U.S. patent application number 12/221626 was filed with the patent office on 2009-04-16 for urine gene expression ratios for detection of cancer.
This patent application is currently assigned to Pacific Edge Biotechnology Limited. Invention is credited to Parry John Guilford.
Application Number | 20090098553 12/221626 |
Document ID | / |
Family ID | 38345418 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090098553 |
Kind Code |
A1 |
Guilford; Parry John |
April 16, 2009 |
Urine gene expression ratios for detection of cancer
Abstract
This invention relates to methods for determining the presence
of cancer in a subject based on the analysis of the expression
levels of an under-expressed tumour marker (TM) and at least one
other TM. Specifically, this invention relates to the determination
of a cancer, particularly bladder cancer, by performing ratio,
regression or classification analysis of the expression levels of
at least one under-expressed TM, particularly an under-expressed
bladder TM (BTM), and at least one over-expressed TM, particularly
an over-expressed BTM. In various aspects, the invention telates to
kits and devices for carrying out these methods.
Inventors: |
Guilford; Parry John;
(Dunedin, NZ) |
Correspondence
Address: |
BORSON LAW GROUP, PC
1320 WILLOW PASS ROAD, SUITE 490
CONCORD
CA
94520-5232
US
|
Assignee: |
Pacific Edge Biotechnology
Limited
Dunedin
NZ
|
Family ID: |
38345418 |
Appl. No.: |
12/221626 |
Filed: |
August 5, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/NZ2007/000029 |
Feb 9, 2007 |
|
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12221626 |
|
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Current U.S.
Class: |
435/6.16 ;
435/26; 435/287.2; 435/4 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 2600/118 20130101; C12Q 1/6886 20130101; Y02A 90/26 20180101;
G16H 50/20 20180101; G01N 33/57407 20130101; Y02A 90/10 20180101;
C12Q 2600/16 20130101 |
Class at
Publication: |
435/6 ; 435/4;
435/26; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12Q 1/00 20060101 C12Q001/00; C12M 1/34 20060101
C12M001/34; C12Q 1/02 20060101 C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2006 |
NZ |
545243 |
Claims
1. A method for determining the presence of a cancer in a subject,
comprising: (a) providing a sample from the subject; (b) detecting
the expression level of at least two tumor marker (TM) family
members in said sample, wherein at least one TM is an
under-expressed TM; (c) establishing whether the patient has cancer
according to a predetermined threshold, wherein the predetermined
threshold is established by determining the ratio of expression of
said TMs, or by performing regression or classification analysis on
the TM expression values.
2. The method of claim 1, wherein the TM is a bladder tumor marker
(BTM).
3. The method of claim 1, wherein said cancer is bladder
cancer.
4. The method of claim 1, wherein at least one of the TMs is an
over-expressed TM.
5. The method according to claim 1, wherein the at least one
under-expressed TM is a BTM selected from the group listed in FIG.
3.
6. The method according to claim 1, wherein the at least one
under-expressed TM is a BTM selected from the group listed in FIG.
4.
7. The method according to claim 4, wherein the over-expressed TM
is BTM selected from the group listed in FIG. 11 or FIG. 12.
8. The method of claim 1, wherein said step of detecting is carried
out by detecting expression of TM mRNA.
9. The method of claim 1, wherein said step of detecting is carried
out by detecting expression of a TM protein.
10. The method of claim 1, wherein said step of detecting is
carried out by detecting expression of a TM peptide.
11. The method of claim 1, wherein the sample is any one of biopsy,
blood, serum, peritoneal washes, cerebrospinal fluid, urine and
stool samples
12. A device for detecting a TM, comprising: a substrate having a
TM capture reagent thereon; and a detector associated with said
substrate, said detector capable of detecting a TM associated with
said capture reagent, wherein the TM is an under-expressed TM.
13. The device of claim 12, wherein the TM is a BTM.
14. The device of claim 12, wherein said TM capture reagent is an
oligonucleotide.
15. The device of claim 12, wherein said TM capture reagent is an
antibody.
16. The device of claim 12, wherein the TM is a BTM selected from
the group listed in FIG. 3.
17. The device of claim 12, wherein the TM is a BTM selected from
the group listed in FIG. 4.
18. A kit for determining the presence of a cancer in a subject,
comprising: a substrate; at least two TM capture reagents, wherein
at least one TM is an under-expressed TM; and instructions for
use.
19. The Kit of claim 18, wherein the TM is a BTM.
20. The kit of claim 18, wherein said TM capture reagent is a
TM-specific oligonucleotide.
21. The kit of claim 18, wherein said TM capture reagent is a
TM-specific antibody.
22. The kit of claim 18, wherein the under-expressed TM is a BTM
selected from the group listed in FIG. 3.
23. The kit of claim 18, wherein the under-expressed TM is a BTM
selected from the group listed in FIG. 4.
24. The kit according to claim 20, wherein at least one TM is an
over-expressed TM.
25. The kit according to claim 263, wherein the over-expressed TM
is a BTM selected from the group listed in FIG. 11 or FIG. 12.
Description
TECHNICAL FIELD
[0001] This invention relates to detection of cancer. Specifically,
the invention relates to the use of markers for the detection of
bladder cancer. More specifically, this invention relates to use of
an under-expressed marker in combination with at least one other
marker for the detection of bladder cancer.
BACKGROUND
[0002] Survival of cancer patients is greatly enhanced when the
cancer is treated early. In the case of bladder cancer, patients
diagnosed with early stage disease have 5-year survival rates of
>90%, compared to approximately 15-30% for patients diagnosed
with advanced disease. Therefore, developments that lead to early
diagnosis of bladder cancer can lead to an improved prognosis for
the patients. The established method for detecting bladder cancer
using urine samples is cytology. However, cytology is known to be
only about 75% sensitive for detecting invasive bladder cancer and
only about 25% sensitive for detecting superficial bladder cancer
(Lotan and Roehrbom, Urology 61, 109-118 (2003)).
[0003] Identification of specific markers for cancer in urine can
provide a valuable approach for the early diagnosis of cancer,
leading to early treatment and improved prognosis. Specific cancer
markers also provide a means for monitoring disease progression,
enabling the efficacy of surgical, radiotherapeutic and
chemotherapeutic treatments to be monitored.
[0004] At present, the most reliable method for detecting bladder
cancer is cystoscopy accompanied by histology of biopsied lesions.
However, this technique is time consuming, invasive and its
sensitivity is only approximately 90%, meaning that about 10
percent of cancers are not detected using these methods. Of the
non-invasive methodologies, urine cytology, which detects
exfoliated malignant cells microscopically, is the current
preferred method. Although cytology has a specificity of about 95%,
it has poor sensitivity (9-25%) for low-grade lesions, is extremely
dependent on sample quality and suffers from high inter-observer
variability.
[0005] Several urine protein markers are known. Tests for these
markers offer better sensitivity than cytology, but tend to suffer
from sub-optimal specificity because elevated levels of these
markers are also commonly observed in patients with non-malignant
diseases including inflammation, urolithiasis and benign prostatic
hyperplasia. For example, NMP22, which detects a specific nuclear
matrix protein, has a sensitivity of 47-87% and a specificity of
58-91%.
[0006] One drawback associated with urine testing is that
individual marker levels can vary significantly with: (i) different
urine collection methods (catheterised, voided, urine pellets);
(ii) the diurnal timing of urine sampling; (iii) the point of
sampling during voiding (e.g. midstream vs end sample); and (iv)
urine concentration associated with varying fluid intake, kidney
function or diseases that affect plasma volume. These variations
have the potential to lead to false positive and false negative
tests. Although some of this variation can be reduced using strict
standard operating procedures, patient compliance with these
procedures can be unreliable. The effect of varying urine
concentration can, in some instances, be accounted for by assessing
marker levels relative to urinary creatinine, however, this
increases the cost and complexity of testing, particularly when
sample preparation or storage methods differ for marker detection
and creatinine measurement.
[0007] There is a need for simple tools for the early detection and
diagnosis of cancer. This invention provides further methods,
devices and kits based on markers, specifically ratios, regression
or classification analysis of bladder cancer markers, to aid in the
detection and diagnosis of bladder cancer.
SUMMARY OF THE INVENTION
[0008] The present invention provides for a method for determining
the presence of a cancer in a subject, comprising:
[0009] (a) providing a sample from the subject;
[0010] (b) detecting the expression level of at least two tumour
marker (TM) family members in said sample, wherein at least one TM
is an under-expressed TM;
[0011] (c) establishing whether the patient has cancer according to
a predetermined threshold.
[0012] Step (c) can be preformed by determining the ratio of
expression of said TMs, or by performing regression or
classification analysis on the TM expression levels.
[0013] The TM can be a BTM. The cancer to be detected can be
bladder cancer, and in certain embodiments at least one of the TMs
is an over-expressed BTM. The over-expressed BTM may be selected
from the group outlined in FIG. 11 or FIG. 12.
[0014] In certain embodiments at least one under-expressed TM is a
BTM selected from the group outlined in FIG. 3 or FIG. 4.
[0015] In other embodiments of the present invention the step of
detecting is carried out by detecting over expression of BTM mRNA,
a BTM protein, or a BTM peptide.
[0016] The sample can be any one of biopsy, blood, serum,
peritoneal washes, cerebrospinal fluid, urine and stool samples
[0017] The present invention also provides for a device for
detecting a TM, comprising:
[0018] a substrate having a TM capture reagent thereon; and
[0019] a detector associated with said substrate, said detector
capable of detecting a TM associated with said capture reagent,
wherein the TM is an under-expressed TM.
