U.S. patent application number 11/758382 was filed with the patent office on 2008-10-02 for use of imp3 as a prognostic marker for cancer.
Invention is credited to Zhong Jiang.
Application Number | 20080242606 11/758382 |
Document ID | / |
Family ID | 38832656 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080242606 |
Kind Code |
A1 |
Jiang; Zhong |
October 2, 2008 |
Use of IMP3 as a Prognostic Marker for Cancer
Abstract
Provided herein are methods and compositions for the prognostic
evaluation of a patient suspected of having, or having, cancer by
assessing the expression of IMP3 in a biological sample of a
patient. Methods can be used at the time of initial diagnosis of
malignant tumors to identify a group of patients with a high
potential to develop progression or metastasis later. Therefore,
methods not only are able to provide very useful prognostic
information for patients but also can help clinicians to select a
candidate patient likely to benefit from early and aggressive
cancer therapy. Methods and compositions for the treatment of
cancer associated with expression of IMP3 are also provided.
Inventors: |
Jiang; Zhong; (Northborough,
MA) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
38832656 |
Appl. No.: |
11/758382 |
Filed: |
June 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60811702 |
Jun 6, 2006 |
|
|
|
60813216 |
Jun 12, 2006 |
|
|
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Current U.S.
Class: |
514/6.9 ;
435/7.23 |
Current CPC
Class: |
C12Q 1/6886 20130101;
Y10T 436/25 20150115; C12Q 2600/118 20130101; C12Q 2600/106
20130101; G01N 33/6893 20130101; C12Q 2600/158 20130101; A61P 35/00
20180101 |
Class at
Publication: |
514/12 ;
435/7.23 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61K 38/30 20060101 A61K038/30 |
Claims
1. A method for determining the prognosis of a subject having renal
cell carcinoma (RCC) or bladder carcinoma, comprising determining
the presence or level of IMP3 in a primary tumor of a subject,
wherein the presence of IMP3 in the primary tumor of the subject
indicates that the prognosis of the subject is poor, whereas an
essentially undetectable level of IMP3 in the primary tumor of the
subject indicates that the prognosis of the subject is good.
2. The method of claim 1, wherein the subject has a renal cell
carcinoma that is a clear cell, papillary or chromophobe RCC.
3. The method of claim 1, wherein the subject has a superficial
urothelial carcinoma of the bladder.
4. The method of claim 1, further comprising determining at least
one other factor, the presence, absence or level of which
reasonably correlates with the prediction of the prognosis of the
subject.
5. The method of claim 4, wherein the factor is tumor stage, grade,
necrosis, ECOG performance status or the presence of a biomarker
other than IMP3.
6. The method of claim 4, further comprising determining the stage
of the primary tumor.
7. The method of claim 6, wherein the cancer is RCC and wherein the
combined level of expression of IMP3 and tumor stage provides the
prognostic set forth in FIG. 9.
8. The method of claim 1, wherein the level of IMP3 is compared to
a control value, wherein the control value is that of the level of
IMP3 in non-cancerous cells of the same origin as those of the
primary tumor or the level of IMP3 in cells of a primary tumor of
the same type as that in the subject from a subject having a good
prognosis.
9. The method of claim 1, wherein determining the presence or level
of IMP3 comprises determining the presence or level of IMP3
protein.
10. The method of claim 9, wherein determining the presence or
level of IMP3 protein comprises immunohistochemical staining.
11. The method of claim 10, comprising using a computerized image
analyzer for quantitative analysis.
12. The method of claim 1, wherein the method further comprises
first obtaining a biopsy of a primary tumor of the subject and the
presence or level of IMP3 is determined in the biopsy.
13. The method of claim 1, further comprising first removing the
primary tumor from the subject and wherein determining the presence
or level of IMP3 is done in the primary tumor that has been
removed.
14. The method of claim 1, wherein the subject has RCC and the
method first comprises conducting radical or partial nephrectomy
and wherein determining the presence or level of IMP3 is done in
the tumor tissue that has been removed.
15. A kit comprising an agent for detecting the presence or level
of IMP3 and a control IMP3 value.
16. The kit of claim 15, wherein the agent for detecting the
presence or level of IMP3 is an antibody or a fragment thereof.
17. The kit of claim 15, further comprising at least one other
agent for detecting the presence or level of a biomarker other than
IMP3.
18. A method for monitoring the progression of cancer in RCC or a
bladder carcinoma in a subject, comprising monitoring the presence
or level of IMP3 in a primary tumor of the subject over time,
wherein an increase in the level of IMP3 in the primary tumor of
the subject indicates that the cancer is progressing.
19. A method for treating a subject having a RCC or a bladder
carcinoma, comprising (i) determining the presence or level of IMP3
in a primary tumor of a subject pursuant to claim 1; and (ii) if
the primary tumor has a detectable level of IMP-3, treating the
subject aggressively, whereas if the primary tumor has an
essentially undetectable level of IMP3, treating the subject less
aggressively.
20. A method for treating a subject having a RCC or bladder
carcinoma and having a primary tumor expressing IMP3, comprising
administering to the subject a therapeutically effective amount of
an agent that reduces the level or activity of IMP3.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
provisional applications 60/811,702 filed Jun. 6, 2006 and
60/813,216, filed Jun. 12, 2006, both of which are specifically
incorporated herein by reference in their entirety.
BACKGROUND
[0002] In spite of numerous advances in medical research, cancer
remains a leading cause of death in the United States. Traditional
modes of clinical care, such as surgical resection, radiotherapy
and chemotherapy, have a significant failure rate, especially for
solid tumors. Failure occurs either because the initial tumor is
unresponsive, or because of recurrence due to regrowth at the
original site and/or metastases. The etiology, diagnosis and
ablation of cancer remain a central focus for medical research and
development.
[0003] While different forms of cancer have different properties,
one factor which many cancers share is the ability to metastasize.
Distant metastasis of all malignant tumors remains the primary
cause of death in patients with the disease. Patients with
metastatic disease are typically treated with systemic therapy,
which is associated with substantial toxicity. Unless the patient
presents with metastatic disease, clinical observation is typically
used to prognose the disease following surgical resection.
Currently, the methods to determine prognosis and select patients
for adjuvant therapy rely mainly on pathological and clinical
staging. However, it is very difficult to predict which localized
tumor will eventuate in distant metastasis.
[0004] Since the chance for complete remission of cancer is, in
most cases, greatly enhanced by accurate prognosis, it is desirable
that physicians be able to determine the metastatic potential of
tumors. However, the metastatic potential of localized cancers is
often unpredictable. The development of methods that permit rapid
and accurate detection of many forms of cancers continues to
challenge the medical community. Thus a major problem in the
treatment of cancer remains detection and prognosis, which enables
appropriate therapeutic treatment resulting in successful treatment
in many cases. Therefore, there is a great need for the
identification of biomarkers that can accurately distinguish
localized tumors with a high probability of metastasis from those
that will remain indolent. Using such biomarkers, one can predict
the patient's prognosis and can effectively target the individuals
who would most likely benefit from adjuvant therapy.
SUMMARY
[0005] The present invention is based at least in part on the
finding that the expression of IMP3 is strongly associated with
tumor metastasis and poor prognosis. IMP3 thus serves as an
independent prognostic biomarker to predict cancer metastasis and a
potential target protein to treat metastatic cancer.
[0006] Provided herein are methods for predicting the prognosis of
a subject having a cancerous tumor. A method may comprise
determining the level of IMP3 in a cancerous tumor of a subject,
wherein a higher level of IMP3 in the cancerous tissue of the
subject, e.g., relative to that in a control indicates that the
prognosis of the subject is poor, whereas an undetectable or a
lower or similar level of IMP3 in the cancerous tissue of the
subject relative to that in the control indicates that the
prognosis of the subject is good. A method may also comprise
determining the presence of IMP3 in a cancerous tissue in a
subject, wherein the presence of IMP3 in the cancerous tissue
indicates that the prognosis of the subject is poor. Determining
the level of IMP3 may comprise determining the level of IMP3
protein, e.g., by immunohistochemical staining, which may be
followed by computerized image analysis for quantitative
immunohistochemistry. A control may be the level of IMP3 in
non-cancerous cells of the same origin as those of the cancerous
tumor or the level of IMP3 in cells of a cancerous tumor that has a
good prognosis. The cancerous tumor may be a renal tumor or a
urinary bladder tumor. The method further comprises first obtaining
a biopsy of a cancerous tumor of the subject.
[0007] A prognostic method may also comprise determining the
presence or level of IMP3 and evaluate another prognostic factor,
e.g., the stage of the tumor.
[0008] Also provided herein are methods for treating a subject
having a cancerous tumor. A method may comprise (i) determining the
level of IMP3 in the cancerous tumor of a subject; and (ii) if the
level of IMP3 in the cancerous tumor of a subject is more similar
to that of a cancerous tumor of a subject having a poor prognosis
rather than that of a subject having a good prognosis, treating the
subject aggressively, whereas if the level of IMP3 in the cancerous
tumor of a subject is more similar to that of a cancerous tumor of
a subject having a good prognosis rather than that of a subject
having a poor prognosis, treating the subject less aggressively.
Instead of determining a level of IMP3 and comparing it to a
control, a method may comprise determining the presence of IMP3,
wherein the presence of IMP3 indicates that the subject should be
treated aggressively.
[0009] Further provided herein are kits, e.g., kits comprising one
or more agents for detecting the level of IMP3. A kit may also
comprise a control. The agent for detecting the level of IMP3 may
be an antibody or a variant, e.g., a fragment, thereof. A control
may be the level of IMP3 in a cancerous tumor having a good
prognosis or it may be a sample of a healthy subject. A kit may
also comprise one or more other biomarkers or reagents for
evaluating other prognostic factors.
[0010] Other methods provided herein are methods for monitoring the
progression of a cancer in a subject. A method may comprise
monitoring the level of IMP3 in a cancerous tumor of the subject
over time, wherein an increase in the level of IMP3 in a cancerous
tumor of the subject indicates that the cancer is progressing.
[0011] Therapeutic methods are also provided herein. An exemplary
method for treating a subject having a cancer associated with high
levels of IMP3, comprises administering to the subject a
therapeutically effective amount of an agent that reduces the level
or activity of IMP3, such as by directly targeting IMP3 protein or
its expression or by targeting an IMP3 target, such as insulin-like
growth factor II (IGF-II).
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Expression of IMP3 in primary and metastatic renal
cell carcinomas. Immunohistochemical stains for IMP3 showing that
metastatic RCC in the bone (A) and primary RCC with subsequent
development of metastasis (B) were positive for IMP3 while primary
RCC without metastasis (C) were negative for IMP3.
[0013] FIG. 2. The percentage of IMP3 expression in patients with
primary RCCs without metastasis (Met), primary RCCs with metastasis
(Met) and metastatic (Met) RCCs.
[0014] FIG. 3. Western blot analysis of IMP3 expression in primary
RCCs. Metastatic RCC cell line (Cell Line) was used for comparison.
Actin was included as a loading control. Primary RCCs with
Met=primary RCCs with metastasis; RCCs without Met=primary RCCs
without metastasis. (Lower) Bar graph showing IMP3 protein levels
in above Western blot analysis (case 1-3=RCCs with Met; case
4-5=primary RCCs without Met) and IMP3 mRNA Levels by quantitative
real-time PCR analysis in the same sample. A ratio of IMP3 protein
expression in Western blot analysis was calculated relative to
actin expression and a ratio of IMP3 mRNA expression in
quantitative real-time PCR analysis was calculated relative to the
housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
expression. IHC=immunohistochemistry; "+"=positive for IMP3
staining by IHC; "-"=negative for IMP3 staining by IHC.
[0015] FIG. 4. Kaplan-Meier analysis of metastasis-free (A) and
overall survivals (B) according to IMP3 status (positive verses
negative) assessed by immunohistochemical analysis in total 371
patients with localized primary RCCs without metastasis during
surgery. P values were calculated by using the log-rank test.
[0016] FIG. 5. Kaplan-Meier analysis of metastasis-free and overall
survivals in stage I patients (n=216, A and B), stage II patients
(n=64, C and D) and stage III patients (n=91, E and F) according to
IMP3 status (positive verses negative) with localized primary RCCs
assessed by immunohistochemical analysis. P values were calculated
by using the log-rank test.
[0017] FIG. 6: Association of IMP3 tumor expression with
progression to distant metastases for 317 patients with localized
papillary and chromophobe RCC. Metastases-free survival rates (SE,
number still at risk) at 5 and 10 years following nephrectomy were
63.9% (8.8%, 17) and 48.6% (9.5%, 12), respectively, for patients
with IMP3-positive tumors compared with 97.7% (0.9%, 223) and 93.5%
(1.9%, 86), respectively, for patients with IMP3-negative
tumors.
[0018] FIG. 7: Association of IMP3 tumor expression with death for
317 patients with localized papillary and chromophobe RCC. Overall
survival rates (SE, number still at risk) at 5 and 10 years
following nephrectomy were 57.9% (9.0%, 17) and 47.1% (9.2%, 12),
respectively, for patients with IMP3-positive tumors compared with
85.0% (2.2%, 223) and 67.4% (3.3%, 86), respectively, for patients
with IMP3-negative tumors.
[0019] FIG. 8 shows the probability of metastasis free survival as
a function of metastasis-free survival time (years) for stage 1, 2
and 3 RCCs.
[0020] FIG. 9 shows the shows the probability of metastasis free
survival as a function of metastasis-free survival time (years
after surgery) for stages 1, 2 and 3 RCCs and negative, low
positive or high positive IMP3 expression.
[0021] FIG. 10. Immunohistochemical stains for IMP3 showing that
high grade UC (A) was positive for IMP3 while low grade UC (B) was
negative for IMP3.
[0022] FIG. 11. Kaplan-Meier analysis of progression-free (A) and
disease-free survival (B) in patients with superficial urothelial
carcinomas (Ta, T1 and Tis). P values were calculated by using the
log-rank test.
[0023] FIG. 12. Kaplan-Meier analysis of progression-free survivals
in patients with Ta superficial urothelial carcinomas (A) and T1
superficial urothelial carcinomas (B). P values were calculated by
using the log-rank test.
[0024] FIG. 13. Kaplan-Meier analysis of metastasis-free survivals
in patients with T1 superficial urothelial carcinomas. P values
were calculated by using the log-rank test.
DETAILED DESCRIPTION
Definitions
[0025] "Cancers of epithelial origin" refers to "carcinoma" that
arise from epithelial cells which include, but are not limited to,
skin cancer, such as squamous cell and basal cell cancers, lung
cancer, breast cancer, prostate cancer, renal cell carcinoma, liver
cancer, urinary bladder cancer, ovarian cancer, cervical cancer,
endometrial cancer, gastrointestinal cancers including esophageal
cancer, small bowel cancer and stomach cancer, colon cancer, and
other known cancers that effect epithelial cells throughout the
body.
[0026] The term "aggressive" or "invasive" with respect to cancer
refers to the proclivity of a tumor for expanding beyond its
boundaries into adjacent tissue (Darnell, J. (1990), Molecular Cell
Biology, Third Ed., W. H. Freeman, N.Y.). Invasive cancer can be
contrasted with organ-confined cancer wherein the tumor is confined
to a particular organ. The invasive property of a tumor is often
accompanied by the elaboration of proteolytic enzymes, such as
collagenases, that degrade matrix material and basement membrane
material to enable the tumor to expand beyond the confines of the
capsule, and beyond confines of the particular tissue in which that
tumor is located.
[0027] The term "metastasis", as used herein, refers to the
condition of spread of cancer from the organ of origin to
additional distal sites in the patient. The process of tumor
metastasis is a multistage event involving local invasion and
destruction of intercellular matrix, intravasation into blood
vessels, lymphatics or other channels of transport, survival in the
circulation, extravasation out of the vessels in the secondary site
and growth in the new location (Fidler, et al, Adv. Cancer Res. 28,
149-250 (1978), Liotta, et al., Cancer Treatment Res. 40, 223-238
(1988), Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988) and
Zetter, N. Eng. J. Med. 322, 605-612 (1990)). Increased malignant
cell motility has been associated with enhanced metastatic
potential in animal as well as human tumors (Hosaka, et al., Gann
69, 273-276 (1978) and Haemmerlin, et al., Int. J. Cancer 27,
603-610 (1981)).
[0028] A "biological sample" refers to a sample of biological
material obtained from a subject, preferably a human subject,
including a tissue, a tissue sample, a cell sample, a tumor sample,
and a biological fluid, e.g., blood, urine, and a nipple aspirate.
A biological sample may be obtained in the form of, e.g., a tissue
biopsy, such as, an aspiration biopsy, a brush biopsy, a surface
biopsy, a needle biopsy, a punch biopsy, an excision biopsy, an
open biopsy, an incision biopsy and an endoscopic biopsy.
[0029] An "isolate" of a biological sample (e.g., an isolate of a
tissue or tumor sample) refers to a material or composition (e.g.,
a biological material or composition) which has been separated,
derived, extracted, purified or isolated from the sample and
preferably is substantially free of undesirable compositions and/or
impurities or contaminants associated with the biological
sample.
[0030] A "tissue sample" includes a portion, piece, part, segment,
or fraction of a tissue which is obtained or removed from an intact
tissue of a subject, preferably a human subject.
[0031] A "tumor sample" includes to a portion, piece, part,
segment, or fraction of a tumor, for example, a tumor which is
obtained or removed from a subject (e.g., removed or extracted from
a tissue of a subject), preferably a human subject.
[0032] A "primary tumor" is a tumor appearing at a first site
within the subject and can be distinguished from a "metastatic
tumor" which appears in the body of the subject at a remote site
from the primary tumor.
[0033] A "patient" refers to any warm-blooded animal, preferably a
human.
Prognostic and Diagnostic Biomarker and Methods
[0034] The present invention provides methods for predicting or
determining the prognosis, e.g., likelihood of metastasis or
aggressive behavior, in subjects with malignant tumors. A method
may comprise measuring the level of expression of IMP3 present in a
biological sample obtained from a patient and comparing the
observed level with one or a range of IMP3 levels normally found in
biological samples (of the same type) of healthy individuals. A
high level of IMP3 expression is indicative of a greater potential
for metastatic activity or aggressive behavior and corresponds to a
poor prognosis, while very low or undetectable levels indicate that
the tumor is less aggressive and correspond to a better
prognosis.
[0035] An exemplary method, e.g., for predicting the prognosis of a
subject having a cancerous tumor, comprises determining the
presence of IMP3 in a cancerous tumor of a subject, wherein the
presence of IMP3 in the cancerous tissue of the subject indicates
that the prognosis of the subject is poor, whereas the absence of
IMP3 in the cancerous tissue of the subject indicates that the
prognosis of the subject is good. A method may also comprise
determining or the level of expression of IMP3 in a cancerous tumor
of a subject, wherein a higher level of expression of IMP3 in the
cancerous tissue of the subject relative to a control value, e.g.,
level in a control, indicates that the prognosis of the subject is
poor, whereas a lower or similar level of expression of IMP3 in the
cancerous tissue of the subject relative to that in the control
indicates that the prognosis of the subject is good. A poor
prognosis indicates that the cancer is of an aggressive or invasive
type, likely to progress fast and/or likely to metastasize.
[0036] IMP3 is an oncofetal protein and is a member of the
insulin-like growth factor II (IGF-II) mRNA binding protein (IMP)
family that consists of IMP 1, IMP2 and IMP3.sup.11. IMP family
members play an important role in RNA trafficking and
stabilization, cell growth, and cell migration during the early
stages of embryogenesis.sup.12. The IMP3 gene is located on
chromosome 7p11.2.+-.11cM.sup.13 and is identical to the KOC (KH
domain containing protein overexpressed in cancer) protein that was
originally cloned from a pancreatic tumor cDNA screen.sup.14. IMP3
is expressed in developing epithelium muscle and placenta during
early stages of human and mouse embryogenesis, but it is expressed
at low or undetectable levels in adult tissues.sup.11,12. The amino
acid sequence of the human IMP3 protein is set forth in GenBank
Accession Nos. AAC35208 and NP.sub.--006538.2 and is encoded by the
nucleotide sequence set forth in GenBank Accession Nos. U97188 and
NM.sub.--006547.2. The protein has several RNA recognition motifs
and K homology RNA-binding domains, type I (see GenBank entries).
The nucleotide and amino acid sequences of the human IMP3 protein
are set forth below:
TABLE-US-00001 (SEQ ID NO: 1)
atgaacaaactgtatatcggaaacctcagcgagaacgccgccccctcgga
cctagaaagtatcttcaaggacgccaagatcccggtgtcgggacccttcc
tggtgaagactggctacgcgttcgtggactgcccggacgagagctgggcc
ctcaaggccatcgaggcgctttcaggtaaaatagaactgcacgggaaacc
catagaagttgagcactcggtcccaaaaaggcaaaggattcggaaacttc
agatacgaaatatcccgcctcatttacagtgggaggtgctggatagttta
ctagtccagtatggagtggtggagagctgtgagcaagtgaacactgactc
ggaaactgcagttgtaaatgtaacctattccagtaaggaccaagctagac
aagcactagacaaactgaatggatttcagttagagaatttcaccttgaaa
gtagcctatatccctgatgaaatggccgcccagcaaaaccccttgcagca
gccccgaggtcgccgggggcttgggcagaggggctcctcaaggcaggggt
ctccaggatccgtatccaagcagaaaccatgtgatttgcctctgcgcctg
ctggttcccacccaatttgttggagccatcataggaaaagaaggtgccac
cattcggaacatcaccaaacagacccagtctaaaatcgatgtccaccgta
aagaaaatgcgggggctgctgagaagtcgattactatcctctctactcct
gaaggcacctctgcggcttgtaagtctattctggagattatgcataagga
agctcaagatataaaattcacagaagagatccccttgaagattttagctc
ataataactttgttggacgtcttattggtaaagaaggaagaaatcttaaa
aaaattgagcaagacacagacactaaaatcacgatatctccattgcagga
attgacgctgtataatccagaacgcactattacagttaaaggcaatgttg
agacatgtgccaaagctgaggaggagatcatgaagaaaatcagggagtct
tatgaaaatgatattgcttctatgaatcttcaagcacatttaattcctgg
attaaatctgaacgccttgggtctgttcccacccacttcagggatgccac
ctcccacctcagggcccccttcagccatgactcctccctacccgcagttt
gagcaatcagaaacggagactgttcatctgtttatcccagctctatcagt
cggtgccatcatcggcaagcagggccagcacatcaagcagctttctcgct
ttgctggagcttcaattaagattgctccagcggaagcaccagatgctaaa
gtgaggatggtgattatcactggaccaccagaggctcagttcaaggctca
gggaagaatttatggaaaaattaaagaagaaaactttgttagtcctaaag
aagaggtgaaacttgaagctcatatcagagtgccatcctttgctgctggc
agagttattggaaaaggaggcaaaacggtgaatgaacttcagaatttgtc
aagtgcagaagttgttgtccctcgtgaccagacacctgatgagaatgacc
aagtggttgtcaaaataactggtcacttctatgcttgccaggttgcccag
agaaaaattcaggaaattctgactcaggtaaagcagcaccaacaacagaa
ggctctgcaaagtggaccacctcagtcaagacggaagtaa (SEQ ID NO: 2)
MNKLYIGNLSENAAPSDLESIFKDAKIPVSGPFLVKTGYAFVDCPDESWA
LKAIEALSGKIELHGKPIEVEHSVPKRQRIRKLQIRNIPPHLQWEVLDSL
LVQYGVVESCEQVNTDSETAVVNVTYSSKDQARQALDKLNGFQLENFTLK
VAYIPDEMAAQQNPLQQPRGRRGLGQRGSSRQGSPGSVSKQKPCDLPLRL
LVPTQFVGAIIGKEGATIRNITKQTQSKIDVHRKENAGAAEKSITILSTP
EGTSAACKSILEIMHKEAQDIKFTEEIPLKILAHNNFVGRLIGKEGRNLK
KIEQDTDTKITISPLQELTLYNPERTITVKGNVETCAKAEEEIMKKIRES
YENDIASMNLQAHLIPGLNLNALGLFPPTSGMPPPTSGPPSAMTPPYPQF
EQSETETVHLFIPALSVGAIIGKQGQHIKQLSRFAGASIKIAPAEAPDAK
VRMVIITGPPEAQFKAQGRIYGKIKEENFVSPKEEVKLEAHIRVPSFAAG
RVIGKGGKTVNELQNLSSAEVVVPRDQTPDENDQVVVKITGHFYACQVAQ
RKIQEILTQVKQHQQQKALQSGPPQSRRK
[0037] Determining the presence or the level of IMP3 (or expression
of IMP3) in a cell or a biological sample includes determining
qualitatively or quantitatively the presence of IMP3 protein or
degradation product thereof, the presence of IMP3 mRNA or pre-mRNA,
or the presence of any biological molecule or product that is
indicative of IMP3 expression, or degradation product thereof. The
level of IMP3 may also be determined by detecting and/or measuring
the level of IMP3 autoantibodies.
