U.S. patent application number 13/475432 was filed with the patent office on 2013-06-27 for chromosome copy number gain as a biomarker of urothelial carcinoma lethality.
This patent application is currently assigned to Dana-Farber Cancer Institute, Inc.. The applicant listed for this patent is Joaquim Bellmunt, Philip Kantoff, Massimo Loda, Jonathan E. Rosenberg. Invention is credited to Joaquim Bellmunt, Philip Kantoff, Massimo Loda, Jonathan E. Rosenberg.
Application Number | 20130165331 13/475432 |
Document ID | / |
Family ID | 47175368 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130165331 |
Kind Code |
A9 |
Rosenberg; Jonathan E. ; et
al. |
June 27, 2013 |
CHROMOSOME COPY NUMBER GAIN AS A BIOMARKER OF UROTHELIAL CARCINOMA
LETHALITY
Abstract
Diagnostic assays for medically classifying cancer patients are
provided. The method comprises assessing a tissue sample of the
patient for the presence of a copy number gain of chromosome
regions 1q23.3 and/or 1q21.2. Copy number gain of chromosome
regions 1q23.3 and/or 1q21.2 is indicative of a less favorable
prognosis as compared to the prognosis if there was no copy number
gain in the same regions.
Inventors: |
Rosenberg; Jonathan E.; (New
York, NY) ; Bellmunt; Joaquim; (Barcelona, ES)
; Kantoff; Philip; (Brookline, MA) ; Loda;
Massimo; (Belmont, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rosenberg; Jonathan E.
Bellmunt; Joaquim
Kantoff; Philip
Loda; Massimo |
New York
Barcelona
Brookline
Belmont |
NY
MA
MA |
US
ES
US
US |
|
|
Assignee: |
Dana-Farber Cancer Institute,
Inc.
Boston
MA
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20120295807 A1 |
November 22, 2012 |
|
|
Family ID: |
47175368 |
Appl. No.: |
13/475432 |
Filed: |
May 18, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61487532 |
May 18, 2011 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/6.11;
435/6.14 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 2600/112 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
506/9 ; 435/6.14;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C40B 30/04 20060101 C40B030/04; G01N 21/64 20060101
G01N021/64 |
Claims
1. A method for diagnosing a subject, comprising obtaining a tissue
sample of the subject, determining from the tissue sample whether
the subject has a copy number gain for a region of the subjects
genome defined by chr:159256737-159407240 and/or
chr1:148747863-149235943, wherein copy number gain is indicative of
a less favorable prognosis than the prognosis if there is no copy
number gain.
2. The method of claim 1, further comprising providing a report
whether the subject has a copy number gain.
3. The method of claim 1, further comprising reporting prognostic
information about the subject based on the existence or
non-existence of copy number gain for the region.
4. The method of claim 1, further comprising providing information,
based on the existence or non-existence of copy number gain of the
region, that assists in medical care of the subject.
5. The method of claim 1, further comprising reporting the
existence or non-existence of copy number gain for the region to
the subject.
6. The method of claim 1, wherein the less favorable prognosis is
(a) a higher risk of developing invasive/metastatic disease, (b) a
higher risk of mortality within six (6) months, or (c) a higher
risk of mortality within two (2) years.
7. The method of claim 1, wherein the tissue sample is
cancerous.
8. The method of claim 1, wherein the tissue sample is a bladder
tissue sample.
9. The method of claim 8, wherein the tissue sample is
cancerous.
10. The method of claim 1, wherein the determining comprises
hybridization.
11. The method of claim 1, wherein the determining comprises
contacting DNA from the tissue sample with a probe.
12. The method of claim 11, wherein the probe is a DNA probe.
13. The method of claim 12, wherein the DNA probe is fluorescently
labeled.
14. The method of claim 13, wherein the probe hybridizes to at
least part of the genome region defined by chr
1:159256737-159407240 and/or chr 1:148747863-149235943.
15. The method of claim 1, wherein the determining comprises
determining a duplication of the entire or a portion of the genome
regions defined by chr 1:159256737-159407240 and/or
chr1:148747863-149235943.
16. The method of claim 1, wherein the determining comprises
determining a duplication of the entire or a portion of the genome
regions defined by the genes located in the genome regions defined
by chr:159256737-159407240 and/or chr1:148747863-149235943.
17. The method of claim 1, wherein the determining comprises
determining a duplication of one or more genes selected from the
group consisting of JAM1, NIT1, PFDN2, PPOX, KAT, USF1, DEDD,
DEDPR01, USP21, F11R, UFC1, PVRL4, KLHC9, ARHGAP30, TSTD1, FCGR and
PBX1.
18. The method of claim 1, wherein the determining comprises
determining a duplication of one or more genes selected from the
group consisting of ARNT, CTSK, CTSS, ECM1, ENSA, MCL1, ANXA9,
SETDB1, LASS2, ADAMTSL4, GOLPH3L, FAM63A, and HORMAD1.
19. The method of claim 1, wherein the subject is known to have
urinary cancer.
20. The method of claim 1, wherein the subject is known to have
invasive or metastatic urinary cancer.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application Ser. No. 61/382,710,
filed Sep. 14, 2010, the content of which is incorporated by
reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Current estimates from the American Cancer Society indicate
that over 70,000 new cases of urothelial carcinoma (UC) are
diagnosed annually in the US alone, and over 14,000 will die from
the disease. While many patients' tumors recur in the urinary tract
only, this relapsing non-invasive disease is usually not
life-threatening. In contrast, other patients develop invasive
and/or metastatic disease, the lethal phenotype of UC. While our
understanding of the molecular changes in UC has rapidly evolved
over the last few decades, the therapeutic arsenal has not.
First-line treatment for advanced disease remains platinum-based
combination chemotherapy, and no FDA-approved second-line treatment
exists. Attempts to improve current therapies have focused on
cytotoxic chemotherapy dose intensity and combination doublet and
triplet regimens, none of which have led to substantial
improvements in survival. Metastatic UC remains incurable in the
vast majority of patients, with a median survival of approximately
8 months without treatment, and 14 months with treatment. Unlike
treatment of other solid tumors, targeted therapies thus far have
failed to advance the standard of care for UC. Furthermore, there
is no clear molecular understanding of what defines the lethal
phenotype of UC. Thus, there is an urgent need for new biomarkers
and treatment approaches.