[0020] The TM can be a BTM.
[0021] The TM capture reagent can be an oligonucleotide or an
antibody.
[0022] In certain embodiments the TM can be a BTM selected from the
group outlined in FIG. 3 or FIG. 4.
[0023] The present invention also provides for a kit for
determining the presence of a cancer in a subject, comprising:
[0024] a substrate;
[0025] at least two TM capture reagents, wherein at least one TM is
an under-expressed TM; and
[0026] instructions for use.
[0027] The TM can be a BTM.
[0028] The TM capture reagent may be a TM-specific oligonucleotide
or a TM-specific antibody.
[0029] The TM detected by the kit may be a BTM selected from the
group outlined in FIG. 3 or FIG. 4.
[0030] At least one of the TMs detected by the kit may be an
over-expressed TM or an over-expressed BTM. The over-expressed BTM
may be selected from the group outlined in FIG. 11 or FIG. 12.
BRIEF DESCRIPTION OF THE FIGURES
[0031] This invention is described with reference to specific
embodiments thereof and with reference to the Figures, in
which:
[0032] FIG. 1 depicts a table showing the characteristics of urine
samples used in the qPCR analyses.
[0033] FIG. 2 depicts a table of primers and oligonucleotide probes
of markers for qPCR analysis of bladder cancer according to the
present invention.
[0034] FIG. 3 depicts a table of under-expressing bladder tumour
markers identified using microarray methods on samples of bladder
cancer.
[0035] FIG. 4 depicts a table of under-expressing bladder tumour
markers identified using microarray methods on samples of bladder
cancer that have insignificant expression in whole blood, but high
expression in normal bladder tissue.
[0036] FIG. 5 depicts box and whisker plots showing the ratios of
three bladder transitional cell carcinoma (TCC) markers (HoxA13,
IGFBP5, and MDK) with the under expressing marker LTB4DH for urine
samples from patients with either non-malignant urological disease
or TCC. The boxes define the 25.sup.th, 50.sup.th and 75.sup.th
percentiles and the horizontal bars mark the adjacent values.
Outliers are shown by circles. The unfilled boxes represent samples
from non-malignant disease controls and the shaded boxes represent
samples from patients with TCC.
[0037] FIG. 6 shows examples of the sensitivities and specificities
of TCC detection for tests that include LTB4DH. (a). single tests;
(b). combination tests using LTB4DH and two of the three markers
HoxA13, IGFBP5, and MDK.
[0038] FIG. 7a-c shows ROC curves for the sensitivity and
specificity of detection of TCC in urine samples using ratios that
include LTB4DH. 7a. IGFBP5/LTB4DH; 7b. MDK/LT4BDH; 7c.
HoxA13/LTB4DH.
[0039] FIG. 8a-f shows scatter plots for combination tests, a-c
using LTB4DH and two of the three markers HoxA13, IGFBP5, and MDK,
and d-f repeated using BAG1 for LTB4DH. 8a. MDK/LTB4DH and
IGFBP51LTB4DH; Bb. MDK/LTB4DH and HoxA13/LTB4DH; IGFBP5/LTB4DH and
HoxA13/LTB4DH; 8d MDK/BAG1 and IGFBP5/BAG1; 8e.MDK/BAG1 and
HoxA13/BAG1; 8f. IGFBP5/BAG1 and HoxA13/BAG1.
[0040] FIG. 9a-b shows scatter plots showing the correlation
between .DELTA.Ct for IGFBP5 and .DELTA.Ct ratios for IGFBP5/LTB4DH
and urine creatinine concentration. 9a. Urine samples from patients
with TCC 9b. Urine samples from patients with non-malignant
disease
[0041] FIG. 10a-f depicts self-self scatter plots showing the
distribution of voided and catheterised urine samples from TCC
patients analysed using the bladder tumour markers MDK, IGFBP5 and
HoxA13 alone or in ratios with LTB4DH.
[0042] FIG. 11 shows known over-expressed markers from invasive
bladder tumours.
[0043] FIG. 12 shows known over-expressed markers from superficial
bladder tumours.
[0044] FIG. 13 shows the clinical characteristics of low grade TCC
samples and controls used in ROC curve analysis.
[0045] FIG. 14 shows the results of a ROC Curve analysis.
Illustration of the increased test accuracy obtained when LTB4DH is
used in ratios with HoxA13 and IGFBP5.
[0046] FIG. 15 shows the results of a Linear Discriminate Analysis
of BTMs, with and without LTB4DH, for the detection of TCC.
DETAILED DESCRIPTION
Definitions
[0047] The term "marker" means a molecule that is associated
quantitatively or qualitatively with the presence of a biological
phenomenon. Examples of "markers" include a polynucleotide, such as
a gene, gene fragment, RNA, or RNA fragment; or a gene product,
including a polypeptide such as a peptide, oligopeptide protein or
protein fragment; or related metabolites, by products or other
identifying molecules, such as antibodies or antibody fragments
whether related directly or indirectly to a mechanism underlying
the phenomenon. The markers of the invention include the nucleotide
sequences (e.g. GenBank sequences) as disclosed herein, in
particular the full length sequences, any coding sequences,
non-coding sequences and fragments, or any compliments thereof, and
any measurable marker thereof as defined above.
[0048] The term "sensitivity" means the proportion of individuals
with the disease who test (by the model) positive. Thus, increased
sensitivity means fewer false negative test results.
[0049] The term "specificity" means the proportion of individuals
without the disease who test (by the model) negative. Thus,
increased specificity means fewer false positive test results.
[0050] The term "expression" includes production of polynucleotides
and polypeptides, in particular, the production of RNA (e.g., mRNA)
from a gene or portion of a gene, and includes the production of a
polypeptide encoded by an RNA or gene or portion of a gene, and
includes appearance of a detectable material associated with
expression. For example, the formation of a complex, for example,
from a polypeptide-polypeptide interaction, polypeptide-nucleotide
interaction, or the like, is included within the scope of the term
"expression". Another example, the binding of a binding ligand,
such as a hybridization probe or antibody, to a gene or other
polynucleotide, a polypeptide or a protein fragment and the
visualization of the binding ligand Thus, the density of a spot on
a microarray, on a hybridization blot such as a Northern blot, or
on an immunoblot, such as a Western blot, or on a bead array, or by
PCR analysis, is included within the term "expression" of the
underlying biological molecule.
[0051] The term "over expression" is used where the expression of a
marker in one cell, or cell type, is greater than that of another
equivalent cell, or cell type.
[0052] The term "under expression" is used where the expression of
a marker in one cell, or cell type, is less than that of another
equivalent cell, or cell type.
[0053] The term "TM" or "tumour marker" or "TM family member" means
a marker that is associated with a particular cancer. The term TM
also includes combinations of individual markers, whose combination
improves the sensitivity and specificity of detecting cancer. It is
to be understood that the term TM does not require that the marker
be specific only for a particular tumour. Rather, expression of TM
can be altered in other types of cells, diseased cells, tumours,
including malignant tumours.
[0054] A TM can be identified by extracting RNA from a tissue
sample from a patient suspected of having bladder cancer, applying
the RNA or cDNA copy to a microarray having a number of
oligonucleotides thereon, permitting the sample RNA to hybridize to
the oligonucleotides on the array, and then quantifying the level
of measured RNA bound to the each array spot. A marker is
considered to be a under expressing TM if its presence is below a
threshold of at least about 1.2 times that found in normal,
non-malignant tissue using microarray methods. Alternatively, the
threshold can be below about 2 times normal, about 3 times less
than normal, 4 times or even about 5 times less than normal. By
"normal" we mean less than the 90.sup.th percentile of the normal
population. In other cases, normal can mean a level of presence of
the 95.sup.th percentile (i.e., about 2 Standard Deviations (SD)
from the mean), and in other cases, less than about 97.5.sup.th
percentile (i.e., about 3 SD) or the 99.sup.th percentile.
[0055] The term "under expressing TM" means a marker that shows
lower expression in bladder tumours than in non-malignant bladder
tissue.
[0056] The term "over expressing TM" means a marker that shows
higher expression in bladder tumours than in non-malignant
tissue.
[0057] The term "BTM" or "bladder tumour marker" or "BTM family
member" means a TM that is associated with bladder cancer. The term
BTM also includes combinations of individual markers, whose
combination improves the sensitivity and specificity of detecting
bladder cancer. It is to be understood that the term BTM does not
require that the marker be specific only for bladder tumours.
Rather, expression of BTM can be altered in other types of cells,
diseased cells, tumours, including malignant tumours.
[0058] The term "under expressing BTM" means a marker that shows
lower expression in bladder tumours than in non-malignant bladder
tissue.
[0059] The term "over expressing BTM" means a marker that shows
higher expression in bladder tumours than in non-malignant
tissue.
[0060] The term "qPCR" means quantitative polymerase chain
reaction. The term "qPCR" or "QPCR" refers to quantative polymerase
chain reaction as described, for example, in PCR Technique:
Quantitative PCR, J. W. Larrick, ed., Eaton Publishing, 1997, and
A-Z of Quantitative PCR, S. Bustin, ed., IUL Press, 2004.
[0061] The term "TCC" means transitional cell carcinoma of the
bladder. TCCs constitute .about.95% of all bladder cancers.