[0038] It may be sufficient to detect the presence of IMP3 rather
than determining its level and comparing it to a standard or
control level, as IMP3 levels are relatively low in normal cells
and tissues and may not be detectable by the usual methods of
detection, e.g., immunohistochemistry. However, when comparing an
IMP3 level to a control level, a control may be a value that
corresponds to the level of IMP3 in a normal or healthy tissue of
the same type as that from which a sample was obtained. A control
may also be an average or mean value of at least 2, 5, 10 or more
values of levels of IMP3 in normal or healthy tissues. A control
may also be a normal control sample, i.e., a sample obtained from a
normal or healthy individual, or an individual that does not have
cancer, or at least not the type of cancer that is being
investigated. A control may also be a value obtained from
non-cancerous tissue that is adjacent to the cancerous tissue from
which a value was obtained.
[0039] In one embodiment, a value obtained from a subject is
compared to other values, e.g., control values, present in computer
readable form. For example, a value obtained from a subject may be
entered into an algorithm or software program comprising one or
more values, e.g., control values, and values from cancerous
specimens, and a comparison is effected by the software program A
software program may also provide a conclusion based on the
comparison, e.g., likelihood of cancer progression or
metastasis.
[0040] For purposes of comparison, a test sample and normal control
sample may be of the same type, that is, obtained from the same
biological source. A normal control sample can also be a standard
sample that contains the same concentration of IMP3 that is
normally found in a biological sample of the same type and that is
obtained from a healthy individual, e.g., an individual who does
not have cancer. For example, there can be a standard normal
control sample for the amounts of IMP3 normally found in a cell or
tissue.
[0041] When comparing an IMP3 level to a control value, a poor
prognosis may be concluded from the presence of at least about 50%,
2 fold, 5 fold, 10 fold, 30 fold, 50 fold, 100 fold or more IMP3 in
the sample of the subject compared to the control value. The term
"higher level" in the context of levels of IMP3 in a sample from a
patient relative to a control value of the level in a tissue from a
healthy subject refers to a level that is statistically significant
or significantly above levels found in the control value or tissue
from the healthy subject. The term "statistically significant" or
"significantly" refers to statistical significance and generally
means a two standard deviation (2SD) above normal, or higher,
concentration of the marker. The levels of IMP3 can be represented
by arbritary units, for example as units obtained from a
densitometer, luminometer, or an ELISA plate reader. Ratios or
differences in number of units from a cancerous sample and number
of units from a control sample, e.g., an adjacent non-cancerous
tissue, may be used to determine the prognosis of the patient from
whom the samples were taken. These ratios and differences are
further discussed herein in the context of particular methods of
detection of IMP3 mRNA or protein.
[0042] Generally, the presence or amount of IMP3 will be determined
in a sample obtained from a tumor. However, it may also be possible
that high IMP3 levels located on cells other than the tumor cells
(e.g., red or white blood cells) is indicative of a bad prognosis
of a cancer, in which case one could determine IMP3 presence or
levels in these other cells. A control value or sample would then
be obtained from the same type of cells.
[0043] A sample may be obtained from a primary tumor.
Alternatively, it may also be obtained from a metastatic tumor. A
sample may be obtained from a surgically removed tumor, e.g., from
a kidney tumor. In one embodiment, a sample is obtained from tissue
removed in a radical or partial nephrectomy. When a nephrectomy is
performed, IMP3 levels may be measured in cancerous tissue as well
as in non-cancerous tissue of the kidney. Similarly, when a tissue
or organ is removed for treating another type of cancer, a
cancerous sample and a control sample may be obtained from the same
tissue or organ.
[0044] The level of IMP3 may be determined directly in a biological
specimen, e.g., a biopsy, obtained from a subject. Depending on the
method used to determine the level of IMP3, it may be desirable to
treat a biological specimen prior to measuring the level of IMP3.
For example, one or more cells may be isolated from the biological
specimen. Fractions of the specimen may be prepared. In other
cases, proteins or specific proteins may be isolated or purified
from of the sample. In other cases, nucleic acids, e.g., RNA may be
isolated or purified from of the sample. For example, in a case
when IMP3 protein is detected or measured, one may wish to isolate
proteins from the sample, and one may even want to isolate IMP3
proteins from most of the other proteins. When IMP3 mRNA is
detected or measured, one may want to isolate total RNA from the
sample, and optionally isolate and/or amplify IMP3 mRNA.
[0045] The methods described herein are useful for predicting the
prognosis of subjects having any one of a variety of cancers. For
example, the cancer can be kidney cancer, e.g., renal cell
carcinoma (RCC). RCC embraces a group of renal cancers, all of
which are derived from the renal tubular epithelium but each with
distinct clinical, pathologic, and genotypic features. Examples of
RCC include clear cell RCC, papillary RCC, chromophobe RCC, and
collecting duct carcinoma. The RCC may be a stage I, II, III or IV.
Stages I-III (T3, N0, M0) tumors define localized RCC, whereas most
of stage IV tumors defines metastasized RCC.
[0046] Other cancers include urogenital cancer, e.g., urothelial
carcinomas in urinary bladder, kidney, pelvic and ureter. An
exemplary bladder carcinoma is urothelial carcinoma of the bladder.
The majority (75%) of these carcinomas are early stages (i.e.,
stage Ta and T1) of superficial urothelial carcinomas and can be
prognosed and diagnosed as described herein. Generally, the
following other tumors or carcinomas of the bladder can also be
prognosed and diagnosed as described herein: urothelial
(transitional cell) carcinoma, including noninvasive urothelial
tumors, invasive urothelial tumors, superficial urothelial tumors,
papillary urothelial tumors (invasive or non invasive), papillomas,
papillary urothelial neoplasms of low malignant potential,
papillary urothelial carcinoma (low grade or high grade), flat
urothelial tumors, noninvasive flat urothelial carcinomas, flat
carcinoma in situ (cis), and flat invasive urothelial
carcinomas.
[0047] Other cancers whose development may potentially be
prognosticated as described herein include melanoma, prostate
carcinoma, lung carcinomas (non-small cell carcinoma, small cell
carcinoma, neuroendocrine carcinoma and carcinoid tumor), breast
carcinomas (ductal carcinoma, lobular carcinoma and mixed ductal
and lobular carcinoma), thyroid carcinomas (papillary thyroid
carcinoma, follicular carcinoma and medullary carcinoma), brain
cancers (meningioma, astrocytoma, glioblastoma, cerebellum tumors,
medulloblastoma, ependymoma), pancreatic carcinoma, ovarian
carcinomas (serous, mucinous and enodmetrioid types), cervical
cancers (squamous cell carcinoma in situ, invasive squamous cell
carcinoma and endocervical adenocarcinoma), uterine endometrial
carcinoma (endometrioid, serous and mucinous types), primary
peritoneal carcinoma, mesothelioma (pleura and peritoneum), eye
cancer (retinoblastoma), muscle (rhapdosarcoma and leiomyosarcoma),
lymphomas, esophageal cancer (adenocarcinoma and squamous cell
carcinoma), gastric cancers (gastric adenocarcinoma and
gastrointestinal stroma tumor), liver cancers (hepatocellular
carcinoma and bile duct cancer), small intestinal tumors (small
intestinal stromal tumor and carcinoid tumor) colon cancer
(adenocarcinoma of the colon, colon high grade dysplasia and colon
carcinoid tumor), and adrenal carcinoma.
[0048] Based at least on the observation that certain primary
tumors have a high IMP3 level (see Examples), determining the
presence or levels of IMP3 in a subject or a biological sample may
also be used to determine whether a subject has or is likely to
develop cancer (diagnostic method). In one embodiment, a method for
determining whether a subject has or is likely to develop cancer
comprises determining the presence of IMP3 in the subject, such as
in cells or tissues of the subject. The method allows the detection
or likelihood of development of any cancer that is associated with
high IMP3 expression, as further described herein. A method may
involve measuring levels of IMP3 in a test sample obtained from a
patient having or suspected of having cancer. A method may further
comprise comparing the observed levels of IMP3 to a control or to
levels of IMP3 found in a normal control sample, for example a
sample obtained from a subject that does not have cancer. The
presence of IMP3 or the presence of IMP3 at levels that are higher
than levels that are observed in the normal control indicate the
presence of cancer or the likelihood of developing cancer.
Exemplary cancers include kidney cancer, such as RCC, and
urothelial cancers, e.g., superficial urothelial carcinoma of the
bladder.
[0049] Additionally, disease progression can be assessed by
following IMP3 levels in individual patients over time.
Accordingly, methods provided herein also include methods for
monitoring the progression of cancer in a subject, comprising,
e.g., monitoring the presence or level of IMP3 in a cell, e.g., in
a cancerous cell or tumor, of the subject over time. An increase of
IMP3 in cancer cells, e.g., over time, indicates that the cancer is
progressing. In another embodiment, a reference reading is taken
after surgical removal of tissue, e.g., cancerous tissue, then
another taken at regular intervals. Any rise in IMP3 levels could
be indicative of a relapse, or possibly metastasis.
[0050] The information provided by the methods described herein may
be used by the physician in determining the most effective course
of treatment. A course of treatment refers to the therapeutic
measures taken for a patient after diagnosis or after treatment for
cancer. For example, a determination of the likelihood for cancer
recurrence, spread, or patient survival, can assist in determining
whether a more conservative or more radical approach to therapy
should be taken, or whether treatment modalities should be
combined. For example, when cancer progression or metastasis is
likely, it can be advantageous to precede or follow surgical
treatment with chemotherapy, radiation, immunotherapy, biological
modifier therapy, gene therapy, vaccines, and the like, or adjust
the span of time during which the patient is treated.
[0051] Also provided herein are methods for treating a subject
having cancer. A method may comprise (i) determining the presence
or level of IMP3 in a cancerous tumor of a subject; and (ii) if
IMP3 is present or is present at a higher level in the cancerous
tumor of the subject relative to a control, treating the subject
aggressively, whereas if no IMP3 is detected or if the level of
IMP3 in the cancerous tumor of a subject is statistically within
the range of a control, treating the subject less aggressively. An
aggressive therapy may comprise surgical removal of cancer cells or
a tissue comprising the cancer cells, e.g., nephrectomy in the case
of kidney cancer, chemotherapy, radiation, or a combination
thereof. Surgical removal may be followed by early systematic
therapy. The treatment may then be followed by monitoring of IMP3
levels.
[0052] In one embodiment, a partial or radical nephrectomy or
cancer tissue resection is conducted in a subject having a kidney
cancer, e.g., RCC, and the presence or level of IMP3 is determined
in the tissue, e.g., tumor tissue obtained by the nephrectomy. If
IMP3 is detected in the cancerous tissue, the subject is then
treated aggressively, e.g., with postoperative adjuvant therapy,
such as with anti-angiogenic agents and/or other drugs, e.g.,
Nexavar.RTM. (sorafenib) and Sutent.RTM. (sunitinib). If IMP3 is
not detected in the cancerous tissue, then the subject may be
spared postoperative adjuvant therapy or other aggressive
treatment.
[0053] IMP3 status in cancer patients, such as in RCC, can be added
in the pathology report with other pathological predictors
including tumor size, grade, subtype, and stage for the patient's
outcome information and clinical treatment.
[0054] In one embodiment, measuring the presence or level of IMP3
is combined with the determination of another prognostic factor,
such that the combined determination results in a more accurate
prognosis then determining either prognostic factor alone. Other
prognostic factors that may be determined include tumor stage,
size, grade, necrosis, histology type (clear cell, papillary and
chromophobe types) and Eastern Cooperative Oncology Group (ECOG)
performance status, and the tumor-node-metastasis (TNM) staging
system. In a preferred embodiment, a prognostic method comprises
determining the presence or level of IMP3 and determining the stage
of the tumor. As described in the Examples, the combination of
these two prognostic factors provides much better prognostic
information compared with TNM stage alone. For example, the stage
and IMP3 level may be compared to the data set forth in the diagram
in FIG. 9. This diagram provides that the best to worst diagnosis
are as follows (starting from best to worst): stage 1 and 2 RCC
with negative IMP3; stage 3 RCC with negative IMP3; stage 1 RCC
with low positive IMP3 and all stage RCC with high positive IMP3
and stage 2 and 3 RCC with low positive IMP3. The meaning of "low
positive" and "high positive" IMP3 levels is provided in the
Examples.
[0055] In addition, other prognostic factors for renal cancer,
e.g., RCC, which may be combined with IMP3 presence of levels
include Von Hippel-Linau gene alteration, DNA ploidy, and the
following biomarkers: carbonic anhydrase IX (CAIX or CA9), adipose
differentiation-related protein (ADFP), p53, mdm2, p27, cyclin A,
cyclin D1, PTEN, Ki-67, proliferating cell nuclear antigen (PCNA);
cadherins, catenins, MMP-9, MMP-E2/E-cadherin, CD44, EpCAM,
vimentin, MUC1, and immunal regulators (e.g., B7-H1 and B7-H4).
These markers are all discussed in Zhong Jiang (2007) Expert Rev.
Mol. Diagn. 7:1-15, which is incorporated by reference herein.
[0056] Determining the presence or level of IMP3 may also be
combined with the detection of one or more other biomarkers for
which increased or decreased expression correlates with cancer. The
selected biomarker can be a general diagnostic or prognostic marker
useful for multiple types of cancer, such as CA 125, CEA or LDH, or
can be a cancer-specific diagnostic or prognostic marker, such as a
colon cancer marker (for example, sialosyl-TnCEA, CA19-9, or LASA),
breast cancer marker (for example, CA 15-2. Her-2/neu and CA
27.29), ovarian cancer marker (for example, CA72-4), lung cancer
(for example, neuron-specific enolase (NSE) and tissue polypeptide
antigen (TPA)), prostate cancer (for example, PSA,
prostate-specific membrane antigen and prostatic acid phosphatase),
melanoma (for example, S-100 and TA-90), as well as other
biomarkers specific for other types of cancer. Those skilled in the
art will be able to select useful diagnostic or prognostic markers
for detection in combination with IMP3. Similarly, three or more,
four or more or five or more or a multitude of biomarkers can be
used together for determining a diagnosis or prognosis of a
patient.
[0057] Also provided herein are kits, e.g., kits for determining
the presence or level of IMP3 in a subject or in a biological
sample of a subject. A kit may comprise any agent useful for
qualitatively or quantitatively detecting IMP3 proteins or mRNA
(including potentially pre-mRNA), such as agents further described
herein. A kit may further comprise a control, such as a control
value or control sample or control tissue. A control may be protein
or RNA attached to a solid support. A kit may also comprise
additional components or reagents necessary for the detection of
IMP3, such as secondary antibodies for use in immunohistochemistry.
A kit may further comprise one or more other biomarkers or reagents
for evaluating other prognostic factors, e.g., tumor stage.
IMP3 Protein Detection Techniques
[0058] Methods for the detection of protein, e.g., IMP3 protein,
are well known to those skilled in the art, and include ELISA
(enzyme linked immunosorbent assay), RIA (radioimmunoassay),
Western blotting, and immunohistochemistry. Immunoassays such as
ELISA or RIA, which can be extremely rapid, are more generally
preferred. These methods use antibodies, or antibody equivalents,
to detect IMP3 protein. Antibody arrays or protein chips can also
be employed, see for example U.S. Patent Application Nos:
20030013208A1; 20020155493A1, 20030017515 and U.S. Pat. Nos.
6,329,209; 6,365,418, herein incorporated by reference in their
entirety.
[0059] ELISA and RIA procedures may be conducted such that a IMP3
standard is labeled (with a radioisotope such as .sup.125I or
.sup.35S, or an assayable enzyme, such as horseradish peroxidase or
alkaline phosphatase), and, together with the unlabelled sample,
brought into contact with the corresponding antibody, whereon a
second antibody is used to bind the first, and radioactivity or the
immobilized enzyme assayed (competitive assay). Alternatively, IMP3
in the sample is allowed to react with the corresponding
immobilized antibody, radioisotope- or enzyme-labeled anti-IMP3
antibody is allowed to react with the system, and radioactivity or
the enzyme assayed (ELISA-sandwich assay). Other conventional
methods may also be employed as suitable.
[0060] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. A "one-step" assay involves
contacting antigen with immobilized antibody and, without washing,
contacting the mixture with labeled antibody. A "two-step" assay
involves washing before contacting, the mixture with labeled
antibody. Other conventional methods may also be employed as
suitable.
[0061] In one embodiment, a method for measuring IMP3 levels
comprises the steps of: contacting a biological specimen with an
antibody or variant (e.g., fragment) thereof which selectively
binds IMP3, and detecting whether said antibody or variant thereof
is bound to said sample and thereby measuring the levels of IMP3. A
method may further comprise contacting the specimen with a second
antibody, e.g., a labeled antibody. The method may further comprise
one or more steps of washing, e.g., to remove one or more
reagents.
[0062] Enzymatic and radiolabeling of IMP3 and/or the antibodies
may be effected by conventional means. Such means will generally
include covalent linking of the enzyme to the antigen or the
antibody in question, such as by glutaraldehyde, specifically so as
not to adversely affect the activity of the enzyme, by which is
meant that the enzyme must still be capable of interacting with its
substrate, although it is not necessary for all of the enzyme to be
active, provided that enough remains active to permit the assay to
be effected. Indeed, some techniques for binding enzyme are
non-specific (such as using formaldehyde), and will only yield a
proportion of active enzyme.
[0063] It may be desirable to immobilize one component of the assay
system on a support, thereby allowing other components of the
system to be brought into contact with the component and readily
removed without laborious and time-consuming labor. It is possible
for a second phase to be immobilized away from the first, but one
phase is usually sufficient.
[0064] It is possible to immobilize the enzyme itself on a support,
but if solid-phase enzyme is required, then this is generally best
achieved by binding to antibody and affixing the antibody to a
support, models and systems for which are well-known in the art.
Simple polyethylene may provide a suitable support.
[0065] Enzymes employable for labeling are not particularly
limited, but may be selected from the members of the oxidase group,
for example. These catalyze production of hydrogen peroxide by
reaction with their substrates, and glucose oxidase is often used
for its good stability, ease of availability and cheapness, as well
as the ready availability of its substrate (glucose). Activity of
the oxidase may be assayed by measuring the concentration of
hydrogen peroxide formed after reaction of the enzyme-labeled
antibody with the substrate under controlled conditions well-known
in the art.
[0066] Other techniques may be used to detect IMP3 according to a
practitioner's preference based upon the present disclosure. One
such technique is Western blotting (Towbin et at., Proc. Nat. Acad.
Sci. 76:4350 (1979)), wherein a suitably treated sample is run on
an SDS-PAGE gel before being transferred to a solid support, such
as a nitrocellulose filter. Anti-IMP3 antibodies (unlabeled) are
then brought into contact with the support and assayed by a
secondary immunological reagent, such as labeled protein A or
anti-immunoglobulin (suitable labels including .sup.125I,
horseradish peroxidase and alkaline phosphatase). Chromatographic
detection may also be used.
[0067] Immunohistochemistry may be used to detect expression of
human IMP3, e.g., in a biopsy sample. A suitable antibody is
brought into contact with, for example, a thin layer of cells,
washed, and then contacted with a second, labeled antibody.
Labeling may be by fluorescent markers, enzymes, such as
peroxidase, avidin, or radiolabelling. The assay is scored
visually, using microscopy. The results may be quantitated, e.g.,
as described in the Examples.
[0068] As further described in the Examples, immunohistochemical
analysis optionally coupled with quantification of the signal may
be conducted as follows. IMP3 expression may be directly evaluated
in the tissue by preparing immunohistochemically stained slides
with, e.g., an avidin-biotinylated peroxidase complex system, as
further described in the Examples. Tumor cells with dark brown
color indicate high levels of IMP3, whereas cells that do not have
a detectable level of IMP3 will not appear brown, but rather blue,
e.g., if cells are hematoxycilin stained. Accordingly, a subject,
e.g., a pathologist, may determine by merely looking at a slide
under a microscope whether cells are brown or not and therefore
whether they contain IMP3, the presence of which would be
indicative of a poor prognosis.
[0069] Evaluation of the presence of brown stain, i.e., IMP3, may
also be done by quantitative immunohistochemical investigation,
e.g., with a computerized image analyzer (e.g., Automated Cellular
Imaging System, ACIS, ChromaVision Medical System Inc., San Juan
Capistrano, Calif.) may be used for evaluation of the levels of
IMP3 expression in the immunostained tissue samples. Using ACIS,
"cytoplasmic staining" may be chosen as program for IMP3 detection.
Different areas of immunostained tumor samples may be analyzed with
the ACIS system. With ACIS, positive staining may be calculated by
applying two thresholds with one recognizing blue background
(hematoxylin stained) cells and another recognizing brown positive
cells. The integrated optical density (IOD) is the sum of pixels
times (multiplied by) the intensity of those pixels. Accordingly,
brown IOD is the sum of brown pixels times the intensity of the
brown pixels and blue IOD is the sum of blue pixels times the
intensity of the blue pixels. ACIS values can be calculated as
brown IOD divided by the sum of the blue area and the brown area,
i.e., divided by (blue IOD+brown IOD). An average of the ACIS
values that is more than 1, e.g., about 1,1, 1.2, 1.3, 1.4, 1.5, 2,
2.5, 3, 5, 10, 30, 100 or more indicates an elevated IMP3
expression and therefore a poor prognosis.
[0070] Other machine or autoimaging systems may also be used to
measure immunostaining results for IMP3. As used herein,
"quantitative" immunohistochemistry refers to an automated method
of scanning and scoring samples that have undergone
immunohistochemistry, to identify and quantitate the presence of a
specified biomarker, such as an antigen or other protein. The score
given to the sample is a numerical representation of the intensity
of the immunohistochemical staining of the sample, and represents
the amount of target biomarker present in the sample. As used
herein, Optical Density (OD) is a numerical score that represents
intensity of staining. As used herein, semi-quantitative
immunohistochemistry refers to scoring of immunohistochemical
results by human eye, where a trained operator ranks results
numerically (e.g., as 1, 2 or 3).
[0071] Various automated sample processing, scanning and analysis
systems suitable for use with immunohistochemistry are available in
the art. Such systems may include automated staining (see, e.g, the
Benchmark.TM. system, Ventana Medical Systems, Inc.) and
microscopic scanning, computerized image analysis, serial section
comparison (to control for variation in the orientation and size of
a sample), digital report generation, and archiving and tracking of
samples (such as slides on which tissue sections are placed).