SUMMARY OF THE INVENTION
[0003] It has been discovered, surprisingly, that copy number gain
of chromosome regions defined by chr 1:159256737-159407240 and/or
chr 1:148747863-149235943 can predict poor prognosis in cancer. The
invention involves obtaining a tissue sample of the subject, and
determining from the tissue sample whether the subject has a copy
number gain for a region of the subjects genome defined by chr
1:159256737-159407240 and/or chr 1:148747863-149235943. A copy
number gain of the regions is indicative of a less favorable
prognosis than the prognosis if there is no copy number gain.
[0004] According to one aspect of the invention, a copy number gain
means determining a duplication of the entire or a portion of the
genome regions defined by chr 1:159256737-159407240 and/or chr
1:148747863-149235943. In some embodiments, a copy number gain
means determining a duplication of the entire or a portion of the
genome regions defined by the genes located in the genome regions
defined by chr 1:159256737-159407240 and/or chr
1:148747863-149235943. In some embodiments, the present invention
involves determining whether the tissue sample from the subject has
a copy number gain of any one or more of the genes located in the
genome regions defined by chr 1:159256737-159407240, such as, JAM1,
NIT1, PFDN2, PPOX, KAT, USF1, DEDD, DEDPR01, USP21, F11R, UFC1,
PVRL4, KLHC9, ARHGAP30, TSTD1, FCGR and PBX1. In some embodiments,
the present invention involves determining whether the tissue
sample from the subject has a copy number gain of any one or more
of the genes located in the genome regions defined by chr
1:148747863-149235943, such as, ARNT, CTSK, CTSS, ECM1, ENSA, MCL1,
ANXA9, SETDB1, LASS2, ADAMTSL4, GOLPH3L, FAM63A, and HORMAD1.
[0005] In some embodiments, the method further comprises providing
a report whether the subject has a copy number gain. In some
embodiments, the method further comprises reporting prognostic
information about the subject based on the existence or
non-existence of copy number gain for the region. In some
embodiments, the method further comprises providing information,
based on the existence or non-existence of copy number gain of the
region, that assists in medical care of the subject. The existence
or non-existence of copy number gain for the region can optionally
be reported to the subject.
[0006] In some embodiments, the less favorable prognosis may be (a)
a higher risk of developing invasive/metastatic disease, (b) a
higher risk of mortality within six (6) months, or (c) a higher
risk of mortality within two (2) years. It is believed also that
copy number gain may predict the likelihood that a subject may
develop cancer.
[0007] In some embodiments, the tissue sample is cancerous. In some
embodiments, the tissue sample is a bladder tissue sample. In some
embodiments, the bladder tissue sample is cancerous.
[0008] According to some aspects of the invention, the determining
step comprises hybridization. In some embodiments, the determining
step comprises contacting DNA from the tissue sample with a probe.
The probe could be a DNA probe, for example a PCR probe, an array
of DNA on a chip or a labeled DNA probe, such as a fluorescently
labeled DNA probe. The probe hybridizes to at least part of the
genome region defined by chr 1:159256737-159407240 and/or chr
1:148747863-149235943.
[0009] The subject (individual) is a human. In some embodiments,
the subject is known to have urinary cancer, or invasive or
metastatic urinary cancer.
[0010] These and other aspects of the inventions, as well as
various advantages and utilities will be apparent with reference to
the Detailed Description. Each aspect of the invention can
encompass various embodiments as will be understood.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows that gain of the 150 kb region of 1q23.3 was
significantly associated with lethal bladder cancer.
[0012] FIG. 2 shows recurrent copy number gains (2A) and losses
(2B) in the Spanish cohort of UC patients as well as recurrent copy
number gains (2C) and losses (2D) in the BW/DF cohort of UC
patients.
[0013] FIG. 3 demonstrates the prognostic utility of 1q23.3
amplification. The figure shows the Kaplan-Meier plots for the
Spanish cohort (FIG. 3A; aCGH), BW/DF cohort (FIG. 3B, SNP array),
and BW/DF and Greek cohorts (FIG. 3C, FISH) stratified by the
1q23.3 amplification.
[0014] FIG. 4 displays the wide peak amplification regions in three
bladder cancer cohorts, in the TCGA primary glioblastoma dataset,
as well as the multi-cancer study by Beroukhim et. al. Peak heights
visualize the statistical significance (GISTIC q-values). Gene
names shown in grey are known cancer genes.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention, in one aspect, relates to the
surprising discovery that copy number gain of particular regions of
chromosomes is associated with poor clinical outcomes. In
particular, the invention is based on the identification of the
association between copy number gain of chromosome regions 1q23.3
and 1q21.2 with a poor prognosis in UC. While previous work has
identified 1q23.3 as gained in UC, and in other malignancies, this
previous work did not make any association between copy number
findings and clinical outcomes. As is common in medicine, markers
are discoveries which are shown to be present in some portion of
subjects with a disease. The presence of the marker, however, is
not established as having any predictive value as to whether a
disease will develop in the first instance (should the marker be
present prior to development of the disease) or how the disease
will progress (should the disease already be present. Instead,
until it is proven otherwise, such markers may be assumed to result
from the disease.
[0016] According to one aspect, the present invention provides
diagnostic assays for medically classifying cancer patients which
comprises assessing a tissue sample of the patient for the presence
of a copy number gain of chromosome regions 1q23.3 and/or 1q21.2.
Copy number gain of chromosome regions 1q23.3 and/or 1q21.2 is
indicative of a less favorable prognosis as compared to the
prognosis if there was no copy number gain in the same regions.