[0062] As used herein "antibodies" and like terms refer to
immunoglobulin molecules and immunologically active portions of
immunoglobulin (Ig) molecules, i.e., molecules that contain an
antigen binding site that specifically binds (immunoreacts with) an
antigen. These include, but are not limited to, polyclonal,
monoclonal, chimeric, single chain, Fc, Fab, Fab', and Fab2
fragments, and a Fab expression library. Antibody molecules relate
to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ
from one another by the nature of heavy chain present in the
molecule. These include subclasses as well, such as IgG1, IgG2, and
others. The light chain may be a kappa chain or a lambda chain.
Reference herein to antibodies includes a reference to all classes,
subclasses, and types. Also included are chimeric antibodies, for
example, monoclonal antibodies or fragments thereof that are
specific to more than one source, e.g., a mouse or human sequence.
Further included are camelid antibodies, shark antibodies or
nanobodies.
[0063] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by abnormal or unregulated cell growth. Cancer and cancer pathology
can be associated, for example, with metastasis, interference with
the normal functioning of neighbouring cells, release of cytokines
or other secretory products at abnormal levels, suppression or
aggravation of inflammatory or immunological response, neoplasia,
premalignancy, malignancy, invasion of surrounding or distant
tissues or organs, such as lymph nodes, etc.
[0064] The term "tumour" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues.
[0065] The term "microarray" refers to an ordered or unordered
arrangement of capture agents, preferably polynucleotides (e.g.,
probes) or polypeptides on a substrate. See, e.g., Microarray
Analysis, M. Schena, John Wiley & Sons, 2002; Microarray
Biochip Technology, M. Schena, ed., Eaton Publishing, 2000; Guide
to Analysis of DNA Microarray Data, S. Knudsen, John Wiley &
Sons, 2004; and Protein Microarray Technology, D. Kambhampati, ed.,
John Wiley & Sons, 2004.
[0066] The term "oligonucleotide" refers to a polynucleotide,
typically a probe or primer, including, without limitation,
single-stranded deoxyribonucleotides, single- or double-stranded
ribonucleotides, RNA: DNA hybrids, and double-stranded DNAs.
Oligonucleotides, such as single-stranded DNA probe
oligonucleotides, are often synthesized by chemical methods, for
example using automated oligonucleotide synthesizers that are
commercially available, or by a variety of other methods, including
in vitro expression systems, recombinant techniques, and expression
in cells and organisms.
[0067] The term "polynucleotide," when used in the singular or
plural, generally refers to any polyribonucleotide or
polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. This includes, without limitation, single- and
double-stranded DNA, DNA including single- and double-stranded
regions, single- and double-stranded RNA, and RNA including single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or include single- and double-stranded regions. Also included are
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
Specifically included are mRNAs, cDNAs, and genomic DNAs, and any
fragments thereof. The term includes DNAs and RNAs that contain one
or more modified bases, such as tritiated bases, or unusual bases,
such as inosine. The polynucleotides of the invention can encompass
coding or non-coding sequences, or sense or antisense sequences. It
will be understood that each reference to a "polynucleotide" or
like term, herein, will include the full-length sequences as well
as any fragments, derivatives, or variants thereof.
[0068] "Polypeptide," as used herein, refers to an oligopeptide,
peptide, or protein sequence, or fragment thereof, and to naturally
occurring, recombinant, synthetic, or semi-synthetic molecules.
Where "polypeptide" is recited herein to refer to an amino acid
sequence of a naturally occurring protein molecule, "polypepide"
and like terms, are not meant to limit the amino acid sequence to
the complete, native amino acid sequence for the full-length
molecule. It will be understood that each reference to a
"polypeptide" or like term, herein, will include the full-length
sequence, as well as any fragments, derivatives, or variants
thereof.
[0069] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridisable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. Additional details and
explanation of stringency of hybridization reactions, are found
e.g., in Ausubel et al., Current Protocols in Molecular Biology,
Wiley Interscience Publishers, (1.995).
[0070] "Stringent conditions" or "high stringency conditions", as
defined herein, typically: (1) employ low ionic strength and high
temperature for washing, for example 0.015 M sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2)
employ a denaturing agent during hybridization, such as formamide,
for example, 50% (v/v) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM
sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times., Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC (sodium chloride/sodium citrate) and 50% formamide
at 55.degree. C., followed by a high-stringency wash comprising
0.1.times.SSC containing EDTA at 55.degree. C.
[0071] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength, and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0072] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology, and
biochemistry, which are within the skill of the art. Such
techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, 2nd edition, Sambrook et
al., 1989; Oligonucleotide Synthesis, M J Gait, ed., 1984; Animal
Cell Culture, R. I. Freshney, ed., 1987; Methods in Enzymology,
Academic Press, Inc.; Handbook of Experimental Immunology, 4th
edition, D. M. Weir & C C. Blackwell, eds., Blackwell Science
Inc., 1987; Gene Transfer Vectors for Mammalian Cells, J. M. Miller
& M. P. Calos, eds., 1987; Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., 1987; and PCR: The Polymerase
Chain Reaction, Mullis et al., eds., 1994.
DESCRIPTION OF EMBODIMENTS OF THE INVENTION
[0073] Using a combination of microarray analysis and quantitative
polymerase chain reaction (qPCR), markers for transitional cell
carcinoma of the bladder (TCC) that are under-expressed in tumours
have been identified. It has surprisingly been found that ratios
between these markers and other bladder tumour markers (BTM),
especially markers that are over expressed in tumours, are
diagnostic for bladder cancer.
[0074] The ratios (rather than measuring an absolute level of a
marker) identifies a simple gene expression `signature` that
typifies bladder cancer cells, and surprisingly is more robust to
variations in sampling techniques or urine concentration. Moreover,
the combination of an under-expressed marker and an over-expressed
marker maximizes the differential between samples from patients and
non-malignant controls, increasing the test reliability. The
under-expressed markers described here have been selected on the
basis of (i) strong and consistent down-regulation in TCC, (ii)
high expression in normal tissue, and (iii) insignificant
expression in whole blood to minimize the risk of false positives
in patients presenting with hematuria.
[0075] As an alternative to determining the ration of the two BTMs,
it has also been found that the under-expressed and over-expressed
BTMs can be analysed in regression analyses or classification
techniques including linear discriminate analysis, and the results
of these analyses are also indicative of the presence of bladder
cancer.
[0076] The test involves the measuring of at least two TM markers,
such as a BTM, in a sample from a patient suspected of having a
cancer or at risk of having cancer, wherein at least one of the TMs
is an under-expressed TM. The ratio of the under-expressed TM and
the other TM is indicative of the presence of cancer. The second TM
can be any TM as known in the art, but preferably is an
over-expressed BTM. FIG. 3 shows a number of under-expressed
markers suitable for use in the present invention.
[0077] The test is best preformed using an under-expressed TM in
combination with an over-expressed TM. Any over-expressed TM can be
used, for example. Known over expressed BTMs identified from
invasive bladder tumours (defined here as tumours.gtoreq.stage 1),
are outlined in FIG. 11, and over-expressed BTMs identified from
superficial bladder tumours (defined here as Stage Ta and T is
tumours) are shown in FIG. 12.
[0078] It has also been surprisingly established that preferred
under-expressed BTMs for use in the present invention are ones that
are not significantly elevated in whole blood, and are present in
sufficiently high copy numbers in both tumour cells and
non-malignant bladder cells. Preferred under-expressed BTMs are
outlined in FIG. 4.
[0079] Cancer markers can be detected in a sample using any
suitable technique, and can include, but are not limited to,
oligonucleotide probes, qPCR or antibodies raised against cancer
markers.
[0080] It will be appreciated that the sample to be tested is not
restricted to a sample of the tissue suspected of being a tumour.
The marker may be secreted into the serum, sloughed from cell
membranes, released from lysed cells or associated with cells lost
into the urine. Therefore, a sample can include any bodily sample,
and includes biopsies, blood, serum, peritoneal washes,
cerebrospinal fluid, urine and stool samples.
[0081] It will also be appreciate that the present invention is not
restricted to the detection of cancer in humans, but is suitable
for the detection of cancer in any animal, including, but not
limited to dogs, cats, horses, cattle, sheep, deer, pigs and any
other animal known to get cancer.
General Approaches to Cancer Detection
[0082] The following approaches are non-limiting methods that can
be used to measure TMs. Following measurement of individual TMs,
ratios between high and low expressing BTM family members are
determined. These ratios are used to predict the presence or
absence cancer.
[0083] Alternatively, the high and low expressing TMs are used in
regression or classification analyses. The results of these
analyses are also used to predict the presence or absence
cancer.
[0084] General methodologies for determining expression levels are
outlined below, although it will be appreciated that any method for
determining expression levels would be suitable.
Quantitative PCR (qPCR)
[0085] Quantitative PCR (qPCR) can be carried out on tumour
samples, on serum, plasma and urine samples using BTM specific
primers and probes. In controlled reactions, the amount of product
formed in a PCR reaction (Sambrook, J., E Fritsch, E. and T
Maniatis, Molecular Cloning: A Laboratory Manual 3.sup.rd. Cold
Spring Harbor Laboratory Press: Cold Spring Harbor (2001))
correlates with the amount of starting template. Quantification of
the PCR product can be carried out by stopping the PCR reaction
when it is in log phase, before reagents become limiting. The PCR
products are then electrophoresed in agarose or polyacrylamide
gels, stained with ethidium bromide or a comparable DNA stain, and
the intensity of staining measured by densitometry. Alternatively,
the progression of a PCR reaction can be measured using PCR
machines such as the Applied Biosystems' Prism 7000 or the Roche
LightCycler which measure product accumulation in real-time.