Cellular imaging systems are commercially available that combine
conventional light microscopes with digital image processing
systems to perform quantitative analysis on cells and tissues,
including immunostained samples. See, e.g., the CAS-200 system
(Becton, Dickinson & Co.).
[0072] Another method that may be used for detecting and
quantitating IMP3 protein levels is Western blotting, e.g., as
described in the Examples. Tumor tissues may be frozen and
homogenized in lysis buffer. Immunodetection can be performed with
an IMP3 antibody using the enhanced chemiluminescence system (e.g.,
from PerkinElmer Life Sciences, Boston, Mass.). The membrane may
then be stripped and re-blotted with a control antibody, e.g.,
anti-actin (A-2066) polyclonal antibody from Sigma (St. Louis,
Mo.). The intensity of the signal may be quantified by densitometry
software (e.g., NIH Image 1.61). After quantification of the IMP3
and control signals (e.g., actin), the relative expression levels
of IMP3 are normalized by amount of the actin in each lane, i.e.,
the value of the IMP3 signal is divided by the value of the control
signal. IMP3 protein expression is considered to be elevated (and
therefore predictive of a poor prognosis) when the value of
IMP3/actin is more than 1, e.g., about 1,1, 1.2, 1.3, 1.4, 1.5, 2,
2.5, 3, 5, 10, 30, 100.
[0073] Anti-IMP3 antibodies may also be used for imaging purposes,
for example, to detect the presence of IMP3 in cells and tissues of
a subject. Suitable labels include radioisotopes, iodine
(.sup.125I, .sup.121I), carbon (.sup.14C), sulphur (.sup.35S),
tritium (.sup.3H), indium (.sup.112In), and technetium (.sup.99
mTc), fluorescent labels, such as fluorescein and rhodamine, and
biotin. Immunoenzymatic interactions can be visualized using
different enzymes such as peroxidase, alkaline phosphatase, or
different chromogens such as DAB, AEC or Fast Red.
[0074] For in vivo imaging purposes, antibodies are not detectable,
as such, from outside the body, and so must be labeled, or
otherwise modified, to permit detection. Markers for this purpose
may be any that do not substantially interfere with the antibody
binding, but which allow external detection. Suitable markers may
include those that may be detected by X-radiography, NMR or MRI.
For X-radiographic techniques, suitable markers include any
radioisotope that emits detectable radiation but that is not
overtly harmful to the patient, such as barium or caesium, for
example. Suitable markers for NMR and MRI generally include those
with a detectable characteristic spin, such as deuterium, which may
be incorporated into the antibody by suitable labeling of nutrients
for the relevant hybridoma, for example.
[0075] The size of the subject, and the imaging system used, will
determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of technetium-99 m. The
labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain IMP3. The labeled
antibody or variant thereof, e.g., antibody fragment, can then be
detected using known techniques.
[0076] Antibodies that may be used to detect IMP3 include any
antibody, whether natural or synthetic, full length or a fragment
thereof, monoclonal or polyclonal, that binds sufficiently strongly
and specifically to the IMP3 to be detected, e.g., human IMP3. An
antibody may have a Kd of at most about 10.sup.-6M, 10.sup.-7M,
10.sup.-8M, 10.sup.-9M, 10.sup.-10M, 10.sup.-11M, 10.sup.-12M. The
phrase "specifically binds" refers to binding of, for example, an
antibody to an epitope or antigen or antigenic determinant in such
a manner that binding can be displaced or competed with a second
preparation of identical or similar epitope, antigen or antigenic
determinant. An antibody may bind preferentially to IMP3 relative
to other proteins, such as related proteins, e.g., IMP1 and
IMP2.
[0077] Antibodies are commercially available, e.g., from DAKO
(L523S) or may be prepared according to methods known in the
art.
[0078] Antibodies and derivatives thereof that may be used
encompasses polyclonal or monoclonal antibodies, chimeric, human,
humanized, primatized (CDR-grafted), veneered or single-chain
antibodies, phase produced antibodies (e.g., from phage display
libraries), as well as functional, i.e., IMP3 binding fragments, of
antibodies. For example, antibody fragments capable of binding to
IMP3 or portions thereof, including, but not limited to Fv, Fab,
Fab' and F(ab').sub.2 fragments can be used. Such fragments can be
produced by enzymatic cleavage or by recombinant techniques. For
example, papain or pepsin cleavage can generate Fab or F(ab')2
fragments, respectively. Other proteases with the requisite
substrate specificity can also be used to generate Fab or
F(ab').sub.2 fragments. Antibodies can also be produced in a
variety of truncated forms using antibody genes in which one or
more stop codons have been introduced upstream of the natural stop
site. For example, a chimeric gene encoding a F(ab').sub.2 heavy
chain portion can be designed to include DNA sequences encoding the
CH, domain and hinge region of the heavy chain.
[0079] Synthetic and engineered antibodies are described in, e.g.,
Cabilly et al., U.S. Pat. No. 4,816,567 Cabilly et al., European
Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss
et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,
WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276
B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No.
0,239,400 B1; Queen et al., European Patent No. 0451216 B1; and
Padlan, E. A. et al., EP 0519596 A1. See also, Newman, R. et al.,
BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody,
and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al.,
Science, 242: 423-426 (1988)) regarding single-chain
antibodies.
[0080] In some embodiments, agents that specifically bind to IMP3
other than antibodies are used, such as peptides. Peptides that
specifically bind to IMP3 can be identified by any means known in
the art. For example, specific peptide binders of IMP3 can be
screened for using peptide phage display libraries.
[0081] Generally, an agent that is capable of detecting an IMP3
polypeptide, such that the presence of IMP3 is detected and/or
quantitated, may be used. As defined herein, an "agent" refers to a
substance that is capable of identifying or detecting IMP3 in a
biological sample (e.g., identifies or detects IMP3 mRNA, IMP3 DNA,
IMP3 protein). In one embodiment, the agent is a labeled or
labelable antibody which specifically binds to IMP3 polypeptide. As
used herein, the phrase "labeled or labelable" refers to the
attaching or including of a label (e.g., a marker or indicator) or
ability to attach or include a label (e.g., a marker or indicator).
Markers or indicators include, but are not limited to, for example,
radioactive molecules, colorimetric molecules, and enzymatic
molecules which produce detectable changes in a substrate.
[0082] In addition, an IMP3 protein may be detected using Mass
Spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid
chromatography-mass spectrometry (LC-MS), gas chromatography-mass
spectrometry (GC-MS), high performance liquid chromatography-mass
spectrometry (HPLC-MS), capillary electrophoresis-mass
spectrometry, nuclear magnetic resonance spectrometry, or tandem
mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for
example, U.S. Patent Application Nos: 20030199001, 20030134304,
20030077616, which are herein incorporated by reference.
[0083] Mass spectrometry methods are well known in the art and have
been used to quantify and/or identify biomolecules, such as
proteins (see, e.g., Li et al. (2000) Tibtech 18:151-160; Rowley et
al. (2000) Methods 20: 383-397; and Kuster and Mann (1998) Curr.
Opin. Structural Biol 8: 393-400). Further, mass spectrometric
techniques have been developed that permit at least partial de novo
sequencing of isolated proteins. Chait et al., Science 262:89-92
(1993); Keough et al., Proc. Natl. Acad. Sci. USA. 96:7131-6
(1999); reviewed in Bergman, EXS 88:133-44 (2000).
[0084] In certain embodiments, a gas phase ion spectrophotometer is
used. In other embodiments, laser-desorption/ionization mass
spectrometry is used to analyze the sample. Modern laser
desorption/ionization mass spectrometry ("LDI-MS") can be practiced
in two main variations: matrix assisted laser desorption/ionization
("MALDI") mass spectrometry and surface-enhanced laser
desorption/ionization ("SELDI"). In MALDI, the analyte is mixed
with a solution containing a matrix, and a drop of the liquid is
placed on the surface of a substrate. The matrix solution then
co-crystallizes with the biological molecules. The substrate is
inserted into the mass spectrometer. Laser energy is directed to
the substrate surface where it desorbs and ionizes the biological
molecules without significantly fragmenting them. However, MALDI
has limitations as an analytical tool. It does not provide means
for fractionating the sample, and the matrix material can interfere
with detection, especially for low molecular weight analytes. See,
e.g., U.S. Pat. No. 5,118,937 (Hillenkamp et al.), and U.S. Pat.
No. 5,045,694 (Beavis & Chait).
[0085] In SELDI, the substrate surface is modified so that it is an
active participant in the desorption process. In one variant, the
surface is derivatized with adsorbent and/or capture reagents that
selectively bind the protein of interest. In another variant, the
surface is derivatized with energy absorbing molecules that are not
desorbed when struck with the laser. In another variant, the
surface is derivatized with molecules that bind the protein of
interest and that contain a photolytic bond that is broken upon
application of the laser. In each of these methods, the
derivatizing agent generally is localized to a specific location on
the substrate surface where the sample is applied. See, e.g., U.S.
Pat. No. 5,719,060 (Hutchens & Yip) and WO 98/59361 (Hutchens
& Yip). The two methods can be combined by, for example, using
a SELDI affinity surface to capture an analyte and adding
matrix-containing liquid to the captured analyte to provide the
energy absorbing material.
[0086] For additional information regarding mass spectrometers,
see, e.g., Principles of Instrumental Analysis, 3rd edition.,
Skoog, Saunders College Publishing, Philadelphia, 1985; and
Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. Vol.
15 (John Wiley & Sons, New York 1995), pp. 1071-1094.
[0087] Detection of the presence of a marker or other substances
will typically involve detection of signal intensity. This, in
turn, can reflect the quantity and character of a polypeptide bound
to the substrate. For example, in certain embodiments, the signal
strength of peak values from spectra of a first sample and a second
sample can be compared (e.g., visually, by computer analysis etc.),
to determine the relative amounts of particular biomolecules.
Software programs such as the Biomarker Wizard program (Ciphergen
Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing
mass spectra. The mass spectrometers and their techniques are well
known to those of skill in the art.
[0088] Any person skilled in the art understands, any of the
components of a mass spectrometer (e.g., desorption source, mass
analyzer, detect, etc.) and varied sample preparations can be
combined with other suitable components or preparations described
herein, or to those known in the art. For example, in some
embodiments a control sample may contain heavy atoms (e.g.
.sup.13C) thereby permitting the test sample to be mixed with the
known control sample in the same mass spectrometry run.
[0089] In one preferred embodiment, a laser desorption
time-of-flight (TOF) mass spectrometer is used. In laser desorption
mass spectrometry, a substrate with a bound marker is introduced
into an inlet system. The marker is desorbed and ionized into the
gas phase by laser from the ionization source. The ions generated
are collected by an ion optic assembly, and then in a
time-of-flight mass analyzer, ions are accelerated through a short
high voltage field and let drift into a high vacuum chamber. At the
far end of the high vacuum chamber, the accelerated ions strike a
sensitive detector surface at a different time. Since the
time-of-flight is a function of the mass of the ions, the elapsed
time between ion formation and ion detector impact can be used to
identify the presence or absence of molecules of specific mass to
charge ratio.
[0090] In some embodiments the relative amounts of one or more
biomolecules present in a first or second sample is determined, in
part, by executing an algorithm with a programmable digital
computer. The algorithm identifies at least one peak value in the
first mass spectrum and the second mass spectrum. The algorithm
then compares the signal strength of the peak value of the first
mass spectrum to the signal strength of the peak value of the
second mass spectrum of the mass spectrum. The relative signal
strengths are an indication of the amount of the biomolecule that
is present in the first and second samples. A standard containing a
known amount of a biomolecule can be analyzed as the second sample
to better quantify the amount of the biomolecule present in the
first sample. In certain embodiments, the identity of the
biomolecules in the first and second sample can also be
determined.
IMP3 RNA Detection Techniques
[0091] Any method for qualitatively or quantitatively detecting
IMP3 RNA, e.g., mRNA, may be used.
[0092] Detection of RNA transcripts may be achieved by Northern
blotting, for example, wherein a preparation of RNA is run on a
denaturing agarose gel, and transferred to a suitable support, such
as activated cellulose, nitrocellulose or glass or nylon membranes.
Radiolabeled cDNA or RNA is then hybridized to the preparation,
washed and analyzed by autoradiography.
[0093] Detection of RNA transcripts can further be accomplished
using amplification methods. For example, it is within the scope of
the present invention to reverse transcribe mRNA into cDNA followed
by polymerase chain reaction (RT-PCR); or, to use a single enzyme
for both steps as described in U.S. Pat. No. 5,322,770, or reverse
transcribe mRNA into cDNA followed by symmetric gap ligase chain
reaction (RT-AGLCR) as described by R. L. Marshall, et al., PCR
Methods and Applications 4: 80-84 (1994).
[0094] In one embodiment, quantitative real-time polymerase chain
reaction (qRT-PCR) is used to evaluate mRNA levels of IMP3 (see
Examples). IMP3 and a control mRNA, e.g.,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels may be
quantitated in cancer tissue and adjacent benign tissues. For this,
frozen tissues may be cut into 5 micron sections and total RNA may
be extracted, e.g., by Qiagen RNeasy Mini Kit (Qiagen, Inc.,
Valencia, Calif.). A certain amount of RNA, e.g., five hundred
nanograms of total RNA, from each tissue may be reversely
transcribed by using, e.g., Qiagen Omniscript RT Kit. Two-step
qRT-PCR may be performed, e.g., with the ABI TaqMan PCR reagent kit
(ABI Inc, Foster City, Calif.), and IMP3 primers and GAPDH primers,
and the probes for both genes on ABI Prism 7700 system. The primers
that may be used are set forth in the Examples. The IMP3 copy
number may then be divided by the GAPDH copy number and multiplied
by 1,000 to give a value for the particular subject. In other
words, the amount of IMP3 mRNA was normalized with the amount of
GAPDH mRNA measured in the same RNA extraction to obtain an
IMP3/GAPDH ratio. A ratio that is equal to or more than 1, e.g.,
about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 5, 10, 30, 100 may be
considered as a high IMP3 expression and therefore a poor
prognosis.
[0095] Other known amplification methods which can be utilized
herein include but are not limited to the so-called "NASBA" or
"3SR" technique described in PNAS USA 87: 1874-1878 (1990) and also
described in Nature 350 (No. 6313): 91-92 (1991); Q-beta
amplification as described in published European Patent Application
(EPA) No. 454-4610; strand displacement amplification (as described
in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996) and European
Patent Application No. 684315; and target mediated amplification,
as described by PCT Publication WO9322461.
[0096] Primers that may be used for amplification of IMP3 nucleic
acid portions are set forth in the Examples.
[0097] In situ hybridization visualization may also be employed,
wherein a radioactively labeled antisense RNA probe is hybridized
with a thin section of a biopsy sample, washed, cleaved with RNase
and exposed to a sensitive emulsion for autoradiography. The
samples may be stained with haematoxylin to demonstrate the
histological composition of the sample, and dark field imaging with
a suitable light filter shows the developed emulsion.
Non-radioactive labels such as digoxigenin may also be used.
[0098] Another method for evaluation of IMP3 expression is to
detect gene amplification by fluorescent in situ hybridization
(FISH). FISH is a technique that can directly identify a specific
region of DNA or RNA in a cell and therefore enables to visual
determination of the IMP3 expression in tissue samples. The FISH
method has the advantages of a more objective scoring system and
the presence of a built-in internal control consisting of the IMP3
gene signals present in all non-neoplastic cells in the same
sample. Fluorescence in situ hybridization is a direct in situ
technique that is relatively rapid and sensitive. FISH test also
can be automated. Immunohistochemistry can be combined with a FISH
method when the expression level of IMP3 is difficult to determine
by immunohistochemistry alone.
[0099] Alternatively, mRNA expression can be detected on a DNA
array, chip or a microarray. Oligonucleotides corresponding to the
IMP3 are immobilized on a chip which is then hybridized with
labeled nucleic acids of a test sample obtained from a patient.
Positive hybridization signal is obtained with the sample
containing IMP3 transcripts. Methods of preparing DNA arrays and
their use are well known in the art. (See, for example U.S. Pat.
Nos. 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S.
20030157485 and Schena et al. 1995 Science 20:467-470; Gerhold et
al. 1999 Trends in Biochem. Sci. 24, 168-173; and Lennon et al.
2000 Drug discovery Today 5: 59-65, which are herein incorporated
by reference in their entirety). Serial Analysis of Gene Expression
(SAGE) can also be performed (See for example U.S. Patent
Application 20030215858).
[0100] To monitor mRNA levels, for example, mRNA is extracted from
the biological sample to be tested, reverse transcribed, and
fluorescent-labeled cDNA probes are generated. The microarrays
capable of hybridizing to IMP3 cDNA are then probed with the
labeled cDNA probes, the slides scanned and fluorescence intensity
measured. This intensity correlates with the hybridization
intensity and expression levels.
[0101] Types of probes for detection of IMP3 RNA include cDNA,
riboprobes, synthetic oligonucleotides and genomic probes. The type
of probe used will generally be dictated by the particular
situation, such as riboprobes for in situ hybridization, and cDNA
for Northern blotting, for example. Most preferably, the probe is
directed to nucleotide regions unique to IMP3 RNA. The probes may
be as short as is required to differentially recognize IMP3 mRNA
transcripts, and may be as short as, for example, 15 bases;
however, probes of at least 17 bases, more preferably 18 bases and
still more preferably 20 bases are preferred. Preferably, the
primers and probes hybridize specifically under stringent
conditions to a DNA fragment having the nucleotide sequence
corresponding to the IMP3 gene. As herein used, the term "stringent
conditions" means hybridization will occur only if there is at
least 95% and preferably at least 97% identity between the
sequences.
[0102] The form of labeling of the probes may be any that is
appropriate, such as the use of radioisotopes, for example,
.sup.32P and .sup.35S. Labeling with radioisotopes may be achieved,
whether the probe is synthesized chemically or biologically, by the
use of suitably labeled bases.
IMP3 Autoantibody Detection
[0103] In one embodiment, the level of IMP3 in a subject is
determined by the level of IMP3 autoantibodies. An exemplary method
comprises determining the presence of IMP3 autoantibodies in a
subject, such as in a bodily fluid or sample of the subject,
wherein the presence of IMP3 autoantibodies in the bodily fluid or
sample of the subject, indicates that the subject has a poor
prognosis. In another embodiment, the method comprises determining
the level of IMP3 autoantibodies in a subject; comparing the level
of autoantibodies to that in a control, e.g., a subject who does
not have a cancer, and optionally to that of one or more subjects
who have a cancer, e.g., a slow progressive cancer or an invasive
cancer and/or a cancer having a high likelihood of metastasis; and
or a poor or a good prognosis. A higher level of IMP3
autoantibodies may be a level that is statistically significant,
e.g., at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 5, 10, 30,
100 times higher.
[0104] Human tumors stimulate the production of autoantibodies
against autologous cellular proteins called tumor associated
antigens (TAAs) (see, e.g., Wang et al. New England J. Med. (2005)
353:1224 and Int. J. Oncol. (2005) 26:311). The level of IMP3
autoantibodies may be detected and/or measured in a bodily fluid of
a subject, e.g., in blood or serum. Antibodies may be detected by
spectrometry, ELISA, PCR, cDNA, peptide phage display, autoantigen
microarray, immunoblotting (see, e.g., Casiano et al. (May 2006)
Mol. Cell. Proteomics). In one embodiment, an IMP3 protein or one
or more peptide thereof is used as an agent to detect IMP3
antibodies.
Therapeutic Applications Using IMP3
[0105] Based at least on the observation that high IMP3 levels in
primary tumors is associated with a higher likelihood of metastasis
and a poor prognosis, it may be possible to prevent the likelihood
of metastasis and progression of the cancer or prevent cancer
altogether by inhibiting or reducing the expression level of IMP3
or IMP3 activity in the tumor or tissue of the subject. In
particular, recent studies have demonstrated that IMP3 promotes
tumor cell proliferation and invasion (Liao et al. (2006) J. Biol.
Chem. 280:18517 and Vikesaa et al. (2006) EMBO J. 25:1456). In one
embodiment, a method for treating or preventing cancer, such as
kidney or a urogenital cancer, comprises reducing the level of
expression of IMP3, reducing the amount of IMP3 protein, or
inhibiting the activity of an IMP3 protein. In a method for
treatment of cancer, one may reduce IMP3 levels or activity in a
tumor, e.g., a primary tumor. In a method for preventing cancer,
one may reduce IMP3 levels or activity in tissue likely to develop
cancer, e.g., tissue that exhibits high levels of IMP3
expression.
[0106] Prophylaxis may be appropriate even at very early stages of
the disease, to prevent metastasis. Thus, administration of an
agent that reduces IMP3 levels or activity may be effected as soon
as cancer is diagnosed, and treatment continued for as long as is
necessary, preferably until the threat of the disease has been
removed. Such treatment may also be used prophylactically in
individuals at high risk for development of certain cancers, e.g.,
prostate or breast.
RNAi Technology
[0107] In one embodiment, IMP3 levels are decreased by
administration of or expression in a subject, e.g., in cells or a
tissue of the subjet, of one or more IMP3 siRNAs.
[0108] The term "short interfering RNAs (siRNA)" as used herein is
intended to refer to any nucleic acid molecule capable of mediating
RNAi or gene silencing. The term siRNA is intended to encompass
various naturally generated or synthetic compounds, with RNAi
function. Such compounds include, without limitation, duplex
synthetic oligonucleotides, of about 21 to 23 base pairs with
terminal overlaps of 2 or 3 base pairs; hairpin structures of one
oligonucleotide chain with sense and complementary, hybridizing,
segments of 21-23 base pairs joined by a loop of, e.g., 3-5 base
pairs; and various genetic constructs leading to the expression of
the preceding structures or functional equivalents. Such genetic
constructs are usually prepared in vitro and introduced in the test
system, but can also include siRNA from naturally occurring siRNA
precursors coded by the genome of the host cell or animal.
[0109] It is not a requirement that the siRNA be comprised solely
of RNA. In one embodiment, the siRNA comprises one or more chemical
modifications and/or nucleotide analogues. The modification and/or
analogue may be any modification and/or analogue, respectively,
that does not negatively affect the ability of the siRNA to inhibit
IMP3 expression. The inclusion of one or more chemical
modifications and/or nucleotide analogues in an siRNA may be
preferred to prevent or slow nuclease digestion, and in turn,
create a more stable siRNA for practical use. Chemical
modifications and/or nucleotide analogues which stabilize RNA are
known in the art. Phosphorothioate derivatives, which include the
replacement of non-bridging phosphoroyl oxygen atoms with sulfur
atoms, are one example of analogues showing increased resistance to
nuclease digestion. Sites of the siRNA which may be targeted for
chemical modification include the loop region of a hairpin
structure, the 5' and 3' ends of a hairpin structure (e.g. cap
structures), the 3' overhang regions of a double-stranded linear
siRNA, the 5' or 3' ends of the sense strand and/or antisense
strand of a linear siRNA, and one or more nucleotides of the sense
and/or antisense strand.
[0110] As used herein, the term siRNA is intended to be equivalent
to any term in the art defined as a molecule capable of mediating
sequence-specific RNAi. Such equivalents include, for example,
double-stranded RNA (dsRNA), micro-RNA (mRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, and
post-transcriptional gene silencing RNA (ptgsRNA) siRNAs may be
introduced into cells to suppress gene expression for therapeutic
or prophylactic purposes as described in International Publication
Number WO 0175164. Publications describing RNAi technology include
but are not limited to the following: U.S. Pat. No. 6,686,463, U.S.
Pat. No. 6,673,611, U.S. Pat. No. 6,623,962, U.S. Pat. No.
