.5 minutes at room
temperature). The slides can then be immersed in PBST (0.2% Tween
20 in PBS) for permeabilization (for example at room temperature
for at least 10 minutes, at least 15 minutes, or at least 20
minutes, such as 5 to 30, 10 to 30 or 15 to 30 minutes, such as 20
minutes), rinsed in DI water and dehydrated in ascending alcohol
series (e.g., 80%, 90% and absolute EtOH, 1 minute each), followed
by air drying. The samples can be stored at -20.degree. C. until
use in airtight boxes.
[0115] Other exemplary fixatives for mounted cell and tissue
preparations are well known in the art and include, without
limitation, 95% alcoholic Bouin's fixative; 95% alcohol fixative;
B5 fixative, Bouin's fixative, formalin fixative, Karnovsky's
fixative (glutaraldehyde), Hartman's fixative, Hollande's fixative,
Orth's solution (dichromate fixative), and Zenker's fixative (see,
e.g., Carson, Histotechology: A Self-Instructional Text,
Chicago:ASCP Press, 1997). In some embodiments, cells are treated
after fixation with enzymes, such as proteases, to open cellular
structures and thereby making targets more available to binding
partners, such as nucleic acid probes or antibodies.
[0116] In some examples the method also includes fixing the CTCs on
the slide, for example using a 10% Neutral Buffered Formalin (NBF)
solution, which is about 3.7% formaldehyde in phosphate buffered
saline, thereby preserving the target nucleic acid molecules within
the CTCs.
[0117] The inventors have identified fixation and permeabilization
processes that prevent the loss of significant numbers of cells.
The loss of cells is detrimental to the analysis of CTCs due to the
very low number of these cells available for analysis. A biased
loss of the CTCs would render a method, as described herein,
relatively unsuitable for the purpose of CTC evaluation. However,
even an unbiased loss of cells on the slide decreases the
sensitivity of the evaluation. Accordingly, considerable
experimentation was devoted to establishing fixation and
permeabilization steps which prevent significant cell loss during
subsequent evaluation. The cell loss of interest is the number of
cells lost from prior to fixation until subsequent to imaging.
According to one measure, significant cell loss is described as
cell loss that is statistically significant in comparison to the
standard deviation across three counted regions having a number of
cells in excess of 1000 cells. According to another measure,
significant cell loss is loss of greater than about 10% of the
cells. Thus, in one example, the disclosed methods retain at least
90% of the CTC cells, at least 95% of the CTC cells, or at least
98% of the CTC cells, such as 90-100%, 90-98%, or 90-95% of the CTC
retained.
[0118] Examples of the effects of different fixation and treatment
conditions are shown in Tables 1-3. Table 1 shows the effects of a
10 minute 10% NBF fixation and a 3 minute methanol fixation on cell
retention subsequent a cytochex (>30 minute) and a 2 minute 0.4%
formalin pre-fixation. The cell retention values were ascertained
by identifying three regions of interest per slide, wherein one
region of interest is a 1 mm.times.1 mm. The number of DAPI stained
nuclei were counted and evaluated for each region of interest at
20.times. magnification. The average and standard deviation is
calculated from the three regions of interest.
[0119] Table 1 shows two of the fixation processes resulted in no
significant cell retention loss. In this case, the loss of cells is
less than the standard deviation of the cell counting process, thus
it is considered insignificant.
TABLE-US-00001 TABLE 1 Effect of fixation on cell retention.
PRE-FIX: Cytochex > 30 min + 0.4% Formalin 2' PRE-FIX: POST-FIX:
POST-FIX: 0.4% Formalin 2' 10% NBF 10' Methanol 3' Methanol 3' Ave
= 5530 5210 4458 4702 Std dev = 967 1114 1763 997
[0120] Table 2 shows two of the fixation processes as the cell loss
is monitored through an IF and FISH procedure with exemplary target
retrieval steps. In this case, the loss of cells is less than the
standard deviation of the cell counting process, thus it is
considered insignificant. The third and fourth lines indicate the
experiment was duplicated on a second slide with the results shown
therein.
TABLE-US-00002 TABLE 2 Cell retention through IF and FISH. PRE-FIX:
Cytochex > 30 min + 0.4% Formalin 2' PRE-FIX: POST-FIX:
POST-FIX: 0.4% Formalin 2' 10% NBF 10' Methanol 3' Methanol 3' Ave
= 5530 5210 4458 4702 Std Dev = 967 1114 1763 997 Ave = 5778 7526
8056 5483 Std Dev = 1300 1632 1685 1704
[0121] Table 3 shows two of the fixation processes as the cell loss
is monitored through an IF and FISH procedure with exemplary target
retrieval steps to monitor the effect of PBST (a phosphate buffered
saline with 0.2% surfactant TWEEN.RTM.-20). In this case, the loss
of cells is less than the standard deviation of the cell counting
process, thus it is considered insignificant.
TABLE-US-00003 TABLE 3 Effect of PBST on cell retention through IF
and FISH. PRE-FIX: Cytochex >30 min + 0.4% Formalin 2' PRE-FIX:
POST-FIX: POST-FIX: 0.4% Formalin 2' 10% NBF 10' Methanol 3'
Methanol 3' PBST+ PBST- PBST+ PBST- PBST+ PBST- PBST+ PBST- Ave
4395 4169 4342 5606 3167 3805 4044 5900 Std Dev 1289 806 1678 297
2056 1500 913 593 Ave 5520 7201 7850 7858 8254 5555 5410 6325 Std
Dev 633 661 2033 477 773 1500 1400 1500
[0122] Thus, in some examples, the CTCs are fixed using a 1-5
minute (such as 1, 2, 3, 4 or 5 minute) formalin pre-fixation (such
as 0.1% to 1% formalin, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, or 1% formalin) followed by a 5-15 minute (such as
5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 minute) NBF fixation (such
as 1% to 20% NBF, for example 1, 2, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, or 20% NBF) and a 1-5 minute methanol fixation (such as 1,
2, 3, 4 or 5 minutes), such as using a 2 minute 0.4% formalin
pre-fixation followed by a 10 minute 10% NBF fixation and a 3
minute methanol fixation.
[0123] E. Target Retrieval
[0124] The method of analyzing a sample containing CTCs using an
automated instrument can include a step of retrieving targets in
the sample. Retrieving targets can include contacting the sample
with a target retrieval reagent. Exemplary target retrieval
reagents for nucleic acid targets (ISH) can include a solution
including ethylenediaminetetraacetic acid (EDTA). Exemplary target
retrieval reagents for protein targets (IHC) can include a cell
conditioning solution such as a boric acid buffer. In one
embodiment, retrieving targets includes contacting the sample with
a tris-based buffer having a slightly basic pH and applying heat so
that covalent bonds resulting from fixation are broken. In another
embodiment, the method includes contacting the sample with a
protease. In another embodiment, the method includes contacting the
sample with a citrate buffer having a slightly acidic pH and
applying heat so that covalent bonds resulting from fixation are
broken. In yet another embodiment, retrieving targets includes
contacting the sample with a tris-based buffer having a slightly
basic pH and applying heat so that covalent bonds resulting from
fixation are broken and contacting the sample with a protease prior
to contacting the sample with CTC identification reagents and the
method further comprises contacting the sample with a citrate
buffer having a slightly acidic pH and applying heat so that
covalent bonds resulting from fixation are broken and contacting
the sample with a protease prior to contacting the sample with CTC
characterization reagents. In one example (for example if the
target is a nucleic acid), the contacting can be done at a
temperature of about 95.degree. C. for between about 2 and about 90
minutes. In one example (for example if the target is a protein),
the contacting can be done at a temperature of about 100.degree. C.
for between about 2 and about 90 minutes. A partial list of
possible reagents appears in Analytical Morphology, Gu, ed., Eaton
Publishing Co. (1997) at pp. 1-40. Sodium dodecyl sulfate (SDS)
and/or ethylene glycol may be included in the conditioning
solution. Furthermore, metal ions or other materials may be added
to these reagents to increase effectiveness of the cell
conditioning. Exemplary cell conditioning solutions are available
from Ventana Medical Systems, Inc., Tucson, Ariz. (Cell
Conditioning 1 (CC1) catalog #: 950-124; Cell Conditioning 2 (CC2)
catalog #: 950-123; SSC (10.times.) catalog #: 950-110; ULTRA Cell
Conditioning (ULTRA CC1) catalog #: 950-224; ULTRA Cell
Conditioning (ULTRA CC2) catalog #: 950-223, Protease 1 catalog #:
760-2018; Protease 2 catalog #: 760-2019; Protease 3 catalog #:
760-2020). In one embodiment, applying the IHC binding reagent or
the in situ hybridization binding reagent occurs subsequent to
applying the cell conditioning reagent and prior to applying the
chromogenic reagent.
[0125] As described herein in association with the fixation and
permeabilization steps, the selection of target retrieval reagents
influences cell retention and target expression. Upon treating a
sample with a target retrieval process that is too aggressive,
significant cell loss from the substrate may occur, the targets may
be denatured so as to be undetectable, or combinations thereof.
Conversely, upon treating a sample with a target retrieval process
that is not adequate, the signals associated with the targets will
not be evident during imaging. Confounding the identification of
appropriate target retrieval steps is that the process is inversely
related to fixation in that greater fixation requires greater
retrieval and thus changes in fixation necessitate redevelopment of
appropriate retrieval processes. Further confounding the
identification of appropriate target retrieval steps is that it was
discovered that protein targets and gene targets routinely require
different retrieval conditions for adequate detection. Since the
present method includes detection of both protein and gene targets,
a retrieval approach that enabled this dual gene-protein analysis
was developed and is disclosed herein. According to one approach,
the proteins are first retrieved. The proteins are then labeled
with a specific binding moiety (e.g. an antibody) prior to
retrieving the gene targets. This approach enables the use of gene
target retrieval reagents that would, without the specific binding
interaction, denature the target to the extent that it could not be
subsequently detected. Conversely, preserving the protein target
would result in the use of gene retrieval reagents that
inadequately expressed the gene targets.
[0126] In illustrative embodiments, a method of analyzing a sample
containing CTCs using an automated instrument includes applying a
rinsing reagent. Between various steps described herein and as part
of the system described herein, rinse steps may be added to remove
unreacted residual reagents from the prior step. Rinse steps may
further include incubations which include maintaining a rinsing
reagent on the sample for a pre-determined time at a pre-determined
temperature with or without mixing. The conditions appropriate for
the rinsing steps may be distinct between the various steps.
Exemplary rinsing reagents are available from Ventana Medical
Systems, Inc., Tucson, Ariz. (Reaction Buffer (10.times.) catalog
#: 950-300; Special Stains Wash (10.times.) catalog #860-015).
[0127] F. Identification of CTCs
[0128] In some examples, the method of analyzing a sample
containing CTCs using an automated instrument includes contacting
the sample with CTC identification reagents using the automated
instrument. CTC identification reagents permit the determination
that a cell is a CTC cell. Exemplary CTC protein markers include
CD45, cytokeratin, ERG, androgen receptor, and PSMA. In one
embodiment, the method includes imaging immunofluorescence of the
CTC identification reagents (such as imaging labeled antibodies
specific for the CTC protein markers). In one embodiment, imaging
the sample includes using multi-spectral bandpass filters. In some
embodiments, the immunofluorescence emanates from antibodies
directly labeled with fluorophores or the immunofluorescence
results from exciting the fluorophores with spectrally filtered
visible light. In another embodiment, the spectrally filtered
visible light includes a first selected range to excite a first
fluorophore and a second selected range to excite a second
fluorophore, wherein the first selected range does not
significantly excite the second fluorophore and the second selected
range does not significantly excite the first fluorophore. One
skilled in the art will appreciate that additional fluorophores can
be excited at the appropriate range, for example if more than two
proteins are to be detected. In another embodiment, imaging the
sample includes acquiring a first immunofluorescence image of the
sample excited by the first selected range and acquiring a second
immunofluorescence image of the sample excited by the second
selected range and locating the CTCs by locating the CTC
identification reagents includes comparing or overlaying the first
immunofluorescence image and the second immunofluorescence image.
One skilled in the art will appreciate that additional images can
be obtained and overlaid, for example if more than two proteins are
to be detected. In one example, imaging the first
immunofluorescence image identifies CK+ cells, and the second
immunofluorescence image identifies CD45+ cells, wherein comparing
or overlaying includes identifying cells that are CK+ and CD45-. In
another embodiment, locating the CTCs by locating the CTC
identification reagents includes algorithmically analyzing the
first immunofluorescence image and the second immunofluorescence
image (or even more images) using a computer. In another
embodiment, algorithmically analyzing includes digitally
interrogating the images to measure cell size, cell compartment
localization of markers, and/or intensity of marker expression. In
one embodiment, spectral imaging the CTCs includes multispectral
imaging.
[0129] The method can include applying a chromogenic reagent so
that the sample is specifically stained. In one embodiment,
specifically staining includes the application of a primary stain
that selectively stains portions of the sample through adhesion
associated with hydrophobicity, intercalation, or other
non-recognition associations. In further illustrative embodiments,
the method includes applying an immunohistochemical (IHC) or
immunofluorescence (IF) binding reagent. As the terms are used
herein, IF and IHC differ only in the manner in which they are
detected. In particular, IF implies the specific interaction is
detected using fluorescence (e.g., using dark-field microscopy) and
IHC implies the specific interaction is detected using chromogens
(e.g., using bright-field microscopy). As used variously herein,
the term "IF" implies "IHC" could be used and vice versa. IHC
includes use of antibodies that specifically bind epitopes of
interest. The epitopes, also referred to as antigens or antigenic
sequences, are portions of proteins that have been established as a
marker of clinical interest. For example, the epitope may be a
mutated form of a protein, a protein-protein binding site, or a
normal protein that is expressed at a concentration either higher
or lower than normal, such as in a control sample. Detection and/or
quantification of epitopes in various biological samples have been
used for a many clinical purposes.