[0017] The tissue sample can be any tissue sample from which a copy
number gain can be determined. In some embodiments, the tissue
sample can be obtained from a local solid tumor or suspected tumor,
or tumor resulting from metastasis. Non-limiting examples of tissue
samples include tumor samples obtained from bladder cancer,
urothelial carcinoma, small cell carcinoma, squamous carcinoma,
adenocarcinoma, leiomyosarcoma (a tumor arising from smooth
muscle), lymphoma (a tumor that usually arises in the lymph nodes),
malignant melanoma (a tumor that usually arises from the skin)
breast cancer, cervical cancer, colon cancer (including colorectal
cancer), esophageal cancer, gastric cancer, head and neck cancer,
liver cancer, lung cancer (both small cell and non-small cell),
melanoma, myeloma, neuroblastoma, ovarian cancer, pancreatic
cancer, prostate cancer, renal cancer, sarcoma (including
osteosarcoma), skin cancer (including squamous cell carcinoma),
stomach cancer, testicular cancer, thyroid cancer, and uterine
cancer. In important embodiments, the tissue sample comprises a
bladder sample of the patient.
[0018] The tissue sample may be a fresh frozen tissue sample, or a
paraffin-embedded tissue sample. The tissue can be genomic tissue,
extra-nuclear tissue (e.g., tissue containing mRNA), or can be
isolated from blood (i.e. tumor tissue resulting from metastasis).
The copy number gain can be determined in the genome itself (i.e.
the location where the copy exists), or the copy number gain can be
determined indirectly. For example, copy number is sometimes
associated with increased levels of mRNA, protein expression or
activity. Thus, copy number gain may be determined from tissues
containing the indirect measure. Non-limiting examples of such
tissue samples include urine sample, blood sample, bone marrow
sample, lymph node sample, and spinal fluid sample.
[0019] According to one aspect of the invention, a tissue sample
obtained from the patient is analyzed. To practice the invention as
claimed, it is not necessary for the person determining the copy
number gain to obtain the tissue sample directly from the subject.
In some embodiments, the tissue sample may be removed from the
subject by a medical practitioner (e.g., a doctor, nurse, or a
clinical laboratory practitioner), and then provided to the person
determining the copy number gain. The tissue sample may be provided
to the person determining the copy number gain by the subject or by
a medical practitioner (e.g., a doctor, nurse, or a clinical
laboratory practitioner). In some embodiments, the person
determining the copy number gain obtains a tissue sample from the
subject by removing the sample from the subject.
[0020] As used herein, a copy number gain means determining a
duplication of the entire or a portion of the genome regions
defined by chr 1:159256737-159407240 and/or
chr1:148747863-149235943. In some embodiments, a copy number gain
means determining a duplication of the entire or a portion of the
genome regions defined by the genes located in the genome regions
defined by chr 1:159256737-159407240 and/or
chr1:148747863-149235943. In some embodiments, a copy number gain
means determining a duplication of any one or more of the genes
located in the genome regions defined by chr 1:159256737-159407240
and/or chr1:148747863-149235943. Thus, in some embodiments, the
present invention involves determining whether the tissue sample
from the subject has a copy number gain of any one or more of the
genes located in the genome regions defined by chr
1:159256737-159407240, such as, JAM1, NIT1, PFDN2, PPOX, KAT, USF1,
DEDD, DEDPR01, USP21, F11R, UFC1, PVRL4, KLHC9, ARHGAP30, TSTD1,
FCGR and PBX1. In some embodiments, the present invention involves
determining whether the tissue sample from the subject has a copy
number gain of any one or more of the genes located in the genome
regions defined by chr1:148747863-149235943, such as, ARNT, CTSK,
CTSS, ECM1, ENSA, MCL1, ANXA9, SETDB1, LASS2, ADAMTSL4, GOLPH3L,
FAM63A, and HORMAD1.
[0021] Methods of determining the copy number gain of a particular
genomic region are well known in the art, and include,
hybridization and amplification based assays. According to the
methods of the invention, DNA copy number gains may be identified
using copy number profiling, such as comparative genomic
hybridization (CGH) (including both dual channel hybridization
profiling and single channel hybridization profiling (e.g.
SNP-CGH)). Other suitable methods including fluorescent in situ
hybridization (FISH), PCR, nucleic acid sequencing, and Southern
blot analysis may be used in accordance with the invention.
[0022] In some embodiments of the invention, the DNA copy number
gains are identified using CGH. In comparative genomic
hybridization methods, a "test" collection of nucleic acids (e.g.
from a tumor or cancerous cells) is labeled with a first label,
while a second collection (e.g. from a normal cell or tissue) is
labeled with a second label. The ratio of hybridization of the
nucleic acids is determined by the ratio of the first and second
labels binding to each fiber in an array. Differences in the ratio
of the signals from the two labels, for example, due to gene
amplification in the test collection, is detected and the ratio
provides a measure of the gene copy number, corresponding to the
specific probe used. A cytogenetic representation of DNA
copy-number variation can be generated by CGH, which provides
fluorescence ratios along the length of chromosomes from
differentially labeled test and reference genomic DNAs.
[0023] In some embodiments of the present invention, the DNA copy
number gains are analyzed by microarray based CGH (array-CGH).
Microarray technology offers high resolution. For example, the
traditional CGH generally has a 20 Mb limited mapping resolution;
whereas in microarray based CGH, the fluorescence ratios of the
differentially labeled test and reference genomic DNAs provide a
locus-by-locus measure of DNA copy-number variation, thereby
achieving increased mapping resolution. Details of various
microarray methods can be found in the literature. See, for
example, U.S. Pat. No. 6,232,068; Pollack et al., Nat. Genet.,
23(1):41-6, (1999), Pastinen (1997) Genome Res. 7: 606614; Jackson
(1996) Nature Biotechnology 14:1685; Chee (1995) Science 274: 610;
WO 96/17958, Pinkel et al. (1998) Nature Genetics 20: 207-211 and
others. High resolution CGH arrays can be performed using the
Agilent or Affymetrix platforms. The DNA used to prepare the CGH
arrays is not critical. For example, the arrays can include genomic
DNA, e.g., overlapping clones that provide a high resolution scan
of the portion of the genome of interest.