6,506,559, U.S. Pat. No. 6,573,099, and U.S. Pat. No. 6,531,644;
U.S. publication Nos: 20030153519, 20030167490, International
Publication Numbers WO04061081; WO04052093; WO04048596; WO04048594;
WO04048581; WO04048566; WO04046320; WO04044537; WO04043406;
WO04033620; WO04030660; WO04028471; WO 0175164. Papers which
describe the methods and concepts for the optimal use of these
compounds include but are not limited to the following: Brummelkamp
Science 296: 550-553 (2002); Caplen Expert Opin. Biol. Ther.
3:575-86 (2003); Brummelkamp, Sciencexpress 21 Mar. 3 1-6 (2003);
Yu Proc Natl Acad Sci USA 99:6047-52 (2002); Paul Nature
Biotechnology 29:505-8 (2002); Paddison Proc Natl Acad Sci USA
99:1443-8 (2002); Brummelkamp Nature 424: 797-801 (2003);
Brummelkamp, Science 296: 550-3 (2003); Sui Proc Natl Acad Sci USA
99: 5515-20 (2002); Paddison, Genes and Development 16:948-58
(2002).
[0111] A composition comprising an siRNA effective to inhibit IMP3
expression may include an RNA duplex comprising a sense sequence of
IMP3. In this embodiment, the RNA duplex comprises a first strand
comprising a sense sequence of IMP3 and a second strand comprising
a reverse complement of the sense sequence of IMP3. In one
embodiment the sense sequence of IMP3 comprises of from 10 to 25
nucleotides in length. More preferably, the sense sequence of IMP3
comprises of from 19 to 25 nucleotides in length. Most preferably,
the sense sequence of IMP3 comprises of from 21 to 23 nucleotides
in length. The sense sequence of IMP3 preferably comprises a
sequence of IMP3 containing a translational start site, and may
comprise a portion of IMP3 sequence within the first 400 nt of the
human IMP3 mRNA.
[0112] In another embodiment, a composition comprising an siRNA
effective to inhibit IMP3 expression may comprise in a single
molecule a sense sequence of IMP3, the reverse complement of the
sense sequence of IMP3, and an intervening sequence enabling duplex
formation between the sense and reverse complement sequences. The
sense sequence of IMP3 may comprise 10 to 25 nucleotides in length,
or more preferably 19 to 25 nucleotides in length, or most
preferably 21 to 23 nucleotides in length.
[0113] It will be readily apparent to one of skill in the art that
an siRNA of the present invention may comprise a sense sequence of
IMP3 or the reverse complement of the sense sequence of IMP3 which
is less than perfectly complementary to each other or to the
targeted region of IMP3. In other words, the siRNA may comprise
mismatches or bulges within the sense or reverse complement
sequence. In one aspect, the sense sequence or its reverse
complement may not be entirely contiguous. The sequence or
sequences may comprise one or more substitutions, deletions, and/or
insertions. The only requirement of the present invention is that
the siRNA sense sequence possess enough complementarity to its
reverse complement and to the targeted region of IMP3 to allow for
RNAi activity. It is an object of the present invention, therefore,
to provide for sequence modifications of an siRNA of the present
invention that retain sufficient complementarity to allow for RNAi
activity. One of skill in the art may predict that a modified siRNA
composition of the present invention will work based on the
calculated binding free energy of the modified sequence for the
complement sequence and targeted region of IMP3. Calculation of
binding free energies for nucleic acids and the effect of such
values on strand hybridization is known in the art.
[0114] A wide variety of delivery systems are available for use in
delivering an siRNA to a target cell in vitro and in vivo. An siRNA
of the present invention may be introduced directly or indirectly
into a cell in which IMP3 inhibition is desired. An siRNA may be
directly introduced into a cell by, for example, injection. As
such, it is an object of the invention to provide for a composition
comprising an siRNA effective to inhibit IMP3 in injectable, dosage
unit form. An siRNA of the present invention may be injected
intravenously or subcutaneously as an example, for therapeutical
use in conjunction with the methods and compositions of the present
invention. Such treatment may include intermittent or continuous
administration until therapeutically effective levels are achieved
to inhibit IMP3 expression in the desired tissue.
[0115] Indirectly, an expressible DNA sequence or sequences
encoding the siRNA may be introduced into a cell, and the siRNA
thereafter transcribed from the DNA sequence or sequences. It is an
object of the present invention, therefore, to provide for
compositions comprising a DNA sequence or sequences which encode an
siRNA effective to inhibit IMP3 expression.
[0116] A DNA composition of the present invention comprises a first
DNA sequence which encodes a first RNA sequence comprising a sense
sequence of IMP3 and a second DNA sequence which encodes a second
RNA sequence comprising the reverse complement of the sense
sequence of IMP3. The first and second RNA sequences, when
hybridized, form an siRNA duplex capable of forming an RNA-induced
silencing complex, the RNA-induced silencing complex being capable
of inhibiting IMP3 expression. The first and second DNA sequences
may be chemically synthesized or synthesized by PCR using
appropriate primers to IMP3. Alternatively, the DNA sequences may
be obtained by recombinant manipulation using cloning technology,
which is well known in the art. Once obtained, the DNA sequences
may be purified, combined, and then introduced into a cell in which
IMP3 inhibition is desired. Alternatively, the sequences may be
contained in a single vector or separate vectors, and the vector or
vectors introduced into the cell in which IMP3 inhibition is
desired.
[0117] Delivery systems available for use in delivering a DNA
composition of the present invention to a target cell include, for
example, viral and non-viral systems. Examples of suitable viral
systems include, for example, adenoviral vectors, adeno-associated
virus, lentivirus, poxvirus, retroviral vectors, vaccinia, herpes
simplex virus, UV, the minute virus of mice, hepatitis B virus and
influenza virus. Non-viral delivery systems may also be used, for
example using, uncomplexed DNA, DNA-liposome complexes, DNA-protein
complexes and DNA-coated gold particles, bacterial vectors such as
salmonella, and other technologies such as those involving VP22
transport protein, Co-X-gene, and replicon vectors. A viral or
non-viral vector in the context of the present invention may
express the antigen of interest.
Antisense Technology
[0118] In another embodiment, the level of IMP3 is reduced or
decreased by administration or the expression of antisense
molecules in a subject or tissue or cell thereof.
[0119] Gene expression can be controlled through triple-helix
formation or antisense DNA or RNA, both of which methods are based
on binding of a polynucleotide to DNA or RNA. An antisense nucleic
acid molecule which is complementary to a nucleic acid molecule
encoding IMP3 can be designed based on the known IMP3 nucleotide
sequences. An antisense nucleic acid molecule can comprise a
nucleotide sequence which is complementary to a coding strand of a
nucleic acid, e.g. complementary to an mRNA sequence, constructed
according to the rules of Watson and Crick base pairing, and can
hydrogen bond to the coding strand of the nucleic acid. The
antisense sequence complementary to a sequence of an mRNA can be
complementary to a sequence in the coding region of the mRNA or can
be complementary to a 5' or 3' untranslated region of the mRNA.
Furthermore, an antisense nucleic acid can be complementary in
sequence to a regulatory region of the gene encoding the mRNA, for
instance a transcription initiation sequence or regulatory element.
Preferably, an antisense nucleic acid complementary to a region
preceding or spanning the initiation codon or in the 3'
untranslated region of an mRNA is used. A nucleic acid is designed
which has a sequence complementary to a sequence of the coding or
untranslated region of the shown nucleic acid. Alternatively, an
antisense nucleic acid can be designed based upon sequences of the
IMP3 gene, which are known or can be identified by screening a
genomic DNA library with an isolated nucleic acid of the invention.
For example, the sequence of an important regulatory element can be
determined by standard techniques and a sequence which is antisense
to the regulatory element can be designed.
[0120] The antisense nucleic acids and oligonucleotides of the
invention can be constructed using chemical synthesis and enzymatic
ligation reactions using procedures known in the art. The antisense
nucleic acid or oligonucleotide can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids e.g. phosphorothioate derivatives
and acridine substituted nucleotides can be used. Alternatively,
the antisense nucleic acids and oligonucleotides can be produced
biologically using an expression vector into which a nucleic acid
has been subcloned in an antisense orientation (i.e. nucleic acid
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest). The antisense
expression vector is introduced into cells in the form of a
recombinant plasmid, phagemid or attenuated virus in which
antisense nucleic acids are produced under the control of a high
efficiency regulatory region, the activity of which can be
determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using
antisense genes see Weintraub, H. et al., Antisense RNA as a
molecular tool for genetic analysis, Reviews--Trends in Genetics,
Vol. 1 (1) 1986.
[0121] In addition, ribozymes can be used to inhibit expression of
IMP3. For example, the nucleic acids of the invention can further
be used to design ribozymes which are capable of cleaving a
single-stranded nucleic acid encoding a IMP3 protein, such as a
IMP3 mRNA transcript. A catalytic RNA (ribozyme) having
ribonuclease activity can be designed which has specificity for an
mRNA encoding IMP3 based upon the sequence of a nucleic acid of the
invention. For example, a derivative of a Tetrahymena L-19 IVS RNA
can be constructed in which the base sequence of the active site is
complementary to the base sequence to be cleaved in a IMP3-encoding
mRNA. See for example Cech, et al., U.S. Pat. No. 4,987,071; Cech,
et al., U.S. Pat. No. 5,116,742. Alternatively, a nucleic acid of
the invention could be used to select a catalytic RNA having a
specific ribonuclease activity from a pool of RNA molecules. See
for example Bartel, D. and Szostak, J. W. Science 261: 1411-1418
(1993). RNA-mediated interference (RNAi) (Fire, et al., Nature 391:
806-811, 1998) may also be used.
IMP3 Blocking Antibodies and Aptamers
[0122] In yet another embodiment, IMP3 levels are reduced by
administration to or expression in a subject or a cell or tissue
thereof, of IMP3 blocking antibodies or aptamers.
[0123] Antibodies, or their equivalents and derivatives, e.g.,
intrabodies, or other IMP3 antagonists may be used in accordance
with the present invention for the treatment or prophylaxis of
cancers. Administration of a suitable dose of the antibody or the
antagonist may serve to block the activity of the protein and this
may provide a crucial time window in which to treat the malignant
growth.
[0124] A method of treatment may involve attachment of a suitable
toxin to IMP3 antibodies which then target the area of the tumor.
Such toxins are well known in the art, and may comprise toxic
radioisotopes, heavy metals, enzymes and complement activators, as
well as such natural toxins as ricin which are capable of acting at
the level of only one or two molecules per cell. It may also be
possible to use such a technique to deliver localized doses of
suitable physiologically active compounds, which may be used, for
example, to treat cancers.
[0125] The antibody (or other inhibitors or intrabody) can be
administered by a number of methods. One preferred method is set
forth by Marasco and Haseltine in PCT WO94/02610, which is
incorporated herein by reference. This method discloses the
intracellular delivery of a gene encoding the antibody. One would
preferably use a gene encoding a single chain antibody. The
antibody would preferably contain a nuclear localization sequence.
One preferably uses an SV40 nuclear localization signal. By this
method one can intracellularly express an antibody, which can block
IMP3 functioning in desired cells.
[0126] Where the present invention provides for the administration
of, for example, antibodies to a patient, then this may be by any
suitable route. If the tumor is still thought to be, or diagnosed
as, localized, then an appropriate method of administration may be
by injection direct to the site. Administration may also be by
injection, including subcutaneous, intramuscular, intravenous and
intradermal injections.
[0127] Aptamers can be produced using the methodology disclosed in
a U.S. Pat. No. 5,270,163 and WO 91/19813.
Other IMP3 Inhibitors
[0128] Other agents, e.g., compounds, that inhibit the activity of
IMP3 may also be used. Such compounds include small molecules,
e.g., molecules that interact with the active site or a binding
site of the protein, e.g., an RNA binding site. For example, an
IMP3 inhibitory agent may be an agent that inhibits binding of IMP3
to its target mRNA. One agent that can be used is a target RNA or a
portion thereof to which IMP3 binds. In one embodiment, a large
amount of such an oligonucleotide or nucleic acid is administered
to a subject to thereby prevent IMP3 to reach its targets in the
cell, and thereby prevent IMP3 activity. An agent may be a portion
of IGF-II mRNA to which IMP3 binds, such as a portion of an IGF-II
leader 3 mRNA (see, e.g., Nielsen et al. Scand J Clin Lab Invest
Suppl. 2001; 234:93). Other agents may be identified according to
methods known in the art.
Pharmaceutical Compositions
[0129] Formulations may be any that are appropriate to the route of
administration, and will be apparent to those skilled in the art.
The formulations may contain a suitable carrier, such as saline,
and may also comprise bulking agents, other medicinal preparations,
adjuvants and any other suitable pharmaceutical ingredients.
Catheters are one preferred mode of administration.
[0130] The term "pharmaceutically acceptable" refers to compounds
and compositions which may be administered to mammals without undue
toxicity. Exemplary pharmaceutically acceptable salts include
mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the
like.
[0131] The antibodies, nucleic acids or antagonists of the
invention may be administered orally, topically, or by parenteral
means, including subcutaneous and intramuscular injection,
implantation of sustained release depots, intravenous injection,
intranasal administration, and the like. Accordingly, antibodies or
nucleic acids of the invention may be administered as a
pharmaceutical composition comprising the antibody or nucleic acid
of the invention in combination with a pharmaceutically acceptable
carrier. Such compositions may be aqueous solutions, emulsions,
creams, ointments, suspensions, gels, liposomal suspensions, and
the like. Suitable carriers (excipients) include water, saline,
Ringer's solution, dextrose solution, and solutions of ethanol,
glucose, sucrose, dextran, mannose, mannitol, sorbitol,
polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen,
Carbopol Registered.TM., vegetable oils, and the like. One may
additionally include suitable preservatives, stabilizers,
antioxidants, antimicrobials, and buffering agents, for example,
BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like.
Cream or ointment bases useful in formulation include lanolin,
Silvadene.RTM. (Marion), Aquaphor.RTM. (Duke Laboratories), and the
like. Other topical formulations include aerosols, bandages, and
other wound dressings. Alternatively one may incorporate or
encapsulate the compounds in a suitable polymer matrix or membrane,
thus providing a sustained-release delivery device suitable for
implantation near the site to be treated locally. Other devices
include indwelling catheters and devices such as the Alzet.RTM.
minipump. Ophthalmic preparations may be formulated using
commercially available vehicles such as Sorbi-care.RTM. (Allergan),
Neodecadron.RTM. (Merck, Sharp & Dohme), Lacrilube.RTM., and
the like, or may employ topical preparations such as that described
in U.S. Pat. No. 5,124,155. Further, one may provide an antagonist
in solid form, especially as a lyophilized powder. Lyophilized
formulations typically contain stabilizing and bulking agents, for
example human serum albumin, sucrose, mannitol, and the like. A
thorough discussion of pharmaceutically acceptable excipients is
available in Remington's Pharmaceutical Sciences (Mack Pub.
Co.).
[0132] The amount of antibody, nucleic acid or inhibitor required
to treat any particular disorder will of course vary depending upon
the nature and severity of the disorder, the age and condition of
the subject, and other factors readily determined by one of
ordinary skill in the art.
Immunotherapy
[0133] In further aspects, the present invention provides methods
for using IMP3 or an immunoreactive polypeptide thereof (or DNA
encoding the protein or polypeptides) for immunotherapy of cancer
in a patient. Accordingly, IMP3 or an immunoreactive polypeptide
thereof may be used to treat cancer or to inhibit the development
of cancer.
[0134] In accordance with this method, the protein, polypeptide or
DNA is generally present within a pharmaceutical composition and/or
a vaccine. Pharmaceutical compositions may comprise the full length
protein or one or more immunogenic polypeptides, and a
physiologically acceptable carrier. The vaccines may comprise the
full length protein or one or more immunogenic polypeptides and a
non-specific immune response enhancer, such as an adjuvant,
biodegradable microsphere (PLG) or a liposome (into which the
polypeptide is incorporated).
[0135] Alternatively, a pharmaceutical composition or vaccine may
contain DNA encoding IMP3 or an immunogenic polypeptide thereof,
such that the full length protein or polypeptide is generated in
situ. In such pharmaceutical compositions and vaccines, the DNA may
be present within any of a variety of delivery systems known to
those of ordinary skill in the art, including nucleic acid
expression systems, bacteria and viral expression systems.
Appropriate nucleic acid expression systems contain the necessary
DNA sequences for expression in the patient (such as a suitable
promoter). Bacterial delivery systems involve the administration of
a bacterium (such as Bacillus-Calmette-Guerrin) that expresses an
epitope of a prostate cell antigen on its cell surface. In a
preferred embodiment, the DNA may be introduced using a viral
expression system (e.g., vaccinia or other pox virus, retrovirus,
or adenovirus), which may involve the use of a non-pathogenic
(defective), replication competent virus. Suitable systems are
disclosed, for example, in Fisher-Hoch et al., PNAS 86:317-321,
1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989;
Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112,
4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB
2,200,651; EP 0,345,242; WO 91/02805; Berkner, iotechniques
6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls
et al., PNAS 91:215-219, 1994; Kass-Eisler et al., PNAS
90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848,
1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques
for incorporating DNA into such expression systems are well known
to those of ordinary skill in the art. The DNA may also be "naked,"
as described, for example, in published PCT application WO
90/11092, and Ulmer et al., Science 259:1745-1749 (1993), reviewed
by Cohen, Science 259:1691-1692 (1993).
[0136] Routes and frequency of administration, as well as dosage,
will vary from individual to individual and may parallel those
currently being used in immunotherapy of other diseases. In
general, the pharmaceutical compositions and vaccines may be
administered by injection (e.g., intracutaneous, intramuscular,
intravenous or subcutaneous), intranasally (e.g., by aspiration) or
orally. Between 1 and 10 doses may be administered over a 3-24 week
period. Preferably, 4 doses are administered, at an interval of 3
months, and booster administrations may be given periodically
thereafter. Alternate protocols may be appropriate for individual
patients. A suitable dose is an amount of polypeptide or DNA that
is effective to raise an immune response (cellular and/or humoral)
against tumor cells, e.g., kidney tumor cells, in a treated
patient. A suitable immune response is at least 10-50% above the
basal (i.e. untreated) level. In general, the amount of polypeptide
present in a dose (or produced in situ by the DNA in a dose) ranges
from about 1 pg to about 100 mg per kg of host, typically from
about 10 pg to about 1 mg, and preferably from about 100 pg to
about 1 .mu.g. Suitable dose sizes will vary with the size of the
patient, but will typically range from about 0.01 mL to about 5
ml.
[0137] IMP3 or an immunogenic polypeptide or immunogenic homolog
thereof can be used in cell based immunotherapies, e.g.,
stimulation of dendritic cells with IMP3 or fusion with IMP3
expressing cells. An "immunogenic homolog" refers to a protein that
is at least about 80%, 85%, 90%, 95%, 98% or 99% identical to a
wildtype IMP3 protein or a fragment thereof. The modified dendritic
cells, once injected into the patient, are a cellular vaccine,
where the dendritic cells activate an immune response against the
IMP3 expressing cancer.
[0138] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration. For parenteral administration, such as
subcutaneous injection, the carrier preferably comprises water,
saline, alcohol, a fat, a wax and/or a buffer. For oral
administration, any of the above carriers or a solid carrier, such
as mannitol, lactose, starch, magnesium stearate, sodium
saccharine, talcum, cellulose, glucose, sucrose, and/or magnesium
carbonate, may be employed. Biodegradable microspheres (e.g.,
polyleptic galactide) may also be employed as carriers for the
pharmaceutical compositions of this invention. Suitable
biodegradable microspheres are disclosed, for example, in U.S. Pat.
Nos. 4,897,268 and 5,075,109.
[0139] Any of a variety of non-specific immune response enhancers
may be employed in the vaccines of this invention. For example, an
adjuvant may be included. Most adjuvants contain a substance
designed to protect the antigen from rapid catabolism, such as
aluminum hydroxide or mineral oil, and a nonspecific stimulator of
immune response, such as lipid A, Bordella pertussis or
Mycobacterium tuberculosis. Such adjuvants are commercially
available as, for example. Freund's Incomplete Adjuvant and
Complete Adjuvant (Difco Laboratories. Detroit, Mich.) and Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).
[0140] All publications, patents, patent applications, and GenBank
Accession numbers mentioned herein are hereby incorporated by
reference to the extent necessary to support that for which they
have been cited herein. In case of conflict, the present
application, including any definitions herein, will control. The
present invention is further illustrated by the following examples
which should not be construed as limiting in any way. The present
invention is further illustrated by the following examples which
should not be construed as limiting in any way.
EXAMPLES
Example 1
The RNA-Binding Protein IMP3: A Novel Biomarker to Predict
Metastasis and Prognosis of Renal Cell Carcinomas
Summary
[0141] Background Distant metastases of renal cell carcinoma (RCC)
remain the primary cause of death in patients with this disease and
the metastatic potential of localized RCC is often unpredictable.
In this study, we investigated whether IMP3, an oncofetal
RNA-binding protein, can serve as a biomarker to predict metastasis
and prognosis of RCC.
[0142] Methods A total of 501 primary and metastatic RCCs were
studied. The 371 patients with localized primary RCCs were further
evaluated for survival analysis. The expression of IMP3 in RCC
tissues was evaluated by immunohistochemistry and selected cases
were also assessed for mRNA and protein expression of IMP3 by
quantitative real-time polymerase chain reaction and Western blot
analysis.
[0143] Findings The expression of IMP3 was significantly increased
not only in the metastatic RCCs but most importantly also in a
subset of primary RCCs that were much more likely to subsequently
develop metastases. Kaplan-Meier plots and log-rank tests showed
that patients without IMP3 expression in their primary localized
RCCs had significant longer metastasis-free survival and overall
survival than patients with IMP3 expression (P<0.0001). In
patients whose localized RCCs were positive for IMP3 versus those
with IMP3 negative RCCs, the 5-year metastasis-free survival were
44% vs. 98% [hazards ratio=17.18 (95% confidence interval:
7.82-37.78), stage I], 41% vs. 94% [10.14 (3.46-29.68), stage II]
and 16% vs. 62% [4.04 (2.23-7.31), stage III], and the 5-year
overall survival rates were 32% vs. 89% [6.44 (3.63-11.42), stage
I], 41% vs. 88% [6.93 (2.63-18.27), stage II] and 14% vs. 58% [3.46
(1.98-6.05), stage III] respectively. Multivariate Cox proportional
hazards regression analysis showed that the hazard ratio of IMP3
status in primary RCCs were 5.84 (metastasis-free survival,
P<0.0001) and 4.01 (overall survival, P<0.0001) respectively,
which were much higher than the hazard ratios associated with the
other independent risk factors.
[0144] Interpretation IMP3 is an excellent independent prognostic
marker that can be used at the time of initial diagnosis of RCC to
identify a group of patients with a high potential to develop
metastasis and who might benefit from early systemic therapy.
Introduction
[0145] Renal cell carcinoma is the most common type of kidney
cancer and accounts for about 85 percent of malignant kidney
tumors.sup.1,2. The incidence of renal cell carcinoma has been
rising steadily.sup.3. It is estimated that there will be about
36,160 new cases of kidney cancer in the United States in the year
2005, and about 12,660 people will die from this disease.sup.4.
[0146] Surgical resection of primary renal cell carcinoma can be a
curative treatment when the disease is localized. However, distant
metastasis remains the primary cause of therapeutic failure and
cancer death.sup.1,2. Patients with metastatic disease are
typically treated with systemic therapy, which is associated with
substantial toxicity.sup.1,2. Therefore, unless the patient
presents with metastatic disease, clinical observation is the
standard of care following nephrectomy. Currently, the methods to
determine prognosis and select patients for postoperative adjuvant
therapy rely mainly on pathological and clinical staging.sup.5-8.