[0130] Both IHC and ISH involve a specific recognition event
between a nucleic acid probe (ISH) or an antibody (IHC) and a
target within the sample. This specific interaction labels the
target. The label can be directly visualized (direct labeling) or
indirectly observed using additional detection chemistries.
Chromogenic detection, which involves the deposition of a
chromogenic substance in the vicinity of the label, involves
further detection steps to amplify the intensity of the signal to
facilitate visualization. Visualization of the amplified signal
(e.g. the use of reporter molecules) allows an observer to localize
targets in the sample.
[0131] Chromogenic detection offers a simple and cost-effective
method of detection. Chromogenic substrates have traditionally
functioned by precipitating when acted on by the appropriate
enzyme. That is, the traditional chromogenic substance is converted
from a soluble reagent into an insoluble, colored precipitate upon
contacting the enzyme. The resulting colored precipitate requires
no special equipment for processing or visualizing. Table 4 is a
non-exhaustive list of chromogen systems useful within the scope of
the present disclosure:
TABLE-US-00004 TABLE 4 Chromogenic detection reagents. Abbr. Name
Color Enzyme DAB 3,3'-diamino-benzidine + H.sub.2O.sub.2 brown-
peroxidase black AEC 3-amino-9-ethyl-carbazole + H.sub.2O.sub.2 red
peroxidase CN 4-chloro-1-naphthol + H.sub.2O.sub.2 blue peroxidase
BCIP/ 5-bromo-4-chloro-3-indolyl- indigo- alkaline NBT phosphate +
nitroblue tetrazolium black phosphatase FAST 4-chloro-2- red
alkaline RED methylbenzenediazonium + phosphatase
3-hydroxy-2-naphthoic acid 2,4- dimethylanilide phosphate FAST
Naphthol AS-MX phosphate blue alkaline BLUE disodium salt + fast
blue BB salt phosphatase hemi(zinc chloride) salt FUCHSIN Naphthol
AS-BI + New Fuchsin red alkaline phosphatase NBT nitroblue
tetrazolium + phenazine blue- dehydrogenase methosulfate purple ALK
3-methyl-1-phenyl-1H-pyrazol-5-yl yellow- alkaline GOLD.dagger.
dihydrogen phosphate + fast gold phosphatase blue BB
[0132] Table 4, while not exhaustive, provides insight into the
varieties of presently available chromogenic substances (see also
WO2012/024185, Kelly et al. "Substrates for Chromogenic detection
and methods of use in detection assays and kits").
[0133] The CTCs on the slide can be identified as those cells that
are CK+ and CD45- (e.g., using pan-CK and CD45 antibodies as
described below). In some examples, the CTCs are also stained with
DAPI.
[0134] G. Characterization of CTCs
[0135] In some examples, the method of analyzing a sample
containing CTCs includes contacting the sample with CTC
characterization reagents using the automated instrument. CTC
characterization reagents are reagents that permit for analysis of
a disorder, such as prognosis or diagnosis of cancer, such as
prostate cancer. In one embodiment, the CTC characterization
reagents include nucleic acid probes directed to genomic markers
(such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genomic markers). The
genomic markers can be analyzed for gene expression and/or genetic
rearrangements/deletions. For example, the genomic markers can
include a gene expression probe and a rearrangement/deletion probe
combination. In a specific embodiment, the genomic markers include
ERG, PTEN, and CEN-10. For example, the nucleic acid probes can be
a 5' ERG probe, a 3' ERG probe, a PTEN probe, and a CEN-10 probe.
In one embodiment, the CTC characterization reagents include
nucleic acid probes directed to genomic markers and the CTC
identification reagents include IHC reagents directed to CTC
protein markers. In a specific embodiment, the genomic markers
include ERG, PTEN, and CEN-10 and the CTC protein markers include
one or more of CD45, CK, ERG, AR, and PSMA (such as 2, 3, 4 or 5 of
these proteins).
[0136] In some embodiments, the methods for analyzing a sample
containing CTCs using an automated instrument includes imaging the
sample. For example, the method can include spectral imaging the
CTCs by location, or spectral imaging luminescence of the CTC
characterization reagents (e.g., the luminescence emanating from
specific binding moieties labeled with quantum dots). The specific
binding moieties can be directly labeled (e.g., with quantum dots),
or are indirectly labeled (e.g., with the quantum dots, the
luminescence emanating from the quantum dots labeling anti-hapten
secondary antibodies, the anti-hapten secondary antibodies being
specific to haptens labeling the specific binding moieties). In one
embodiment, the specific binding moieties are nucleic acid probes
and/or antibodies. In one embodiment, spectral imaging the CTCs
includes exciting the quantum dots with radiation, such as UV or
near-UV radiation. In one embodiment, the radiation has a spectral
emission profile with a maximum between 300 and 400 nm. In another
embodiment, spectral imaging the CTCs includes hyperspectral
imaging. In one embodiment, spectral imaging is guided by the step
of locating the CTCs to regions of interest to the exclusion of
regions devoid of interest. In another embodiment, regions of
interest include CTCs. In another embodiment, the spectrally
filtered visible light does not result in significant quantum dot
luminescence. In yet another embodiment, imaging the sample
includes multi-spectral imaging immunofluorescence of the CTC
identification reagents and spectral imaging the CTCs includes
hyper-spectral imaging the CTC characterization reagents. In one
embodiment, the multi-spectral and hyper-spectral imaging
differentiates at least about four CTC identification reagents
and/or CTC characterization reagents, at least about five CTC
identification reagents and/or CTC characterization reagents, or at
least about six CTC identification reagents and/or CTC
characterization reagents. In a specific embodiment, the
multi-spectral imaging differentiates at least about two CTC
identification reagents and the hyper-spectral imaging
differentiates at least about four CTC characterization
reagents.
[0137] For example, the method may include contacting the CTCs
present on the glass slide (or other substrate) with one or more
nucleic acid probes specific for ERG, PTEN, and CEN-10 (such as 3,
4, or 5 different probes), under conditions sufficient for the
probes to hybridize to their complementary target sequence in the
CTCs (if present), and contacting the cells present on the glass
slide (or other substrate) with one or more antibodies specific for
at least two of CD45, CK, ERG, AR, and PSMA (to confirm that the
cell is a CTC cell, as described herein). The probes can be a
single nucleic acid probe, or a population of several different
nucleic acid probes. The use of probe specific for ERG, PTEN, or
CEN-10 permit detection of alternations in the genomic sequence of
a CTC cell, such as rearrangement of at least one ERG allele,
amplification of at least one ERG allele, fusion of at least one
ERG allele, deletion of at least one PTEN allele, or combinations
thereof. ERG and PTEN can be detected at the genomic level, for
example by detecting alteration(s) in the genomic sequence(s) of
ERG or PTEN, such as detecting a TMPRSS-ERG rearrangement or PTEN
deletion. The use of antibodies specific for two or more of CD45,
CK, ERG, AR, and PSMA permits for the identification of CTC
cells.
[0138] The nucleic acid probes may include quantum dots to permit
detection of the hybridization. For example, the nucleic acid
probes specific for ERG, PTEN, and CEN-10 can each be labeled with
a different quantum dot (or other detectable label), such that the
signal from each quantum dot (or label) is distinguishable. For
example, a 5'-end ERG probe can be labeled with a first quantum dot
(such as a quantum dot that emits at 565 nm), a 3'-end ERG probe
can be labeled with a second quantum dot (such as a quantum dot
that emits at 655 nm), a PTEN probe can be labeled with a third
quantum dot (such as a quantum dot that emits at 605 nm), and a
CEN-10 probe can be labeled with a fourth quantum dot (such as a
quantum dot that emits at 585 nm). Thus, when a nucleic acid probe
hybridizes to its corresponding complementary sequence in the CTCs,
its presence is indicated by emission of a characteristic signal
that indicates hybridization of a particular nucleic acid probe
with a particular target (e.g., ERG, PTEN, or CEN-10).
[0139] Similarly, as noted herein, the antibodies may include
quantum dots or other labels to permit detection of the specific
binding. For example, the antibodies specific for CD45, CK, ERG,
AR, and PSMA can each be labeled with a different quantum dot (or
other detectable label), such that the signal from each quantum dot
(or label) is distinguishable. For example, an antibody specific
for CD45 can be labeled with a first quantum dot, an antibody
specific for CK can be labeled with a second quantum dot, and so
forth. Thus, when an antibody binds to its target protein in the
CTCs, its presence is indicated by emission of a characteristic
signal that indicates specific binding of a particular antibody
with a particular target (e.g., CD45, CK, ERG, AR, or PSMA).
[0140] In one example, the one or more nucleic acid probes specific
for ERG includes two probes or population of probes: one specific
for the 5'-end of ERG and one specific for the 3'-end of ERG. Such
a probe can be referred to as a break-apart probe. For example, if
an ERG rearrangement is absent, the signals from the 5'-end ERG
probe and the 3'-end ERG probe will substantially overlap. For
example, if the 5'-end ERG probe includes a quantum dot that emits
a green signal (e.g., 565 nm) and the 3'-end ERG probe includes a
quantum dot that emits a red signal (e.g., 655 nm), if an ERG
rearrangement is absent the red and green signals will generally
overlap, while if an ERG rearrangement is present the red and green
signals will be substantially separated. Thus, for ERG there are
four measurable events, (1) two co-localized red and green signals
is a normal ERG, (2) one co-localization plus one split of red and
green is an ERG rearrangement through insertion, (3) one
co-localization plus a red signal (no green) is an ERG
rearrangement through 5' deletion, and (4) one co-localization and
multiple red signals is an ERG deletion and amplification). In one
example, the 5'-end ERG probe and the 3'-end ERG probe each include
a population of a plurality of nucleic acid probes, each of which
are at least 50 kb, such as at least 100 kb or at least 150 kb,
such as at least 170 kb. In one example the 5'-end ERG probe covers
about 370 kb at the 5'-end of the ERG gene, and the 3'-end ERG
probe covers about 317 kb at the 3'-end of the ERG gene.
[0141] In one example, the one or more nucleic acid probes specific
for PTEN includes a population of a plurality of nucleic acid
probes, each of which are at least 50 kb, such as at least 100 kb,
at least 150 kb, at least 200 kb, or at least 240 kb, such as about
200 to 300 kb. In particular examples, the PTEN probe does not
specifically hybridize to a PTEN coding region.
[0142] In one example, the one or more nucleic acid probes specific
for CEN-10 is a plasmid probe, such as pA10RP8 (available from
American Type Culture Collection).
[0143] In some examples, the method also includes exposing the CTCs
to ultraviolet light, under conditions sufficient to excite the
quantum dots on the nucleic acid probes that have hybridized to the
nucleic acid targets in the CTCs, resulting in emission of a unique
signal for each type of quantum dot.
[0144] The method also includes detecting signals from the one or
more quantum dots on the one or more nucleic acid probes or
antibodies, for example using spectral imaging or fluorescence
microscopy or both. In one example, signals from 400-700 nm are
acquired at 5 nm increments. In some examples, the signals from the
one or more quantum dots are detected simultaneously or
contemporaneously. Based on the signals detected, a determination
is made as to whether the CTCs obtained from the subject have an
ERG rearrangement, PTEN gene deletion, and whether CEN-10 is
detected. If the CEN-10 probe is not detected, the results are
disregarded. If the CEN-10 probe is detected, the results are
determined. In addition, based on the signals detected, a
determination is made as to whether CD45, CK, ERG, AR, and/or PSMA
protein is present in the cells.
[0145] For example if the CEN-10 probe is detected, but no PTEN
probe signal is detected, this indicates both PTEN genes are
deleted. If the CEN-10 probe is detected, but only one PTEN probe
signal is detected, this indicates one PTEN gene is deleted. If the
CEN-10 probe is detected, and both PTEN probe signals are detected,
this indicates both of the PTEN genes are intact. If the CEN-10
probe is detected, and the 5'-end ERG probe and the 3'-end ERG
probe produce two co-localized signals, this indicates two normal
ERG genes. If the CEN-10 probe is detected, and the 5'-end ERG
probe and the 3'-end ERG probe produce one co-localized signal and
one split of each signal (split of the signal from the 5'-end ERG
probe and the signal from the 3'-end ERG probe) this is an ERG
rearrangement through insertion. If the CEN-10 probe is detected,
and the 5'-end ERG probe and the 3'-end ERG probe produce one
co-localized signal, no signal from the 5'-end ERG probe but a
signal from the 3'-end ERG probe, this is an ERG rearrangement
through a 5' deletion of ERG. If the CEN-10 probe is detected, and
the 5'-end ERG probe and the 3'-end ERG probe produce one
co-localized signal, and multiple signals from the 3'-end ERG
probe, this is an ERG deletion and amplification.