[0024] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system that multiplies
the target nucleic acid being detected. Examples of such systems
include the polymerase chain reaction (PCR) system and the ligase
chain reaction (LCR) system. Other suitable methods include are the
nucleic acid sequence based amplification (NASBAO, Cangene,
Mississauga, Ontario) and Q Beta Replicase systems. In one
embodiment of the invention, the DNA copy number alterations in a
genome are determined by single channel profiling, such as single
nucleotide polymorphism (SNP)-CGH. Traditional CGH data consists of
two channel intensity data corresponding to the two alleles. The
comparison of normalized intensities between a reference and
subject sample is the foundation of traditional array-CGH. Single
channel profiling (such as SNP-CGH) is different in that a
combination of two genotyping parameters are analyzed: normalized
intensity measurement and allelic ratio. Collectively, these
parameters provide a more sensitive and precise profile of
chromosomal aberrations. SNP-CGH also provides genetic information
(haplotypes) of the locus undergoing aberration.
[0025] In some embodiments, NanoString direct DNA and/or mRNA
quantification is used to determine the copy number gain (Nat
Biotechnol. 2008 March; 26(3):293-4). NanoString technology
requires less DNA as compared to CGH-array, and allows precise
measurement of copy number gain.
[0026] In some embodiments, the Panomics CNV DNA service may be
used to determine the copy number gain
(www.nanostring.com/products/subpage.asp?id=485).
[0027] In another embodiment, FISH is used to determine the DNA
copy number alterations in a genome. Fluorescence in situ
hybridization (FISH) is known to those of skill in the art (see
Angerer, 1987 Meth. Enzymol., 152: 649). Generally, in situ
hybridization comprises the following major steps: (1) fixation of
tissue or biological structure to be analyzed; (2) prehybridization
treatment of the biological structure to increase accessibility of
target DNA, and to reduce nonspecific binding; (3) hybridization of
the mixture of nucleic acids to the nucleic acid in the biological
structure or tissue; (4) post-hybridization washes to remove
nucleic acid fragments not bound in the hybridization, and (5)
detection of the hybridized nucleic acid fragments.
[0028] In a typical in situ hybridization assay, cells or tissue
sections are fixed to a solid support, typically a glass slide. If
a nucleic acid is to be probed, the cells are typically denatured
with heat or alkali. The cells are then contacted with a
hybridization solution at a moderate temperature to permit
annealing of labeled probes specific to the nucleic acid sequence
encoding the protein. The targets (e.g., cells) are then typically
washed at a predetermined stringency or at an increasing stringency
until an appropriate signal to noise ratio is obtained.
[0029] The probes used in such applications are typically labeled,
for example, with radioisotopes or fluorescent reporters. Preferred
probes are sufficiently long, for example, from about 50, 100, or
200 nucleotides to about 1000 or more nucleotides, to enable
specific hybridization with the target nucleic acid(s) under
stringent conditions.
[0030] In some applications it is necessary to block the
hybridization capacity of repetitive sequences. Thus, in some
embodiments, tRNA, human genomic DNA, or Cot-1 DNA is used to block
non-specific hybridization.
[0031] In another embodiment, Southern blotting is used to
determine the DNA copy number alterations in a genome. Methods for
doing Southern blotting are known to those of skill in the art (see
Current Protocols in Molecular Biology, Chapter 19, Ausubel, et
al., Eds., Greene Publishing and Wiley-Interscience, New York,
1995, or Sambrook et al., Molecular Cloning: A Laboratory Manual,
2d Ed. vol. 1-3, Cold Spring Harbor Press, NY, 1989). In such an
assay, the genomic DNA (typically fragmented and separated on an
electrophoretic gel) is hybridized to a probe specific for the
target region. Comparison of the intensity of the hybridization
signal from the probe for the target region with control probe
signal from analysis of normal genomic DNA (e.g., genomic DNA from
the same or related cell, tissue, organ, etc.) provides an estimate
of the relative copy number of the target nucleic acid.
[0032] In one embodiment, amplification-based assays, such as PCR,
are used to determine the DNA copy number alterations in a genome.
In such amplification-based assays, the genomic region where a copy
number alteration occurred serves as a template in an amplification
reaction. In a quantitative amplification, the amount of
amplification product will be proportional to the amount of
template in the original sample. Comparison to appropriate controls
provides a measure of the copy number of the genomic region.
Methods of "quantitative" amplification are well known to those of
skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that may
be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR are provided, for example, in Innis et al. (1990)
PCR Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.
[0033] Real time PCR can be used in the methods of the invention to
determine DNA copy number alterations. (See, e.g., Gibson et al.,
Genome Research 6:995-1001,1996; Heid et al., Genome Research
6:986-994, 1996). Real-time PCR evaluates the level of PCR product
accumulation during amplification. To measure DNA copy number,
total genomic DNA is isolated from a sample. Real-time PCR can be
performed, for example, using a Perkin Elmer/Applied Biosystems
(Foster City, Calif.) 7700 Prism instrument. Matching primers and
fluorescent probes can be designed for genes of interest using, for
example, the primer express program provided by Perkin
Elmer/Applied Biosystems (Foster City, Calif). Optimal
concentrations of primers and probes can be initially determined by
those of ordinary skill in the art, and control (for example,
beta-actin) primers and probes may be obtained commercially from,
for example, Perkin Elmer/Applied Biosystems (Foster City, Calif.).
To quantify the amount of the specific nucleic acid of interest in
a sample, a standard curve is generated using a control. Standard
curves may be generated using the Ct values determined in the
real-time PCR, which are related to the initial concentration of
the nucleic acid of interest used in the assay. Standard dilutions
ranging from 10-106 copies of the gene of interest are generally
sufficient. In addition, a standard curve is generated for the
control sequence. This permits standardization of initial content
of the nucleic acid of interest in a tissue sample to the amount of
control for comparison purposes.