However, as there are remarkable differences in the biological
behavior of renal cell carcinomas classified in the same stage, it
is very difficult to predict which localized tumor will eventuate
in distant metastasis. Approximately twenty percent of patients
with localized tumors develop metastasis and the median survival
for patients with metastatic disease is approximately 13
months.sup.2,9,10. Therefore, there is a great need for biomarkers
that can accurately distinguish localized tumors with a high
probability of metastasis from those that will remain indolent.
Using such biomarkers, one can predict the patient's prognosis and
can effectively target the individuals who would most likely
benefit from adjuvant therapy. Recently, molecular biomarkers are
an area of interest for studying renal cell carcinoma. Various
protein markers and gene expression profiles based on DNA
microarray analysis have demonstrated potential in predicting
disease outcome in renal cell carcinoma.sup.11,12.
[0147] IMP3 is a member of the insulin-like growth factor II
(IGF-II) mRNA binding protein (IMP) family that consists of IMP1,
IMP2 and IMP3. IMP family members play an important role in RNA
trafficking and stabilization, cell growth, and cell migration
during the early stages of embryogenesis.sup.14. The IMP3 gene is
located on chromosome 7p11.2.+-.11cM.sup.15 and is identical to the
KOC (KH domain containing protein overexpressed in cancer) protein
that was originally cloned from a pancreatic tumor cDNA
screen.sup.16. IMP3 is expressed in developing epithelium, muscle
and placenta during early stages of human and mouse embryogenesis,
but it is expressed at low or undetectable levels in adult
tissues.sup.13,14. The expression of IMP3/KOC is also found in
malignant tumors including pancreas, lung, stomach, and colon
cancers, and soft tissue sarcomas but it is not detected in
adjacent benign tissues.sup.13,16-18. Moreover, a recent study has
demonstrated that IMP3 promotes human leukemia cell
proliferation.sup.19. These findings indicate that IMP3 is an
oncofetal protein that may have a critical role in the regulation
of cell proliferation. However, the expression of IMP3 in renal
cell carcinomas and the relationship between IMP3 and tumor
metastasis are unknown. In this study, we investigated whether IMP3
could serve as an independent biomarker to predict metastasis and
prognosis in patients with renal cell carcinoma.
Methods
Patients and Tumor Specimens:
[0148] Formalin-fixed, paraffin-embedded samples from 406 patients
with primary renal cell carcinomas, who underwent radical or
partial nephrectomy, were obtained from the archival files at the
University of Massachusetts Medical Center (UMMC, n=159), the
Massachusetts General Hospital (MGH, n=152) and the City of Hope
National Medical Center (CHNMC, n=95). The data from these sources
represented all patients for whom archival tissues and adequate
clinical follow-up information were readily available. All cases
were collected between January of 1989 and December 2003 and the
diagnoses were confirmed by at least two pathologists. Staging was
based on pathological findings following the American Joint
Committee on Cancer (AJCC) staging manual, sixth edition, 2002. Two
hundred sixteen patients (pT1a or b) were stage I, 64 patients
(pT2) were stage II, 98 patients (pT3a, n=62; pT3b, n=29; pT3, N1,
n=7) were stage III and 28 patients (pT2, N2 or M1, n=5; pT3, N2 or
M1, n=12; pT4, N2 or M1, n=7; pT4, n=4) were stage IV. Follow-up
for this retrospective study was carried out by researchers (Z J, P
G C, and C L W) reviewing the patient clinical records. Metastasis
was found in 119 of 406 (29%; UMMC: 27%; MGH: 29%; CHNMC: 34%)
patients with primary RCCs during nephrectomy (N=30) or after
surgery (N=89). One hundred fifty nine patients out of 406 patients
(39%; UMMC: 42%; MGH: 35%; CHNMC:42%) with primary RCC expired. An
additional 95 metastatic renal cell carcinomas (26 of the
metastatic RCCs were from the same patients with primary tumors and
the others were from the biopsy or resection of metastatic tumors
only), which were obtained from lung (n=22), lymph nodes (n=10),
gastrointestinal organs including liver, intestines, pancreas, and
gallbladder (n=7), bone (n=23), brain (n=8), adrenal, thyroid and
ovary (n=10), head and neck area (n=2), soft tissues (n=6),
diaphragm, pleura, retroperitoneum and omentum (n=7) at the three
institutions, were also examined by immunohistochemistry (IHC) and
compared to the primary RCCs. The Institutional Review Board at
each institution approved this study.
Immunohistochemical Analysis
[0149] Immunohistochemical studies were performed on 5 .mu.m
sections of formalin-fixed, paraffin-embedded tissue from
nephrectomy specimens by using an avidin-biotinylated peroxidase
complex system as a previously published protocol 18 on the DAKO
Autostainer (DAKO Corporation, Carpinteria, Calif.). Sections of
pancreatic carcinoma with known positivity of IMP3 were used as
positive controls for the L523S mouse monoclonal antibody (MAb)
specific for IMP3/KOC (Corixa Corporation, Seattle, Wash.)
staining. Negative controls were performed by replacing the primary
antibody with nonimmune IgG. Positive staining of IMP3 was defined
as dark brown cytoplasm (FIGS. 1A and B, IMP3 positive), while
negative staining of IMP3 was defined as no staining at all (FIG.
1C, IMP3 negative). The status of IMP3 was assessed by a
genitourinary pathologist (Z J) without knowledge of the clinical
and pathological features of the cases or the clinical outcome. To
assess the reproducibility of the immunohistochemical test for IMP3
expression, 50 cases were randomly chosen for independent analysis
in terms of positive or negative staining by three other
pathologists (C W, P C, and C L). There was complete concurrence of
the results by all pathologists. Each positive case was also
further evaluated for the percentage of the cells that stained
positively and was scored as focal: .ltoreq.30%, or diffuse:
>30%.
Quantitative Analysis of Immunostaining
[0150] A total of 270 different areas (15 different areas per case)
from 9 RCC IMP3 positive cases and 9 RCC IMP3 negative cases were
quantitatively analyzed by a pathologist (Z J) using a computerized
image analyzer (Automated Cellular Imaging System, ACIS, Chroma
Vision Medical System Inc., San Juan Capistrano, Calif.) to
evaluate the IHC results. With ACIS, positive staining is
calculated by applying two thresholds with one recognizing blue
background (hematoxylin stained) cells and another recognizing
brown positive cells. The integrated optical density (IOD) is that
the sum of brown pixels times brown intensity of those pixels. The
ACIS values were calculated as IOD was divided by the sum of the
blue area and the brown area.
Western Blotting Analysis of IMP3 Expression:
[0151] Primary RCC frozen tissues and a metastatic renal cell
cancer cell line (ATCC Global Bioresource Center, Manassas, Va.,
ATCC.RTM. Number: HTB-46) were homogenized in 3 volumes of lysis
buffer. Immunodetection was performed with IMP3 MAb (L523S) at a 1
ug/ml dilution using the enhanced chemiluminescence system
(PerkinElmer Life Sciences, Boston, Mass.). The membrane was
stripped and re-blotted with anti-actin (A-2066) polyclonal
antibody from Sigma (St. Louis, Mo.). Intensity of the signal was
quantified by densitometry software (NIH Image 1.61) and relative
expression levels of IMP3 were normalized by amount of the actin in
each lane.
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
[0152] IMP3 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, as
an internal reference) mRNA levels in RCC tissues were quantified
by qRT-PCR. Frozen tissues were cut into 5 micron sections and
total RNAs were extracted by Qiagen RNeasy Mini Kit (Qiagen, Inc.,
Valencia, Calif.). Two-step qRT-PCR was performed with the ABI
TaqMan PCR reagent kit (ABI Inc, Foster City, Calif.), and IMP3
primers and GAPDH primers, and the probes for both genes on ABI
Prism 7700 system. The primers were used as follow: IMP3 forward
primer, 5'-GCT AAA GTG AGG ATG GTG ATT ATC ACT-3' (SEQ ID NO: 3);
IMP3 reverse primer, 5'-ACT AAC AAA GTT TTC TTC TTT AAT TTT TCC
AT-3' (SEQ ID NO: 4); IMP3 probe, 5' FAM-ACC AGA GGC TCA GTT CAA
GGC TCA GGG AA-TAMRA 3' (SEQ ID NO: 5); GAPDH forward primer,
5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO: 6); GAPDH reverse primer,
5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 7); GAPDH probe, 5'
FAM-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 8). The expression of
IMP3 mRNA was normalized with GAPDH mRNA expression measured in the
same RNA extraction and calculated as the numbers of IMP3/GAPDH
ratio.
Statistical Analysis:
[0153] Overall survival was measured from the date of nephrectomy
to the date of death or was censored as of the date of the last
follow-up visit for survivors. Metastasis-free survival was
measured from the date of surgery to the date of first clinical
evidence of metastasis, and was censored at the date of death or
the date of the last follow-up visit for survivors. The median
follow-up was 63 months (range=1-174 months). Age, sex, size of the
tumor, tumor stage, grade and histological type, and IMP3 status
were collected as baseline variables. The distribution of each
baseline variable was compared for IMP3-positive and IMP3-negative
subgroups with the Wilcoxon rank sum test for continuous variables
and the Fisher's exact test for categorical variables. Thirty-one
(stage III: N=7 and Stage IV, N=24) of 406 patients with metastatic
disease found during nephrectomy were excluded from the overall and
metastasis-free survival study, as the aim of this study was to
evaluate the risk of metastases after surgery in patients initially
presenting with localized disease. By AJCC TNM staging criteria,
most of the stage IV patients (24 of 28, 86%) in this study were
found with metastasis (M1: n=13; N2: n=10; N2 and M1: n=1) during
surgery. Only four patients with stage IV disease were found
without metastasis during nephrectomy and they were excluded from
the prognostic analysis, as such a small number of patients would
not be informative for the prognostic analysis of patients with
stage IV RCCs. Therefore, a total of 371 patients with stage I, II
and III disease and without metastasis during surgery were included
in our survival analysis. Overall survival and metastasis-free
survival of 371 patients were estimated by the Kaplan-Meier method
and evaluated with the use of log-rank test for univariate
analysis. The Cox proportional-hazard model was used to assess the
simultaneous contribution of the following baseline covariates of
age, sex, size of the tumor, tumor stage, grade and histological
type, and IMP3 status. A two-sided P-value of less than 0.05 was
considered to indicate the statistical significance.
[0154] Institutional funds supported this study and funding source
had no role in making decisions including study design, data
collection, analysis, and interpretation, writing of the report,
and decision to submit.
Results
Expression of IMP3 in all Primary and Metastatic Renal Cell
Carcinomas
[0155] IMP3 protein was observed in the cytoplasm of tumor cells
(FIGS. 1A and 1B). Expression of IMP3 was found in 62% (59 of 95)
of metastatic RCCs, in 50% (60 of 119) of primary RCCs with
metastases during and after nephrectomy, and in 4% (11 of 287)
metastasis-free primary RCCs (FIG. 2). In 71 positive primary RCCs,
the IMP3 positivity was detected in .ltoreq.30% tumor cells (focal)
in 38 cases, and >30% of tumor cells (diffuse) in 33 cases. No
expression of IMP3 was found in benign kidney tissue adjacent to
the tumors. The results of quantitative immunohistochemistry showed
significant differences in IMP3 staining values between positive
and negative cases. The average of the ACIS values was 44.3 in the
IMP3 positive RCCs and 0.01 in the IMP3 negative RCCs
(P=0.0025).
[0156] In addition, the reactivity of L523S MAb with IMP3/KOC
protein produced by RCCs was determined by Western blot analysis
(FIG. 3). L523S MAb reacted with a protein at the expected
molecular weight for IMP3 (approximately 65 kilodalton). FIG. 3
(upper) showed that IMP3 was highly expressed in the metastatic RCC
cell line and three IHC positive cases of primary RCCs with
subsequent development of metastasis. In contrast, IMP3 exhibited
no expression in two IHC negative cases of primary RCCs without the
subsequent development of metastasis.
[0157] The over expression of IMP3 mRNA was also detected in
primary RCCs. The results of qRT-PCR performed to quantitatively
assess the expression levels of IMP3 mRNA in the primary RCCs,
which were subject to Western blot analysis, are shown in FIG. 3
(lower). The three IHC positive primary RCCs with subsequent
development of metastasis were found to over express IMP3 mRNA as
compared to IHC negative primary RCCs without development of
metastasis (FIG. 3, lower). Analysis of the agreement between the
Western blot test and the real-time PCR analysis of the same
samples showed complete agreement between the tests (data not
shown). However, since this analysis was based on only five
samples, a larger study is needed to show conclusive results.
Characteristics of the Patients
[0158] Table 1 provides the relevant clinical characteristics of
the 406 patients with primary RCCs. Age was not correlated with the
positivity for IMP3 (P=0.28). Male patients were more likely than
female patients to show IMP3 expression in their tumors (P=0.015).
The expression of IMP3 was strongly associated with standard
pathologic predictors of clinical outcome. IMP3 expression was
found predominately in large tumors, and tumors with higher grade
and stage. Only 10-24% of stage I, II and III RCCs expressed IMP3,
whereas 50% of stage IV tumors were positive for IMP3. Expression
of IMP3 was found predominately in high-grade (grade 3 and 4)
tumors and all grade 1 RCCs were negative for IMP3. The IMP3
positivity rate was slightly increased in conventional (clear cell)
type carcinomas (23%) as compared with papillary (11%) and
chromophobe (15%) RCCs. Two cases of unclassified RCCs were all
positive for IMP3. IMP3 expression in primary RCCs was associated
with a significant increase in the risk of death. Ninety percent
(64 of 71) of patients with expression of IMP3 died and 28% (95 of
335) of patients without IMP3 expression expired.
TABLE-US-00002 TABLE 1 Clinicopathological Characteristics of Renal
Cell Carcinoma Patients IMP3+ IMP3- P Characteristic (N = 71) (N =
335) Value Sex: Female 17 (11%) 132 (89%) 0.015 Male 54 (21%) 203
(79%) Age - yr 61.8 .+-. 11.2* 59.5 .+-. 14.1 0.28 Tumor stage I 22
(10%) 194 (90%) <0.0001 II 11 (17%) 53 (83%) III 24 (24%) 74
(76%) IV 14 (50%) 14 (50%) Tumor Size 8.5 .+-. 4.9* 6.1 .+-. 3.7
<0.0001 Tumor Grade 1 0 (0%) 30 (100%) <0.0001 2 17 (9%) 169
(91%) 3 36 (24%) 111 (76%) 4 18 (42%) 25 (58%) Histological types
Clear cell type 58 (23%) 256 (74%) 0.04 Papillary 7 (11%) 56 (89%)
Chromophobe 4 (15%) 23 (85%) Unclassified 2 (100%) 0 (0%)
*Plus-minus values are means .+-. SD.
Expression of IMP3 and Prognosis in Patients with Localized Renal
Cell Carcinoma During Nephrectomy
[0159] A total of 371 patients who did not have metastasis at the
time of surgery were included in follow-up analysis. The percentage
of metastasis after surgery was significantly different between the
IMP3 positive patients and the IMP3 negative patients in their
primary RCCs. Eighty percent (43 of 54) of patients with IMP3
positivity in their primary RCCs subsequently developed metastasis
(median follow-up=38 months, range=1-155 months), whereas 13% (41
of 317) of patients without expression of IMP3 in their primary
tumors were found to have metastases after surgery (median
follow-up=70 months, range=1-174 months).
[0160] Kaplan-Meier plots and log-rank tests in all patients
(n=371) with localized disease at the time of surgery and in these
371 patients separated into each stage (Stage I, n=216; Stage II,
n=64; Stage III, n=91) showed that patients without IMP3 expression
in their primary RCCs had significant longer metastasis-free
survival and overall survival than patients with IMP3 expression.
FIG. 4A showed that the 5-year metastasis-free survival rate was
89% in IMP3 negative patients versus 33% in IMP3 positive patients.
The 5-year overall survival rate was 82% in patients without
expression of IMP3 versus 27% in patients with IMP3 expression
(FIG. 4B). In patients with stage I (FIGS. 5A and B), II (FIGS. 5C
and D), and III (FIGS. 5E and F) renal cell cancers, the status of
IMP3 expression was also significantly associated with increased
risk of metastasis and was strongly linked to poor overall survival
rate. Univariate analysis showed that hazard ratios (HR) of IMP3
expression were 9.22 (for metastasis-free survival; 95% confidence
interval, 6.01-14.14; P<0.0001) and 5.66 (for overall survival;
95% confidence interval, 3.93-8.16; P<0.0001) respectively
(Table 2). In patients whose localized RCCs were positive for IMP3
versus those with IMP3 negative RCCs, the 5-year metastasis-free
survival were 44% vs. 98% [hazards ratio=17.18 (95% confidence
interval: 7.82-37.78), stage 1], 41% vs. 94% [10.14 (3.46-29.68),
stage II] and 16% vs. 62% [4.04 (2.23-7.31), stage III], and the
5-year overall survival rates were 32% vs. 89% [6.44 (3.63-11.42),
stage I], 41% vs. 88% [6.93 (2.63-18.27), stage II] and 14% vs. 58%
[3.46 (1.98-6.05), stage III] respectively.
TABLE-US-00003 TABLE 2 Univariate Cox Proportional Hazard
Regression Analysis for Metastasis-free and Overall Survival
Metastasis-Free Survival Overall Survival Hazard Ratio P Hazard
Ratio P Variable (95% CI) Value (95% CI) Value IMP3 STATUS 9.22
(6.01-14.14) <0.0001 5.66 (3.93-8.16) <0.0001 (+ VS. -) Age
1.01 (1.00-1.03) 0.167 1.03 (1.02-1.04) <0.0001 Sex (F vs. M)
0.77 (0.48-1.21) 0.250 0.94 (0.65-1.34) 0.722 Tumor Size 1.16
(1.12-1.21) <0.0001 1.10 (1.06-1.15) <0.0001 Tumor Stage II
vs. I 1.85 (0.96-3.53) 0.065 1.21 (0.72-2.02) 0.470 III vs. I 6.05
(3.71-9.84) <0.0001 3.39 (2.32-4.96) <0.0001 Tumor Grade 2
vs. 1 4.40 (0.60-32.4) 0.146 1.22 (0.52-2.87) 0.646 3 vs. 1 11.37
(1.57-82.6) 0.016 2.59 (1.12-6.01) 0.027 4 vs. 1 18.44 (2.44-139.6)
0.005 4.42 (1.78-10.96) 0.001 Histological type Papillary vs. Clear
0.48 (0.23-0.99) 0.047 0.66 (0.39-1.12) 0.121 Chromophobe vs. Clear
0.90 (0.39-2.08) 0.808 0.75 (0.35-1.61) 0.454
[0161] The IMP3 positive staining patterns (focal vs. diffuse) in
the primary RCCs did not alter the patients' prognosis. There were
no significant differences in metastasis-free survival (P=0.732)
and overall survival (P=0.728) between patients with focal IMP3
staining and patients with diffuse IMP3 staining in their primary
RCCs. No significant difference of overall survival is found
between IMP3 positive and IMP3 negative patients with stage IV
disease (P=0.14).
Multivariable Analysis
[0162] The results of the multivariable analysis, which was
stratified by three different centers in this study, for
metastasis-free survival and overall survival in the 371 patients
with localized disease at the time of surgery are presented in
Table 3. For these analyses, all factors shown in table 1 were
initially included in the model as potential risk factors.
Multivariable Cox proportional hazards regression analysis showed
that the expression of IMP3 in primary RCCs was a strong
independent predictor of the patients' clinical outcome. The hazard
ratios were 5.84 (for metastasis-free survival; 95% confidence
interval, 3.60 to 9.49; P<0.0001) and 4.01 (for overall
survival; 95% confidence interval, 2.66 to 6.05; P<0.0001)
respectively, which were much higher than the hazard ratios
associated with all other independent risk factors (Table 3). In
addition to IMP3 status, age and tumor stage III (compared to stage
I) were also observed as significant risk factors for overall
survival, and tumor size and tumor stage III (compared to stage I)
were also observed as significant factors for metastasis-free
survival (Table 3).
TABLE-US-00004 TABLE 3 Multivariable Cox Proportional Hazard
Regression Analysis for Metastasis-free and Overall Survival
Metastasis-Free Survival Overall Survival Hazard Ratio P Hazard
Ratio P Variable (95% CI) Value (95% CI) Value IMP3 STATUS 5.84
(3.60-9.49) <0.0001 4.01 (2.66-6.05) <0.0001 (+ VS. -) Age
1.00 (0.98-1.02) 0.752 1.02 (1.01-1.04) 0.006 Sex 1.08 (0.66-1.77)
0.747 1.08 (0.73-1.59) 0.694 Tumor Size 1.11 (1.04-1.18) 0.001 1.05
(0.99-1.11) 0.081 Tumor Stage II vs. I 0.73 (0.30-1.77) 0.484 0.87
(0.44-1.71) 0.677 III vs. I 3.15 (1.72-5.77) <0.0001 2.08
(1.30-3.33) 0.002 Tumor Grade 2 vs. 1 2.02 (0.27-15.37) 0.496 0.89
(0.37-2.16) 0.797 3 vs. 1 4.43 (0.59-33.54) 0.149 1.57 (0.64-3.84)
0.319 4 vs. 1 3.22 (0.40-25.80) 0.272 1.27 (0.47-3.44) 0.634
Histological type Papillary vs. 0.50 (0.22-1.11) 0.090 0.71
(0.41-1.25) 0.236 Clear Chromophobe 1.29 (0.54-3.08) 0.564 0.95
(0.44-2.09) 0.908 vs. Clear
Discussion
[0163] Distant metastasis of renal cell carcinomas is the primary
cause of death and therapeutic failure.sup.1,2. Although tumor
stage, grade and subtype provide some prognostic information, the
metastatic potential of localized RCC is often unpredictable. Since
a well-characterized antibody to IMP3, but not IMP1 or IMP2, was
available for immunohistochemical study, we investigated IMP3
expression in primary RCC and metastatic RCC as part of our program
to develop biomarkers for clinical use. After we found that the
expression of IMP3 was significantly increased in metastatic RCCs
as compared to primary RCCs, we examined the relationship between
IMP3 expression and progression in primary RCCs. Our findings
demonstrate that the expression of IMP3 in primary renal cell
carcinomas can predict tumor metastasis and provide important
prognostic information in patients with localized disease who
undergo nephrectomy.
[0164] IMP3 displays several features that make it an attractive
prognostic marker for renal cell carcinoma. First, the expression
of IMP3 is correlated with other known pathological indicators of
aggressive RCCs. Our results showed that the expression of IMP3 was
strongly related to higher tumor grade and stage, and larger tumor
size.
[0165] Second, the expression of IMP3 in primary RCCs is
independently linked to poor clinical outcome. Overall survival of
patients with expression of IMP3 was extremely poor as compared to
that of patients without IMP3 expression in primary localized RCCs
and this was independent of tumor stage. Patients with IMP3
expression died at a rate 4 times greater than patients without
IMP3 expression. Remarkably, the multivariate Cox analysis showed
that the hazard ratios for death in patients with positive IMP3 in
the primary tumor was much higher than the hazard ratios associated
with any other clinical and pathological predictors including age,
sex, and tumor stage, size, grade and subtype.
[0166] Third, the expression of IMP3 is an independent predictor of
tumor metastasis. A decreased overall survival rate was strongly
associated with a high metastatic rate in IMP3 positive patients.
Out data showed that the expression of IMP3 was significantly
increased not only in metastastic renal cell carcinomas but most
importantly also in patients with primary renal cell cancers who
developed metastatic disease as compared with renal cell cancers
without metastasis. We found that 80% of patients with IMP3
positivity in their localized RCCs developed metastasis, whereas
only 13% of patients without expression of IMP3 in their primary
tumors developed metastases. In patients with stage III disease,
almost all of IMP3 positive patients developed metastases after
nephrectomy. In the multivariable Cox analysis, patients with IMP3
expression in their primary RCCs developed metastasis at a rate,
which is 5.84 times greater than patients without expression of
IMP3 adjusting for other well-known clinical variables.