Real-time PCR measures either the fluorescence of DNA intercalating
dyes such as Sybr Green into the synthesized PCR product, or the
fluorescence released by a reporter molecule when cleaved from a
quencher molecule; the reporter and quencher molecules are
incorporated into an oligonucleotide probe which hybridizes to the
target DNA molecule following DNA strand extension from the primer
oligonucleotides. The oligonucleotide probe is displaced and
degraded by the enzymatic action of the Taq polymerase in the next
PCR cycle, releasing the reporter from the quencher molecule. In
one variation, known as Scorpion.RTM., the probe is covalently
linked to the primer.
Reverse Transcription PCR (RT-PCR)
[0086] RT-PCR can be used to compare RNA levels in different sample
populations, in normal and tumour tissues, with or without drug
treatment, to characterize patterns of expression, to discriminate
between closely related RNAs, and to analyze RNA structure.
[0087] For RT-PCR, the first step is the isolation of RNA from a
target sample. The starting material is typically total RNA
isolated from human tumours or tumour cell lines, and corresponding
normal tissues or cell lines, respectively. RNA can be isolated
from a variety of samples, such as tumour samples from breast,
lung, colon (e.g., large bowel or small bowel), colorectal,
gastric, esophageal, anal, rectal, prostate, brain, liver, kidney,
pancreas, spleen, thymus, testis, ovary, uterus, bladder etc.,
tissues, from primary tumours, or tumour cell lines, and from
pooled samples from healthy donors. If the source of RNA is a
tumour, RNA can be extracted, for example, from frozen or archived
paraffin-embedded and fixed (e.g., formalin-fixed) tissue
samples.
[0088] The first step in gene expression profiling by RT-PCR is the
reverse transcription of the RNA template into cDNA, followed by
its exponential amplification in a PCR reaction. The two most
commonly used reverse transcriptases are avian myeloblastosis virus
reverse transcriptase (AMV-RT) and Moloney murine leukaemia virus
reverse transcriptase (MMLV-RT). The reverse transcription step is
typically primed using specific primers, random hexamers, or
oligo-dT primers, depending on the circumstances and the goal of
expression profiling. For example, extracted RNA can be
reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer,
Calif., USA), following the manufacturer's instructions. The
derived cDNA can then be used as a template in the subsequent PCR
reaction.
[0089] Although the PCR step can use a variety of thermostable
DNA-dependent DNA polymerases, it typically employs the Taq DNA
polymerase, which has a 5'-3' nuclease activity but lacks a 3'-5'
proofreading endonuclease activity. Thus, TaqMan (q) PCR typically
utilizes the 5' nuclease activity of Taq or Tth polymerase to
hydrolyze a hybridization probe bound to its target amplicon, but
any enzyme with equivalent 5' nuclease activity can be used.
[0090] Two oligonucleotide primers are used to generate an amplicon
typical of a PCR reaction. A third oligonucleotide, or probe, is
designed to detect nucleotide sequence located between the two PCR
primers. The probe is non-extendible by Taq DNA polymerase enzyme,
and is labeled with a reporter fluorescent dye and a quencher
fluorescent dye. Any laser-induced emission from the reporter dye
is quenched by the quenching dye when the two dyes are located
close together as they are on the probe. During the amplification
reaction, the Taq DNA polymerase enzyme cleaves the probe in a
template-dependent manner. The resultant probe fragments
disassociate in solution, and signal from the released reporter dye
is free from the quenching effect of the second fluorophore. One
molecule of reporter dye is liberated for each new molecule
synthesized, and detection of the unquenched reporter dye provides
the basis for quantitative interpretation of the data.
[0091] TaqMan RT-PCR can be performed using commercially available
equipment, such as, for example, ABI PRISM 7700 Sequence Detection
System (Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA),
or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany).
In a preferred embodiment, the 5' nuclease procedure is run on a
real-time quantitative PCR device such as the ABI PRISM 7700tam
Sequence Detection System. The system consists of a thermocycler,
laser, charge-coupled device (CCD), camera, and computer. The
system amplifies samples in a 96-well format on a thermocycler.
During amplification, laser-induced fluorescent signal is collected
in real-time through fibre optics cables for all 96 wells, and
detected at the CCD. The system includes software for running the
instrument and for analyzing the data.
[0092] 5' nuclease assay data are initially expressed as Ct, or the
threshold cycle. As discussed above, fluorescence values are
recorded during every cycle and represent the amount of product
amplified to that point in the amplification reaction. The point
when the fluorescent signal is first recorded as statistically
significant is the threshold cycle.
Real-Time Quantitative PCR (qPCR)
[0093] A more recent variation of the RT-PCR technique is the real
time quantitative PCR, which measures PCR product accumulation
through a dual-labeled fluorigenic probe (i.e., TaqMan probe). Real
time PCR is compatible both with quantitative competitive PCR and
with quantitative comparative PCR. The former uses an internal
competitor for each target sequence for normalization, while the
latter uses a normalization gene contained within the sample, or a
housekeeping gene for RT-PCR. Further details are provided, e.g.,
by Held et al., Genome Research 6: 986-994 (1996).
[0094] Expression levels can be determined using fixed,
paraffin-embedded tissues as the RNA source. According to one
aspect of the present invention, PCR primers and probes are
designed based upon intron sequences present in the gene to be
amplified. In this embodiment, the first step in the primer/probe
design is the delineation of intron sequences within the genes.
This can be done by publicly available software, such as the DNA
BLAT software developed by Kent, W. J., Genome Res. 12 (4): 656-64
(2002), or by the BLAST software including its variations.
Subsequent steps follow well established methods of PCR primer and
probe design.
[0095] In order to avoid non-specific signals, it is useful to mask
repetitive sequences within the introns when designing the primers
and probes. This can be easily accomplished by using the Repeat
Masker program available on-line through the Baylor College of
Medicine, which screens DNA sequences against a library of
repetitive elements and returns a query sequence in which the
repetitive elements are masked. The masked sequences can then be
used to design primer and probe sequences using any commercially or
otherwise publicly available primer/probe design packages, such as
Primer Express (Applied Biosystems); MGB assay-by-design (Applied
Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000)
Primer3 on the WWW for general users and for biologist programmers
in: Krawetz S, Misener S (eds) Bioinformatics Methods and
Protocols: Methods in Molecular Biology. Humana Press, Totowa,
N.J., pp 365-386).
[0096] The most important factors considered in PCR primer design
include primer length, melting temperature (Tm), and G/C content,
specificity, complementary primer sequences, and 3' end sequence.
In general, optimal PCR primers are generally 17-30 bases in
length, and contain about 20-80%, such as, for example, about
50-60% G+C bases. Melting temperatures between 50 and 80.degree.
C., e.g., about 50 to 70.degree. C., are typically preferred. For
further guidelines for PCR primer and probe design see, e.g.,
Dieffenbach, C. W. et al., General Concepts for PCR Primer Design
in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory
Press, New York, 1995, pp. 133-155; Innis and Gelfand, Optimization
of PCRs in: PCR Protocols, A Guide to Methods and Applications, CRC
Press, London, 1994, pp. 5-11; and Plasterer, T. N. Primerselect
Primer and probe design. Methods Mol. Biol. 70: 520-527 (1997), the
entire disclosures of which are hereby expressly incorporated by
reference.
Microarray Analysis
[0097] Differential expression can also be identified, or confirmed
using the microarray technique. Thus, the expression profile of
CCPMs can be measured in either fresh or paraffin-embedded tumour
tissue, using microarray technology. In this method, polynucleotide
sequences of interest (including cDNAs and oligonucleotides) are
plated, or arrayed, on a microchip substrate. The arrayed sequences
(i.e., capture probes) are then hybridized with specific
polynucleotides from cells or tissues of interest (i.e., targets).
Just as in the RT-PCR method, the source of RNA typically is total
RNA isolated from human tumours or tumour cell lines, and
corresponding normal tissues or cell lines. Thus RNA can be
isolated from a variety of primary tumours or tumour cell lines. If
the source of RNA is a primary tumour, RNA can be extracted, for
example, from frozen or archived formalin fixed paraffin-embedded
(FFPE) tissue samples and fixed (e.g., formalin-fixed) tissue
samples, which are routinely prepared and preserved in everyday
clinical practice.
[0098] In a specific embodiment of the microarray technique, PCR
amplified inserts of cDNA clones are applied to a substrate. The
substrate can include up to 1, 2, 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, or 75 nucleotide sequences. In other aspects, the substrate
can include at least 10,000 nucleotide sequences. The microarrayed
sequences, immobilized on the microchip, are suitable for
hybridization under stringent conditions. As other embodiments, the
targets for the microarrays can be at least 50, 100, 200, 400, 500,
1000, or 2000 bases in length; or 50-100, 100-200, 100-500,
100-1000, 100-2000, or 500-5000 bases in length. As further
embodiments, the capture probes for the microarrays can be at least
10, 15, 20, 25, 50, 75, 80, or 100 bases in length; or 10-15,
10-20, 10-25, 10-50, 10-75, 1080, or 20-80 bases in length.
[0099] Fluorescently labeled cDNA probes may be generated through
incorporation of fluorescent nucleotides by reverse transcription
of RNA extracted from tissues of interest. Labeled cDNA probes
applied to the chip hybridize with specificity to each spot of DNA
on the array. After stringent washing to remove non-specifically
bound probes, the chip is scanned by confocal laser microscopy or
by another detection method, such as a CCD camera. Quantitation of
hybridization of each arrayed element allows for assessment of
corresponding mRNA abundance. With dual colour fluorescence,
separately labeled cDNA probes generated from two sources of RNA
are hybridized pairwise to the array. The relative abundance of the
transcripts from the two sources corresponding to each specified
gene is thus determined simultaneously. An exemplary protocol for
this is described in detail in Example 4.