[0034] Methods of real-time quantitative PCR using TaqMan probes
are well known in the art. Detailed protocols for real-time
quantitative PCR are provided, for example, for RNA in: Gibson et
al., 1996, A novel method for real time quantitative RT-PCR. Genome
Res., 10:995-1001; and for DNA in: Heid et al., 1996, Real time
quantitative PCR. Genome Res., 10:986-994.
[0035] A TaqMan-based assay also can be used to quantify a
particular genomic region for DNA copy number alterations. TaqMan
based assays use a fluorogenic oligonucleotide probe that contains
a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes
to a PCR product, but cannot itself be extended due to a blocking
agent at the 3' end. When the PCR product is amplified in
subsequent cycles, the 5' nuclease activity of the polymerase, for
example, AmpliTaq, results in the cleavage of the TaqMan probe.
This cleavage separates the 5' fluorescent dye and the 3' quenching
agent, thereby resulting in an increase in fluorescence as a
function of amplification (see, for example,
www.perkinelmer.com).
[0036] Other suitable amplification methods include, but are not
limited to ligase chain reaction (LCR) (see Wu and Wallace (1989)
Genomics 4:560, Landegren et al. (1988) Science 241:1077, and
Barringer et al. (1990) Gene 89:117), transcription amplification
(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173),
self-sustained sequence replication (Guatelli et al. (1990) Proc.
Nat. Acad. Sci. USA 87:1874), dot PCR, and linker adapter PCR, etc.
In one embodiment, DNA sequencing is used to determine the DNA copy
number alterations in a genome. Methods for DNA sequencing are
known to those of skill in the art.
[0037] In one embodiment, karyotyping (such as spectral
karyotyping, SKY) is used to determine the chromosomal structural
aberrations in a genome. Methods for karyotyping are known to those
of skill in the art. For example, for SKY, a collection of DNA
probes, each complementary to a unique region of one chromosome,
may be prepared and labeled with a fluorescent color that is
designated for a specific chromosome. DNA amplification,
translocations or other structural abnormalities may be determined
based on fluorescence emission of the probes.
[0038] Copy number gain is sometimes associated with a mutation
leading to increased levels of mRNA, protein expression or
activity. In some embodiments, DNA copy number gain is determined
by quantifying the increase in the levels of mRNA or protein
expression and activity. mRNA levels can be measured using methods
well known in the art, such as Northern blot analysis, and real
time PCR. Protein levels can be measured using standard protein
assays, using immunologic-based assays (such as ELISAs and related
techniques), or using activity assays.
[0039] As used herein, the term prognosis refers to a prediction of
the probable outcome of a medical condition, such as cancer. In the
case of the present invention, the prognosis is typically performed
based on the gain in copy number of all or part of the genome
regions defined by chr 1:159256737-159407240 and/or chr
1:148747863-149235943. Suitably, the prognosis may be favorable,
i.e. the absence of copy number gain reflects an improved prognosis
of the cancer indicating that the subject is at a lower risk of
developing invasive/metastatic disease, or has a lower risk of
mortality within six months or two years. Alternatively, the
presence of copy number gain may reflect that the prognosis is less
favorable (i.e. the subject is at a higher risk of developing
invasive/metastatic disease, or has a higher risk of mortality
within six months or two years).
[0040] A report summarizing the results of the analysis, i.e. the
presence or absence of the copy number gain in the subject's genome
and any other information pertaining to the analysis could
optionally be generated as part of the analysis (which may be
interchangeably referred to herein as "providing" a report,
"producing" a report, or "generating" a report). Examples of
reports may include, but are not limited to, reports in paper (such
as computer-generated printouts of test results) or equivalent
formats and reports stored on computer readable medium (such as a
CD, computer hard drive, or computer network server, etc.).
Reports, particularly those stored on computer readable medium, can
be part of a database (such as a database of patient records, which
may be a "secure database" that has security features that limit
access to the report, such as to allow only the patient and the
patient's medical practitioners to view the report, for example).
In addition to, or as an alternative to, generating a tangible
report, reports can also be displayed on a computer screen (or the
display of another electronic device or instrument).
[0041] A report can further be transmitted, communicated or
reported (these terms may be used herein interchangeably), such as
to the individual who was tested, a medical practitioner (e.g., a
doctor, nurse, clinical laboratory practitioner, genetic counselor,
etc.), a healthcare organization, a clinical laboratory, and/or any
other party intended to view or possess the report. The act of
`transmitting` or `communicating` a report can be by any means
known in the art, based on the form of the report. Furthermore,
"transmitting" or "communicating" a report can include delivering a
report ("pushing") and/or retrieving ("pulling") a report. For
example, reports can be transmitted/communicated by such means as
being physically transferred between parties (such as for reports
in paper format), such as by being physically delivered from one
party to another, or by being transmitted electronically or in
signal form (e.g., via e-mail or over the internet, by facsimile,
and/or by any wired or wireless communication methods known in the
art), such as by being retrieved from a database stored on a
computer network server, etc.
[0042] As used herein, the term "reporting prognostic information"
refers to providing information regarding the impact of the
presence of cancer (e.g., as determined by the diagnostic methods
of the present invention) on a subject's future health (e.g.,
expected morbidity or mortality, the likelihood of getting cancer
or, and the risk of metastasis) based on the existence or
non-existence of copy number gain of the region. This information
may assist in the medical care of the subject. Medical care as
defined herein includes both medical diagnosis and therapeutic
treatment of the subject. For example, the copy number gain can
identify subjects who are destined to develop invasive and/or
metastatic cancer, and who, therefore, require aggressive
therapy.
[0043] The present invention is further illustrated by the
following Example, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Materials and Methods
[0044] Patients: Three cohorts of patients were identified for this
study. To be eligible, all patients had to have developed
metastatic UC, and have primary tumor tissue available for
analysis. All specimens were formalin-fixed and paraffin embedded
from three sources: Spain (primary tumors from patients who
developed metastatic disease), Greece (HECOG specimens from an
aborted phase III study of dose-dense gemcitabine and cisplatin or
dose-dense M-VAC chemotherapy), and Dana-Farber/Brigham and Women's
Cancer Center. Slides were evaluated and tumor-bearing 0.6 mm cores
were punched for DNA extraction by standard methodology and/or
tissue microarray (TMA) construction. All cases were collected
under IRB approved protocols at the different institutions,
de-identified, and approved for use via an exemption by the
Dana-Farber/Harvard Cancer Center institutional review board.