[0167] Fourth, IMP3 immunohistochemical staining is a simple,
inexpensive and reliable assay. As localized renal cell cancers are
usually treated by partial or radical nephrectomy, tumor tissue is
routinely available for immunohistochemical staining with the
monoclonal L523S antibody. Our study showed that pathologists can
readily analyze IMP3 immunohistochemistry without inter observer
variation to determine positive and negative staining, which can be
easily applied in routine clinical practice in all patients with
nephrectomy. A computerized image analyzer for quantitative
immunohistochemistry (ACIS), which has been clinically used for
evaluation of Her2/neu gene expression, also confirmed the accuracy
of the evaluation of the IMP3 immunostaining results by
pathologists. In this study, we mainly focused on evaluation of
IMP3 immunostaining by pathologists and used ACIS as a confirmatory
test. A further study is necessary to determine how quantitative
image analysis of immunostaining results correlates with patient
outcome.
[0168] Fifth, as patients whose tumors express IMP3 have a high
potential to develop metastasis, IMP3 provides a marker that not
only can identify a subgroup of the patients who might benefit from
a different follow-up approach after nephrectomy, but also can be
used at initial diagnosis which would be the optimal time for
considering early systemic therapy. Although the expression of IMP3
was found in multiple malignant tumors but not in adjacent benign
tissues.sup.11,12,15,16, and a recent study demonstrated that IMP3
may have a critical role in the regulation of cell
proliferation.sup.17, little is known of the biological function of
IMP3 in tumor pathogenesis. Our findings raise the possibility that
IMP3, as an oncofetal protein, may play a direct role in the
metastasis and/or more lethal behavior of renal cell carcinoma.
Interestingly, Yaniv et al found that IMP3 in Xenopus laevis.sup.13
is required for the migration of cells forming the roof plate of
the neural tube and, subsequently, for neural crest
migration.sup.20. The findings indicated that IMP3 plays an
important role in promoting cell migration. Further study is
required to investigate whether IMP3 plays a direct role in the
biological behavior of metastatic RCC.
[0169] In summary, IMP3 serves as an excellent independent
prognostic marker for renal cell carcinoma. IMP3 expression status
in primary renal cell cancers may identify a subgroup of patients,
particularly in patients with early-stage disease, who have a high
potential to develop metastasis after surgery and die from the
disease. The findings may have therapeutic implications in a group
of patients who may benefit from early systematic therapy after
nephrectomy.
REFERENCES
[0170] 1. Motzer R J, Bander N H, Nanus D M. Renal-cell carcinoma.
[see comment]. [Review][150 refs]. New England Journal of Medicine
1996; 335(12):865-75. [0171] 2. Cohen H T, McGovern F J. Renal-cell
carcinoma. [Review][103 refs]. New England Journal of Medicine
2005; 353(23):2477-90. [0172] 3. Chow W H, Devesa S S, Warren J L,
Fraumeni J F, Jr. Rising incidence of renal cell cancer in the
United States. [see comment]. Jama 1999; 281(17):1628-31. [0173] 4.
Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. Ca: a
Cancer Journal for Clinicians 2005; 55(1):10-30. [0174] 5. Tsui K
H, Shvarts O, Smith R B, Figlin R A, deKernion J B, Belldegrun A.
Prognostic indicators for renal cell carcinoma: a multivariate
analysis of 643 patients using the revised 1997 TNM staging
criteria. Journal of Urology 2000; 163(4):1090-5. [0175] 6. Sene A
P, Hunt L, McMahon R F, Carroll R N. Renal carcinoma in patients
undergoing nephrectomy: analysis of survival and prognostic
factors. British Journal of Urology 1992; 70(2): 125-34. [0176] 7.
Couillard D R, deVere White R W. Surgery of renal cell carcinoma.
[Review][83 refs]. Urologic Clinics of North America 1993;
20(2):263-75. [0177] 8. Thrasher J B, Paulson D F. Prognostic
factors in renal cancer. [Review][66 refs]. Urologic Clinics of
North America 1993; 20(2):247-62. [0178] 9. Rabinovitch R A,
Zelefsky M J, Gaynor J J, Fuks Z. Patterns of failure following
surgical resection of renal cell carcinoma: implications for
adjuvant local and systemic therapy. Journal of Clinical Oncology
1994; 12(1):206-12. [0179] 10. Sandock D S, Seftel A D, Resnick M
I. A new protocol for the followup of renal cell carcinoma based on
pathological stage. Journal of Urology 1995; 154(1):28-31. [0180]
11. Lam J S, Shvarts O, Leppert J T, Figlin R A, Belldegrun A S.
Renal cell carcinoma 2005: new frontiers in staging,
prognostication and targeted molecular therapy. [Review][75 refs].
Journal of Urology 2005; 173(6): 1853-62. [0181] 12. Zhao H,
Ljungber B, Grankvist K, Rasmuson T, Tibshirani R, Brooks J D. Gene
Expression Profiling Predicts Survival in Conventional Renal Cell
Carcinoma. PLoS Med 2006; 3(1):e13. [0182] 13. Nielsen J,
Christiansen J, Lykke-Andersen J, Johnsen A H, Wewer U M, Nielsen F
C. A family of insulin-like growth factor II mRNA-binding proteins
represses translation in late development. Molecular & Cellular
Biology 1999; 19(2):1262-70. [0183] 14. Mueller-Pillasch F, Pohl B,
Wilda M, et al. Expression of the highly conserved RNA binding
protein KOC in embryogenesis. Mechanisms of Development 1999;
88(1):95-9. [0184] 15. Monk D, Bentley L, Beechey C, et al.
Characterisation of the growth regulating gene IMP3, a candidate
for Silver-Russell syndrome. Journal of Medical Genetics 2002;
39(8):575-81. [0185] 16. Mueller-Pillasch F, Lacher U, Wallrapp C,
et al. Cloning of a gene highly overexpressed in cancer coding for
a novel KH-domain containing protein. Oncogene 1997;
14(22):2729-33. [0186] 17. Wang T, Fan L, Watanabe Y, et al. L523S,
an RNA-binding protein as a potential therapeutic target for lung
cancer. British Journal of Cancer 2003; 88(6):887-94. [0187] 18.
Yantiss R K, Woda B A, Fanger G R, et al. KOC (K homology domain
containing protein overexpressed in cancer): a novel molecular
marker that distinguishes between benign and malignant lesions of
the pancreas. American Journal of Surgical Pathology 2005; 29(2):
188-95. [0188] 19. Liao B, Hu Y, Herrick D J, Brewer G. The
RNA-binding Protein IMP-3 Is a Translational Activator of
Insulin-like Growth Factor II Leader-3 mRNA during Proliferation of
Human K562 Leukemia Cells. J. Biol. Chem. 2005;
280(18):18517-18524. [0189] 20. Yaniv K, Fainsod A, Kalcheim C,
Yisraeli J K. The RNA-binding protein Vg1 RBP is required for cell
migration during early neural development. Development 2003;
130(23):5649-61.
Example 2
Association of IMP3 Tumor Expression with Clinicopathologic
Features and Patient Outcome Among Patients with Papillary and
Chromophobe Renal Cell Carcinoma
Introduction
[0190] IMP3 is a member of the insulin-like growth factor-II
(IGF-II) mRNA binding protein family. Although IMP3 is expressed
within developing epithelia, myocytes, and placenta during human
and mouse embryogenesis, its expression is low or undetectable in
post-natal tissues and virtually absent in adult tissues. IMP3 is
thought to participate in the protection and intracellular
distribution of IGF-II mRNA and, thus, has been implicated in
regulating the production of IGF-II. Interestingly, some reports
suggest that renal cell carcinoma (RCC) tumors, especially
aggressive tumors exhibiting sarcomatoid differentiation, express
increased IGF-II expression. Moreover, overexpression of IGF-IR, a
cognate cell-surface receptor for IGF-I and II, has also been
observed in RCC tumors and implicated as a feature of aggressive
tumor behavior. Jiang et al..sup.1 systematically studied the
expression of IMP3 in a cohort of 371 patients with localized
tumors of the clear cell, papillary, or chromophobe RCC subtypes.
In this study, Jiang et al..sup.1 reported that tumor cell IMP3
expression was significantly associated with progression to distant
metastases and death, even after multivariate adjustment for
patient age, sex, tumor size, stage, grade and histologic subtype.
These findings were recently independently validated using a cohort
of 629 consecutively-treated patients with localized clear cell RCC
(Hoffmann et al, under review at Cancer Research). The various RCC
subtypes including papillary RCC and chromophobe RCC have distinct
genetic and morphologic characteristics..sup.2-5 Papillary and
chromophobe RCCs accounts for approximately 25% of RCCs. Clear cell
RCCs has worse cancer specific survival compared to papillary and
chromophobe RCCs (ref 4). However, there is no large study for IMP3
expression in these various RCCs. The goal of the current study is
to evaluate the association of IMP3 expression with
clinicopathologic features and outcome using a multi-institutional
cohort of patients with the papillary and chromophobe RCC
subtypes.
Materials and Methods
Patient Selection
[0191] Using the combined resources of the Mayo Clinic, the
University of Massachusetts Medical Center (UMMC), Massachusetts
General Hospital (MGH), and City of Hope National Medical Center
(CHNMC), we identified 334 patients treated with radical
nephrectomy or nephron-sparing surgery for papillary or chromophobe
RCC. The Mayo Clinic patients (N=246) were treated between 1990 and
1999, while the UMMC(N=39), MGH(N=38), and CHNMC (N=11) patients
were treated between 1989 and 2003.1 There were 254 (76.0%)
patients with papillary RCC and 80 (24.0%) with chromophobe
RCC.
Clinicopathologic Features
[0192] The clinicopathologic features studied included age, sex,
histologic subtype classified according to the Union Internationale
Contre le Cancer, American Joint Committee on Cancer, and
Heidelberg guidelines.sup.2,3, tumor size, primary tumor
classification, regional lymph node involvement, distant
metastases, the TNM stage groupings, and nuclear grade.
IMP3 Immunohistochemical Staining
[0193] Immunohistochemical studies were performed by the Department
of Pathology at the UMMC on 5-um sections of formalin-fixed,
paraffin-embedded tissue as previously described..sup.1 Antigen
retrieval was carried out with 0.01 mol/L citrate buffer at pH 6.0,
in an 800-W microwave oven for 15 minutes before immunostaining.
The slides were stained on the DAKO Autostainer (DAKO Corporation,
Carpinteria, Calif.) using the EnVision (Dako) staining reagents.
The sections were first blocked for endogenous protein binding and
peroxidase activity with an application of Dual Endogenous Block
(Dako) for 10 minutes, followed by a buffer wash. The sections were
then incubated with a mouse monoclonal antibody specific for IMP3
(L523S, Corixa, Seattle, Wash.) at a 2.0 .mu.g/ml concentration for
30 minutes, followed again by a buffer wash. Sections were then
incubated with the EnVision+Dual Link reagent (a polymer conjugated
with goat-anti-mouse-Ig, and horseradish peroxidase) for 30
minutes. The sections were then washed, and treated with
diaminobenzidine (DAB) and hydrogen peroxide, which reacted to
visualize the end product. A toning solution (DAB Enhancer, Dako)
was used to enrich the final color. The sections were
counterstained with hematoxylin, dehydrated, and coverslipped with
permanent media. Sections of urothelial carcinoma with known
positivity of IMP3 were used as positive controls for the L523S
mouse monoclonal antibody (MAb) specific for IMP3/KOC (Corixa
Corporation, Seattle, Wash.) staining. Negative control sections
were stained by replacing the primary antibody with non-immune
mouse IgG (Vector, Burlingame Calif.) at 2.0 ug/ml.
IMP3 Quantitation
[0194] IMP3 tumor expression was recorded as negative or positive
after visual assessment by a genitourinary pathologist (Z J)
without knowledge of patient outcome.
Statistical Methods
[0195] Associations of IMP3 expression with clinicopathologic
features were evaluated using Wilcoxon rank sum, chi-square, and
Fisher's exact tests. Kaplan-Meier curves were used to visualize
the associations of IMP3 expression with outcome. The magnitude of
these associations were evaluated using Cox proportional hazards
regression models and summarized with risk ratios and 95%
confidence intervals (CI). Statistical analyses were performed
using the SAS software package (SAS Institute; Cary, N.C.). All
tests were two-sided and p-values <0.05 were considered
statistically significant.
Results
[0196] There were 294 (88.0%) papillary or chromophobe RCC tumors
with negative IMP3 expression and 40 (12.0%) with positive IMP3
expression. Comparisons of clinicopathologic features by IMP3
expression are summarized in Table 4. Positive IMP3 tumor
expression was significantly associated with later tumor stage and
higher tumor grade. For example, 70% of the IMP3-positive tumors
were high grade (i.e., grade 3 or 4) compared with only 37% of the
IMP3-negative tumors (p<0.001).
TABLE-US-00005 TABLE 4 Comparison of Clinicopathologic Features by
IMP3 Tumor Expression for 334 Patients with Papillary and
Chromophobe RCC Tumor IMP3 Expression Negative Positive Feature N =
294 N = 40 P-value Median (Range) Age at Surgery (Years) 65 (21-89)
63 (44-80) 0.883 Tumor Size (cm) 4.1 (0.3-15.0) 5.5 (0.7-25.0)
0.090 N (%) Sex Female 68 (23.1) 9 (22.5) 0.929 Male 226 (76.9) 31
(77.5) RCC Histologic Subtype Papillary 228 (77.6) 26 (65.0) 0.081
Chromophobe 66 (22.5) 14 (35.0) Primary Tumor Classification pT1
213 (72.5) 21 (52.5) 0.008 pT2 49 (16.7) 7 (17.5) pT3 31 (10.5) 12
(30.0) pT4 1 (0.3) 0 Regional Lymph Node Involvement pNX and pN0
289 (98.3) 35 (87.5) 0.003 pN1 and pN2 5 (1.7) 5 (12.5) Distant
Metastases at Presentation pM0 289 (98.3) 37 (92.5) 0.058 pM1 5
(1.7) 3 (7.5) 2002 TNM Stage Groupings I 211 (71.8) 21 (52.5) 0.002
II 48 (16.3) 6 (15.0) III 28 (9.5) 7 (17.5) IV 7 (2.4) 6 (15.0)
Nuclear Grade 1 9 (3.1) 0 <0.001 2 176 (59.9) 12 (30.0) 3 103
(35.0) 19 (47.5) 4 6 (2.0) 9 (22.5)
[0197] There were 17 patients with papillary or chromophobe RCC who
had extrarenal disease at nephrectomy, including 9 with regional
lymph node involvement, 7 with distant metastases, and 1 with both.
As such, associations of IMP3 expression with patient outcome were
evaluated using the 317 patients with localized disease at
nephrectomy (i.e., pNX/pN0; pM0; stage groups I, II, or III). In
this subset, there were 284 (89.6%) papillary or chromophobe RCC
tumors with negative IMP3 expression and 33 (10.4%) with positive
IMP3 expression.
[0198] Twenty-eight of the 317 patients with localized disease
progressed to distant metastases at a median of 3.1 years following
nephrectomy (range 0-10).
[0199] Patients with localized IMP3-positive tumors were over 10
times more likely to progress to distant metastases compared with
patients with localized IMP3-negative tumors (risk ratio 11.38; 95%
CI 5.40-23.96; p<0.001; Table 5). In fact, 15 (45.5%) of the 33
patients with IMP3-positive tumors progressed compared with only 13
(4.6%) of the 284 patients with IMP3-negative tumors.
Metastases-free survival rates (SE, number still at risk) at 5 and
10 years following nephrectomy were 63.9% (8.8%, 17) and 48.6%
(9.5%, 12), respectively, for patients with IMP3-positive tumors
compared with 97.7% (0.9%, 223) and 93.4% (1.9%, 86), respectively,
for patients with IMP3-negative tumors (FIG. 6). In multivariate
analysis adjusting for the TNM stage groupings and nuclear grade,
patients with IMP3-positive tumors were still over 10 times more
likely to progress compared with patients with IMP3-negative tumors
(risk ratio 13.45; 95% CI 6.00-30.14; p<0.001; Table 5). IMP3
expression was univariately significantly associated with
progression to distant metastases among patients with papillary RCC
(risk ratio 9.14; 95% CI 3.39-24.64; p<0.001) as well as among
patients with chromophobe RCC (risk ratio 11.91; 95% CI 3.58-39.61;
p<0.001), although there were too few patients who progressed in
these subsets to evaluate these associations in a multivariate
setting.
TABLE-US-00006 TABLE 5 Associations of IMP3 Tumor Expression with
Outcome for 317 Patients with Localized Papillary and Chromophobe
RCC Overall Survival Metastases-free Survival Risk Ratio P-
Univariate Risk Ratio (95% CI) P-value (95% CI) value IMP3
Expression Negative 1.0 (reference) 1.0 (reference) Positive 11.38
(5.40-23.96) <0.001 1.91 (1.13-3.22) 0.016 Multivariate TNM
Stage Groupings I 1.0 (reference) 1.0 (reference) II 4.38
(1.69-11.36) 0.002 0.97 (0.55-1.68) 0.900 III 10.94 (4.18-28.68)
<0.001 2.28 (1.29-4.04) 0.005 Nuclear Grade 1 and 2 1.0
(reference) 1.0 (reference) 3 and 4 5.30 (1.94-14.49) 0.001 1.38
(0.93-2.06) 0.112 IMP3 Expression Negative 1.0 (reference) 1.0
(reference) Positive 13.45 (6.00-30.14) <0.001 1.95 (1.15-3.31)
0.013
[0200] At last follow-up 103 patients had died at a median of 4.5
years following nephrectomy (range 0-16). Among the 214 patients
who were still alive at last follow-up, the median duration of
follow-up was 8.8 years (range 0-16). Overall survival rates (SE,
number still at risk) at 5 and 10 years following nephrectomy were
82.2% (2.2%, 240) and 65.4% (3.1%, 98), respectively. There was not
a statistically significant difference in overall survival between
patients with localized papillary and chromophobe RCC (p=0.997;
log-rank test).
[0201] Univariately, patients with localized IMP3-positive tumors
were nearly twice as likely to die compared with patients with
localized IMP3-negative tumors (risk ratio 1.91; 95% CI 1.13-3.22;
p=0.016; Table 5). Seventeen (51.5%) of the 33 patients with
IMP3-positive tumors died compared with 86 (30.3%) of the 284
patients with IMP3-negative tumors. Overall survival rates (SE,
number still at risk) at 5 and 10 years following nephrectomy were
57.9% (9.0%, 17) and 47.1% (9.2%, 12), respectively, for patients
with IMP3-positive tumors compared with 85.0% (2.2%, 223) and 67.4%
(3.3%, 86), respectively, for patients with IMP3-negative tumors
(FIG. 7). In multivariate analysis adjusting for the TNM stage
groupings and nuclear grade, patients with IMP3-positive tumors
were still nearly twice as likely to die compared with patients
with IMP3-negative tumors (risk ratio 1.95; 95% CI 1.15-3.31;
p=0.013; Table 5).
REFERENCES
[0202] 1. Jiang Z, Chu P G, Woda B A, et al. Analysis of
RNA-binding protein IMP3 to predict metastasis and prognosis or
renal-cell carcinoma: a retrospective study. Lancet Oncology 2006;
7:556-564. [0203] 2. Storkel S, Eble J N, Adlakha K, et al.
Classification of renal cell carcinoma: Workgroup No. 1. Union
Internationale Contre le Cancer (UICC) and the American Joint
Committee on Cancer (AJCC). Cancer 1997; 80:987-989. [0204] 3.
Kovacs G, Akhtar M, Beckwith B J, et al. The Heidelberg
classification of renal cell tumors. Journal of Pathology 1997;
183:131-133. [0205] 4. Cheville J C, Lohse C M, Zincke H, et al.
Comparisons of outcome and prognostic features among histologic
subtypes of renal cell carcinoma. American Journal of Surgical
Pathology 2003; 27:612-624. [0206] 5. Lohse C M and Cheville J C. A
review of prognostic pathologic features and algorithms for
patients treated surgically for renal cell carcinoma. Clinics in
Laboratory Medicine 2005; 25:433-464.
Example 3
Combination of Expression of IMP3 and Tumor Staging: A New System
to Predict Metastasis for Patients with Localized Renal Cell
Carcinomas
Abstract
[0207] Purpose: We investigate whether the levels of expression of
a new prognostic biomarker (IMP3) combined with tumor staging can
serve as a new system to predict metastasis of localized renal cell
carcinoma.
[0208] Design: The 369 patients with localized RCCs from three
institutions were investigated by use of survival analysis. The
expression of IMP3 was evaluated by immunohistochemistry and a
computerized image analyzer (Automated Cellular Imaging System).
Combining quantitative IMP3 results with tumor staging (QITS
system) generated four distinct groups of patients.
[0209] Result: Four groups of patients in QITS system showed
significant differences for their metastasis-free (P<0.0001) and
overall survivals (P<0.0001). Almost all patients of group IV
with localized RCCs developed metastasis (sub clinical metastasis)
and died after nephrectomy. The 5 and 10 year metastasis-free
survival rates for the QITS groups were as follows for groups: I,
97% and 91%, II, 62% and 55%, III, 46% and 19%, and V, 17% and 4%,
respectively. The 5 and 10 year overall survival rates for the QITS
groups were as follows for groups: I, 89% and 72%, II, 58% and 41%,
III, 38% and 17%, and V, 14% and 4%, respectively.
[0210] Conclusion: Our findings suggest that combining quantitative
IMP3 expression and tumor staging provide a unique, simple and
accurate system to predict tumor metastasis. This system will
provide important prognostic information for patients with
localized RCCs and will help physicians to select high risk
patients to start systematic therapy right after nephrectomy.
Introduction
[0211] Renal cell carcinoma accounts for about 85% of all malignant
kidney tumors in the United States, making it the most common type
of kidney cancer.sup.1,2. The incidence of this type of carcinoma
has been rising steadily.sup.3. It was expected that about 38,890
new cases of kidney cancer would be diagnosed in the U.S. in 2006
with approximately 12,840 mortalities.sup.4.
[0212] Currently surgical resection of tumor (nephrectomy) is the
standard of care for almost all patients with renal cell
carcinoma.sup.1,2,5. After nephrectomy, patients with metastatic
disease typically receive systemic treatment (e.g. immunotherapy),
which is associated with significant toxic side effects.sup.1,2,5.
In order to avoid the multiple toxicities associated with
treatment, watchful waiting is the standard of care following
nephrectomy unless the patient presents with clearly metastatic
disease.sup.1,2,5. Recently, two new drugs, Nexavar.RTM.
(sorafenib) and Sutent.RTM. (sunitinib), the multi kinase receptor
inhibitors, which can block the signal cascade of the vascular
endothelial growth factor (VEGF) 2, 3 and R1, as well as
platelet-derived growth factor (PDGF) that are critical to
angiogenesis, have been used for treatment of patients with
metastatic renal cell carcinoma.sub.6-9. However, these new drugs
were only evaluated in patients with clinically metastatic
RCC.sup.6-9.
[0213] The metastastic potential of localized tumors is often
unpredictable. Currently, evaluation of patients for
post-nephrectomy adjuvant therapy relies almost entirely on
clinical and pathological staging.sup.10-13. Renal cell cancers
that are typically classified at the same stage exhibit markedly
different biological behavior.sup.10-13. Consequently, 30% of
patients with localized tumor during surgery will subsequently
recur and metastasize and the survival rates of these are typically
less than 10%.sup.14,15. Therefore, there is still a great need for
biomarkers to predict patients' metastasis, particularly for early
stages (stage I, II and III) of RCC. A reliable biomarker that can
at the time of initial diagnosis of localized disease distinguish
between tumors which will remain indolent following nephrectomy
from those with early stage of tumor and a high probability for
metastasis will allow clinicians to early target those individual
patients who are most likely to benefit from adjuvant therapy,
particularly from sorafenib and sunitinib therapy while sparing
patients who are less likely to suffer metastasis from the side
effects of systemic treatment.