[0146] Based on the determination as to whether one or more ERG
genes are rearranged, and/or whether one or more PTEN genes are
deleted, the prostate cancer is characterized. For example,
characterizing a prostate cancer can include predicting the
likelihood that the prostate cancer will respond to a particular
therapy, such as treatment with a poly-(ADP) ribose polymerase
(PARP) inhibitor (e.g., olaparib, MK4827, and iniparib), treatment
with an agent that blocks the hormone pathway (e.g., abiraterone),
or treatment with radiotherapy; predicting the likelihood of
disease recurrence after prostatectomy; predicting the likelihood
of prostate cancer progression; predicting the likelihood of
prostate cancer metastasis; predicting likelihood survival time; or
combinations thereof. Thus in some example, the method is
prognostic, in that it predicts the likelihood that the prostate
cancer is more or less aggressive, for example the likelihood that
the cancer will progress, metastasize, or recur (for example after
prostatectomy), or the likelihood of survival. In other examples,
the method is diagnostic, in that it determines that the prostate
cancer is more or less aggressive, for example the likelihood that
the cancer will progress, metastasize, or recur (for example after
prostatectomy), or the likelihood of survival (such as likelihood
of surviving at least 1 year, at least 3 years, or at least 5
years).
[0147] For example, the method can be used to predict that the
prostate cancer will respond to a PARP inhibitor when the CTCs have
an ERG rearrangement and/or a PTEN deletion; predict that the
prostate cancer will respond to a AKT or PI3K inhibitors when the
CTCs have a PTEN deletion; predict that the prostate cancer will
not respond to a radiotherapy when the CTCs have an ERG
rearrangement and/or a PTEN deletion; predict that the prostate
cancer will respond to a hormone pathway inhibitor (such as
abiraterone) when the CTCs have an ERG rearrangement and/or a PTEN
deletion; predict that the prostate cancer has a higher likelihood
of recurring after a treatment (such as recurrence within 5 years
of the prostatectomy or other therapy) when the CTCs have an ERG
rearrangement and/or a PTEN deletion; predict that the prostate
cancer has a higher likelihood of progressing when the CTCs have an
ERG rearrangement and/or a PTEN deletion; predict that the prostate
cancer is more likely to metastasize when the CTCs have an ERG
rearrangement and/or a PTEN deletion; predict a survival time of
less than 5 years when the CTCs have an ERG rearrangement and/or a
PTEN deletion; or combinations thereof.
[0148] In some examples, the method can further include detecting
one or more housekeeping molecules in the CTCs (or in a separate
prostate cancer sample), for example using labeled nucleic acid
probes or antibodies specific for such housekeeping molecules. In
some examples, the detected housekeeping molecule is compared
expression of the one or more housekeeping molecules in the CTC or
prostate cancer sample to a control representing expression of the
one or more housekeeping molecules expected in a normal prostate
sample. Housekeeping molecules are known in the art, and include
those whose expression is similar (e.g., varies by less than 10%,
less than 5%, or less than 1%) in a prostate cancer sample relative
to a normal prostate cancer sample. Examples include but are not
limited to: GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA
(succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl
transferase 1), HBS1L (HBS1-like protein), .beta.-actin, and AHSP
(alpha haemoglobin stabilizing protein).
[0149] In illustrative embodiments, the method of analyzing a
sample containing CTCs includes contacting the sample with CTC
characterization reagents that includes a reagent directed towards
one or more housekeeping molecules in the CTCs, wherein analyzing
the sample includes comparing expression of the one or more
housekeeping molecules in the CTCs to a control representing the
one or more housekeeping molecules expected in a normal prostate
sample. In another embodiment, analyzing the sample includes
detecting one or more other prostate cancer-related molecules in
the CTCs and comparing expression of the one or more other prostate
cancer related molecules in the CTCs to a control the one or more
other prostate cancer-related molecules expected in a normal
prostate sample.
[0150] H. Prostate Cancer Probes
[0151] Nucleic acid probes specific for ERG, PTEN, and CEN-10 can
be used in the disclosed methods. Such probes permit one to
determine whether one or more ERG alleles are rearranged (for
example by insertion or 5' deletion) or amplified, whether one or
more PTEN genes are deleted, whether centromere-10 (CEN-10) is
detected, or combinations thereof. The disclosure is not limited to
the use of specific probe sequences. In one example, the assay is
multiplexed so more than one nucleic acid is detected in the same
sample, such as in the same CTC or populations of CTCs (such as
those present on a glass slide or other substrate). For example,
ERG, PTEN, and CEN-10 can be detected in the same CTC (or
population of CTCs on a slide), such as detecting an ERG
rearrangement or amplification, PTEN gene deletion, CEN-10, or
combinations thereof, in the same CTC.
[0152] Thus, after isolating CTCs from a patient, such as one with
CRPC or mCRPC, the CTCs are fixed and then contacted or incubated
with one or more probes specific for ERG, PTEN and CEN-10, and
allowed to hybridize at pertinent temperature, and excess probe is
washed away. In some examples the probes are directly labeled, for
example with a quantum dot, so that the probe's location and
quantity in the cells can be determined (e.g., using spectral
imaging). In some examples, for example if the probe is not
labeled, the method further includes incubation with a labeled
complementary probe (such as labeled with a radioactive,
fluorescent or antigenic tag), so that the probe's location and
quantity in the CTC can be determined.
[0153] Gene alterations in the genome (e.g., gene amplification,
gene deletion, gene fusion, or other chromosomal rearrangements or
chromosome duplications (e.g., polysomy) or loss of one or more
chromosomes) can be determined using any technique known in the
art. Exemplary techniques include, for example, methods based on
hybridization analysis of polynucleotides (e.g., genomic nucleic
acid sequences) as well as methods based on sequencing of
polynucleotides.
[0154] Accordingly, in some embodiments, genomic alterations are
detected, for example by using in situ hybridization of
gene-specific genomic probes. The making of gene-specific genomic
probes is well known in the art (see, e.g., U.S. Pat. Nos.
5,447,841, 5,756,696, 6872817, 6596479, 6500612, 6607877, 6344315,
6475720, 6132961, 7115709, 6280929, 5491224, 5663319, 5776688,
5663319, 5776688, 6277569, 6569626, 7,763,421, U.S. patent
application Ser. No. 11/849,060, and PCT Appl. Nos. PCT/US07/77444
and PCT/US2010/062485). In some exemplary methods, detection of
genomic alterations is facilitated by comparing the number of
binding sites for a gene-specific genomic probe to a control
genomic probe (e.g., a genomic probe specific for the centromere of
the chromosome upon which the gene of interest is located, such as
centromere 10). In some examples, gene deletion may be determined
by the ratio of the gene-specific genomic probe to a control (e.g.,
centromeric) probe. For example, a ratio less than one indicates
deletion of the gene (or the chromosomal region) to which the
gene-specific probe binds.
[0155] In some examples, genomic alterations or regions of a genome
are detected using in situ hybridization techniques, such as
fluorescence in situ hybridization (FISH). In these methods,
specific binding partners, such as probes labeled with a quantum
dot or fluorophore specific for a target gene (e.g., a ERG or PTEN
gene) or region of a chromosome (e.g., a CEN-10) is contacted with
a sample, such as CTCs mounted on a substrate (e.g., glass slide).
The specific binding partners form specific detectable interactions
(e.g., hybridized to) their cognate targets in the sample. For
example, hybridization between the probes and the target nucleic
acid can be detected, for example by detecting a label associated
with the probe. In some examples, spectral imaging or fluorescence
microscopy is used.
[0156] In some examples, the means used to detect genomic
alternations of PTEN and ERG, and the presence of CEN-10, is a
nucleic acid molecule, such as a probe or primer. For example,
nucleic acid probes specific for PTEN, ERG, and CEN-10 can be
obtained from a commercially available source (such as Ventana
Medical Systems (Tucson, Ariz.) and Vysis (IL)) or prepared using
techniques common in the art (such as those described in
PCT/US2010/062485 or U.S. Pat. No. 7,763,421). Nucleic acid probes
and primers are nucleic acid molecules capable of hybridizing with
a target nucleic acid molecule (e.g., genomic target nucleic acid
molecule or centromere). For example, probes specific for PTEN or
ERG, when hybridized to the target, are capable of being detected
either directly or indirectly, for example by a label (such as a
quantum dot) present on the probe. Thus probes and primers permit
the detection, and in some examples quantification, of a target
nucleic acid molecule, such as PTEN, ERG, and CEN-10.
[0157] Probes and primers can "hybridize" to a target nucleic acid
sequence by forming base pairs with complementary regions of the
target nucleic acid molecule (e.g., DNA or RNA, such as genomic
DNA, cDNA or mRNA), thereby forming a duplex molecule.
Hybridization conditions resulting in particular degrees of
stringency will vary depending upon the nature of the hybridization
method and the composition and length of the hybridizing nucleic
acid sequences. Generally, the temperature of hybridization and the
ionic strength (such as the Na.sup.+ concentration) of the
hybridization buffer will determine the stringency of
hybridization. Calculations regarding hybridization conditions for
attaining particular degrees of stringency are discussed in
Sambrook et al., (1989) Molecular Cloning, second edition, Cold
Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). The
following is an exemplary set of hybridization conditions and is
not limiting:
[0158] Very High Stringency (Detects Sequences that Share at Least
90% Identity)
[0159] Hybridization: 5.times. SSC at 65.degree. C. for 16
hours
[0160] Wash twice: 2.times. SSC at room temperature (RT) for 15
minutes each
[0161] Wash twice: 0.5.times. SSC at 65.degree. C. for 20 minutes
each
[0162] High Stringency (Detects Sequences that Share at Least 80%
Identity)
[0163] Hybridization: 5.times.-6.times. SSC at 65.degree.
C.-70.degree. C. for 16-20 hours
[0164] Wash twice: 2.times. SSC at RT for 5-20 minutes each
[0165] Wash twice: 1.times. SSC at 55.degree. C.-70.degree. C. for
30 minutes each
[0166] Low Stringency (Detects Sequences that Share at Least 50%
Identity)
[0167] Hybridization: 6.times. SSC at RT to 55.degree. C. for 16-20
hours
[0168] Wash at least twice: 2.times.-3.times. SSC at RT to
55.degree. C. for 20-30 minutes each.
[0169] One of skill in the art can identify conditions sufficient
for a probe to specifically hybridize to a target gene, such as an
ERG gene, PTEN gene or CEN-10 present in a CTC sample. For example,
one of skill in the art can determine experimentally the features
(such as length, base composition, and degree of complementarity)
that will enable a nucleic acid probe to hybridize to its target
nucleic acid (e.g., an ERG gene, PTEN gene or CEN-10) under
conditions of selected stringency, while minimizing non-specific
hybridization to other substances or molecules. Typically, the
nucleic acid sequence of a probe will have sufficient
complementarity to the corresponding gene to enable it to hybridize
under selected stringent hybridization conditions, for example
hybridization at about 37.degree. C. or higher (such as about
37.degree. C., 42.degree. C., 44.degree. C., 50.degree. C.,
55.degree. C., 60.degree. C., 65.degree. C., 70.degree. C., or
higher, for example at about 37.degree. C. to 70.degree. C.,
37.degree. C. to 50.degree. C., such as 44.degree. C.).
[0170] Methods of generating a probe or primer specific for a
target nucleic acid (e.g., PTEN, ERG, or CEN-10) are routine in the
art. In one example, the nucleic acid probe is a population of
nucleic acid probes, such as a population that includes fragments
of the target sequence (such as fragments of at least 50
nucleotides (nt), at least 75 nt, at least 100 nt, at least 200 nt,
at least 500 nt, such as 50 to 1000 nt or 100 to 500 nt), wherein
the fragments are amplified and then randomly ligated together. For
example the resulting population of probes can include individual
probes that are at least 10 kb, such as at least 20 kb, at least 50
kb, or at least 100 kb, such as 10 to 1000 kb, 10 to 500 kb, or 20
to 200 kb. Thus for example an ERG, PTEN or CEN-10 probe can
include a population of nucleic acid probes that includes fragments
of an ERG gene, PTEN gene or CEN-10 (such as fragments of at least
50 nt, at least 75 nt, at least 100 nt, at least 200 nt, at least
500 nt, such as 50 to 1000 nt or 100 to 500 nt of an ERG gene, PTEN
gene or CEN-10, respectively), and the resulting population of
probes can include individual probes that are at least 10 kb, such
as at least 20 kb, at least 50 kb, or at least 100 kb, such as 10
to 1000 kb, 10 to 500 kb, or 20 to 200 kb. In one example, nucleic
acid probes specific for SEQ ID NOS: 1 or 3, can be specific for at
least 12, at least 50, at least 100, or at least 1000 contiguous
nucleotides of such sequence (or its complementary strand). In some
examples the fragments are sufficiently deleted of repeat
sequences. The probe can be coupled directly or indirectly to a
"label," which renders the probe detectable. For example, labeled
nucleotides can be incorporated into a probe by a variety of
methods including in vitro transcription with SP6, T3 or T7 RNA
polymerase, 3' end labeling with Terminal Deoxynucleotidyl
Transferase (TdT), T4 DNA polymerase or T7 DNA polymerase, random
primed DNA labeling with Klenow fragment, cDNA labeling with AMV or
M-MuLV reverse transcriptase, nick translation labeling with DNAse
1 and DNA Polymerase 1, and PCR labeling with thermophilic DNA
polymerases like Taq or Pfu. In some examples, probes are labeled
using nick translation (using hapten-labeled nucleotides for
example), followed by a labeled anti-hapten antibody, such as those
labeled with a quantum dot. Exemplary hapten-labeled nucleotides
include but are not limited to those that include nitropyrazole
(NP) or tyro sulfonamide (TS).