[0045] Spanish Cohort. All patients in this cohort were treated for
metastatic disease with platinum-based cytotoxic chemotherapy.
Median overall survival for this cohort was 15 months. DNA was
hybridized to Agilent Oligonucleotide human genome 180 k array CGH
arrays. Normal male 46, XY genomic DNA was obtained from Promega
(Madison, Wis.). Genomic DNA was extracted using the QIAamp DNA
FFPE Tissue Kit (Qiagen, Valencia, Calif.). The Genomic DNA ULS
labeling kit for FFPE Samples (Agilent) was used to chemically
label 500 ng of genomic DNA with either ULS-Cy5 (tumor) or ULS-Cy3
dye (normal/reference DNA) according to the manufacturer's protocol
(AgilentTechnologies, Inc., Palo Alto, Calif.). Samples were
hybridized to the Agilent SurePrint G3 Human CGH+SNP Microarray
4.times.180K. Each slide contains four identical arrays consisting
of approximately 170,334 in situ synthesized 60-mer oligonucleotide
probes that span coding and non-coding sequences with an average
spatial resolution of 13 kb. The sample was applied to the array
using an Agilent microarray hybridization chamber and hybridization
was carried out for 40 hours at 65.degree. C. in a Robbins
Scientific rotating oven at 20 rpm. The arrays were then
disassembled according to the manufacturer's protocol. Slides were
dried and scanned using an Agilent DNA microarray scanner. CGH
Analytics software version 3.4 (Agilent Technologies, Calif.) was
used to analyze the aCGH data.
[0046] BW/DF Cohort. We obtained data on n=47 patients from the
Dana-Farber/Harvard Cancer Center. All patients in this cohort went
on to develop metastatic disease and had primary tumor tissue
available. DNA was hybridized to Affymetrix OncoScanTM FFPE Express
2.0 SNP molecular inversion probe arrays with 334,183 copy number
and SNP probes. Median probe spacing was 9 kb. Matched normals were
available for 41 samples. Copy numbers were estimated with the
NEXUS software. Quality control was carried out by Affymetrix and
only samples that passed their metrics (MAPD value of .ltoreq.0.6)
were considered. A tissue microarray was stained with a FISH probe
targeting the region of 1q23.3 identified in the Spanish cohort,
and was scored for copy number gain or amplification in 33 patients
from the BW/DF cohort by a single.
[0047] Greek Cohort. We obtained data on n=93 patients from an
unfinished randomized phase III study from Greece. Patients all had
metastatic UC and ECOG performance status (PS) 0 or 1, and a
majority was randomized to either dose-dense MVAC or dose-dense
Gem-Cis. After randomization was stopped, additional patients
received dose-dense MVAC; this group of patients was also included
in the cohort. Patients with ECOG PS 0-2 were included in this
latter group. Overall survival for this cohort of patients was 16
months. A tissue microarray was stained with a FISH probe targeting
the region of 1q23.3 identified in the Spanish cohort, and was
scored for copy number gain or amplification in all patients from
the Greek cohort by a single cytogeneticist (AL).
[0048] TCGA Cohort. We further obtained copy number data from The
Cancer Genome Atlas (TCGA). We acquired segmented data from the
TCGA bladder cancer cohort (n=35), and the TCGA primary
glioblastoma (GBM) cohort (n=518). [0049] Clinical Endpoints: The
primary endpoint was overall survival (OS) from the start of
front-line chemotherapy for detection of metastatic disease in the
Spanish cohort. For the BW/DF cohort, overall survival was defined
as starting from the diagnosis date of metastatic recurrence, due
to the unavailability of initial treatment dates in this cohort for
most patients. For the Greek cohort and all 33 BW/DF patients that
were tested using FISH, treatment dates were available and the
primary endpoint in the FISH study was the overall survival from
the start of chemotherapy. [0050] Recurrent Copy Number
Aberrations. Normalized copy number data was first segmented using
GLAD with default parameters as available in GenePattern version
3.3.3. The Genomic Identification of Significant Targets in Cancer
(GISTIC) software (v2.0.12) was then used to identify regions of
the genome that were significantly amplified or deleted across a
set of samples. Each region was assigned a score that considers the
amplitude of the region as well as the frequency of its occurrence
across samples. The software estimated false discovery rates
(q-values) as well as potential targets (drivers) of the
aberrations. For the latter, a "wide peak" region was determined
that contained the target with a specified confidence level. We
used GISTIC with a confidence level of 0.95 and otherwise default
parameters. Copy number alterations were defined as broad if more
than 50% of a chromosome arm was lost or gained, otherwise as
focal. Copy numbers of the altered regions as reported by GISTIC
corresponded to the maximum copy numbers within the wide peak
regions. To compare the GISTIC results of our bladder cancer
specimens with findings from other cancer types, we extracted
q-values from wide peak regions determined in a pooled analysis of
3,131 TCGA specimens from 26 histologic types. [0051] Survival
analysis. Copy numbers of significantly amplified or deleted
regions (q-value<0.25) were dichotomized based on the standard
GISTIC cutoffs for amplifications or deletion (log base 2
ratio>0.9 or <-1.3, respectively). Cox proportional hazard
models were then used to assess the associations of these regions
and overall survival. Cox p-values were adjusted for multiple
comparisons using the Benjamini and Hochberg method. Association of
1q23.3 amplification and overall survival was further adjusted for
ECOG performance status (>0) and visceral metastasis.