[0100] The miniaturized scale of the hybridization affords a
convenient and rapid evaluation of the expression pattern for large
numbers of genes. Such methods have been shown to have the
sensitivity required to detect rare transcripts, which are
expressed at a few copies per cell, and to reproducibly detect at
least approximately two-fold differences in the expression levels
(Schena et al., Proc. Natl. Acad. Sci. USA 93 (2): 106-149 (1996)).
Microarray analysis can be performed by commercially available
equipment, following manufacturer's protocols, such as by using the
Affymetrix GenChip technology, Illumina microarray technology or
Incyte's microarray technology. The development of microarray
methods for large-scale analysis of gene expression makes it
possible to search systematically for molecular markers of cancer
classification and outcome prediction in a variety of tumour
types.
RNA Isolation, Purification, and Amplification
[0101] General methods for mRNA extraction are well known in the
art and are disclosed in standard textbooks of molecular biology,
including Ausubel et al., Current Protocols of Molecular Biology,
John Wiley and Sons (1997). Methods for RNA extraction from
paraffin embedded tissues are disclosed, for example, in Rupp and
Locker, Lab Invest 56: A67 (1987), and De Sandres et al.,
BioTechniques 18: 42044 (1995). In particular, RNA isolation can be
performed using purification kit, buffer set, and protease from
commercial manufacturers, such as Qiagen, according to the
manufacturer's instructions. For example, total RNA from cells in
culture can be isolated using Qiagen RNeasy mini-columns. Other
commercially available RNA isolation kits include MasterPure
Complete DNA and RNA Purification Kit (EPICENTRE (D, Madison,
Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total
RNA from tissue samples can be isolated using RNA Stat-60
(Tel-Test). RNA prepared from tumour can be isolated, for example,
by cesium chloride density gradient centrifugation.
[0102] The steps of a representative protocol for profiling gene
expression using fixed, paraffin-embedded tissues as the RNA
source, including mRNA isolation, purification, primer extension
and amplification are given in various published journal articles
(for example: T. E. Godfrey et al. J. Molec. Diagnostics 2: 84-91
(2000); K. Specht et al., Am. J. Pathol. 15B: 419-29 (2001)).
Briefly, a representative process starts with cutting about 10
.mu.m thick sections of paraffin-embedded tumour tissue samples.
The RNA is then extracted, and protein and DNA are removed. After
analysis of the RNA concentration, RNA repair and/or amplification
steps may be included, if necessary, and RNA is reverse transcribed
using gene specific promoters followed by RT-PCR. Finally, the data
are analyzed to identify the best treatment option(s) available to
the patient on the basis of the characteristic gene expression
pattern identified in the tumour sample examined.
Immunohistochemistry and Proteomics
[0103] Immunohistochemistry methods are also suitable for detecting
the expression levels of the proliferation markers of the present
invention. Thus, antibodies or antisera, preferably polyclonal
antisera, and most preferably monoclonal antibodies specific for
each marker, are used to detect expression. 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 horse radish peroxidase or
alkaline phosphatase. Alternatively, 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. Immunohistochemistry protocols and kits are
well known in the art and are commercially available.
[0104] Proteomics can be used to analyze the polypeptides present
in a sample (e.g., tissue, organism, or cell culture) at a certain
point of time. In particular, proteomic techniques can be used to
assess the global changes of polypeptide expression in a sample
(also referred to as expression proteomics). Proteomic analysis
typically includes: (1) separation of individual polypeptides in a
sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of
the individual polypeptides recovered from the gel, e.g., by mass
spectrometry or N-terminal sequencing, and (3) analysis of the data
using bioinformatics. Proteomics methods are valuable supplements
to other methods of gene expression profiling, and can be used,
alone or in combination with other methods, to detect the products
of the proliferation markers of the present invention.
Hybridization Methods Using Nucleic Acid Probes Selective for a
Marker
[0105] These methods involve binding the nucleic acid probe to a
support, and hybridizing under appropriate conditions with RNA or
cDNA derived from the test sample (Sambrook, J., E Fritsch, E. and
T Maniatis, Molecular Cloning: A Laboratory Manual 3rd. Cold Spring
Harbor Laboratory Press: Cold Spring Harbor (2001)). These methods
can be applied to BTM derived from a tumour tissue or fluid sample.
The RNA or cDNA preparations are typically labeled with a
fluorescent or radioactive molecule to enable detection and
quantification. In some applications, the hybridizing DNA can be
tagged with a branched, fluorescently labeled structure to enhance
signal intensity (Nolte, F. S., Branched DNA signal amplification
for direct quantitation of nucleic acid sequences in clinical
specimens. Adv. Clin. Chem. 33, 201-35 (1998)). Unhybridized label
is removed by extensive washing in low salt solutions such as
0.1.times.SSC, 0.5% SDS before quantifying the amount of
hybridization by fluorescence detection or densitometry of gel
images. The supports can be solid, such as nylon or nitrocellulose
membranes, or consist of microspheres or beads that are hybridized
when in liquid suspension. To allow washing and purification, the
beads may be magnetic (Haukanes, B-I and Kvam, C., Application of
magnetic beads in bioassays. Bio/Technology 11, 60-63 (1993)) or
fluorescently-labeled to enable flow cytometry (see for example:
Spiro, A., Lowe, M. and Brown, D., A Bead-Based Method for
Multiplexed Identification and Quantitation of DNA Sequences Using
Flow Cytometry. Appl. Env. Micro. 66, 4258-4265 (2000)).
[0106] A variation of hybridization technology is the QuantiGene
Plex.RTM. assay (Genospectra, Fremont) which combines a fluorescent
bead support with branched DNA signal amplification. Still another
variation on hybridization technology is the Quantikine.RTM. mRNA
assay (R&D Systems, Minneapolis). Methodology is as described
in the manufacturer's instructions. Briefly the assay uses
oligonucleotide hybridization probes conjugated to Digoxigenin.
Hybridization is detected using anti-Digoxigenin antibodies coupled
to alkaline phosphatase in colorometric assays.
[0107] Additional methods are well known in the art and need not be
described further herein.
Enzyme-Linked Immunological Assays (ELISA)
[0108] Briefly, in sandwich ELISA assays, a polyclonal or
monoclonal antibody against the BTM is bound to a solid support
(Crowther, J. R. The ELISA guidebook. Humana Press: New Jersey
(2000); Harlow, E. and Lane, D., Using antibodies: a laboratory
manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor
(1999)) or suspension beads. Other methods are known in the art and
need not be described herein further. Monoclonal antibodies can be
hybridoma-derived or selected from phage antibody libraries (Hust
M. and Dubel S., Phage display vectors for the in vitro generation
of human antibody fragments. Methods Mol Biol. 295:71-96 (2005)).
Non-specific binding sites are blocked with non-target protein
preparations and detergents. The capture antibody is then incubated
with a preparation of urine or tissue containing the BTM antigen.
The mixture is washed before the antibody/antigen complex is
incubated with a second antibody that detects the target BTM. The
second antibody is typically conjugated to a fluorescent molecule
or other reporter molecule that can either be detected in an
enzymatic reaction or with a third antibody conjugated to a
reporter (Crowther, Id). Alternatively, in direct ELISAs, the
preparation containing the BTM can be bound to the support or bead
and the target antigen detected directly with an antibody-reporter
conjugate (Crowther, Id.).
[0109] Methods for producing monoclonal antibodies and polyclonal
antisera are well known in the art and need not be described herein
further.
Immunodetection
[0110] The methods can also be used for immunodetection of marker
family members in sera or plasma from bladder cancer patients taken
before and after surgery to remove the tumour, immunodetection of
marker family members in patients with other cancers, including but
not limited to, colorectal, pancreatic, ovarian, melanoma, liver,
oesophageal, stomach, endometrial, and brain and immunodetection of
marker family members in urine and stool from bladder cancer
patients.
[0111] BTMs can also be detected in tissues or urine using other
standard immunodetection techniques such as immunoblotting or
immunoprecipitation (Harlow, E. and Lane, D., Using antibodies: a
laboratory manual. Cold Spring Harbor Laboratory Press: Cold Spring
Harbor (1999)). In immunoblotting, protein preparations from tissue
or fluid containing the BTM are electrophoresed through
polyacrylamide gels under denaturing or non-denaturing conditions.
The proteins are then transferred to a membrane support such as
nylon. The BTM is then reacted directly or indirectly with
monoclonal or polyclonal antibodies as described for
immunohistochemistry. Alternatively, in some preparations, the
proteins can be spotted directly onto membranes without prior
electrophoretic separation. Signal can be quantified by
densitometry.
[0112] In immunoprecipitation, a soluble preparation containing the
BTM is incubated with a monoclonal or polyclonal antibody against
the BTM. The reaction is then incubated with inert beads made of
agarose or polyacrylamide with covalently attached protein A or
protein G. The protein A or G beads specifically interact with the
antibodies forming an immobilized complex of antibody-BTM-antigen
bound to the bead. Following washing the bound BTM can be detected
and quantified by immunoblotting or ELISA.
Threshold Determination
[0113] For tests using down-regulated BTMs in either ratios or
regression analyses, thresholds will be derived that will enable a
sample to be called either positive or negative for TCC. These
thresholds will be determined by the analysis of cohorts of
patients who are being investigated for the presence of TCC.