[0171] In one example the probe is a ISH probe of at least 1000 bp,
such as at least 2000, at least 3000, at least 4000, at least 5000,
or at least 6000, such as 1000 to 6000 bp, that covers from about
300 kb to 800 kb.
[0172] In one example, the one or more nucleic acid probes specific
for ERG includes two probes or population of probes: one specific
for the 5'-end of ERG and one specific for the 3'-end of ERG. Such
a probe can be referred to as a break-apart probe. In one example,
the 5'-end ERG probe and the 3'-end ERG probe each include a
population of a plurality of nucleic acid probes. In one example,
the population of probes for the 5'-end ERG probe are each about at
least 50 kb, such as at least 100 kb, at least 150 kb, such as at
least 188 kb. In one example the 5'-end ERG probe covers about
370.38 kb at the 5'-end of the ERG gene. In one example the 5'-end
ERG probe is labeled using digoxygenin-nucleotides specifically
bound to labeled anti-hapten antibodies (labeled with quantum dot
565). In one example, the population of probes for the 3'-end ERG
probe are each about at least 50 kb, such as at least 100 kb, at
least 150 kb, such as at least 177 kb. In one example the 3'-end
ERG probe covers about 317.176 kb at the 3'-end of the ERG gene. In
one example the 3'-end ERG probe is labeled using 2,4-dinitrophenyl
(DNP)-nucleotides specifically bound to labeled anti-hapten
antibodies (labeled with quantum dot 655).
[0173] In one example, a nucleic acid probe specific for PTEN
includes a population of PTEN genomic fragments that cover about
765 kb in the area of chromosome region 10q23.31. In one example,
the one or more nucleic acid probes specific for PTEN includes a
population of a plurality of nucleic acid probes, each of which are
at least 50 kb, such as at least 100 kb, at least 150 kb, at least
200 kb, or at least 240 kb, such as about 200 to 300 kb. In
particular examples, the PTEN probe does not specifically hybridize
to a PTEN coding region (and thus the probe is not complementary to
a PTEN coding sequence). In one example the PTEN probe is labeled
using hapten tyro sulfonimide-nucleotides specifically bound to
labeled anti-hapten antibodies (labeled with quantum dot 605).
[0174] In one example, a nucleic acid probe specific for CEN-10
includes a population of CEN-10 genomic fragments that cover about
1.36 kb in the area of chromosome region 10q11.1-q.11.1. In one
example, the nucleic acid probe specific for CEN-10 is a plasmid
probe, such as pA10RP8 (available from American Type Culture
Collection). In one example the CEN-10 probe is labeled using
hapten nitropyrazole labeled-nucleotides specifically bound to
labeled anti-hapten antibodies (labeled with quantum dot 585). SEQ
ID NO: 5 shows an exemplary CEN-10 probe that can be used to
confirm a PTEN deletion (if PTEN is intact 2 pairs of CEN-10 and
PTEN are expected on chromosome 10; if PTEN is heterozygous, 1 pair
of PTEN/CEN-10 and a single CEN-10 on the other copy of chromosome
10 is expected; if PTEN is homozygous only 2 single CEN-10
signals--one on each arm of chromosome 10).
[0175] I. Detectable Labels
[0176] Detectable labels suitable for the methods provided herein
include those detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels include biotin for staining with labeled streptavidin
conjugate, magnetic beads (for example DYNABEADS.TM.), fluorescent
dyes (for example, fluorescein, Texas red, rhodamine, green
fluorescent protein, and the like), quantum dots, radiolabels (for
example, .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P),
enzymes (for example, horseradish peroxidase, alkaline phosphatase
and others commonly used in an ELISA), and colorimetric labels such
as colloidal gold or colored glass or plastic (for example,
polystyrene, polypropylene, latex, etc.) beads. Means of detecting
such labels are also well known. Thus, for example, radiolabels may
be detected using photographic film or scintillation counters,
fluorescent markers may be detected using a photodetector to detect
emitted light. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
[0177] In a specific example, the label is a nanoparticle, such as
a gold particle or a semiconductor nanocrystal). Chromogenic and/or
fluorescent semiconductor nanocrystals, also often referred to as
quantum dots, can be used as detectable labels. Nanocrystalline
quantum dots are semiconductor nanocrystalline particles, and
without limiting the present disclosure to use with particle light
emitters of a particular size, typically range from 2-10 nm in
size. Quantum dots typically are stable fluorophores, often are
resistant to photo bleaching, and have a wide range of excitation
wavelengths with a narrow emission spectrum. Quantum dots having
particular emission characteristics, such as emissions at
particular wavelengths, can be selected such that plural different
quantum dots having plural different emission characteristics can
be used to identify plural different targets. Quantum dot
bioconjugates are characterized by quantum yields comparable to the
brightest traditional fluorescent dyes available. Additionally,
these quantum dot-based fluorophores absorb 10-1000 times more
light than traditional fluorescent dyes. Emission from the quantum
dots is narrow and symmetric, which means that overlap with other
colors is minimized, resulting in minimal bleed-through into
adjacent detection channels and attenuated crosstalk, which can
lead to the simultaneous multiplexing of differentially emitting
quantum dots for detection purposes. Symmetrical and tunable
emission spectra can be varied according to the size and material
composition of the particles, which allows flexible and close
spacing of different quantum dots without substantial spectral
overlap. In addition, their absorption spectra are broad, which
makes it possible to excite all quantum dot color variants
simultaneously using a single excitation wavelength, thereby
minimizing sample autofluorescence. Furthermore, it has been found
that pegylation, the introduction of polyethylene glycol groups
onto the quantum dot conduits, can substantially decrease
non-specific protein:quantum dot interaction. Certain quantum dots
are commercially available, such as Qdot.TM. products from Life
Technologies, Inc.
[0178] Exemplary working embodiments utilize quantum dot
nanoparticles, such as Qdot.TM. 565 and Qdot.TM. 655 nanocrystals,
where the number used in such nomenclature refers to the
approximate wavelength of the nanoparticle's emission maximum. For
example, a Qdot.TM. 565 nanocrystal emits light having a wavelength
of 565 nm and produces a light-green color. Thus, quantum dots can
be selected to provide a detectable signal at a particular
wavelength. Detection is performed through a variety of means, for
example a fluorescent microscope, fluorometer, fluorescent scanner,
etc., depending on a given application.
[0179] For example, quantum dot fluorescent immunohistochemical
analysis can be performed with nucleic acid probes. Image analysis
can be performed by initially capturing image cubes on a spectral
imaging camera. Excitation can be conducted with a UV (mercury)
light source. The image cubes can then be analyzed. Briefly, image
cubes can be retrieved in the application and data can be extracted
and reported based on the pixel intensities of quantum dots
expected to emit at the wavelength specific for the quantum dots
used on the probes (such as 585 nm, 565 nm, 605 nm and 655 nm).
[0180] As an example, fluorescence can be measured with the
multispectral imaging system (such as those available from Ventana,
Tucson, Ariz.; Nuance.TM., Cambridge Research &
Instrumentation, Woburn, Mass.; or SpectrView.TM., Applied Spectral
Imaging, Vista, Calif.). Multispectral imaging is a technique in
which spectroscopic information at each pixel of an image is
gathered and the resulting data analyzed with spectral
image-processing software. For example, a series of images at
different wavelengths (such as 5 nm segments from 400 to 700 nm)
can be obtained that are electronically and continuously selectable
and then utilized with an analysis program designed for handling
such data. Quantitative information from multiple labels can be
obtained simultaneously, even when the spectra of the dyes are
highly overlapping or when they are co-localized, or occurring at
the same point in the sample, provided that the spectral curves are
different. Many biological materials autofluoresce, or emit
lower-energy light when excited by higher-energy light. This signal
can result in lower contrast images and data. High-sensitivity
cameras without multispectral imaging capability only increase the
autofluorescence signal along with the fluorescence signal.
Multispectral imaging can unmix, or separate out, autofluorescence
from tissue and, thereby, increase the achievable signal-to-noise
ratio.
[0181] A variety of commercially available peripheral equipment and
software is available for digitizing, storing and analyzing a
digitized video or digitized optical image, e.g., using PC
(Intelx86 or Pentium chip-compatible DOS.TM., OS2.TM. WINDOWS.TM.,
WINDOWS NT.TM. or WINDOWS7.TM. based computers), MACINTOSH.TM., or
UNIX based (for example, a SUN.TM., a SGI.TM., or other work
station) computers.
[0182] J. Detection Outputs
[0183] Following the detection of ERG, PTEN and CEN-10 probes,
whether ERG and PTEN genomic alterations are present and whether
CEN-10 was detected, as well as the assay results, findings,
diagnoses, predictions and/or treatment recommendations can be
recorded and communicated to technicians, physicians and/or
patients, for example. In certain embodiments, computers will be
used to communicate such information to interested parties, such
as, patients and/or the attending physicians. For example, the
results can be displayed to a monitor, printed, or stored in
memory. Based on the measurement, the therapy administered to a
subject can be modified.
[0184] In one embodiment, a diagnosis, prediction and/or treatment
recommendation based on whether ERG is rearranged or PTEN is
deleted (or in combination with other diagnostics, such as those on
a prostate cancer nomogram, such as PSA value and Gleason grade) is
communicated to interested parties as soon as possible after the
assay is completed and the diagnosis and/or prediction is
generated. The results and/or related information may be
communicated to the subject by the subject's treating physician.
Alternatively, the results may be communicated directly to
interested parties by any means of communication, including
writing, such as by providing a written report, electronic forms of
communication, such as email, or telephone. Communication may be
facilitated by use of a computer, such as in case of email
communications. In certain embodiments, the communication
containing results of a diagnostic test and/or conclusions drawn
from and/or treatment recommendations based on the test, may be
generated and delivered automatically to interested parties using a
combination of computer hardware and software which will be
familiar to artisans skilled in telecommunications. One example of
a healthcare-oriented communications system is described in U.S.
Pat. No. 6,283,761; however, the present disclosure is not limited
to methods which utilize this particular communications system. In
certain embodiments of the methods of the disclosure, all or some
of the method steps, including the assaying of CTCs, diagnosing or
prognosis of prostate cancer, and communicating of assay results or
diagnoses or prognosis, may be carried out in diverse (e.g.,
foreign) jurisdictions.
[0185] 1. Detection
[0186] Any suitable method can be used to detect the probes that
have hybridized to their genomic target. In some examples, the
probes include a detectable label and detecting the presence of the
probe(s) includes detecting the detectable label. In some examples,
the probes are labeled with different detectable labels. For
example, a 5'-ERG probe can include a first label, a 3'-ERG probe
can include a second label, a PTEN probe can include a third label,
and a CEN-10 probe can include a fourth label. In a specific
example, a 5'-ERG probe can include a first quantum dot (such as
one that emits at 565 nm), a 3'-ERG probe can include a second
quantum dot (such as one that emits at 655 nm), a PTEN probe can
include a third quantum dot (such as one that emits at 605 nm), and
a CEN-10 probe can include a fourth quantum dot (such as one that
emits at 585 nm). In other examples, the probes are detected
indirectly, for example by hybridization with a labeled nucleic
acid. Thus for example, by examining images of CTCs incubated with
such probes, one can determine which colored dots are present
(wherein each color is specific for a particular probe) and
determine the arrangement of such dots, thereby permitting for a
determination as to whether one or more ERGs are rearranged or
amplified, one or more PTEN genes is deleted, or combinations
thereof.
[0187] K. Prostate Cancer Biomarkers
[0188] 1. Ets Related Gene (ERG) (OMIM: 165080)
[0189] The human Ets related gene (ERG) (also known as erg-3 and
p55) is located on chromosome 21 (21q22.3) and is a member of the
ETS family of transcription factors. ERG sequences are publically
available, for example from GenBank.RTM. (e.g., accession numbers
NP.sub.--001129626 and NP 598420.1 (proteins) and NM 133659.2,
NM.sub.--001136154.1, and NM.sub.--001838708.2 (nucleic
acids)).
[0190] ERG protein (see, e.g., SEQ ID NO: 2) is a transcriptional
regulator that binds purine-rich sequences. Fusion of the ERG gene
with other genes has been shown to be associated with different
cancers. For example, the t (16; 21) (p11; q22) translocation of
the ERG gene fused with the fused in sarcoma (FUS) gene is
associated with human myeloid leukemia. EWS-ERG fusions are
associated with the Ewing family of tumors. ERG fusion with the 5'
untranslated region of transmembrane protease, serine 2 (TMPRSS2)
(located on human chromosome 21) are associated with prostate
cancer. The TMPRS22 and ERG genes are arranged tandemly on
chromosome 21q22. The TMPRSS2/ERG fusion joins TMPRSS2 exons 1 or 2
usually to ERG exons 2, 3 or 4, which results in activation of the
ERG transcription factor. TMPRSS/ERG rearrangements occur in about
50% of prostate cancers and 20% of high-grade prostatic
intraepithelial neoplasia (HGPIN) lesions, resulting in
upregulation of ERG. TMPRSS/ERG rearrangement results in a PIN like
lesion which can be converted to an invasive state by up-regulation
of the PI3K pathway.
[0191] ERG antibodies are publicly available, for example from
Ventana (Epitomics EPR 3864) and Biocare (clone 9F4).