Results
EXAMPLE 1
[0052] A cohort of 94 patients was identified. These patients all
developed metastatic UC, the lethal phenotype of this cancer for
which they subsequently received platinum-based combination
chemotherapy. Clinical data was tabulated in collaboration with the
study biostatistician. Overall survival data, defined as the time
from start of chemotherapy for metastatic disease, was collected on
these patients (Discovery cohort, Table 1). Tumor-bearing
formalin-fixed, paraffin embedded (FFPE) tissue blocks from
transurethral resection (TUR) or cystectomy specimens were
evaluated by a single UC pathologist to identify malignant tissue.
Multiple 0.6 mm cores were taken from tumor tissue and genomic DNA
and total RNA was extracted. Tissue microarrays were also
constructed from this cohort. Using the genomic DNA, high
resolution CGH arrays were performed using Agilent 4.times.180 k
chips with an average resolution of 13 kb to assess copy number
alterations. Gain and Loss Analysis of DNA (GLAD) 16 was used to
segment the array output, and Genomic Identification of Significant
Targets in Cancer (GISTIC) v217 was used to assess the significance
of gains and losses. Thresholds for gain and loss were set at log
base 2 as greater than 0.9 and less than -1.3, respectively. It is
critical to note that the aCGH experiments were done without
knowledge of survival (i.e. when initially calling a gain or loss).
False discovery rates (FDR) less than 0.2 were considered
significant. In univariate analysis, copy number gain of 1q23.3 was
significantly associated with poor survival (HR 2.4 for death,
p=0.01, FDR=0.12). These data were then evaluated in a
multivariable Cox model for survival controlling for known
prognostic variables such as performance status and visceral
metastasis, and confirmed with bootstrap analysis (500
resamplings). Gain of the 150 kb region of 1q23.3 was significantly
associated with lethal bladder cancer (HR 2.94 for death, p=0.007)
(FIG. 1 and Table 2). Bootstrapping (500 re-samples) internally
validated the findings (HR=3.2, 95% CI 1.25-7.2). Of note, the
minimum gained region is not known to contain significant germline
copy number variants that span the 150 kb region (UCSC Genome
Browser). The exact chromosome locus is chr 1:159256737-159407240
in human genome build 18. Very similar results are noted for a
nearby but distinct region at 1q21.2 (chr 1:148747863-149235943),
which is independently prognostic for outcome.
TABLE-US-00001 TABLE 1 Discovery (n = 94) ECOG PS N % 0 34 36% 1 58
62% 2 2 2% 3 -- -- Missing -- -- Visceral 33 35% metastasis Number
of 46 49% deaths
TABLE-US-00002 TABLE 2 p- HR (95% CI) value 1q23.3 gain (yes vs.
no) 2.94 (1.35, 0.007 6.44) ECOG PS (1, 2 vs. 0) 1.93 (1.00, 0.05
3.79) Visceral Disease (yes 2.40 (1.25, 0.008 vs. no) 4.47)
EXAMPLE 2
[0053] Median overall survival for the Spanish aCGH, Greek FISH,
BW/DF FISH, and BW/DF MIP are 15.1, 18.0, 12.2 and 10.5 months,
respectively, and median follow-up for these cohorts was 23.1,
50.1, 131, and 60.9 months. There were significant differences
between the cohorts in the percentage of patients with visceral
metastases and poor performance status. Table 3 summarizes patient
characteristics in all three cohorts.
[0054] First the GISTIC algorithm was applied to the data to
identify regions of recurrent copy number alteration. This approach
identified 95 focal and 21 broad (>50% of the chromosome arm)
events in the Spanish cohort (FIG. 2A-B) and 91 focal and 16 broad
events in the BW/DF cohort (FIG. 2C-D). The results of the broad
alteration analysis were largely consistent with the literature,
with frequent losses of chromosomes 5q (43%), 8p (69%), 9 (p: 48%;
q: 41%), 10q (41%), 11p (49%), 17p (51%), and 22q (40%) and
recurrent gains of chromosomes 3q (46%), 5p (48%), 8q (48%), 19q
(34%), and 20 (60%). The overlap of the focal amplifications
between the cohorts is displayed in Table 4. The most frequent
focal loss is 15q11.2 (59%), which was previously reported in
aggressive UCs and other histologies but harbors no known tumor
suppressor. CDKN2A/B are deleted in 52% of all patients. The tumor
suppressor MAP2K4 is the likely target of a focal amplification at
17p11-12, found in 49% of all patients. Also frequently amplified
are the SOX4 (50%), ERBB2 (48%) and PPARG (44%) loci. All
percentages for both broad and focal events refer to the larger
Spanish cohort and GISTIC cutoffs of -0.1 and 0.1.
[0055] Next, any association of the broad or focal SCNA events with
survival in the Spanish cohort was tested. No broad event was found
to be significantly associated with overall survival. With regard
to focal events, the chromosome bands 1q21.2 and 1q23.3 (chromosome
1:147.645-159.407Mb) were observed to be significantly associated
with overall survival, even after adjusting for ECOG performance
status and visceral metastasis (FDR 0.12). The locus 1q23.3
(adjusted Hazard Ratio [HR] 2.96; 95% CI, 1.35 to 6.48; P=0.007)
represented the GISTIC peak region with the lowest q-value and
largest frequency of high copy number gains among all patients
(FIG. 3). This locus was also significantly associated with overall
survival after recurrence in the BW/DF validation cohort (n=31;
adj. HR 3.73; 95% CI, 1.09 to 12.77; P=0.036). Fluorescence in situ
hybridization (FISH) was then performed on subset patients from the
Greek and BW/DF cohorts (n=113). After adjusting for ECOG
performance status and visceral disease, 1q23.3 amplification, as
quantified by FISH, was significantly associated with overall
survival (adj. HR 1.71; 95% CI, 1.03 to 2.85, P=0.038).
[0056] Finally, potential target genes of the 1q23.3 amplification
were identified. The GISTIC wide peak regions in the Spanish,
BW/DF, and TCGA cohorts were examined. We identified three
different peak regions in total, each of which was observed in at
least two UC patient cohorts (FIG. 4). Peak 1, used in the survival
analyses, was the highest peak in the Spanish and the TCGA cohorts.