Thresholds may vary for different test applications; for example,
thresholds for use of the test in population screening will be
determined using cohorts of patients who are largely free of
urological symptoms, and these thresholds may be different to those
used in tests for patients who are under surveillance for TCC
recurrence, or those being investigated for the presence of
urological symptoms such as hematuria. A threshold could be
selected to provide a practical level of test specificity in the
required clinical setting; that is, a specificity that allows
reasonable sensitivity without excessive numbers of patients
receiving false positive results. This specificity may be within
the range of 80-90%. An alternative method to obtain a test
threshold is to plot sensitivity against specificity for different
test thresholds (ROC curves) then select the point of inflexion of
the curve.
[0114] As an alternative to single thresholds, the test may use
test intervals which provide different degrees of likelihood of
presence of disease and which have different clinical consequences
associated with them. For example, a test may have three intervals;
one associated with a high (e.g. 90%) risk of the presence of TCC,
a second associated with a low risk of TCC and a third regarded as
being suspicious of disease. The "suspicious" interval could be
associated with a recommendation for a repeat test in a defined
period of time.
Methods for Detecting Bladder Cancer Markers in Urine
[0115] In several embodiments, assays for BTM can be desirably
carried out on urine samples. In general, methods for assaying for
oligonucleotides, proteins and peptides in these fluids are known
in the art. However, for purposes of illustration, urine levels of
a BTM can be quantified using a sandwich-type enzyme-linked
immunosorbent assay (ELISA). For plasma or serum assays, a 5 .mu.L
aliquot of a properly diluted sample or serially diluted standard
BTM and 75 .mu.L of peroxidase-conjugated anti-human BTM antibody
are added to wells of a microtiter plate. After a 30-minute
incubation period at 30.degree. C., the wells are washed with 0.05%
Tween 20 in phosphate-buffered saline (PBS) to remove unbound
antibody. Bound complexes of BTM and anti-BTM antibody are then
incubated with o-phenylendiamine containing H.sub.2O.sub.2 for 15
minutes at 30.degree. C. The reaction is stopped by adding 1 M
H.sub.2SO.sub.4, and the absorbance at 492 nm is measured with a
microtiter plate reader. It can be appreciated that anti-BTM
antibodies can be monoclonal antibodies or polyclonal antisera.
[0116] Because many proteins are either (1) secreted by cells, (2)
cleaved from cell membranes, (3) lost from cells upon cell death or
(4) contained within sloughed cells, it will be appreciated that
BTMs may also be detected in the urine. Additionally, diagnosis of
bladder cancer can be determined by measuring either expression of
BTMs in a sample, or accumulation of BTMs in a sample. Prior art
methods of diagnosis include cystoscopy, cytology and examination
of cells extracted during these procedures. Such methods have
relied upon identification of tumour cells in the urine or in a
brush sample of urothelium, or in other cases, in biopsy specimens
of the bladder wall. These methods suffer from several types of
errors, including sampling error, errors in identification between
observers, and the like.
Antibodies to Bladder Tumour Markers
[0117] In additional aspects, this invention includes manufacture
of antibodies against BTMs. Using methods described herein, novel
BTMs can be identified using microarray and/or qPCR methods. Once a
putative marker is identified, it can be produced in sufficient
amount to be suitable for eliciting an immunological response. In
some cases, a full-length BTM can be used, and in others, a peptide
fragment of a BTM may be sufficient as an immunogen. The immunogen
can be injected into a suitable host (e.g., mouse, rabbit, etc) and
if desired, an adjuvant, such as Freund's complete adjuvant,
Freund's incomplete adjuvant can be injected to increase the immune
response. It can be appreciated that making antibodies is routine
in the immunological arts and need not be described herein further.
As a result, one can produce antibodies against BTMs identified
using methods described herein.
[0118] In yet further embodiments, antibodies can be made against
the protein or the protein core of the tumour markers identified
herein or against an oligonucleotide sequence unique to a BTM.
Although certain proteins can be glycosylated, variations in the
pattern of glycosylation can, in certain circumstances, lead to
mis-detection of forms of BTMs that lack usual glycosylation
patterns. Thus, in certain aspects of this invention, BTM
immunogens can include deglycosylated BTM or deglycosylated BTM
fragments. Deglycosylation can be accomplished using one or more
glycosidases known in the art. Alternatively, BTM cDNA can be
expressed in glycosylation-deficient cell lines, such as
prokaryotic cell lines, including E. coli and the like.
[0119] Vectors can be made having BTM-encoding oligonucleotides
therein. Many such vectors can be based on standard vectors known
in the art. Vectors can be used to transfect a variety of cell
lines to produce BTM-producing cell lines, which can be used to
produce desired quantities of BTM for development of specific
antibodies or other reagents for detection of BTMs or for
standardizing developed assays for BTMs.
Kits
[0120] Based on the discoveries of this invention, several types of
test kits can be envisioned and produced. First, kits can be made
that have a detection device pre-loaded with a detection molecule
(or "capture reagent"). In embodiments for detection of BTM mRNA,
such devices can comprise a substrate (e.g., glass, silicon,
quartz, metal, etc) on which oligonucleotides as capture reagents
that hybridize with the mRNA to be detected is bound. In some
embodiments, direct detection of mRNA can be accomplished by
hybridizing mRNA (labeled with cy3, cy5, radiolabel or other label)
to the oligonucleotides on the substrate. In other embodiments,
detection of mRNA can be accomplished by first making complementary
DNA (cDNA) to the desired mRNA. Then, labeled cDNA can be
hybridized to the oligonucleotides on the substrate and
detected.
[0121] Antibodies can also be used in kits as capture reagents. In
some embodiments, a substrate (e.g., a multiwell plate) can have a
specific BTM capture reagent attached thereto. In some embodiments,
a kit can have a blocking reagent included. Blocking reagents can
be used to reduce non-specific binding. For example, non-specific
oligonucleotide binding can be reduced using excess DNA from any
convenient source that does not contain BTM oligonucleotides, such
as salmon sperm DNA. Nor-specific antibody binding can be reduced
using an excess of a blocking protein such as serum albumin. It can
be appreciated that numerous methods for detecting oligonucleotides
and proteins are known in the art, and any strategy that can
specifically detect BTM associated molecules can be used and be
considered within the scope of this invention.
[0122] In addition to a substrate, a test kit can comprise capture
reagents (such as probes), washing solutions (e.g., SSC, other
salts, buffers, detergents and the like), as well as detection
moieties (e.g., cy3, cy5, radiolabels, and the like). Kits can also
include instructions for use and a package.
BTM Ratios Used for Detection of Bladder Cancer I
[0123] In one series of embodiments, reagents for the testing the
BTM LTBDH4 in combination with over-expressing BTMs can be
incorporated into a kit for the testing of unfractionated urine or
urine cell sediments to detect bladder cancer. The urine samples
could be collected from patients with diagnosed bladder cancer who
require monitoring for disease progression or treatment response,
individuals with urological symptoms including macroscopic or
microscopic hematuria, or asymptomatic individuals. For patients or
individuals being tested with a kit that measures the BTMs in
unfractionated urine, approximately 2 mls of urine can be taken for
testing. For tests on the urine pellet, >10 mls of urine can be
collected.
[0124] A suitable kit includes: (i) instructions for use and result
interpretation, (ii) software for interpretation of multiple gene
analyses, including any regression analysis classifier or formula
(iii) reagents for the stabilization and purification of RNA from
unfractionated urine or urine pellets, (iv) reagents for the
synthesis of cDNA including dNTPs and reverse transcriptase, and
(v) reagents for the quantification of the BTM cDNA. In one form,
these reagents would be used for quantitative PCR and would include
specific exon-spanning oligonucleotide primers, a third
oligonucleotide labeled with a probe for detection, Taq polymerase
and the other buffers, salts and dNTPs required for PCR. The kit
can also use other methods for detection of the transcripts such as
direct hybridization of the BTM RNA with labeled probes or branched
DNA technology; and (vi) oligonucleotides and probe for the
detection of transcripts from a highly transcribed gene, such as
.beta.actin, to serve as a quality control measure.
Evaluation of Progression of Bladder Cancer Using BTM Ratios
[0125] To evaluate the progression of bladder tumours, samples of
tissue are obtained by biopsy of bladder wall or samples of urine
are collected over time from a patient having bladder cancer.
Evaluation of the ratio of BTMs or combinations thereof are made
for samples taken at different times. BTM ratios within a specified
range are indicative of progression of bladder cancer.
Evaluation of Therapy of Bladder Cancer Using BTM Ratios
[0126] To evaluate the efficacy of therapy for bladder tumours,
samples of tissue and/or urine are obtained before treatment is
initiated. The baseline levels of one or more BTMs are determined,
as are ratios of various BTMs with respect to each other. Treatment
is initiated, and can include any therapy known in the art,
including surgery, radiation therapy or chemotherapy as appropriate
to the type and stage of the disease. During the course of therapy,
samples of tissue and/or urine are collected and analyzed for the
presence and amount of BTMs. Ratios of various BTMs are determined
and results are compared to: (1) the patient's baseline levels
before treatment or (2) normal values obtained from a population of
individuals not having bladder cancer.
Use of BTM Ratios to Monitor the Progression of TCC Therapies
[0127] In addition to the rapid diagnosis and early detection of
TCC, BTM marker ratios detected in either tissue, serum or urine
can be used to monitor a patient's response to therapy. In these
applications, urine and/or serum samples can be taken at intervals
following the initiation of systemic, intravesicular or
intravascular chemotherapy, radiotherapy or immunotherapy. A change
in marker ratio can indicate a reduction in tumour size, indicative
of effective treatment. The rate of change can be used to predict
the optimum therapeutic dose for each patient or treatment.