[0192] 2. Phosphatase and Tensin Homolog (PTEN) (OMIM 601728)
[0193] The human PTEN gene is located on chromosome 10 (10q23.31)
and the mouse PTEN gene is located on chromosome 19. PTEN sequences
(both wild-type and mutant) are publically available, for example
from GenBank.RTM. (e.g., accession numbers NP.sub.--000305.3,
AAD13528.1, EAW50174.1, EAW50173.1, EAW50172.1, AAH05821.1 and
NP.sub.--032986.1 (proteins) and NM.sub.--000314.4 and
NM.sub.--008960.2 (nucleic acids)).
[0194] PTEN, also referred to as MMAC (mutated in multiple advanced
cancers) phosphatase, is a tumor suppressor gene implicated in a
wide variety of human cancers. The PTEN protein (e.g., SEQ ID NO:
4) is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase,
which includes a tensin-like domain as well as a catalytic domain.
Unlike most protein tyrosine phosphatases, PTEN preferentially
dephosphorylates phosphoinositide substrates. PTEN negatively
regulates intracellular levels of
phosphatidylinositol-3,4,5-trisphosphate in cells and functions as
a tumor suppressor by negatively regulating Akt/PKB signaling
pathway. Mutations and deletions of PTEN have been shown to be
associated with cancers.
[0195] PTEN antibodies are publicly available, for example from
Santa Cruz Biotechnology, Inc. (catalog numbers sc-7974; sc-133197;
sc-133242) and Cell Signaling Technology (clone 138G6).
[0196] 3. Variant Sequences
[0197] In addition to the specific sequences provided herein (e.g.,
SEQ ID NOS: 1-4), and the sequences which are currently publically
available for ERG, PTEN, CEN-10, EpCAM, CD45, and CK, one skilled
in the art will appreciate that variants of such sequences may be
present in a particular subject. For example, polymorphisms for a
particular gene or protein may be present. In addition, a sequence
may vary between different organisms. In particular examples, a
variant sequence retains the biological activity of its
corresponding native sequence. For example, a ERG, PTEN, EpCAM,
CD45, or CK gene sequence present in a particular subject may
encode conservative amino acid changes (such as, very highly
conserved substitutions, highly conserved substitutions or
conserved substitutions), such as 1 to 5 or 1 to 10 conservative
amino acid substitutions. Exemplary conservative amino acid
substitutions are shown in Table 5.
TABLE-US-00005 TABLE 5 Exemplary conservative amino acid
substitutions. Highly Conserved Very Highly- Substitutions
Conserved Substitutions Original Conserved (from the (from the
Residue Substitutions Blosum90 Matrix) Blosum65 Matrix) Ala Ser
Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg Lys Gln, His, Lys Asn,
Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Arg, Asp, Gln, Glu,
His, Lys, Ser, Thr Lys, Ser, Thr Asp Glu Asn, Glu Asn, Gln, Glu,
Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, Arg, Asn, Asp, Glu,
His, His, Lys, Met Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn,
Asp, Gln, His, Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn,
Gln, Tyr Arg, Asn, Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu,
Met, Phe, Val Leu Ile; Val Ile, Met, Phe, Val Ile, Met, Phe, Val
Lys Arg; Gln; Glu Arg, Asn, Gln, Glu Arg, Asn, Gln, Glu, Ser, Met
Leu; Ile Gln, Ile, Leu, Val Gln, Ile, Leu, Phe, Val Phe Met; Leu;
Tyr Leu, Trp, Tyr Ile, Leu, Met, Trp, Tyr Ser Thr Ala, Asn, Thr
Ala, Asn, Asp, Gln, Glu, Gly, Lys, Thr Thr Ser Ala, Asn, Ser Ala,
Asn, Ser, Val Trp Tyr Phe, Tyr Phe, Tyr Tyr Trp; Phe His, Phe, Trp
His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala, Ile, Leu, Met,
Thr
[0198] In some embodiments, an ERG, PTEN, EpCAM, CD45, or CK
sequence is a sequence variant of a native ERG, PTEN, EpCAM, CD45,
or CK sequence, respectively, such as a nucleic acid or protein
sequence that has at least 99%, at least 98%, at least 95%, at
least 92%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 65%, or at least 60% sequence identity to
the sequences set forth in SEQ ID NOS: 1-4 (or such amount of
sequence identity to a GenBank.RTM. accession number referred to
herein) wherein the resulting variant retains ERG, PTEN, EpCAM,
CD45, or CK biological activity. "Sequence identity" is a phrase
commonly used to describe the similarity between two amino acid or
nucleic acid sequences). Sequence identity typically is expressed
in terms of percentage identity; the higher the percentage, the
more similar the two sequences.
[0199] Methods for aligning sequences for comparison and
determining sequence identity are well known in the art. Various
programs and alignment algorithms are described in: Smith and
Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J.
Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad.
Sci. USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988;
Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al., Nucleic
Acids Research, 16:10881-10890, 1988; Huang, et al., Computer
Applications in the Biosciences, 8:155-165, 1992; Pearson et al.,
Methods in Molecular Biology, 24:307-331, 1994; Tatiana et al.,
FEMS Microbiol. Lett., 174:247-250, 1999. Altschul et al. present a
detailed consideration of sequence-alignment methods and homology
calculations (J. Mol. Biol., 215:403-410, 1990).
[0200] The National Center for Biotechnology Information (NCBI)
Basic Local Alignment Search Tool (BLAST.TM., Altschul et al., J.
Mol. Biol., 215:403-410, 1990) is publicly available from several
sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the Internet, for use in
connection with the sequence-analysis programs blastp, blastn,
blastx, tblastn and tblastx. A description of how to determine
sequence identity using this program is available on the internet
under the help section for BLAST.TM..
[0201] For comparisons of amino acid sequences of greater than
about 15 amino acids, the "Blast 2 sequences" function of the
BLAST.TM. (Blastp) program is employed using the default BLOSUM62
matrix set to default parameters (cost to open a gap [default=5];
cost to extend a gap [default=2]; penalty for a mismatch
[default=3]; reward for a match [default=1]; expectation value (E)
[default=10.0]; word size [default=3]; and number of one-line
descriptions (V) [default=100]. When aligning short peptides (fewer
than around 15 amino acids), the alignment should be performed
using the Blast 2 sequences function "Search for short nearly exact
matches" employing the PAM30 matrix set to default parameters
(expect threshold=20000, word size=2, gap costs: existence=9 and
extension=1) using composition-based statistics.
[0202] L. Characterizing a Prostate Cancer
[0203] This disclosure demonstrates that ERG rearrangements (such
as via insertions or deletions and/or amplifications), PTEN
deletions, or combinations thereof can be detected in the same CTC,
for example simultaneously. Based on these results, the disclosed
methods can be used to prognose and diagnose prostate cancer (such
as CRPC or mCRPC) based on whether CTCs isolated from such patients
have one or more ERG rearrangements and PTEN deletions. Based on
the determination as to whether one or more ERG genes are
rearranged, and/or whether one or more PTEN genes are deleted, the
prostate cancer is characterized. In one example, a diagnosis or
prognosis that the cancer is more aggressive indicates that the
prostate cancer is predicted to recur within 1 year, within 3 years
or within 5 years, for example within such time of a prostatectomy.
In one example, a diagnosis or prognosis that the cancer is more
aggressive indicates that the prostate cancer is predicted to
metastasize within 1 year, within 3 years or within 5 years, for
example within such time of a prostatectomy. In one example, a
diagnosis or prognosis that the cancer is more aggressive indicates
that the patient is expected to die from the prostate cancer within
1 year, within 3 years or within 5 years, for example within such
time of a prostatectomy.
[0204] In one example, a diagnosis or prognosis that the cancer is
less aggressive (e.g., ERG is not rearranged PTEN is not deleted)
indicates that the prostate cancer is predicted to not recur within
1 year, within 3 years or within 5 years, for example within such
time of a prostatectomy. In one example, a diagnosis or prognosis
that the cancer is less aggressive indicates that the prostate
cancer is not predicted to metastasize within 1 year, within 3
years or within 5 years, for example within such time of a
prostatectomy. In one example, a diagnosis or prognosis that the
cancer is less aggressive indicates that the patient is not
expected to die from the prostate cancer within 1 year, within 3
years or within 5 years, for example within such time of a
prostatectomy.
[0205] In one example, the method includes predicting the
likelihood that the prostate cancer will respond to a particular
therapy, such as treatment with inhibitors of the enzyme poly-(ADP)
ribose polymerase (PARP). Exemplary PARP inhibitors, include, but
are not limited to, olaparib, Rucaparib, Veliparib, CEP 9722,
BMN-673, 3-aminobenzamide, MK4827, and Iniparib. If one or more ERG
genes are rearranged, and/or one or more PTEN genes are deleted,
(such as presence of ERG gene rearrangement and PTEN deletion) it
is predicted that the prostate cancer (such as CRPC or mCRPC) will
respond to PARP inhibitors. In contrast, if the ERG genes are
normal (e.g., not rearranged), and/or the PTEN genes are intact
(not deleted), (such as an absence of ERG gene rearrangements and
PTEN deletions) it is predicted that the prostate cancer (such as
CRPC or mCRPC) will not respond to PARP inhibitors.
[0206] In one example, the method includes predicting the
likelihood that the prostate cancer will respond to a treatment
with inhibitors of the hormonal pathway, such as androgen receptor
inhibitors (or inhibitors of the AR pathway such as a CYP17A1
inhibitor). Examples of such inhibitors include, but are not
limited to: MDV3100
(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimid-
azolidin-1-yl)-2-fluoro-N-methylbenzamide), abiraterone (inhibitor
of CYP17A1), TAK-700 (Orteronel,
6-(7-Hydroxy-6,7-dihydro-5H-pyrrolo[1,2-d]midazol-7-yl)-N-methylnaphthale-
ne-2-carboxamide; inhibitor of CYP17A1), and TOK-001 (Galeterone,
inhibitor of CYP17A1). If one or more ERG genes are rearranged,
and/or one or more PTEN genes are deleted, such as presence of ERG
gene rearrangement and PTEN deletion, it is predicted that the
prostate cancer (such as CRPC or mCRPC) will respond to such
inhibitors. In contrast, if the ERG genes are normal (e.g., not
rearranged), and/or the PTEN genes are intact (not deleted), such
as absence of ERG gene rearrangements and PTEN deletions, it is
predicted that the prostate cancer (such as CRPC or mCRPC) will not
respond to PARP inhibitors.
[0207] In one example, the method includes predicting the
likelihood that the prostate cancer will respond to a treatment
with radiotherapy. Examples of radiotherapy include, but are not
limited to: electron beam radiation therapy (EBRT), 3-dimensional
conformal radiation therapy (3D-CRT), intensity modulated radiation
(IMRT), proton beam radio therapy, and radio-bone therapy with
radium 223. If one or more ERG genes are rearranged, and/or one or
more PTEN genes are deleted, such as presence of ERG gene
rearrangement and PTEN deletion, it is predicted that the prostate
cancer (such as CRPC or mCRPC) will not respond to radiotherapy. In
contrast, if the ERG genes are normal (e.g., not rearranged),
and/or the PTEN genes are intact (not deleted), such as presence of
normal ERG and PTEN genes, it is predicted that the prostate cancer
(such as CRPC or mCRPC) will respond to radiotherapy.
[0208] In one example, the method includes predicting the
likelihood that the prostate cancer will respond to a treatment
with immunotherapy. Examples of immunotherapy include, but are not
limited to: Ipilimumab (an CTLA-4 antibody) and Sipuleucel-T. If
one or more ERG genes are rearranged, and/or one or more PTEN genes
are deleted, it is predicted that the prostate cancer (such as CRPC
or mCRPC) will not respond to immunotherapy, such as presence of
ERG gene rearrangement and PTEN deletion. In contrast, if the ERG
genes are normal (e.g., not rearranged), and/or the PTEN genes are
intact (not deleted), such as presence of normal ERG and PTEN
genes, it is predicted that the prostate cancer (such as CRPC or
mCRPC) will respond to immunotherapy.
[0209] In one example, the method includes predicting the
likelihood that the prostate cancer will respond to a treatment
with chemotherapy. Examples of chemotherapy include, but are not
limited to: docetaxel and Cabazitaxel. If one or more PTEN genes
are deleted, it is predicted that the prostate cancer (such as CRPC
or mCRPC) will not respond to chemotherapy. In contrast, if the ERG
genes are rearranged or normal and/or the PTEN genes are intact
(not deleted), such as presence of normal ERG and PTEN genes, it is
predicted that the prostate cancer (such as CRPC or mCRPC) will
respond to chemotherapy.
[0210] In one example, the method includes predicting the
likelihood that the prostate cancer will respond to a treatment
with an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/AKT
pathway. Examples of inhibitors of the PI3K/AKT pathway include,
but are not limited to: XL147 (Exelixis), BEZ235 (Novartis),
GDG0941 (Genentech), isoformselective AKT catalytic-domain
inhibitors, inhibitors of the PH domain, perifosine, G5K690693
(GlaxoSmithKline), MK2206 (Merck, Inc), PI-103, XL765, and BEZ-235.
If one or more PTEN genes are deleted, it is predicted that the
prostate cancer (such as CRPC or mCRPC) will respond to inhibitors
of the PI3K/AKT pathway. In contrast, if the PTEN genes are intact
(not deleted), it is predicted that the prostate cancer (such as
CRPC or mCRPC) will not respond to inhibitors of the PI3K/AKT
pathway.