This peak harbors, among others, the DEDD gene, over-expression of
which has been shown to decrease rates of apoptosis. The second
peak contains the FCGR gene cluster, involved in the mediation of
immune responses and apoptosis and with frequently reported
over-expression as result of 1q23 chromosomal alterations in
hematological malignancies. The third peak involves the PBX1 locus,
over-expression of which was recently shown to be associated with
reduced metastasis-free survival in ER.alpha.-positive breast
cancer. The FCGR peak was also observed in GBM; similarly, the
1q23.3 locus in general is frequently altered in many cancer types.
Peaks 2 and 3 were also tested for association with survival and
found to have weaker associations as compared to peak 1.
TABLE-US-00003 TABLE 3 Patient Demographics and Characteristics.
Cohorts Spanish BW/DF Greek N % N % N % 94 47 93 Pathological stage
Stage 0 10 11% 1 2% (Ta) Stage I 5 5% 4 9% (T1) Stage II 45 48% 7
15% (T2) Stage III 28 30% 13 28% (T3, T4) Stage IV 5 5% 13 28% (L,
M) Missing 1 1% 9 19% Visceral disease Yes 34 36% 29 62% 55 59% No
60 64% 18 38% 31 33% Unknown 7 8% ECOG Performance Status (PS) 0 34
36% 29 62% 49 53% 1 58 61% 5 11% 35 38% 2+ 2 2% 11 23% 2 2% Unknown
2 4% 7 8% Survival Dead 46 49% 27 96% 66 71% Alive 48 51% 1 4% 27
29%
TABLE-US-00004 TABLE 4 Focal Amplifications. The table shows
significant (q-value <0.25) GISTIC focal aberrations in the
Spanish and BW/DF cohorts. Multiple peaks in one chromosome band
were combined and the average q-value is shown. If known cancer
genes were located in the peaks, only these are reported;
otherwise, the intersection of the GISTIC target genes in the two
cohorts is reported. Chr Type Freq. (Spanish) q-value (Spanish)
Freq. (BW/DF) q-value (BW/DF) Possible Target Genes 15q11.2 Del 55
(58.5%) <0.001 20 (42.6%) <0.001 LOC283755, A26B1, LOC283767
6p22.3 Amp 47 (50%) <0.001 21 (44.7%) <0.001 SOX4, CDKAL1
17q12 Amp 45 (47.9%) <0.001 24 (51.1%) <0.001 ERBB2 9p21.3
Del 49 (52.1%) <0.001 34 (72.3%) <0.001 CDKN2A, CDKN2B 3p25.1
Amp 41 (43.6%) <0.001 30 (63.8%) <0.001 PPARG 1q23.3 Amp 58
(61.7%) <0.001 27 (57.4%) 0.001 PBX1, FCGR2B 8q22.3 Amp 63 (67%)
<0.001 32 (68.1%) <0.001 PABPC1, SNX31 8p12 Amp 31 (33%)
<0.001 18 (38.3%) <0.001 FGFR1, WHSC1L1 1p34.2 Amp 16 (17%)
<0.001 23 (48.9%) <0.001 MYCL1 10p14 Amp 43 (45.7%) <0.001
23 (48.9%) 0.002 GATA3 14q11.2 Del 24 (25.5%) <0.001 20 (42.6%)
0.018 OR4N2, OR4K2, A26C2, OR11H12, OR4Q3, OR4M1 19q13.2 Amp 36
(38.3%) <0.001 28 (59.6%) 0.001 AKT2 12q15 Amp 26 (27.7%)
<0.001 15 (31.9%) 0.007 MDM2 1q21.2 Amp 56 (59.6%) <0.001 26
(55.3%) 0.018 ARNT 20q11.21 Amp 59 (62.8%) 0.004 36 (76.6%) 0.074
ASXL1 6p22.1 Amp 42 (44.7%) 0.005 18 (38.3%) 0.219 HIST1H1E,
HIST1H1T, HIST1H2AE, HIST1H2AD, HIST1H2BD, HIST1H2AC, HIST1H2BG,
HIST1H2BF, HIST1H2BE, HIST1H2BC, HIST1H3D, HIST1H4D, HIST1H4E
7p21.1 Amp 49 (52.1%) 0.017 26 (55.3%) <0.001 AHR 18p11.31 Amp
36 (38.3%) 0.029 19 (40.4%) 0.062 DLGAP1, FLJ35776 17q11.2 Amp 39
(41.5%) 0.032 18 (38.3%) 0.183 SUZ12 17p11.2 Del 46 (48.9%) 0.033
22 (46.8%) 0.166 MAP2K4 5p15.33 Amp 38 (40.4%) 0.037 23 (48.9%)
0.017 SDHA, SLC9A3, TERT, TRIP13, PDCD6, SLC12A7, TPPP, EXOC3,
CEP72, AHRR, BRD9, ZDHHC11, CLPTM1L, NKD2, LOC116349, CCDC127,
PLEKHG4B, SLC6A19, SLC6A18, LOC389257 3p12.1 Amp 33 (35.1%) 0.047
15 (31.9%) 0.110 VGLL3
TABLE-US-00005 TABLE 5 Association between 1q23.3 amplification and
survival. The table shows the hazard ratios for 1q23.3
amplification in all cohorts. Hazard Ratio (95% CI) Spanish cohort
BW/DF cohort Greek and BW/DF FISH Model Characteristic (n = 94) (n
= 32) (n = 120) Unadjusted 1q23.3 Amp Yes vs 2.4 (1.18-4.86) 4.72
(1.4-15.86) 1.4 (0.87-2.25) No Adjusted 1q23.3 Amp Yes vs 2.96
(1.35-6.48) 3.73 (1.09-12.77) 1.71 (1.03-2.85) No ECOG PS >1 vs
0 1.95 (1-3.81) 1.93 (0.82-4.55) 2.38 (1.51-3.75) visceral Yes vs
2.39 (1.26-4.51) 1.48 (0.65-3.38) 1.21 (0.77-1.9) disease No
* * * * *
References