[0214] IMP3 is a member of a family of conserved RNA-binding
proteins that consists of IMP1, IMP2 and IMP3.sup.16. These IMP of
proteins all contain two RNA recognition motifs and 4 K-homology
domains that allow them to bind RNAs strongly and specifically,
however only a few specific RNA targets, such as insulin-like
growth factor II (IGF-II) have thus far been identified.sup.17,18.
IMP3 has also been called KOC (K-homology domain protein over
expressed in cancer) and L523S.sup.17,19. IMP3 is expressed in
developing epithelium, muscle and placenta during early stages of
human and mouse embryogenesis.sup.16,18. In contrast, it is
expressed at low or undetectable levels in adult tissues. Certain
cancers also express IMP3.sup.17,19,20. In fact, it was identified
in screens for genes over expressed in pancreatic and lung
cancers.sup.17,19,20. It has been reported to be also expressed in
carcinomas of the stomach and colon and soft tissue
sarcomas.sup..noteq.. Thus, IMP3 is an oncofetal antigen.
[0215] Recently, we have discovered that IMP3 is expressed in a
subset of renal cell carcinomas and its expression predicts
remarkably well the RCCs that progress to metastasis.sup.21. In
previous study, we have demonstrated IMP3 status and tumor stage
are the two most important risk factors for predicting metastasis
of localized RCC. In this study, we used a computerized image
analyzer [Automated Cellular Imaging System (ACIS)] to quantitative
analysis of the immunohistochemistry IMP3 in localized RCCs and
determined whether tumors with higher levels of IMP3 may progress
more rapidly than those with lower levels of this molecule and
whether combining the levels of IMP3 expression of IMP3 and tumor
stage can serve as a new system to more accurately predict
metastasis of localized renal cell carcinoma.
Materials and Methods
[0216] Patients and Tumor Specimens: Formalin-fixed,
paraffin-embedded samples from 369 patients with localized primary
renal cell carcinomas, who underwent radical or partial
nephrectomy, were obtained from the archival files at the
University of Massachusetts Medical Center (UMMC, n=144), the
Massachusetts General Hospital (MGH, n=147) and the City of Hope
National Medical Center (CHNMC, n=78). The data from these sources
represented all patients for whom archival tissues and adequate
clinical follow-up information were readily available.
[0217] All cases were collected between January of 1989 and
December 2003 and the diagnoses were confirmed by at least two
pathologists. Staging was based on pathological findings following
the American Joint Committee on Cancer (AJCC) staging manual, sixth
edition, 2002. Two hundred fifteen patients (pT1a or b) were stage
I, 63 patients (pT2) were stage II, 91 patients (pT3a, N0, n=62;
pT3b, N0, n=29) were stage III. Follow-up for this retrospective
study was carried out by reviewing the patient clinical records.
Overall survival was measured from the date of nephrectomy to the
date of death or was censored as of the date of the last follow-up
visit for survivors. Metastasis-free survival was measured from the
date of surgery to the date of first clinical evidence of
metastasis, and was censored at the date of death or the date of
the last follow-up visit for survivors. The median follow-up was 63
months (range=1-174 months). The Institutional Review Board at each
institution approved this study.
[0218] Immunohistochemical Analysis Immunohistochemical studies
were performed on 5 .mu.m sections of formalin-fixed,
paraffin-embedded tissue from nephrectomy specimens by using an
avidin-biotinylated peroxidase complex system as a previously
published protocol.sup.20 on the DAKO Autostainer (DAKO
Corporation, Carpinteria, Calif.). Sections of pancreatic carcinoma
with known positivity of IMP3 were used as positive controls for
the L523S mouse monoclonal antibody (MAb) specific for IMP3/KOC
(Corixa Corporation, Seattle, Wash.) staining. Negative controls
were performed by replacing the primary antibody with nonimmune
IgG.
[0219] Quantitative Analysis of Immunostaining: A total of 1,845
tumor areas (5 different areas/case) from all RCC tissues were
quantitatively analyzed by a computerized image analyzer (Automated
Cellular Imaging System, ACIS, ChromaVision Medical System Inc.,
San Juan Capistrano, Calif.) to evaluate the IHC results. With
ACIS, positive staining is calculated by applying two thresholds
with one recognizing blue background (hematoxylin stained) cells
and another recognizing brown positive cells. The integrated
optical density (IOD) is that the sum of brown pixels times brown
intensity of those pixels. The ACIS values were calculated as IOD
was divided by the sum of the blue area and the brown area. IMP3
expression in RCCs was considered to be negative (average ACIS
value/case: <1) and positive (average ACIS value/case:
.gtoreq.1; low levels of expression: ACIS values=1 to 10, and high
levels of expression: ACIS values >10).
[0220] Statistical Analysis: Age, sex, size of the tumor, tumor
stage and grade, and IMP3 status were collected as baseline
variables. The distribution of each baseline variable was compared
for IMP3-positive and IMP3-negative subgroups with the Wilcoxon
rank sum test for continuous variables and the Fisher's exact test
for categorical variables. Overall survival and metastasis-free
survival of 369 patients were estimated by the Kaplan-Meier method
and evaluated with the use of log-rank test for univariate
analysis. The Cox proportional-hazard model was used to assess the
simultaneous contribution of the following baseline covariates of
age, sex, size of the tumor, tumor stage and grade, and IMP3
status. A two-sided P-value of less than 0.05 was considered to
indicate the statistical significance.
[0221] Based on a Cox proportional-hazard model, IMP3 status and
tumor stage were the two most important independent risk factors
for predicting metastasis of localized RCC, the levels (low versus
high) of IMP3 expression from ACIS analysis and tumor stage were
divided into five subgroups each of which had a significantly
increasing risk of metastasis and death over the previous one.
Results
[0222] Patients based on IMP3 status of their RCCs including
negative, low and high expression, and tumor stage were classified
into four groups (Tables 6 and 7). This QITS system showed
significant differences for their metastasis-free (P<0.0001) and
overall survivals (P<0.0001). FIG. 9 showed significant
stratification of high risk patients compared to TNM staging alone
(FIG. 8). Almost all patients of group IV with localized RCCs
developed metastasis (sub clinical metastasis) and died after
nephrectomy (FIG. 9). The 5 and 10 year metastasis-free survival
rates for the QITS groups were as follows for groups: I, 97% and
91%, II, 62% and 55%, III, 46% and 19%, and V, 17% and 4%,
respectively (Table 7). The 5 and 10 year overall survival rates
for the QITS groups were as follows for groups: I, 89% and 72%, II,
58% and 41%, III, 38% and 17%, and V, 14% and 4%, respectively
(Table 8).
TABLE-US-00007 TABLE 6 Quantitative IMP3 Status of RCC Combined
with Tumor Stage (QITS) System QITS Groups IMP3 Status Tumor Stage
I Negative Stage 1 and 2 II Negative Stage 3 III Low Expression
Stage 1 IV Low Expression Stage 2 and 3 High Expression Stage 1, 2
and 3
TABLE-US-00008 TABLE 7 QITS and TNM Systems with 5- and 10 Year
Metastasis-free Survivals QITS Groups I II III VI 5-Year Metastasis
97% 63% 46% 17% free Survival 10-Year Metastasis 91% 55% 19% 4%
free Survival TNM Stage I II III 5-Year Metastasis 92% 85% 51% free
Survival 10-Year Metastasis 84% 69% 40% free Survival
TABLE-US-00009 TABLE 8 QITS and TNM Systems with 5- and 10 Year
Overall Survivals QITS Groups I II III VI 5-Year Metastasis 89% 58%
38% 14% free Survival 10-Year Metastasis 72% 41% 17% 4% free
Survival TNM Stage I II III 5-Year Metastasis 83% 80% 47% free
Survival 10-Year Metastasis 67% 58% 30% free Survival
Example 4
The RNA-Binding Protein IMP3: A Novel Molecular Marker to Predict
Aggressive Superficial Urothelial Carcinoma of the Bladder, A
Retrospective Study
Abstract
[0223] Background: The majority (75%) of urothelial carcinomas (UC)
of the bladder are superficial (early stage) tumors. The biological
behaviors of superficial UCs are variable, spanning a spectrum from
indolent to aggressive. Identification of patients with aggressive
superficial bladder tumors will allow better personal follow-up
strategies, and more importantly the opportunity to provide early
treatment. In this study, we investigated whether the RNA binding
protein-3 (IMP3), an oncofetal protein, can serve as a new
biomarker to predict progression and metastasis of superficial UC
of the bladder.
[0224] Methods: The expression of IMP3 in 214 patients with the
diagnosis of superficial UC of the bladder [Ta (non-invasive
papillary carcinoma), n=171; Tis (Carcinoma in situ), n=14; T1
(tumor invades laminar propria without evidence of muscularis
propria invasion), n=29] was evaluated by immunohistochemistry
(IHC). An additional 28 metastatic UCs were also included in the
study to evaluate this IMP3 expression. Tumor progression was
measured as the date when tumors were demonstrated on biopsy to
have evolved to a higher stage (deeper invasion) or when metastases
were proven by clinical or pathologic diagnosis. No progression was
defined as negative for tumor or no change from the original T
stage in a follow up biopsy, and/or negative cystoscopic findings
with at least two negative cytology results at the last follow up
date.
[0225] Results: Twenty percent (42 of 214) of primary superficial
urothelial carcinomas and 93% (26 of 28) of metastatic urothelial
carcinomas expressed IMP3, and the non-malignant epithelium from
the adjacent bladder was IMP3 negative. The expression of IMP3 was
significantly increased not only in higher stage (P=0.016) and
grade (P<0.0001) UCs but most importantly also in a subset of
superficial UCs that was much more likely to subsequently progress.
Kaplan-Meier plots and log-rank tests showed that patients without
IMP3 expression in their superficial UCs had significant longer
progression-free survival (P=0.0002) and disease-free survival
(P=0.0067) than patients with IMP3 expression. The 5-year
progression-free survival rate was 91% in IMP3 negative patients
versus 64% in IMP3 positive patients. The 5-year disease-free
survival rate was 94% in patients without expression of IMP3 versus
76% in patients with IMP3 expression. In Ta disease, 19% (5 of 27)
of IMP3 positive patients (median follow up=38 months, range=6-125
months) versus 5% (7 of 144) of IMP3 negative patients (median
follow up=47 months, range=2-146 months) exhibited progression
(P=0.0098). In T1 disease, 60% (6 of 10) of IMP3 positive patients
(median follow up=15 months, range=3-72 months) versus 21% (4 of
19) of IMP3 negative patients (median follow up=17 months,
range=2-88 months) were found to have tumor progression (P=0.03).
Sixty percent (6 of 10) of IMP3 positive patients with T1 disease
at initial diagnosis went on to develop metastases while no
metastasis was found in IMP3 negative patients (P=0.0017).
Multivariable Cox proportional hazards regression analysis showed
that the expression of IMP3 in superficial UCs was an independent
predictor of the patients' clinical outcome. The hazard ratios of
IMP3 status in superficial UCs were 4.94 (95% CI 1.77-13.83,
P=0.002) for progression-free survival and 2.59 (95% CI 1.12-5.99,
P=0.027) for disease-free survival.
[0226] CONCLUSIONS: IMP3 is an independent prognostic marker that
can be used at the time of initial diagnosis of superficial UCs to
identify a group of patients with a high potential to develop
progression and who might benefit from early aggressive
therapy.
Introduction
[0227] Urothelial carcinoma is the most common type of urinary
bladder cancer and accounts for about 95 percent of malignant
bladder tumors. Each year nearly 63,000 new cases of urothelial
cancer are diagnosed with almost 13,000 people dying of the
disease(1, 2). Seventy five percent of urothelial carcinomas of the
bladder are superficial tumors including the tumor, node, and
metastasis (TNM) categories of Ta: papillary tumors confined to the
epithelium; Tis: flat carcinoma in situ (CIS) also confined to the
epithelium; and T1: tumors invading the lamina propria(2, 3). The
biological behavior of these superficial urothelial cancers is
significantly different, ranging from the relatively benign
noninvasive papillary tumor to the highly aggressive tumor with a
significant mortality rate(2). Currently, identification of
patients with aggressive superficial UC and determination of their
treatment are mainly based on the tumor grade and stage(3).
Treatment for the majority of low grade and Ta superficial UCs is
local resection of the tumor with close observation or intravesical
therapy, while more aggressive therapies including cystectomy
and/or radiation/chemotherapy are often reserved for patients with
high-grade or T1 tumors who have a higher chance to progress to
deeply invasive cancer with a much higher mortality(3-8). However,
the tumor grade and stage have limited ability to predict tumor
progression. As superficial UCs show unpredictable behavior, an
important clinical problem is how to accurately assess individual
risk of progression and to stratify patients for treatment.
Therefore, there is a great need for biomarkers that can accurately
distinguish superficial tumors with a high probability of
progression from those that will remain indolent. Using such
biomarkers, one could predict the patient's prognosis and
effectively target the individuals who would most likely benefit
from the therapy.
[0228] Recently, we identified IMP3 as an independent prognostic
biomarker whose expression predicts aggressive behavior of renal
cell carcinomas(9). IMP3 is a member of the insulin-like growth
factor II (IGF-II) mRNA binding protein (IMP) family that consists
of IMP1, IMP2 and IMP3(10). IMP family members play an important
role in RNA trafficking and stabilization, cell growth, and cell
migration during the early stages of embryogenesis(11). The IMP3
gene is located on chromosome 7p11.2(12) and is identical to the
KOC (KH domain containing protein overexpressed in cancer) protein
that was originally cloned from a pancreatic tumor cDNA screen(13).
IMP3 is expressed in developing epithelium, muscle and placenta
during early stages of human and mouse embryogenesis, but it is
expressed at low or undetectable levels in adult tissues(10, 11).
The expression of IMP3/KOC is also found in malignant tumors
including pancreas, lung, stomach, and colon cancers, and soft
tissue sarcomas but it is not detected in adjacent benign
tissues(10, 13-15). Moreover, a recent study has demonstrated that
IMP3 promotes tumor cell proliferation and invasion (16, 17). These
findings indicate that IMP3 is an oncofetal protein that may have a
critical role in the regulation of cell proliferation and invasion.
In this study, we investigated whether IMP3 could serve as an
independent biomarker to predict progression and metastasis of
superficial urothelial carcinoma.
Materials and Methods
[0229] Patients and Tumor Specimens: Formalin-fixed,
paraffin-embedded samples of bladder biopsies from 214 patients
with a diagnosis of urothelial carcinoma of the bladder [Ta
(non-invasive papillary carcinoma), n=171; Tis (Carcinoma in situ),
n=14; T1 (tumor invades lamina propria without evidence of
muscularis propria invasion), n=29] were obtained at the UMass
Memorial Medical Center from years 1992 to 2000. All T1 tumors were
confirmed by tissue diagnosis. These biopsies showed the presence
of muscularis propria with no tumor presence in muscularis propria.
The cases with lamina propria invasion, but without the presence of
muscularis propria were excluded to rule out potential higher stage
of urothelial tumors. The cases with T1 diagnosis in original
biopsy, but rebiopsy within 3 months showing tumor invasion in
muscularis propria (>T1 tumor) were also excluded in our study.
An additional 28 metastatic urothelial bladder carcinoma specimens
were included in the study and evaluated for the expression of the
IMP3. Tumor grade and stage were based on pathological findings
following the World Health Organization grading system (Mostofi,
Geneva: World Health Organization; 1973) and the American Joint
Committee on Cancer (AJCC) staging manual, sixth edition, 2002,
respectively. The data from these sources represented all patients
for whom archival tissues and adequate clinical follow-up
information were readily available. The Institutional Review Board
approved this study.
[0230] Immunohistochemical Analysis Immunohistochemical studies
were performed on 5 .mu.m sections of formalin-fixed,
paraffin-embedded tissue from bladder biopsy specimens by using an
avidin-biotinylated peroxidase complex system as a previously
published protocol(15) on the DAKO Autostainer (DAKO Corporation,
Carpinteria, Calif.). Sections of pancreatic carcinoma with known
positivity of IMP3 were used as positive controls for the L523S
mouse monoclonal antibody (MAb) specific for IMP3/KOC (Corixa
Corporation, Seattle, Wash.) staining. Negative controls were
performed by replacing the primary antibody with nonimmune IgG.
Positive staining of IMP3 was defined as dark brown cytoplasm while
negative staining of IMP3 was defined as no staining at all. The
status of IMP3 was assessed by researchers L S and Z J) without
knowledge of the clinical and pathological features of the cases or
the clinical outcome.
[0231] Statistical Analysis Tumor progression was measured as the
date when tumors were demonstrated on biopsy to have evolved to a
higher stage (deeper invasion) or when metastases were proven by
clinical or pathologic diagnosis. No progression was defined as
negative for tumor or no change from the original T stage in a
follow up biopsy, and/or negative cystoscopic findings with at
least two negative cytology results at the last follow up date.
Recurrence was defined as the appearance of recurrent UC without
alteration from the original stage. Progression-free survival was
measured from the date of the biopsy to the date of follow-up
tissue biopsy/resection that demonstrated tumor progression to a
higher stage of tumor as compared to the previous biopsy or to the
date of the last negative cystoscopy with negative cytology or the
last tissue diagnosis proved without progression. Metastasis-free
survival was measured from the date of the biopsy to the date of
first clinical evidence of metastasis, and was censored at the date
of death or last follows up visit for survivors. The patient's
disease-free survival was measured from the date of the diagnostic
biopsy to the date of clinical evidence of remaining urothelial
carcinoma, and was censored at the date of death without disease or
last follow up for survivors. Overall survival was measured from
the date of biopsy to the date of death, and was censored from the
date of last follow up for survivors. Data on various treatments
including transurethial resection of the tumor in the bladder, BCG
treatment, intravesicle therapy, radiation and chemotherapy, and
cystectomy were collected during the time of each follow-up
category. The patients' age, sex, grade, stage, tumor size,
multiplicity, recurrence and various treatment modalities were
collected as baseline variables. The distribution of each variable
was compared for IMP3-positive and IMP3-negative subgroups with the
Wilcoxon rank sum test for continuous variables and the Fisher's
exact test for categorical variables. Progression-free survival,
metastasis-free survival, disease-free survival and overall
survival were estimated by Kaplan-Meier method and evaluated with
the use of log-rank test for univariate analysis. The Cox
proportional-hazard model was used to assess simultaneous
contribution of baseline covariates in univariate and multivariable
analysis. A two-sided P-value of less than 0.05 was considered to
be statistically significant.
Results
Expression of IMP3 in All Primary and Metastatic Urothelial
Carcinomas
[0232] When detected by IHC, IMP3 protein was observed in the
cytoplasm of tumor cells yielding a dark brown staining pattern
(FIG. 10A). In contrast, cases that were scored as having no
expression of IMP3 were completely lacked the brown stain (no
staining, FIG. 10B). No IMP3 expression was found in benign tissue
adjacent to cancer. Expression of IMP3 was found in 20% (42 of 214)
of primary superficial urothelial carcinoma while 93% (26 of 28) of
metastatic urothelial carcinomas were positive for IMP3
staining.
Characteristics of the Patients
[0233] Table 8 shows the relevant clinical characteristics of the
214 patients with superficial urothelial carcinoma of the bladder.
The expression of IMP3 was strongly associated with tumor stage
(P=0.016), grade (P<0.0001), recurrent rate (P=0.029) and
treatment (P=0.001) while the patient's age (P=0.903), sex
(P=0.497) and tumor size (P=0.237) and multiplicity (P=0.692) were
not linked with IMP3 positivity. The IMP3 positivity rate was
increased in T1 (35%) and urothelial carcinoma in situ (Tis, 36%)
as compared with Ta tumors (16%, P=0.016). Only 7% of grade I UCs
expressed IMP3, whereas 23-40% of grade II, III and CIS tumors were
positive for IMP3.
TABLE-US-00010 TABLE 9 Clinicopathological Characteristics of
Patients with Superficial Urothelial Carcinomas Total IMP3+ IMP3- P
Characteristic (N = 214) (N = 42) (N = 172) Value Sex Female 64 12
(19%) 52 (81%) 0.497 Male 150 30 (20%) 120 (80%) Age - yr 68.7 .+-.
12.0.sup.1 68.9 .+-. 11.3 68.6 .+-. 12.2 0.903 Tumor stage Ta 171
27 (16%) 144 (84%) 0.016 Tis 14 5 (36%) 9 (64%) T1 29 10 (35%) 19
(65%) Tumor Grade G1 86 6 (7%) 80 (93%) <0.0001 G2 84 19 (23%)
65 (77%) G3 30 12 (40%) 18 (60%) CIS 14 5 (33%) 9 (67%) Tumor Size
1.4 .+-. 1.7.sup.1 1.7 .+-. 1.4 1.3 .+-. 1.7 0.237 Multiplicity of
tumor Yes 54 9 (17%) 45 (83%) 0.692 No 160 33 (21%) 127 (79%)
Treatment Resection only 96 9 (9%) 87 (91%) 0.001 BCG 109 33 (30%)
76 (70%) Additional 9 1 (11%) 8 (89%) Therapies Unknown 4 0 4
Recurrence Yes 129 33 (26%) 96 (74%) 0.029 No 63 8 (13%) 55 (87%)
Unknown 22 1 21 .sup.1The value = mean .+-. standard deviation
.sup.2Unknown cases do not include in the statistic analysis.
Expression of IMP3 and Tumor Prognosis
[0234] The progression-free survival was significantly different
between patients with IMP3 positive tumors versus IMP3 negative
ones. By the end of the follow-up, 26% (11 of 42) of patients with
IMP3 positivity in their superficial urothelial cancers
subsequently developed either deeper invasion or metastasis (median
follow-up=35 months, range=3-125 months), whereas only 7% (12 of
172) of patients without expression of IMP3 were found to show
progression (median follow-up=45 months, range=2-146 months).
Twenty-two patients, whose status (progression versus non
progression) was unknown (no follow-up tissue biopsy), were all
negative for IMP3 in their original tumors. In Ta disease, 19% (5
of 27) of IMP3 positive patients (median follow up=38 months,
range=6-125 months) versus 5% (7 of 144) of IMP3 negative patients
(median follow up=47 months, range=2-146 months) showed progressed
(P=0.0098). In T1 disease, 60% (6 of 10) of IMP3 positive patients
(median follow up=15 months, range=3-72 months) versus 21% (4 of
19) of IMP3 negative patients (median follow up=17 months,
range=2-88 months) were found to have tumor progression
(P=0.03).
[0235] Kaplan-Meier plots and log-rank tests in patients with
superficial urothelial cancer showed that patients without IMP3
expression in their superficial urothelial carcinomas had
significant longer progression-free survival (P=0.0002, FIG. 11A)
and disease-free survival (P=0.0067, FIG. 11B) than patients with
IMP3 expression. The 5-year progression-free survival rate was 91%
in IMP3 negative patients versus 64% in IMP3 positive patients. The
5-year disease-free survival rate was 94% in patients without
expression of IMP3 versus 76% in patients with IMP3 expression. In
patients with superficial urothelial cancers at stage Ta and T1,
the status of IMP3 expression was significantly associated with an
increased risk of progression (Ta: FIG. 12A, P=0.0098; T1: FIG.
12B, P=0.03). Metastasis was not found in patients with Ta and Tis
disease.