[0211] In one example, the method includes predicting the
likelihood of disease recurrence. Recurrence means the prostate
cancer has returned after an initial (or subsequent) treatment(s),
for example recurrence within 1, 2, 3, 4, or 5 years of the
treatment). Representative initial treatments include radiation
treatment, chemotherapy, anti-hormone treatment, surgery (e.g.,
prostatectomy), immune therapy, focal therapy, cryotherapy, and/or
radiotherapy. If one or more ERG genes are rearranged, and/or one
or more PTEN genes are deleted, such as presence of ERG gene
rearrangement and PTEN deletion, it is predicted that the prostate
cancer (such as CRPC or mCRPC) will likely recur after treatment.
Thus for example, the methods can be used to predict a higher
likelihood that an aggressive treatment (e.g., prostatectomy) will
fail, and an increased need for a non-surgical or alternate
treatment for the prostate cancer. In contrast, if the ERG genes
are normal (e.g., not rearranged), and/or the PTEN genes are intact
(not deleted), such as presence of normal ERG and PTEN genes, it is
predicted that the prostate cancer (such as CRPC or mCRPC) will not
likely recur after treatment. Typically after an initial prostate
cancer treatment PSA levels in the blood decrease to a stable and
low level and, in some instances, eventually become almost
undetectable. In some examples, recurrence of the prostate cancer
is marked by rising PSA levels (e.g., greater than 2.0-2.5 ng/mL)
and/or by identification of prostate cancer cells in the blood,
prostate biopsy or aspirate, in lymph nodes (e.g., in the pelvis or
elsewhere) or at a metastatic site (e.g., muscles that help control
urination, the rectum, the wall of the pelvis, in bones or other
organs). Serum PSA levels may be characterized as follows (although
some variation of the following ranges is common in the art):
TABLE-US-00006 Normal Range 0 to 2.5 ng/mL Slightly to Moderately
Elevated 2.6 to 10 ng/mL Moderately Elevated 10 to 19.9 ng/mL
Significantly Elevated 20 ng/mL or more
[0212] In one example, the method includes predicting the
likelihood of prostate cancer progression, such as metastasis.
Prostate cancer progression means that one or more indices of
prostate cancer (e.g., serum PSA levels) show that the disease is
advancing independent of treatment. In some examples, prostate
cancer progression is marked by rising PSA levels (e.g., greater
than 2.0-2.5 ng/mL) and/or by identification of (or increasing
numbers of) prostate cancer cells in the blood, prostate biopsy or
aspirate, in lymph nodes (e.g., in the pelvis or elsewhere) or at a
metastatic site (e.g., muscles that help control urination, the
rectum, the wall of the pelvis, in bones or other organs). If one
or more ERG genes are rearranged, and/or one or more PTEN genes are
deleted, such as presence of ERG gene rearrangement and PTEN
deletion, it is predicted that the prostate cancer (such as CRPC or
mCRPC) will likely progress or metastasize. An increased likelihood
of prostate cancer progression or prostate cancer recurrence can be
quantified by any known metric. For example, an increased
likelihood can mean at least a 10% chance of occurring (such as at
least a 25% chance, at least a 50% chance, at least a 60% chance,
at least a 75% chance or even greater than an 80% chance of
occurring). An increased likelihood of prostate cancer progression
or prostate cancer recurrence can indicate the presence of a
more-aggressive prostate cancer, which may indicate a worse
prognosis for the patient (e.g., decreased survival time), an
increased likelihood of disease progression (e.g., metastasis),
failure (or inadequacy) of treatment, and/or a need for alternative
(or additional) treatments. In contrast, if the ERG genes are
normal (e.g., not rearranged), and/or the PTEN genes are intact
(not deleted), such as presence of normal ERG and PTEN genes, it is
predicted that the prostate cancer (such as CRPC or mCRPC) will not
likely progress or metastasize. A decreased likelihood of prostate
cancer progression or prostate cancer recurrence can be quantified
by any known metric. For example, a decreased likelihood can mean
less than a 50% chance of recurring (such as less than a 25%
chance, less than a 20% chance, less than a 10% chance, less than a
5% chance or even less than a 1% chance of recurring). This can
indicate the presence of a less- or non-aggressive prostate cancer,
which may indicate a better prognosis for the patient (e.g.,
increased survival time), a decreased likelihood of disease
progression (e.g., metastasis), success of treatment, and/or a need
for less aggressive (or even no) treatments.
[0213] In one example, the method includes predicting the
likelihood of survival time. If one or more ERG genes are
rearranged, and/or one or more PTEN genes are deleted, such as
presence of ERG gene rearrangement and PTEN deletion, it is
predicted that the patient with prostate cancer (such as CRPC or
mCRPC) will have a shorter survival time (such as less than 5
years, less than 4 years, less than 3 years, less than 2 years, or
less than 1 year), and thus a poor prognosis. A poor (or poorer)
prognosis is likely for a subject with a more aggressive cancer. In
some method embodiments, a poor prognosis is less than 5 year
survival (such as less than 1 year survival or less than 2 year
survival) of the patient after initial diagnosis of the prostate
cancer. In contrast, if the ERG genes are normal (e.g., not
rearranged), and/or the PTEN genes are intact (not deleted), (such
as both ERG genes are normal and both PTEN genes are intact), it is
predicted that the patient with prostate cancer (such as CRPC or
mCRPC) will have a longer survival time (such as at least 5 years,
at least 6 years, at least 7 years, at least 8 years, at least 9
years, or at least 10 years), and thus a good prognosis. In some
method embodiments, a good prognosis is greater than 2-year
survival (such as greater than 3-year survival, greater than 5-year
survival, or greater than 7-year survival) of the patient after
initial diagnosis of the prostate cancer.
[0214] In some examples, the disclosed methods can be used to
identify those subjects that will benefit from a more or less
aggressive therapy. For example, if a patient is diagnosed or
prognosed with an aggressive form of prostate cancer (one or more
ERG genes are rearranged, and/or one or more PTEN genes are
deleted), the patient can be selected for more aggressive treatment
and frequent monitoring. By contrast, if a patient is diagnosed or
prognosed with a less aggressive form of prostate cancer (ERG and
PTEN genes are normal), the patient can be selected for less
aggressive treatment and/or less frequent monitoring. For example,
such diagnostic or prognostic methods can be performed prior to the
subject undergoing treatment. In other examples, these methods are
utilized to predict subject survival or the efficacy of a given
treatment, or combinations thereof. Thus, the methods of the
present disclosure are valuable tools for practicing physicians to
make quick treatment decisions regarding how to treat prostate
cancer, such as CRPC or mCRPC. These treatment decisions can
include the administration of an agent for treating prostate cancer
and decisions to monitor a subject for recurrence or metastasis of
a prostate cancer.
[0215] M. Use in Combination with Other Diagnostic and Prognostic
Assays
[0216] The disclosed methods can be used in combination with one or
more other assays that are used to diagnose or prognose prostate
cancer outcomes. For example, a prostate cancer patient's Gleason
scores based on a histopathological review, PSA scores, and
nomograms (such as the Partin Coefficient Tables) can be used in
combination with the disclosed methods to allow for enhanced
diagnostic and prognostic capabilities of prostate cancer, such as
those that have metastasized.
[0217] For example, prostate cancer nomograms can be used to
predict the probability that a patient's cancer will recur (for
example after radical prostatectomy), that is, the probability at
two, five, seven and 10 years that the patient's serum PSA level
will become detectable and begin to rise steadily. Nomograms
include information on one or more of the patient's pre-treatment
PSA, age, Gleason grade (primary, secondary and sum), year of
prostatectomy, months free of cancer, whether or not the surgical
margins were positive, whether or not there was extra capsular
extension (penetration); whether or not there was seminal vesicle
involvement, whether or not there was lymph node involvement,
whether or not the patient receive neoadjuvant hormones, and
whether or not the patient receive radiation therapy before the
radical prostatectomy. Thus, the patient's ERG rearrangement and
PTEN deletion status can be incorporated into currently available
nomograms to further increase the accuracy of such predictions.
[0218] N. Kits
[0219] Disclosed herein are methods that permit simultaneous or
contemporaneous detection of ERG rearrangements, PTEN deletions,
and CEN-10, in the same CTC (or population of isolated CTCs). Such
methods permit characterization of patients with prostate cancer.
Accordingly, kits that facilitate such detection in CTCs are now
enabled.
[0220] In one embodiment, a kit is provided for detecting PTEN
deletions, ERG rearrangements, and centromere 10, for example in
combination with one to ten (e.g., 1, 2, 3, 4, or 5) housekeeping
genes (e.g., .beta.-actin, GAPDH, SDHA, HPRT1, HBS1L, AHSP or
combinations thereof) and/or in combination with one or more (e.g.,
1, 2, 3, 4, or 5) other prostate cancer related genes (e.g., AR).
In yet other specific examples, kits are provided for detecting
only PTEN deletions, ERG rearrangements, and centromere 10. In
particular examples, the kit includes one or more probes for
detecting ERG rearrangements (such as a 5'-ERG probe and a 3'-ERG
probe), one or more probes for detecting PTEN deletions, one or
more probes for detecting centromere 10, or combinations thereof.
In one examples, the kit includes 1 2, 3, 4, 5, 6, 7, 8, 9, or 10
different probes for detecting ERG rearrangements (such as a 5'-ERG
probe and a 3'-ERG probe), 1 2, 3, 4, 5, 6, 7, 8, 9, or 10
different probes for detecting PTEN deletions, and 1 2, 3, 4, 5, 6,
7, 8, 9, or 10 different probes for detecting CEN-10. Such probes
can be packaged in separate containers or vials (e.g., the 5'-ERG
probe, 3'-ERG probe, PTEN probe, and CEN-10 probe can each be in a
separate container).
[0221] Exemplary kits can include at least one probe for detecting
each of PTEN deletions, ERG rearrangements, and CEN-10 (for example
in combination with other prostate cancer related genes or
housekeeping genes) such as, at least two, at least three, at least
four, or at least five different probes). In some examples, such
kits can further include at least one probe for detecting one or
more (e.g., one to three) housekeeping genes. In some examples,
such kits can further include at least one probe for detecting one
or more (e.g., one to three) other prostate cancer related genes,
such as androgen receptor (AR).
[0222] In one example a kit can further include means for isolating
or confirming the presence of CTCs, such as one or more antibodies
specific for EpCAM, ERG, PTEN, PSMA, AR, CD45, CK, or combinations
thereof. Such antibodies can be packaged in separate containers or
vials (e.g., the EpCAM antibody, ERG antibody, PTEN antibody, PSMA
antibody, AR antibody, CD45 antibody, and CK antibody can each be
in a separate container). In one example the kit includes labeled
CD45 and CK antibodies, in separate vials. In some examples, such
antibodies are directly labeled, for example with a fluorophore or
Qdot.RTM.. Other kit embodiments will include secondary detection
means; such as secondary antibodies (e.g., goat anti-rabbit
antibodies, rabbit anti-mouse antibodies, anti-hapten antibodies)
or non-antibody hapten-binding molecules (e.g., avidin or
streptavidin). In some such instances, the secondary detection
means will be directly labeled with a detectable moiety. In other
instances, the secondary (or higher order) antibody will be
conjugated to a hapten (such as biotin, DNP, and/or FITC), which is
detectable by a detectably labeled cognate hapten binding molecule
(e.g., streptavidin (SA) horseradish peroxidase, SA alkaline
phosphatase, and/or SA QDot.RTM. Nanocrystals.TM.). Some kit
embodiments may include colorimetric reagents (e.g., DAB, and/or
AEC) in suitable containers to be used in concert with primary or
secondary (or higher order) detection means (e.g., antibodies) that
are labeled with enzymes for the development of such colorimetric
reagents.
[0223] In one example a kit can further include a solid substrate
to which isolated CTCs can be attached, such as a microscope slide
or multi-well plate.
[0224] Particular kit embodiments can include, for instance, one or
more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) detection means
selected from a nucleic acid probe specific for an ERG
rearrangement, a nucleic acid probe specific for a PTEN deletion, a
nucleic acid probe specific for CEN-10, an antibody specific for an
EpCAM protein, an antibody specific for a CD45 protein, and an
antibody specific for a CK protein. Particular kit embodiments can
further include, for instance, one or more nucleic acid probes
specific for one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10)
housekeeping genes. Exemplary housekeeping genes/proteins include
GAPDH, SDHA, HPRT1, HBS1L, .beta.-actin, and AHSP. In some
examples, kits can further include, for instance, one or more
nucleic acid probes specific for one or more (such as 2, 3, 4, 5,
6, 7, 8, 9 or 10) prostate cancer related genes. Exemplary prostate
cancer related genes include AR; GAS1; WNT5A; TK1; BRAF; ETV4;
tumor protein p63; BCL-2; Ki67; ERK5; and PSA.
[0225] In some kit embodiments, the detection means (e.g., nucleic
acid probe or antibody) can be directly labeled, e.g., with a
Qdot.RTM., fluorophore, chromophore, or enzyme capable of producing
a detectable product (such as alkaline phosphates, horseradish
peroxidase and others commonly know in the art).
[0226] In some embodiments, a kit includes positive or negative
control samples, such as a cell line or tissue known to or known
not to have a PTEN deletion, ERG rearrangement, CEN-10, or
combinations thereof. Exemplary control samples include but are not
limited to normal (e.g., non cancerous) cells or tissues,
lymphocytes, prostate cancer samples (such as from a patient having
mCRPC or CRPC).