Use of BTM Regression Analyses
[0128] In addition to the BTM ratios, regression or classification
analyses that include high and low expressing BTM family members
can be used for the applications described above.
[0129] Markers evaluated are selected from known human genes. The
genes evaluated are indicated in FIGS. 3 and 4. Included in FIGS. 3
and 4 are the name of the gene, the HUGO identifier, MWG oligo
number, NCBI mRNA reference sequence number and the protein
reference number. The full length sequences can be found at
http://www.ncbi.nim.nih.gov/entrez/.
EXAMPLES
[0130] The examples described herein are for purposes of
illustrating embodiments of the invention and are not intended to
limit the scope of the invention. Other embodiments, methods and
types of analyses are within the scope of persons of ordinary skill
in the molecular diagnostic arts and need not be described in
detail hereon. Other embodiments within the scope of the art that
are based on the teachings herein are considered to be part of this
invention.
Methods
Tumour Collection
[0131] Bladder tumour samples and non-malignant urothelium samples
were collected from surgical specimens resected at Kyoto University
Hospital, Japan.
Urine Collection
[0132] Urine samples from non-malignant controls and bladder cancer
patients were obtained from Kyoto University Hospital, Japan.
Healthy control samples were obtained from Japanese volunteers
(FIG. 1).
RNA Extraction
[0133] Tumour tissues were homogenized in a TriReagent:water (3:1)
mix, then chloroform extracted. Total RNA was then purified from
the aqueous phase using the RNeasy.TM. procedure (Qiagen). RNA was
also extracted from 16 cancer cell lines and pooled to serve as a
reference RNA.
[0134] RNA was extracted from urine by mixing the urine sample with
an equal volume of lysis buffer (5.64M guanidine-HCl, 0.5%
sarkosyl, 50 mM sodium acetate (pH 6.5) and 1 mM
.beta.-mercaptoethanol; pH adjusted to 7.0 with 1.5M Hepes pH 8).
Due to the low amounts of RNA in urine, 7.5 ugs of total bacterial
RNA was added to the urine/lysis buffer mix to act as a carrier.
Total RNA was then extracted using Trizol and the RNeasy.TM.
procedure. RNA preparations were further purified prior to cDNA
synthesis using the Qiagen QIAquick.TM. PCR purification kit.
[0135] RNA was extracted from the blood of three healthy volunteers
by performing a Trizol/RNeasy.TM. extraction on cells enriched from
whole blood using sedimentation in 3.6% dextran.
Microarray Slide Preparation
[0136] Epoxy coated glass slides (MWG Biotech) were printed with
.about.30,000 50mer oligonucleotides (MWG Biotech) using a Gene
Machines microarraying robot, according to the manufacturer's
protocol.
RNA Labeling and Hybridization
[0137] cDNA was transcribed from 5 .mu.g total RNA using
Superscript II.TM. reverse transcriptase (Invitrogen) in reactions
containing 5-(3-aminoallyl) 2' deoxyuridine-5'-4-triphosphate. The
reaction was then de-ionised in a Microcon column before being
incubated with Cy3 or Cy5 in bicarbonate buffer for 1 hour at room
temperature. Unincorporated dyes were removed using a Qiaquick
column (Qiagen) and the sample concentrated to 15 .mu.l in a
SpeedVac. Cy3 and Cy5 labeled cDNAs were then mixed with Ambion
ULTRAhyb.TM. buffer, denatured at 100.degree. C. for 2 min and
hybridized to the microarray slides in hybridisation chambers at
42.degree. C. for 16 hours. The slides were then washed and scanned
twice in an Axon 4000A.TM. scanner at two power settings.
Microarray Analysis of Cancer Marker Genes
[0138] RNA from 53 bladder tumours and 20 non-malignant ("normal")
bladder tissue samples were labeled with Cy5 and hybridized in
duplicate or triplicate with Cy3 labeled reference RNA. After
normalization, the change in expression in each of 29,718 genes was
then estimated by fold change and statistical probability.
Normalisation Procedure
[0139] Median fluorescence intensities detected by Genepix.TM.
software were corrected by subtraction of the local background
intensities. Spots with a background corrected intensity of less
than zero were excluded. To facilitate normalization, intensity
ratios and overall spot intensities were log-transformed. The
logged intensity ratios were corrected for dye and spatial bias
using local regression implemented in the LOCFIT.TM. package.
Logged intensity ratios were regressed simultaneously with respect
to overall spot intensity and location. The residuals of the local
regression provided the corrected logged fold changes. For quality
control, ratios of each normalized microarray were plotted in
respect to spot intensity and localization. The plots were
subsequently visually inspected for any remaining artifacts.
Additionally, an ANOVA model was applied for the detection of
pin-tip bias. All results and parameters of the normalization were
inserted into a Postgres-database for statistical analysis.
Statistical Analysis
[0140] To improve the comparison of measured fold changes between
arrays, log 2 (ratios) were scaled to have the same overall
standard deviation per array. This standardization reduced the
average within-tissue class variability. A rank-test based on fold
changes was then used to improve the noise robustness. This test
consists of two steps: (i) calculation of the rank of fold change
(Rfc) within arrays and ii) subtraction of the median (Rfc) for
normal tissue from the median (Rfc) for tumour tissue. The
difference of both median ranks defines the score of the fold
change rank. Three additional statistical tests were also performed
on standardized data: 1) Two sample student's t-test, 2) the
Wilcoxon test and 3) Statistical Analysis of Microarrays (SAM). The
most significantly down-regulated genes determined by each of the
statistical methods (rank fold change, t-test, Wilcoxon test, and
SAM) were given a rank score for each test. All rank scores were
then added into one summated rank score.
cDNA Synthesis from Urine RNA
[0141] Total urine RNA was annealed to gene-specific primers for
each of the bladder tumour markers by incubating at 70.degree. C.
then cooling on ice for 2 mins in 50 ul reactions containing
forward primers at 0.01 .mu.g/.mu.l. Each cDNA reaction contained
annealed RNA and 4 .mu.l of 5.times. Superscript II reverse
transcriptase buffer (Invitrogen, USA), 2 .mu.l of 0.1M DTT
(Invitrogen, USA), 0.5 .mu.l of RNase out (40 U/.mu.L),
(Invitrogen, USA), 4 .mu.l of 10 mM dNTP (Invitrogen, USA) and 0.5
.mu.l of Superscript II reverse transcriptase (200 U/.mu.l),
(Invitrogen, USA) in a final volume of 20 .mu.l. Reactions were
incubated at 42.degree. C. for 1 hour, 10 minutes at 70.degree. C.
and 1 minute at 80.degree. C. Reactions were cleaned prior to qPCR
with Qiagen QIAquick PCR purification columns (Qiagen, Victoria,
Australia) and stored at -80.degree. C.
Quantitative Real-Time PCR
[0142] Real-time or quantitative PCR (qPCR) is used for absolute or
relative quantitation of PCR template copy number. Taqman.TM. probe
and primer sets were designed using Primer Express V 2.0.TM.
(Applied Biosystems). Where possible, all potential splice variants
were included in the resulting amplicon, with amplicon preference
given to regions covered by the MWG-Biotech-derived microarray
oligonucleotide. Primer and probe sequences are shown in FIG. 2.
Alternatively, if the target gene was represented by an
Assay-on-Demand.TM. expression assay (Applied Biosystems) covering
the desired amplicons, these were used. In the in-house designed
assays, primer concentration was titrated using a SYBR green
labeling protocol and cDNA made from the reference RNA.
Amplification was carried out on an ABI Prism.TM. 7000 sequence
detection system under standard cycling conditions. When single
amplification products were observed in the dissociation curves,
standard curves were generated over a 625 fold concentration range
using optimal primer concentrations and 5'FAM-3TAMRA phosphate
Taqman.TM. probe (Proligo) at a final concentration of 250 nM.
Assays giving standard curves with regression coefficients over
0.98 were used in subsequent analyses.
[0143] Assays were performed in 96 well plates. Each plate
contained a reference cDNA standard curve, over a 625-fold
concentration range. For the urine qPCR, total RNA extracted from
.about.0.5 mls unfractionated urine was used in each reaction. The
.DELTA.Ct (target gene Ct--mean reference cDNA Ct) was calculated
for each marker, and used in subsequent ratios, regression or
classification analysis.
Expression of Markers in Blood
[0144] The expression of the markers shown in FIGS. 3 and 4 in
whole blood was determined in silico. Microarray probes were linked
to UniGene clusters via the GenBank accession numbers of their
target mRNAs, and the tissue expression profile from UniGene used
to determine the number of expressed sequence tags (ESTs) in blood
libraries. Only genes with 0 or 1 expressed sequence tags (EST) are
shown in FIG. 4. To confirm the low expression of LTB4DH in whole
blood, RT-qPCR was carried out on total RNA extracted from whole
blood using the primers and probes shown in FIG. 2. No significant
expression was observed (results not shown).
Identification of Down Regulated Bladder Cancer Markers
[0145] To identify down-regulated markers of bladder cancer, we
performed microarray studies on RNA from 53 bladder tumours and 20
non-malignant bladder tissue samples using 30,000 oligonucleotide
chips. FIG. 3 shows the statistical analysis of microarray data for
300 genes that show significant downregulation in bladder cancer
tissue compared to non-malignant tissue. FIG. 3 includes the HUGO
gene name and symbol, the protein reference sequence number, the
NCBI mRNA reference sequence number, the MWG Biotech probe
oligonucleotide number, the median fold change in gene expression
between tumour and non-malignant tissue, the results of an original
unadjusted Student's t-test, the results of the 2-sample Wilcoxon
test, the results of the SAM test, and the summated rank score.