[0236] FIG. 13 showed that IMP3 positive patients with T1 disease
had significant poorer metastasis-free survival compared to IMP3
negative patients with T1 disease (P=0.0017). There were no
significant differences in overall survival (P=0.39) between
patients with IMP3 positive staining and patients with IMP3
negative staining. No significant difference in disease free
survival was found between IMP3 positive and IMP3 negative patients
with T1 disease (P=0.20).
Multivariate Analysis
[0237] The results of the multivariate analysis for
progression-free survival and disease-free survival in the 214
patients with superficial UCs are presented in Table 10. For these
analyses, risk factors shown in Table 9 were initially included in
the model as potential risk factors. Multivariate Cox proportional
hazards regression analysis showed that the expression of IMP3 in
superficial UCs was a strong independent predictor of the patients'
clinical outcome. The hazard ratio for progression-free survival
was 4.94 (95% confidence interval, 1.77-13.83; P=0.002) for the
patients with IMP3 expression. The hazard ratio for disease-free
survival was 2.59 (95% confidence interval, 1.12-5.99; P=0.027) for
the patients with IMP3 expression. In addition to IMP3 status, age
and tumor stage T1 (compared to stage Ta) were also observed as
significant risk factors for progression-free survival, and age,
tumor stage T1 (compared to stage Ta) and BCG treatment were also
observed as significant factors for disease-free survival (Table
10). Note: Due to the limited number of patients, the risk factor
of coexistent CIS (N=7), and patients' treatment including
radiation therapy (N=5), cystectomy (N=9) and systematic
chemotherapy (N=4) were not included in the analysis.
TABLE-US-00011 TABLE 10 Multivariable Analysis for Progression-free
and Disease-Free Survivals of Superficial Urothelial Carcinoma
Progression-Free Survival Disease-Free Survival Hazard Ratio P
Hazard Ratio P Variable (95% CI) Value (95% CI) Value IMP3 STATUS
4.94 (1.77-13.83) 0.002 2.59 (1.12-5.99) 0.027 (+ VS. -) Age 1.06
(1.02-1.12) 0.006 1.07 (1.02-1.11) 0.001 Sex (F vs. M) 1.55
(0.57-4.17) 0.391 1.64 (0.79-3.43) 0.187 Tumor Size 0.91
(0.64-1.30) 0.614 0.98 (0.75-1.26) 0.848 Multiplicity 1.66
(0.61-4.50) 0.321 0.95 (0.39-2.30) 0.905 Tumor Stage T1 vs. Ta 4.65
(1.69-12.81) 0.003 2.66 (1.08-6.51) 0.032 Tis vs. Ta 0.92
(0.10-8.32) 0.939 0.28 (0.03-2.31) 0.239 Tumor Grade 2 vs. 1 0.36
(0.07-1.86) 0.227 0.89 (0.36-2.19) 0.804 2 vs. 3 2.75 (0.97-7.77)
0.057 1.47 (0.51-4.20) 0.469 BCG (Treated or not) 0.62 (0.23-1.65)
0.341 2.36 (1.05-5.30) 0.037
Discussion
[0238] Different cases of morphologically identical superficial
bladder cancer can behave very differently. In this study, we have
shown that IMP3 is expressed much more frequently in metastatic
urothelial carcinoma than in primary urothelial carcinoma and that
IMP3 expression is associated with tumor progression. Therefore,
IMP3 is a biomarker that can provide important prognostic
information in patients with superficial urothelial cancers.
[0239] Many tumor markers have been studied for their potential use
in assessing the prognosis of UC. However, a recent international
consensus panel on prognostic markers for bladder cancer concluded
that although certain markers, such as p53, appear to be promising
in predicting progression of bladder cancer, the data were still
heterogeneous (18). None of the markers have been adopted in
clinical practice (18). Although markers such as p53 have been
extensively studied and some of them have shown promising results,
a recent meta-analysis and review indicates that evidence is not
sufficient to conclude whether alterations in p53 expression act as
markers of outcome in patients with bladder cancer (19). Thus we
still lack prognostic biomarkers for urothelial carcinoma.
[0240] IMP3 as a prognostic marker exhibits several attractive
features for superficial urothelial carcinoma. First, the
expression of IMP3 is correlated with other known aggressive
indicators for superficial UC. Our results showed that the
expression of IMP3 was strongly related to higher tumor grade and
stage and tumor recurrence. Second, the expression of IMP3 in
biopsies of superficial UCs is an independent predictor of tumor
progression. We found significantly increased tumor progression in
patients with superficial UCs expressing of IMP3 as compared to
those without IMP3 expression. This was independent of tumor grade
and stage. In the multivariable Cox analysis, patients with IMP3
expression in their superficial UCs subsequently developed invasive
lesions or metastasis at a rate, which was about 5 times greater
than cases without expression of IMP3 adjusting for other
well-known clinical variables. Therefore, IMP3 status in the
initial tumor biopsies is a potentially important new risk factor
that may able to be used in addition to tumor stage, grade, size,
multiplicity and coexistent CIS to guide the decision of adjuvant
courses of intravesical therapy after tumor resection. Third, IMP3
expression is associated with metastasis of urothelial carcinoma.
Out data showed that the expression of IMP3 was significantly
increased not only in metastastic urothelial carcinoma but most
importantly also in patients with primary T1 tumors who
subsequently developed metastatic disease. We found that 60% of
patients with IMP3 positivity in their primary T1 UCs developed
metastasis, whereas none of patients without expression of IMP3 in
their primary T1 tumors developed metastases. Currently, the
decision on whether to attempt bladder conservation with
intravesicle therapy or to perform a cystectomy is the most
difficult issue in the management of superficial bladder cancer.
Results from early cystectomy for high risk superficial UCs are
generally excellent with 5-year cancer specific survival in
exceeding 90% (6, 7). However, as patients who undergo cystectomy
have a significantly unpleasant lifestyle and in the absence of
better prognostic tools, many patients who would not have
progressed are subjected to these potential side effects (3, 20,
21). Therefore, accurately identifying the high risk patients,
particularly in T1 tumors with poor prognosis, becomes a very
important clinical issue. The ability of IMP3 to identify patients
presenting at stage T1 that will progress and present with
metastatic disease would provide very important clinical
information. Presumably, if this subset of patients could be
identified prospectively (with IMP3), they would benefit from
earlier definitive treatment (cystectomy) and this concept could be
tested in future clinical trials.
[0241] Fourth, IMP3 immunohistochemical staining is a simple,
inexpensive and reliable assay, which can be used in routine
clinical practice. As superficial UCs are usually treated by
resection, tumor tissue is routinely available for
immunohistochemical staining. There are also two important factors
to reduce inconsistent or variable results of IHC: 1) The IMP3
monoclonal antibody specifically binds its target with very low or
no background in immunohistochemical staining. 2) The utilization
of a stand protocol and automated instrument minimizes variations
in the staining results. Subjective interpretation and variable
criteria for cutoff points (considering a positive or negative
case) may also give variable results in immunohistochemical study
of biomarkers. In our study, we used negative (no stain at all) and
positive (staining with brown color, even with focal positive
staining) criteria for determining IMP3 status. There is no cutoff
point, e.g. 5% or 20% of tumor cell staining considering as
positive criteria. Our criteria significantly reduced the
subjectivity of interpretation.
[0242] Although there are significant differences in disease
progression and disease free survivals between patients with IMP3
positive staining and patients with IMP3 negative staining, we did
not find significant differences in overall survival. One of the
potential reasons for this difference is that most patients with
superficial urothelial carcinomas have a long survival and may die
from diseases other than bladder cancer. We did not find a
significant difference in disease free survival between IMP3
positive and IMP3 negative patients with T1 disease, presumably
because of the limited number of patients. However, our results
provide a very strong rationale for initiating a much larger
clinical study, particularly for patients with T1 disease, to
further validate this marker.
[0243] Our findings raise the possibility that IMP3 may play a
direct role in the progression and metastasis of UC. There are
emerging data that IMP3 may play a role in the growth of malignant
cells and cellular de-adhesion (3, 20, 21). Interestingly, Yaniv et
al found that IMP3 in Xenopus laevis is required for the migration
of cells forming the roof plate of the neural tube and,
subsequently, for neural crest migration (22), which suggested that
IMP3 may play an important role in promoting cell migration. These
findings could explain why IMP3 is associated with tumor
progression and metastasis. Further study is required to
investigate whether IMP3 plays a direct role in the biological
behavior of urothelial carcinoma.
[0244] In summary, our findings provide evidence that IMP3 is an
independent prognostic marker that can be used at the time of
initial diagnosis of superficial UCs to identify a group of
patients with a high potential to develop progression and
metastasis, and who might benefit from early aggressive
therapy.
REFERENCES
[0245] 1. Jemal, A., Murray, T., Ward, E., Samuels, A., Tiwari, R.
C., Ghafoor, A., Feuer, E. J., and Thun, M. J. Cancer statistics,
2005. Ca: a Cancer Journal for Clinicians, 55: 10-30, 2005. [0246]
2. Kirkali, Z., Chan, T., Manoharan, M., Algaba, F., Busch, C.,
Cheng, L., Kiemeney, L., Kriegmair, M., Montironi, R., Murphy, W.
M., Sesterhenn, I. A., Tachibana, M., and Weider, J. Bladder
cancer: epidemiology, staging and grading, and diagnosis.
[Review][202 refs]. Urology, 66: 4-34, 2005. [0247] 3. Sengupta, S,
and Blute, M. L. The management of superficial transitional cell
carcinoma of the bladder. [Review][90 refs]. Urology, 67: 48-54,
2006. [0248] 4. Joudi, F. N., Smith, B. J., O'Donnell, M. A., and
Konety, B. R. Contemporary management of superficial bladder cancer
in the United States: a pattern of care analysis. Urology, 62:
1083-1088, 2003. [0249] 5. Holzbeierlein, J. M. and Smith, J A.,
Jr. Surgical management of noninvasive bladder cancer (stages
Ta/T1/CIS). [Review][25 refs]. Urologic Clinics of North America,
27: 15-24, 2000. [0250] 6. Amling, C. L., Thrasher, J. B., Frazier,
H. A., Dodge, R. K., Robertson, J. E., and Paulson, D. F. Radical
cystectomy for stages Ta, Tis and T1 transitional cell carcinoma of
the bladder. Journal of Urology, 151: 31-35, 1994. [0251] 7.
Freeman, J. A., Esrig, D., Stein, J. P., Simoneau, A. R., Skinner,
E. C., Chen, S. C., Groshen, S., Lieskovsky, G., Boyd, S. D., and
Skinner, D. G. Radical cystectomy for high risk patients with
superficial bladder cancer in the era of orthotopic urinary
reconstruction. Cancer, 76: 833-839, 1995. [0252] 8. Malkowicz, S.
B., Nichols, P., Lieskovsky, G., Boyd, S. D., Huffman, J., and
Skinner, D. G. The role of radical cystectomy in the management of
high grade superficial bladder cancer (PA, P1, P1S and P2)[see
comment]. Journal of Urology, 144: 641-645, 1990. [0253] 9. Jiang,
Z., Chu, P. G., Woda, B. A., Rock, K. L., Liu, Q., Hsieh, C. C.,
Li, C., Chen, W., Duan, H. O., McDougal, S., and Wu, C. L. Analysis
of RNA-binding protein IMP3 to predict metastasis and prognosis of
renal-cell carcinoma: a retrospective study. Lancet Oncology, 7:
556-564, 2006. [0254] 10. Nielsen, J., Christiansen, J.,
Lykke-Andersen, J., Johnsen, A. H., Wewer, U. M., and Nielsen, F.
C. A family of insulin-like growth factor II DNA-binding proteins
represses translation in late development. Molecular & Cellular
Biology, 19: 1262-1270, 1999. [0255] 11. Mueller-Pillasch, F.,
Pohl, B., Wilda, M., Lacher, U., Beil, M., Wallrapp, C., Hameister,
H., Knochel, W., Adler, G., and Gress, T. M. Expression of the
highly conserved RNA binding protein KOC in embryogenesis.
Mechanisms of Development, 88: 95-99, 1999. [0256] 12. Monk, D.,
Bentley, L., Beechey, C., Hitchins, M., Peters, J., Preece, M. A.,
Stanier, P., and Moore, G. E. Characterisation of the growth
regulating gene IMP3, a candidate for Silver-Russell syndrome.
Journal of Medical Genetics, 39: 575-581, 2002. [0257] 13.
Mueller-Pillasch, F., Lacher, U., Wallrapp, C., Micha, A.,
Zimmerhackl, F., Hameister, H., Varga, G., Friess, H., Buchler, M.,
Beger, H. G., Vila, M. R., Adler, G., and Gress, T. M. Cloning of a
gene highly overexpressed in cancer coding for a novel KH-domain
containing protein. Oncogene, 14: 2729-2733, 1997. [0258] 14. Wang,
T., Fan, L., Watanabe, Y., McNeill, P. D., Moulton, G G., Bangur,
C., Fanger, G. R., Okada, M., Inoue, Y., Persing, D. H., and Reed,
S. G. L523S, an RNA-binding protein as a potential therapeutic
target for lung cancer. British Journal of Cancer, 88: 887-894,
2003. [0259] 15. Yantiss, R. K., Woda, B. A., Fanger, G. R., Kalos,
M., Whalen, G. F., Tada, H., Andersen, D. K., Rock, K. L., and
Dresser, K. KOC (K homology domain containing protein overexpressed
in cancer): a novel molecular marker that distinguishes between
benign and malignant lesions of the pancreas. American Journal of
Surgical Pathology, 29: 188-195, 2005. [0260] 16. Liao, B., Hu, Y.,
Herrick, D J., and Brewer, G. The RNA-binding Protein IMP-3 Is a
Translational Activator of Insulin-like Growth Factor II Leader-3
mRNA during Proliferation of Human K562 Leukemia Cells. J. Biol.
Chem., 280: 18517-18524, 2005. [0261] 17. Vikesaa, J., Hansen, T.
V., Jonson, L., Borup, R., Wewer, U. M., Christiansen, J., and
Nielsen, F. C. RNA-binding IMPs promote cell adhesion and
invadopodia formation. EMBO Journal, 25: 1456-1468, 2006. [0262]
18. Habuchi, T., Marberger, M., Droller, M. J., Hemstreet, G. P.,
3rd, Grossman, H. B., Schalken, J A., Schmitz-Drager, B. J.,
Murphy, W. M., Bono, A. V., Goebell, P., Getzenberg, R. H.,
Hautmann, S. H., Messing, E., Fradet, Y., and Lokeshwar, V. B.
Prognostic markers for bladder cancer: International Consensus
Panel on bladder tumor markers. [106 refs]. Urology, 66: 64-74,
2005. [0263] 19. Malats, N., Bustos, A., Nascimento, C. M.,
Fernandez, F., Rivas, M., Puente, D., Kogevinas, M., and Real, F.
X. P53 as a prognostic marker for bladder cancer: a meta-analysis
and review. [Review][74 refs]. Lancet Oncology, 6: 678-686, 2005.
[0264] 20. Herr, H. W. and Sogani, P. C. Does early cystectomy
improve the survival of patients with high risk superficial bladder
tumors? Journal of Urology, 166: 1296-1299, 2001. [0265] 21.
Stockle, M., Alken, P., Engelmann, U., Jacobi, G. H., Riedmiller,
H., and Hohenfellner, R. Radical cystectomy--often too late?
European Urology, 13: 361-367, 1987. [0266] 22. Yaniv, K. and
Yisraeli, J K. The involvement of a conserved family of RNA binding
proteins in embryonic development and carcinogenesis. [Review][29
refs]. Gene, 287: 49-54, 2002.
Equivalents
[0267] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
811740DNAHomo sapiens 1atgaacaaac tgtatatcgg aaacctcagc gagaacgccg
ccccctcgga cctagaaagt 60atcttcaagg acgccaagat cccggtgtcg ggacccttcc
tggtgaagac tggctacgcg 120ttcgtggact gcccggacga gagctgggcc
ctcaaggcca tcgaggcgct ttcaggtaaa 180atagaactgc acgggaaacc
catagaagtt gagcactcgg tcccaaaaag gcaaaggatt 240cggaaacttc
agatacgaaa tatcccgcct catttacagt gggaggtgct ggatagttta
300ctagtccagt atggagtggt ggagagctgt gagcaagtga acactgactc
ggaaactgca 360gttgtaaatg taacctattc cagtaaggac caagctagac
aagcactaga caaactgaat 420ggatttcagt tagagaattt caccttgaaa
gtagcctata tccctgatga aatggccgcc 480cagcaaaacc ccttgcagca
gccccgaggt cgccgggggc ttgggcagag gggctcctca 540aggcaggggt
ctccaggatc cgtatccaag cagaaaccat gtgatttgcc tctgcgcctg
600ctggttccca cccaatttgt tggagccatc ataggaaaag aaggtgccac
cattcggaac 660atcaccaaac agacccagtc taaaatcgat gtccaccgta
aagaaaatgc gggggctgct 720gagaagtcga ttactatcct ctctactcct
gaaggcacct ctgcggcttg taagtctatt 780ctggagatta tgcataagga
agctcaagat ataaaattca cagaagagat ccccttgaag 840attttagctc
ataataactt tgttggacgt cttattggta aagaaggaag aaatcttaaa
900aaaattgagc aagacacaga cactaaaatc acgatatctc cattgcagga
attgacgctg 960tataatccag aacgcactat tacagttaaa ggcaatgttg
agacatgtgc caaagctgag 1020gaggagatca tgaagaaaat cagggagtct
tatgaaaatg atattgcttc tatgaatctt 1080caagcacatt taattcctgg
attaaatctg aacgccttgg gtctgttccc acccacttca 1140gggatgccac
ctcccacctc agggccccct tcagccatga ctcctcccta cccgcagttt
1200gagcaatcag aaacggagac tgttcatctg tttatcccag ctctatcagt
cggtgccatc 1260atcggcaagc agggccagca catcaagcag ctttctcgct
ttgctggagc ttcaattaag 1320attgctccag cggaagcacc agatgctaaa
gtgaggatgg tgattatcac tggaccacca 1380gaggctcagt tcaaggctca
gggaagaatt tatggaaaaa ttaaagaaga aaactttgtt 1440agtcctaaag
aagaggtgaa acttgaagct catatcagag tgccatcctt tgctgctggc
1500agagttattg gaaaaggagg caaaacggtg aatgaacttc agaatttgtc
aagtgcagaa 1560gttgttgtcc ctcgtgacca gacacctgat gagaatgacc
aagtggttgt caaaataact 1620ggtcacttct atgcttgcca ggttgcccag
agaaaaattc aggaaattct gactcaggta 1680aagcagcacc aacaacagaa
ggctctgcaa agtggaccac ctcagtcaag acggaagtaa 17402579PRTHomo sapiens
2Met Asn Lys Leu Tyr Ile Gly Asn Leu Ser Glu Asn Ala Ala Pro Ser1 5
10 15Asp Leu Glu Ser Ile Phe Lys Asp Ala Lys Ile Pro Val Ser Gly
Pro20 25 30Phe Leu Val Lys Thr Gly Tyr Ala Phe Val Asp Cys Pro Asp
Glu Ser35 40 45Trp Ala Leu Lys Ala Ile Glu Ala Leu Ser Gly Lys Ile
Glu Leu His50 55 60Gly Lys Pro Ile Glu Val Glu His Ser Val Pro Lys
Arg Gln Arg Ile65 70 75 80Arg Lys Leu Gln Ile Arg Asn Ile Pro Pro
His Leu Gln Trp Glu Val85 90 95Leu Asp Ser Leu Leu Val Gln Tyr Gly
Val Val Glu Ser Cys Glu Gln100 105 110Val Asn Thr Asp Ser Glu Thr
Ala Val Val Asn Val Thr Tyr Ser Ser115 120 125Lys Asp Gln Ala Arg
Gln Ala Leu Asp Lys Leu Asn Gly Phe Gln Leu130 135 140Glu Asn Phe
Thr Leu Lys Val Ala Tyr Ile Pro Asp Glu Met Ala Ala145 150 155
160Gln Gln Asn Pro Leu Gln Gln Pro Arg Gly Arg Arg Gly Leu Gly
Gln165 170 175Arg Gly Ser Ser Arg Gln Gly Ser Pro Gly Ser Val Ser
Lys Gln Lys180 185 190Pro Cys Asp Leu Pro Leu Arg Leu Leu Val Pro
Thr Gln Phe Val Gly195 200 205Ala Ile Ile Gly Lys Glu Gly Ala Thr
Ile Arg Asn Ile Thr Lys Gln210 215 220Thr Gln Ser Lys Ile Asp Val
His Arg Lys Glu Asn Ala Gly Ala Ala225 230 235 240Glu Lys Ser Ile
Thr Ile Leu Ser Thr Pro Glu Gly Thr Ser Ala Ala245 250 255Cys Lys
Ser Ile Leu Glu Ile Met His Lys Glu Ala Gln Asp Ile Lys260 265
270Phe Thr Glu Glu Ile Pro Leu Lys Ile Leu Ala His Asn Asn Phe
Val275 280 285Gly Arg Leu Ile Gly Lys Glu Gly Arg Asn Leu Lys Lys
Ile Glu Gln290 295 300Asp Thr Asp Thr Lys Ile Thr Ile Ser Pro Leu
Gln Glu Leu Thr Leu305 310 315 320Tyr Asn Pro Glu Arg Thr Ile Thr
Val Lys Gly Asn Val Glu Thr Cys325 330 335Ala Lys Ala Glu Glu Glu
Ile Met Lys Lys Ile Arg Glu Ser Tyr Glu340 345 350Asn Asp Ile Ala
Ser Met Asn Leu Gln Ala His Leu Ile Pro Gly Leu355 360 365Asn Leu
Asn Ala Leu Gly Leu Phe Pro Pro Thr Ser Gly Met Pro Pro370 375
380Pro Thr Ser Gly Pro Pro Ser Ala Met Thr Pro Pro Tyr Pro Gln
Phe385 390 395 400Glu Gln Ser Glu Thr Glu Thr Val His Leu Phe Ile
Pro Ala Leu Ser405 410 415Val Gly Ala Ile Ile Gly Lys Gln Gly Gln
His Ile Lys Gln Leu Ser420 425 430Arg Phe Ala Gly Ala Ser Ile Lys
Ile Ala Pro Ala Glu Ala Pro Asp435 440 445Ala Lys Val Arg Met Val
Ile Ile Thr Gly Pro Pro Glu Ala Gln Phe450 455 460Lys Ala Gln Gly
Arg Ile Tyr Gly Lys Ile Lys Glu Glu Asn Phe Val465 470 475 480Ser
Pro Lys Glu Glu Val Lys Leu Glu Ala His Ile Arg Val Pro Ser485 490
495Phe Ala Ala Gly Arg Val Ile Gly Lys Gly Gly Lys Thr Val Asn
Glu500 505 510Leu Gln Asn Leu Ser Ser Ala Glu Val Val Val Pro Arg
Asp Gln Thr515 520 525Pro Asp Glu Asn Asp Gln Val Val Val Lys Ile
Thr Gly His Phe Tyr530 535 540Ala Cys Gln Val Ala Gln Arg Lys Ile
Gln Glu Ile Leu Thr Gln Val545 550 555 560Lys Gln His Gln Gln Gln
Lys Ala Leu Gln Ser Gly Pro Pro Gln Ser565 570 575Arg Arg
Lys327DNAArtificialPrimer 3gctaaagtga ggatggtgat tatcact
27432DNAArtificialPrimer 4actaacaaag ttttcttctt taatttttcc at
32529DNAArtificialProbe 5accagaggct cagttcaagg ctcagggaa
29619DNAArtificialPrimer 6gaaggtgaag gtcggagtc
19720DNAArtificialPrimer 7gaagatggtg atgggatttc
20820DNAArtificialProbe 8caagcttccc gttctcagcc 20
* * * * *