[0227] In some embodiments, a kit includes instructional materials
disclosing, for example, means of use of a nucleic acid probe that
specifically hybridizes to PTEN or ERG or CEN-10, or means of use
for a particular primer or probe. The instructional materials may
be written, in an electronic form (e.g., computer diskette or
compact disk) or may be visual (e.g., video files). The kits may
also include additional components to facilitate the particular
application for which the kit is designed. Thus, for example, the
kit can include buffers and other reagents routinely used for the
practice of a particular disclosed method. Such kits and
appropriate contents are well known to those of skill in the
art.
[0228] Certain kit embodiments can include a carrier means, such as
a box, a bag, a satchel, plastic carton (such as molded plastic or
other clear packaging), wrapper (such as, a sealed or sealable
plastic, paper, or metallic wrapper), or other container. In some
examples, kit components will be enclosed in a single packaging
unit, such as a box or other container, which packaging unit may
have compartments into which one or more components of the kit can
be placed. In other examples, a kit includes a one or more
containers, for instance vials, tubes, and the like that can
retain, for example, one or more biological samples to be
tested.
[0229] Other kit embodiments include, for instance, syringes,
cotton swabs, blood collection vials, or latex gloves, which may be
useful for handling, collecting and/or processing a biological
sample. Kits may also optionally contain implements useful for
moving a biological sample from one location to another, including,
for example, droppers, syringes, and the like. Still other kit
embodiments may include disposal means for discarding used or no
longer needed items (such as subject samples, etc.). Such disposal
means can include, without limitation, containers that are capable
of containing leakage from discarded materials, such as plastic,
metal or other impermeable bags, boxes or containers.
[0230] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit a disclosed invention to the particular
features or embodiments described.
EXAMPLES
Example 1
Detection of ERG and PTEN in Prostate Cancer Cell Lines
[0231] This example describes methods used to show that ERG gene
rearrangement and PTEN gene deletions can be detected in prostate
cancer cell lines when the cells are applied to a glass slide and
labeled with antibodies and probes, to mimic how circulating tumor
cells would be processed.
[0232] Prostate cancer cell lines (LNCaP and VCaP) were applied
onto a glass slide using cytocentrifugation. Briefly, a Shandon
Cytospin 4 (Shandon Thermo Scientific) was used at 400 rpm/4 min
setting. Either EZ Megafunnel (Shandon Thermo Scientific) or
Shandon Clipped funnel (single) was assembled with positively
charged glass slides, and 1 mL or 200 .mu.L cell suspension was
pipetted into the funnels, respectively. After the set cytospin
cycle was completed, the slides were removed and air-dried for 30
min at ambient temperature. The cells were then fixed in 10% NBF at
room temperature for 20 minutes, followed by rinsing in phosphate
buffered saline (PBS). The slides were then immersed in PBST (0.2%
Tween 20 in PBS) for permeabilization, rinsed in de-ionized water
and dehydrated in ascending alcohol series (80%, 90% and Abs.
EtOH), followed by air drying. The samples were stored at
-20.degree. C. until use in airtight boxes.
[0233] The mounted and fixed CTCs were then stained utilizing the
automated Benchmark staining platform (Ventana) with
pan-Cytokeritin (CK) (Anti-AE1 and AE3, labeled with quantum dot
that emits at 605 nm), CD45 (labeled with quantum dot that emits at
705 nm), and Androgen Receptor (AR) antibodies through fluorescent
or Quantum Dot IHC. These IHC stains are used to identify
circulating tumor cells (CTC). CTC cells have a positive CK
staining, negative CD45 staining, AR location (in CTC cells from
androgen sensitive prostate cancers, the AR is in the nucleus; in
CTC cells from androgen insensitive prostate cancers, the AR is in
the cytoplasm), and AR protein overexpression. Using these IHC
stains, a manual or software identification of the CTC can be
recorded and utilized for subsequent stainings. As shown in FIG. 1,
LNCaP cells were positive for CK, but negative for CD45.
[0234] After this location was identified, multiplexed quantum dot
FISH was performed by hybridization of 5'-end ERG, 3'-end ERG, PTEN
and CEN-10 probes to the prostate cancer cell lines, with the
associated quantum dot detection. The multiplexed FISH assay
includes hybridization of the probes to the target DNA, and
detection of the hybrids. These steps were also performed on the
automated staining platform (BenchMark series). The samples were
denatured together with the probes at 80.degree. C. and hybridized
at 44.degree. C. for 6-8 hours. The post hybridization washes were
completed at 68-74.degree. C. The detection of the hybrids was
performed with quantum dots. IHC and FISH signals were visualized
utilizing spectral imaging (Ventana).
[0235] It was demonstrated that in these cell lines, either ERG
gene rearrangement with normal PTEN status (VCAP), or normal ERG
gene status with PTEN deletion (LN CAP) was observed.
Example 2
Detection of ERG Rearrangement and PTEN Deletion in Circulating
Tumor Cells (CTCs)
[0236] This example describes methods used to show that ERG gene
rearrangements and PTEN gene deletions could be detected in CTCs
from mCRPC patients.
[0237] CTCs were isolated from whole blood samples from mCRPC
patients using the CellSearch.TM. system (Veridex) Profile kit. The
isolated CTCs were transferred to glass slides, fixed in NBF,
treated with protease, essentially as described in Example 1. The
CTCs were hybridized to probes utilizing the automated Benchmark
staining platform (Ventana) with multiplexed Quantum Dot FISH
inclusive of probes for 5'-end ERG, 3'-end ERG, PTEN, and CEN-10
(labeled with quantum dots that emit at 655 nm, 565 nm, 605 nm, and
585 nm, respectively) as described in Example 1. Cells were also
stained with DAPI. FISH signals were visualized utilizing spectral
imaging (Ventana) as described in Example 1, for example as shown
in FIGS. 2 and 3.
[0238] As more therapeutics are approved for use in mCRPC patients,
the ability to molecularly characterize the type of CTCs driving
tumor burden will play a significant role in optimizing patient
management by determining the type and the sequence of future
targeted therapies. It is shown herein for the first time a method
to molecularly characterize patient CTCs on an automated platform
that is amenable to standard clinical use.
Example 3
In Situ Hybridization to Detect ERG and PTEN Gene Status
[0239] This example provides exemplary methods that can be used to
detect ERG gene rearrangements and PTEN gene deletions using in
situ hybridization, such as FISH. Although particular materials and
methods are provided, one skilled in the art will appreciate that
variations can be made.
[0240] CTCs (e.g., isolated from the blood of patients with
prostate cancer (such as CRPC or mCRPC), are isolated and mounted
onto a microscope slide, under conditions that permit detection of
nucleic acid molecules present in the sample. In some examples, the
cells are fixed. In one example, genomic DNA in the sample can be
detected.
[0241] The slide is incubated with nucleic acid probes that are of
sufficient complementarity to hybridize to genomic DNA in the CTCs
under very high or high stringency conditions. Probes can be RNA or
DNA. Separate probes that are specific for ERG and PTEN genomic DNA
(e.g., human sequences), as well as CEN-10 are incubated with the
sample simultaneously or sequentially. For example, each probe can
include a different quantum dot to permit differentiation between
the probes. After contacting the probes with the CTCs under
conditions that permit hybridization of the probe to its gene
target, unhybridized probe is removed (e.g., washed away), and the
remaining signal detected, for example using spectral aquisition.
In some examples, the signal is quantified.
[0242] In some examples, prior to incubation with the probes for
ERG, PTEN and CEN-10, the CTCs are incubated with antibodies
specific for CK and CD45, to ensure that the CTCs analyzed are CK+
and CD45-. Only CTCs that are CK+ and CD45- are analyzed.
[0243] The resulting hybridization signals for CEN-10, ERG and PTEN
are detected. If CEN-10 is detected, the analysis proceeds. If no
CEN-10 signal is detected, the sample is disregarded. If CEN-10 is
detected, a determination made as to whether ERG is rearranged
and/or PTEN is deleted. PTEN deletion is confirmed if signal from
the PTEN probe is not detected. It is possible that a single CTC
may have one PTEN gene intact (signal detected), and another PTEN
gene deleted (signal absent). For ERG, there are four possible
events, assuming a 5'-end probe (e.g., green) and a 3-end probe
(e.g., red) are used: 1) two co-localized red and green signals is
a normal ERG, (2) one co-localization plus one split of red and
green is an ERG rearrangement through insertion, (3) one
co-localization plus a red signal (no green) is an ERG
rearrangement through 5' deletion, and (4) one co-localization and
multiple red signals is an ERG deletion and amplification.
[0244] In view of the many possible embodiments to which the
principles of the disclosure may be applied, it should be
recognized that the illustrated embodiments are only examples of
the disclosure and should not be taken as limiting the scope of the
invention. Rather, the scope of the disclosure is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Sequence CWU 1
1
511461DNAHomo sapiensCDS(1)..(1461) 1atg att cag act gtc ccg gac
cca gca gct cat atc aag gaa gcc tta 48Met Ile Gln Thr Val Pro Asp
Pro Ala Ala His Ile Lys Glu Ala Leu 1 5 10 15 tca gtt gtg agt gag
gac cag tcg ttg ttt gag tgt gcc tac gga acg 96Ser Val Val Ser Glu
Asp Gln Ser Leu Phe Glu Cys Ala Tyr Gly Thr 20 25 30 cca cac ctg
gct aag aca gag atg acc gcg tcc tcc tcc agc gac tat 144Pro His Leu
Ala Lys Thr Glu Met Thr Ala Ser Ser Ser Ser Asp Tyr 35 40 45 gga
cag act tcc aag atg agc cca cgc gtc cct cag cag gat tgg ctg 192Gly
Gln Thr Ser Lys Met Ser Pro Arg Val Pro Gln Gln Asp Trp Leu 50 55
60 tct caa ccc cca gcc agg gtc acc atc aaa atg gaa tgt aac cct agc
240Ser Gln Pro Pro Ala Arg Val Thr Ile Lys Met Glu Cys Asn Pro Ser
65 70 75 80 cag gtg aat ggc tca agg aac tct cct gat gaa tgc agt gtg
gcc aaa 288Gln Val Asn Gly Ser Arg Asn Ser Pro Asp Glu Cys Ser Val
Ala Lys 85 90 95 ggc ggg aag atg gtg ggc agc cca gac acc gtt ggg
atg aac tac ggc 336Gly Gly Lys Met Val Gly Ser Pro Asp Thr Val Gly
Met Asn Tyr Gly 100 105 110 agc tac atg gag gag aag cac atg cca ccc
cca aac atg acc acg aac 384Ser Tyr Met Glu Glu Lys His Met Pro Pro
Pro Asn Met Thr Thr Asn 115 120 125 gag cgc aga gtt atc gtg cca gca
gat cct acg cta tgg agt aca gac 432Glu Arg Arg Val Ile Val Pro Ala
Asp Pro Thr Leu Trp Ser Thr Asp 130 135 140 cat gtg cgg cag tgg ctg
gag tgg gcg gtg aaa gaa tat ggc ctt cca 480His Val Arg Gln Trp Leu
Glu Trp Ala Val Lys Glu Tyr Gly Leu Pro 145 150 155 160 gac gtc aac
atc ttg tta ttc cag aac atc gat ggg aag gaa ctg tgc 528Asp Val Asn
Ile Leu Leu Phe Gln Asn Ile Asp Gly Lys Glu Leu Cys 165 170 175 aag
atg acc aag gac gac ttc cag agg ctc acc ccc agc tac aac gcc 576Lys
Met Thr Lys Asp Asp Phe Gln Arg Leu Thr Pro Ser Tyr Asn Ala 180 185
190 gac atc ctt ctc tca cat ctc cac tac ctc aga gag act cct ctt cca
624Asp Ile Leu Leu Ser His Leu His Tyr Leu Arg Glu Thr Pro Leu Pro
195 200 205 cat ttg act tca gat gat gtt gat aaa gcc tta caa aac tct
cca cgg 672His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln Asn Ser
Pro Arg 210 215 220 tta atg cat gct aga aac aca ggg ggt gca gct ttt