Identification of Preferred Under-Expressed Bladder Tumour Markers
for Use in Urine Tests for Bladder Cancer
[0146] Because urinary hematuria is a common co-occurrence with
bladder cancer, it is an advantage that bladder cancer markers are
not significantly elevated in whole blood. In addition, because the
downregulated markers are being used in ratios, regression or
classification analysis, it is an advantage that they be present in
sufficiently high copy numbers in both tumour cells and
non-malignant bladder cells to enable reliable detection in urine.
To identify suitable markers, we screened the genes in FIG. 3 for a
subset that had little or no representation in blood EST libraries,
and had higher than median expression in non-malignant tissue.
Median expression was estimated by ranking the 30,000
oligonucleotides on the array by their median intensity in the
samples analysed in the microarray study. Markers that met the
criteria are shown in FIG. 4. FIG. 4 includes the HUGO gene name
and symbol, the protein reference sequence number, the NCBI mRNA
reference sequence number, the median fold change, the rank score,
the median rank of microarray spot intensity in tumour tissue and
non-malignant tissue, and the number of ESTs present in blood EST
libraries.
[0147] The down regulation observed in the array data was validated
by qPCR for three genes shown in FIG. 4, LTB4DH, BAG1 and FLJ21511.
These genes were tested on total RNA from 10 tumour samples and 10
non-malignant samples. LTB4DH, BAG1 and FLJ21511 showed an average
downregulation in bladder tumours compared to bladder non-malignant
tissue of 2.5 fold, 1.4 fold and 6.1 fold, respectively, in these
samples.
qPCR Analysis of Urine Using LTB4DH
[0148] Urine from TCC patients and controls with non-malignant
urological conditions was collected by either voiding or
catheterisation. Total RNA was extracted from the voided urine of
42 controls and the voided or catheterised urine of 37 TCC patients
and used in quantitative RT-PCR using primers and probes for LTB4DH
and three over-expressed markers, IGFBP5, MDK and HoxA13. The
.DELTA.Ct ratios were determined for IGFBP5/LTB4DH, MDK/LTB4DH and
HoxA13/LTB4DH. This data is illustrated by the box plots in FIG. 5,
which show a clear difference in the spread of data between the
urine samples from controls and TCC patients for each of the three
tests. The most accurate test was IGFBP5/LTB4DH which demonstrated
sensitivity and specificity of 87% and 88% in this sample cohort,
respectively (FIG. 6a). To illustrate the correspondence between
sensitivity and specificity for each of these tests, ROC curves are
shown in FIG. 7. The areas under the curve for IGFBP5/LTB4DH,
MDK/LTB4DH and HoxA13/LTB4DH are 0.9223, 0.9199, and 0.7497,
respectively. These areas, which measure test accuracy, indicate
that all three ratios with LTB4DH are useful tests, in particular
IGFBP5/LTB4DH and MDK/LTB4DH.
[0149] To increase the sensitivity and specificity of TCC
detection, combinations of two tests were used. The optimal
sensitivities and specificities of these test combinations are
shown in FIG. 6b. FIG. 8a-f shows the separation of data in 2
dimensional space for each of the three tests using LTB4DH and
BAG1. This data shows that combinations of two or more tests that
include either of the downregulated BTMs LTB4DH or BAG1, are able
to achieve sensitivities and specificities of over 90%. Moreover,
because these tests are measuring simple gene expression signatures
and not absolute levels of markers, they will be robust to
variations in urine concentration.
[0150] To demonstrate the robustness of tests involving ratios with
LTB4DH to urine concentration, the levels of IGFBP5 alone
(.DELTA.Ct) and IGFBP5/LTB4DH were plotted as a function of urine
concentration (FIG. 9a-b) and trendlines fitted to the data. It can
be seen that for both urine samples from non-malignant controls and
patients with TCC, there is a decrease in the IGFBP5 .DELTA.Ct with
increasing urine concentration that is absent in the IGFBP5/LTB4DH
ratio. The effect is most pronounced with the non-malignant samples
because of the absence of other influences such as tumour size and
tumour heterogeneity in the expression of IGFBP5 and LTB4DH.
[0151] In some instances, when single markers are used in bladder
cancer assays, the method of urine sample collection can affect the
amount of marker detected due to variations in the number of
exfoliated bladder cells collected. This bias could lead to false
positive or false negative results in a small proportion of
samples. The use of ratios including LTB4DH or other low-expressing
genes should provide a method to compensate for different methods.
To test this hypothesis, samples collected from TCC patients by
either simple voiding (nine samples) or catheterisation (28
samples) were tested for the presence of TCC markers and LTB4DH.
Analysis of the TCC markers alone showed that the voided samples
were more heavily represented at the lower end of the range of data
(higher Ct), consistent with a lower average number of exfoliated
cells in these samples compared to the catheterised samples. This
is illustrated in the self-self scatter plots for IGFBP5, MDK and
HoxA13 in FIG. 10a-c. In contrast, when ratios between these
markers and LTB4DH were calculated, the voided and catheterised
samples were spread over similar ranges of Ct ratios (FIG. 10d-f),
illustrating that the calculation of gene expression signatures
between high expressing markers and low expressing markers such as
LTB4DH compensate for variations in marker levels introduced by
different urine sampling methodologies.
[0152] Urine samples from patients with low grade tumours are often
borderline in their accumulation of BTMs due to the presence of
only small numbers of exfoliated cells in these samples. These
samples are therefore at high risk of being incorrectly classified
due to variations in sampling method or urine concentration.
[0153] The utility of gene expression ratios that incorporate
down-regulated genes for the detection of TCC is therefore likely
to be pronounced when applied to the detection of low grade
TCC.
[0154] To demonstrate this effect, a cohort of voided 43 urine
samples from patients with low grade TCC and 123 controls were
tested with the markers IGFBP5, HoxA13 and LTB4DH. The clinical
characteristics of the cohort are summarised in FIG. 13. The qPCR
data for IGFBP5 and HoxA13 were analysed alone and in ratios with
LTB4DH using the area under the ROC curve as a measure of test
accuracy (STATA statistics package). The results are summarized in
FIG. 14. Using the IGFBP5 marker, LTB4DH increased the accuracy of
detection of low grade (grade 1-2) stage Ta TCCs by 9% and low
grade TCCs of any stage by 8%. The accuracy of HoXA13 testing of
low grade stage Ta TCCs was increased by 3%.
Linear Discriminate Analysis of qPCR Data using LTB4DH
[0155] Linear discriminate analysis (LDA) is a statistical
technique (Fisher R. A. "The Use of Multiple Measurements in
Taxonomic Problems", Annals of Eugenics 7 179 (1936)) in which a
linear combination of variables is generated, such that there is
maximal separation between two or more groups. This linear
combination of variables is termed the "linear discriminant", which
is a linear function of the input variables that maximally
separates the classes of the data set. The ability of LDA (or any
other classification technique) to characterise a particular
dataset, such as qPCR data, can be tested using cross-validation.
In this method, part of the dataset is used to generate a
classifier, and part of the dataset is used to measure the
effectiveness of that classifier. The partitioning of the dataset
into training and testing sets can be repeated multiple times (each
time generating a new classifier). In k-fold cross-validation, the
dataset is split k-wise, and each subset is used as the testing set
in one of k rounds of training and validation. This can be extended
to leave-one-out cross-validation (LOOCV) where each sample is
classified according to a classifier generated from the remaining
samples in the dataset ("leaving one out"; leaving out the sample
which is being tested).
[0156] LDA and LOOCV were used to illustrate the utility of the
downregulated BTM, LTB4DH, in improving the diagnosis of TCC. qPCR
was first carried out on the cohort of control and TCC urine
samples described in FIG. 13 which were supplemented with an
additional 30 grade 3 tumours (5>stage 1, 13=stage 1, 4=T is,
and 8=Ta). Combinations of the six genes LTB4DH, MDK, IGFBP5,
HOXA13, TOP2a and CDC2 were tested for classifier performance, as
judged by LOOCV. The posterior probability (that the sample "left
out" was a TCC sample) was used to generate ROC curves using the
ROCR package of the R statistical programming environment. The
sensitivity of the classifier for a given specificity was obtained
by reference to the appropriate ROC curve.
[0157] The sensitivity of detection of TCC using combinations of
upregulated BTMs with and without LTB4DH was determined at a
specificity of 85%. The results of this analysis are shown in FIG.
15. It can be seen that the addition of LTB4DH to assays including
combinations of the upregulated BTMs MDK, IGFBP5, Top2a, cdc2 and
HoxA13 increased the overall sensitivity by 1-2% and the
sensitivity of detection of Stage Ta tumours, grade 1-2 tumours and
grade 3 tumours by up to 3%.
[0158] Wherein in the foregoing description reference has been made
to integers or components having known equivalents, such
equivalents are herein incorporated as if individually set
fourth.
[0159] Although the invention has been described by way of example
and with reference to possible embodiments thereof, it is to be
appreciated that improvements and/or modifications may be made
without departing from the scope or the spirit thereof.
INDUSTRIAL APPLICABILITY
[0160] Methods for detecting BTM family members include detection
of nucleic acids, proteins and peptides using microarray and/or
real time PCR methods. The compositions and methods of this
invention are useful in diagnosis of disease, evaluating efficacy
of therapy, and for producing reagents and test kits suitable for
measuring expression of BTM family members.
* * * * *
References