att ttc cca aat 720Leu Met His Ala Arg Asn Thr Gly Gly Ala Ala Phe
Ile Phe Pro Asn 225 230 235 240 act tca gta tat cct gaa gct acg caa
aga att aca act agg cca gat 768Thr Ser Val Tyr Pro Glu Ala Thr Gln
Arg Ile Thr Thr Arg Pro Asp 245 250 255 tta cca tat gag ccc ccc agg
aga tca gcc tgg acc ggt cac ggc cac 816Leu Pro Tyr Glu Pro Pro Arg
Arg Ser Ala Trp Thr Gly His Gly His 260 265 270 ccc acg ccc cag tcg
aaa gct gct caa cca tct cct tcc aca gtg ccc 864Pro Thr Pro Gln Ser
Lys Ala Ala Gln Pro Ser Pro Ser Thr Val Pro 275 280 285 aaa act gaa
gac cag cgt cct cag tta gat cct tat cag att ctt gga 912Lys Thr Glu
Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu Gly 290 295 300 cca
aca agt agc cgc ctt gca aat cca ggc agt ggc cag atc cag ctt 960Pro
Thr Ser Ser Arg Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu 305 310
315 320 tgg cag ttc ctc ctg gag ctc ctg tcg gac agc tcc aac tcc agc
tgc 1008Trp Gln Phe Leu Leu Glu Leu Leu Ser Asp Ser Ser Asn Ser Ser
Cys 325 330 335 atc acc tgg gaa ggc acc aac ggg gag ttc aag atg acg
gat ccc gac 1056Ile Thr Trp Glu Gly Thr Asn Gly Glu Phe Lys Met Thr
Asp Pro Asp 340 345 350 gag gtg gcc cgg cgc tgg gga gag cgg aag agc
aaa ccc aac atg aac 1104Glu Val Ala Arg Arg Trp Gly Glu Arg Lys Ser
Lys Pro Asn Met Asn 355 360 365 tac gat aag ctc agc cgc gcc ctc cgt
tac tac tat gac aag aac atc 1152Tyr Asp Lys Leu Ser Arg Ala Leu Arg
Tyr Tyr Tyr Asp Lys Asn Ile 370 375 380 atg acc aag gtc cat ggg aag
cgc tac gcc tac aag ttc gac ttc cac 1200Met Thr Lys Val His Gly Lys
Arg Tyr Ala Tyr Lys Phe Asp Phe His 385 390 395 400 ggg atc gcc cag
gcc ctc cag ccc cac ccc ccg gag tca tct ctg tac 1248Gly Ile Ala Gln
Ala Leu Gln Pro His Pro Pro Glu Ser Ser Leu Tyr 405 410 415 aag tac
ccc tca gac ctc ccg tac atg ggc tcc tat cac gcc cac cca 1296Lys Tyr
Pro Ser Asp Leu Pro Tyr Met Gly Ser Tyr His Ala His Pro 420 425 430
cag aag atg aac ttt gtg gcg ccc cac cct cca gcc ctc ccc gtg aca
1344Gln Lys Met Asn Phe Val Ala Pro His Pro Pro Ala Leu Pro Val Thr
435 440 445 tct tcc agt ttt ttt gct gcc cca aac cca tac tgg aat tca
cca act 1392Ser Ser Ser Phe Phe Ala Ala Pro Asn Pro Tyr Trp Asn Ser
Pro Thr 450 455 460 ggg ggt ata tac ccc aac act agg ctc ccc acc agc
cat atg cct tct 1440Gly Gly Ile Tyr Pro Asn Thr Arg Leu Pro Thr Ser
His Met Pro Ser 465 470 475 480 cat ctg ggc act tac tac taa 1461His
Leu Gly Thr Tyr Tyr 485 2486PRTHomo sapiens 2Met Ile Gln Thr Val
Pro Asp Pro Ala Ala His Ile Lys Glu Ala Leu 1 5 10 15 Ser Val Val
Ser Glu Asp Gln Ser Leu Phe Glu Cys Ala Tyr Gly Thr 20 25 30 Pro
His Leu Ala Lys Thr Glu Met Thr Ala Ser Ser Ser Ser Asp Tyr 35 40
45 Gly Gln Thr Ser Lys Met Ser Pro Arg Val Pro Gln Gln Asp Trp Leu
50 55 60 Ser Gln Pro Pro Ala Arg Val Thr Ile Lys Met Glu Cys Asn
Pro Ser 65 70 75 80 Gln Val Asn Gly Ser Arg Asn Ser Pro Asp Glu Cys
Ser Val Ala Lys 85 90 95 Gly Gly Lys Met Val Gly Ser Pro Asp Thr
Val Gly Met Asn Tyr Gly 100 105 110 Ser Tyr Met Glu Glu Lys His Met
Pro Pro Pro Asn Met Thr Thr Asn 115 120 125 Glu Arg Arg Val Ile Val
Pro Ala Asp Pro Thr Leu Trp Ser Thr Asp 130 135 140 His Val Arg Gln
Trp Leu Glu Trp Ala Val Lys Glu Tyr Gly Leu Pro 145 150 155 160 Asp
Val Asn Ile Leu Leu Phe Gln Asn Ile Asp Gly Lys Glu Leu Cys 165 170
175 Lys Met Thr Lys Asp Asp Phe Gln Arg Leu Thr Pro Ser Tyr Asn Ala
180 185 190 Asp Ile Leu Leu Ser His Leu His Tyr Leu Arg Glu Thr Pro
Leu Pro 195 200 205 His Leu Thr Ser Asp Asp Val Asp Lys Ala Leu Gln
Asn Ser Pro Arg 210 215 220 Leu Met His Ala Arg Asn Thr Gly Gly Ala
Ala Phe Ile Phe Pro Asn 225 230 235 240 Thr Ser Val Tyr Pro Glu Ala
Thr Gln Arg Ile Thr Thr Arg Pro Asp 245 250 255 Leu Pro Tyr Glu Pro
Pro Arg Arg Ser Ala Trp Thr Gly His Gly His 260 265 270 Pro Thr Pro
Gln Ser Lys Ala Ala Gln Pro Ser Pro Ser Thr Val Pro 275 280 285 Lys
Thr Glu Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu Gly 290 295
300 Pro Thr Ser Ser Arg Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu
305 310 315 320 Trp Gln Phe Leu Leu Glu Leu Leu Ser Asp Ser Ser Asn
Ser Ser Cys 325 330 335 Ile Thr Trp Glu Gly Thr Asn Gly Glu Phe Lys
Met Thr Asp Pro Asp 340 345 350 Glu Val Ala Arg Arg Trp Gly Glu Arg
Lys Ser Lys Pro Asn Met Asn 355 360 365 Tyr Asp Lys Leu Ser Arg Ala
Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile 370 375 380 Met Thr Lys Val His
Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe His 385 390 395 400 Gly Ile
Ala Gln Ala Leu Gln Pro His Pro Pro Glu Ser Ser Leu Tyr 405 410 415
Lys Tyr Pro Ser Asp Leu Pro Tyr Met Gly Ser Tyr His Ala His Pro 420
425 430 Gln Lys Met Asn Phe Val Ala Pro His Pro Pro Ala Leu Pro Val
Thr 435 440 445 Ser Ser Ser Phe Phe Ala Ala Pro Asn Pro Tyr Trp Asn
Ser Pro Thr 450 455 460 Gly Gly Ile Tyr Pro Asn Thr Arg Leu Pro Thr
Ser His Met Pro Ser 465 470 475 480 His Leu Gly Thr Tyr Tyr 485
31212DNAHomo sapiensCDS(1)..(1212) 3atg aca gcc atc atc aaa gag atc
gtt agc aga aac aaa agg aga tat 48Met Thr Ala Ile Ile Lys Glu Ile
Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 caa gag gat gga ttc gac
tta gac ttg acc tat att tat cca aac att 96Gln Glu Asp Gly Phe Asp
Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 att gct atg gga
ttt cct gca gaa aga ctt gaa ggc gta tac agg aac 144Ile Ala Met Gly
Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 aat att
gat gat gta gta agg ttt ttg gat tca aag cat aaa aac cat 192Asn Ile
Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55 60
tac aag ata tac aat ctt tgt gct gaa aga cat tat gac acc gcc aaa
240Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys
65 70 75 80 ttt aat tgc aga gtt gca caa tat cct ttt gaa gac cat aac
cca cca 288Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn
Pro Pro 85 90 95 cag cta gaa ctt atc aaa ccc ttt tgt gaa gat ctt
gac caa tgg cta 336Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu
Asp Gln Trp Leu 100 105 110 agt gaa gat gac aat cat gtt gca gca att
cac tgt aaa gct gga aag 384Ser Glu Asp Asp Asn His Val Ala Ala Ile
His Cys Lys Ala Gly Lys 115 120 125 gga cga act ggt gta atg ata tgt
gca tat tta tta cat cgg ggc aaa 432Gly Arg Thr Gly Val Met Ile Cys
Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 ttt tta aag gca caa gag
gcc cta gat ttc tat ggg gaa gta agg acc 480Phe Leu Lys Ala Gln Glu
Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 aga gac aaa
aag gga gta act att ccc agt cag agg cgc tat gtg tat 528Arg Asp Lys
Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 tat
tat agc tac ctg tta aag aat cat ctg gat tat aga cca gtg gca 576Tyr
Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185
190 ctg ttg ttt cac aag atg atg ttt gaa act att cca atg ttc agt ggc
624Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly
195 200 205 gga act tgc aat cct cag ttt gtg gtc tgc cag cta aag gtg
aag ata 672Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val
Lys Ile 210 215 220 tat tcc tcc aat tca gga ccc aca cga cgg gaa gac
aag ttc atg tac 720Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp
Lys Phe Met Tyr 225 230 235 240 ttt gag ttc cct cag ccg tta cct gtg
tgt ggt gat atc aaa gta gag 768Phe Glu Phe Pro Gln Pro Leu Pro Val
Cys Gly Asp Ile Lys Val Glu 245 250 255 ttc ttc cac aaa cag aac aag
atg cta aaa aag gac aaa atg ttt cac 816Phe Phe His Lys Gln Asn Lys
Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 ttt tgg gta aat aca
ttc ttc ata cca gga cca gag gaa acc tca gaa 864Phe Trp Val Asn Thr
Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 aaa gta gaa
aat gga agt cta tgt gat caa gaa atc gat agc att tgc 912Lys Val Glu
Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 agt
ata gag cgt gca gat aat gac aag gaa tat cta gta ctt act tta 960Ser
Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310
315 320 aca aaa aat gat ctt gac aaa gca aat aaa gac aaa gcc aac cga
tac 1008Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg
Tyr 325 330 335 ttt tct cca aat ttt aag gtg aag ctg tac ttc aca aaa
aca gta gag 1056Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys
Thr Val Glu 340 345 350 gag ccg tca aat cca gag gct agc agt tca act
tct gta aca cca gat 1104Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr
Ser Val Thr Pro Asp 355 360 365 gtt agt gac aat gaa cct gat cat tat
aga tat tct gac acc act gac 1152Val Ser Asp Asn Glu Pro Asp His Tyr
Arg Tyr Ser Asp Thr Thr Asp 370 375 380 tct gat cca gag aat gaa cct
ttt gat gaa gat cag cat aca caa att 1200Ser Asp Pro Glu Asn Glu Pro
Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 aca aaa gtc tga
1212Thr Lys Val 4403PRTHomo sapiens 4Met Thr Ala Ile Ile Lys Glu
Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 Gln Glu Asp Gly Phe
Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 Ile Ala Met
Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 Asn
Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55
60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys
65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn
Pro Pro 85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu
Asp Gln Trp Leu 100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile
His Cys Lys Ala Gly Lys 115 120 125 Gly Arg Thr Gly Val Met Ile Cys
Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 Phe Leu Lys Ala Gln Glu
Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys
Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 Tyr
Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185
190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly
195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val
Lys Ile 210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp
Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro
Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu 245 250 255 Phe Phe
His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His 260 265 270
Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275
280 285 Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile
Cys 290 295 300 Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val
Leu Thr Leu 305 310 315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys
Asp Lys Ala Asn Arg Tyr 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys
Leu Tyr Phe Thr Lys Thr Val Glu 340 345 350 Glu Pro Ser Asn Pro Glu
Ala Ser Ser Ser Thr Ser Val Thr Pro Asp 355 360 365 Val Ser Asp Asn
Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp 370 375 380 Ser Asp
Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395
400 Thr Lys Val 51365DNAArtificial Sequenceprobe for centromere 10
5cgacttttaa tatgaagata tttccatgtc taagattggc gtcaaatcgc ttgaaatctc
60cacttgcaaa ttccacaaaa agtgtttttc aaaactgctc tgaataaagg aaggttccac
120tctgtgagtt gaatacacac aacacaaagg atttactgag aattcttctg
tctagcagta 180aatgagaaat cccgcttcca acgaaggcct caaacgggtc
taactaatca cttgcagact 240ttacagacag agtctttcca aactgctcta
tgaagagaaa ggtgaaactc tgtgaactga 300acgcacagat gacaaagcag
tttctgagaa tgcttctgtg tagtttttac acgaagatat 360ttccatttca
aagattagcc tcaaatcgct tgaaatctcc acttgcaaac tccacagaaa
420gaatttttca aaactgctct gtctaaagga aggttcaact ctgtgacttg
aatacacaca 480acacaaagaa gtgactgaga attcttctgt ctagcattat
atgaagaaat cccgtttcca 540acgaaggcct caatgaagtc caaaaaagca
cttgcaggct ttacaaacag agtgtttcca 600aactgctcta tgaaaagaaa
ggttaaactc tgtgagttga acgcacacat cacaaagtag 660ttcttgagaa
tgattctgtg tagtttttat acgaagatat ttcgttttct gccataggcc
720tagaagcgct tgaaatctgc acttgcaaat tccaaaaaca gagtgtttca
aatctgctct 780ctctaaagga aggttcaaat ctgtgagttg aatacaaaca
acacaaagac gttactgaga 840attcttctgt ctagcattat atgaggaaat
cccgtttcca acgaagggct caaagagggc 900caattatcca cctgcagact
tacaaagagt gtatttccaa actgctcgat taaagaaagg 960ttaaactctg
tgagttgaac acacacatca caaagtgttt tctgagaatg attttgtcta
1020gttttaatac gaagatatat cctttcctat cactgtcttc gaagcgtttg
aaatctgcac 1080ttgcaaattc caaaaacaga gtgtttcaac tctgctctct
ctcaagaaag gttcaactct 1140gtgagttgaa tacacacaac acaaagaagt
tactgagaat tcttctgtct agtgttgtat 1200gaagaaatcc cgtttccaac
gaaggcctca aagaggtcca aatatccact tgcagacttt 1260agaaatagag
tgtttccaaa ctgctctatg aaaagaaagg ttaaactctg tgagttgaag
1320gcacacatca caaactagtt tctacgaatg actctgtgtc gagct 1365
* * * * *