U.S. patent application number 13/318174 was filed with the patent office on 2012-02-23 for biomarkers of therapeutic responsiveness.
This patent application is currently assigned to MESO SCALE TECHNOLOGIES, LLC. Invention is credited to Eli N. Glezer, Anu Mathew.
Application Number | 20120046197 13/318174 |
Document ID | / |
Family ID | 43032765 |
Filed Date | 2012-02-23 |
United States Patent
Application |
20120046197 |
Kind Code |
A1 |
Glezer; Eli N. ; et
al. |
February 23, 2012 |
BIOMARKERS OF THERAPEUTIC RESPONSIVENESS
Abstract
The present invention relates to methods of diagnosing a kidney
disorder in a patient, as well as methods of monitoring the
progression of a kidney disorder and/or methods of monitoring a
treatment protocol of a therapeutic agent or a therapeutic regimen.
The invention also relates to assay methods used in connection with
the diagnostic methods described herein.
Inventors: |
Glezer; Eli N.; (Del Mar,
CA) ; Mathew; Anu; (North Potomac, MD) |
Assignee: |
MESO SCALE TECHNOLOGIES,
LLC
Gaithersburg
MD
|
Family ID: |
43032765 |
Appl. No.: |
13/318174 |
Filed: |
April 29, 2010 |
PCT Filed: |
April 29, 2010 |
PCT NO: |
PCT/US2010/032879 |
371 Date: |
October 31, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61215071 |
May 1, 2009 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/7.1;
435/7.4; 436/501; 506/7 |
Current CPC
Class: |
G01N 2800/52 20130101;
G01N 33/543 20130101; G01N 33/57438 20130101 |
Class at
Publication: |
506/9 ; 506/7;
436/501; 435/7.1; 435/7.4 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/566 20060101 G01N033/566; G01N 33/573 20060101
G01N033/573; C40B 30/00 20060101 C40B030/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
[0001] This invention was made with federal support under
HHSN261200700051C awarded by the National Cancer Institute. The
U.S. government has certain rights in the invention.
Claims
1. A method for evaluating the efficacy of a treatment regimen in a
patient diagnosed with renal cell carcinoma (RCC), said method
comprising (a) measuring a level of a biomarker in a test sample
obtained from a patient undergoing said treatment regimen for RCC,
wherein said biomarker is selected from the group consisting of
total Akt, total Erk1/2, total Met, total GSK3b, total Hif1a, total
p21, total AMPKa1, total VEGF, total PlGF, total VEGFR-1/Flt-1,
phosphorylated Akt, phosphorylated Erk1/2, phosphorylated. Met,
phosphorylated STAT3, phosphorylated GSK3b, and phosphorylated
AMPKa1; and (b) evaluating from said level whether said patient is
responsive to said treatment regimen.
2. The method of claim 1 wherein said method comprises measuring
levels of two or more biomarkers.
3. The method of claim 2 wherein said measuring step comprises
measuring levels of a first biomarker and an additional biomarker,
wherein said first biomarker is a total form of a biomarker and
said additional biomarker is a phosphorylated form of said
biomarker.
4. The method of claim 3 wherein said measuring step comprises
measuring levels of a pair of first and additional biomarkers
selected from the group consisting of (a) total Akt and
phosphorylated Akt; (b) total Erk1/2 and phosphorylated Erk1/2; (c)
total Met and phosphorylated Met; (d) total GSK3b and
phosphorylated GSK3b; and (e) total AMPKa1 and phosphorylated
AMPKa1.
5. A method of claim 1 wherein said biomarker is selected from the
group consisting of total Akt, phosphorylated. STAT3, total p21,
total VEGF, total PlGF, total VEGFR-1/Flt-1, phosphorylated Erk1/2,
p21 and phosphorylated AMPKa1.
6. A method of claim 5 wherein said phosphorylated AMPKa1 is
phosphorylated at amino acid 174.
7. A method of any one of claims 2 to 5 wherein said level(s)
reflect responsiveness or non-responsiveness to said treatment
regimen.
8. A method of claim 1 wherein said treatment regimen comprises
administration of a therapeutic agent that modulates one or more
biological activities and/or one or more signaling pathways and the
level(s) of said one or more biomarkers indicate modulation of said
biological activities and/or said signaling pathways by said
therapeutic agent.
9. A method of claim 8 wherein said signaling pathways are selected
from the group consisting of the VEGF-signaling pathway and
Raf/Ras/Erk/Mek signaling pathway.
10. A method of claim 9 wherein said therapeutic agent is an
agonist of said signaling pathways.
11. A method of claim 9 wherein said therapeutic agent is an
antagonist of said signaling pathways.
12. A method of claim 9 wherein said signaling pathway is the
VEGF-signaling pathway.
13. A method of claim 9 wherein said therapeutic agent is a
multi-kinase inhibitor.
14. A method of claim 13 wherein said therapeutic agent is selected
from the group consisting of sorafenib, sunitinib, and
cediranib.
15. A method of claim 14 wherein said therapeutic agent is
sorafenib.
16. A method of any one of claims 14 to 15 wherein said therapeutic
agent causes a reduction in tumor vasculature.
17. A method of claim 1 wherein said measuring and evaluating steps
are carried out by a point of care device and said method further
comprises providing a report to an end-user, by said device,
whether said patient is responsive to said therapeutic regimen.
18. A method of claim 1 further comprising measuring a baseline
level(s) of said biomarker before said therapeutic regimen is
initiated, and said evaluating step further comprises comparing
said level and said baseline level.
19. A method of claim 18 further comprising measuring an interim
level of said biomarker during said therapeutic regimen and said
evaluating step further comprises comparing said level, said
interim level and said baseline level.
20. The method of claim 1, wherein said evaluating step comprises
comparing said level of said biomarker to a detection cut-off
level, wherein said level above said detection cut-off level is
indicative of RCC.
21. The method of claim 1, wherein said evaluating step comprises
comparing said level of said biomarker to a detection cut-off
level, wherein said level below said detection cut-off level is
indicative of RCC.
22. The method of claim 1 further comprising determining from said
level of said biomarker the disease progression of RCC.
23. The method of any one of the preceding claims wherein said
measuring step(s) are conducted on a single sample.
24. The method of any one of the preceding claims wherein said
measuring step(s) employ a multiplexed assay.
25. The method of any one of the preceding claims wherein said
measuring step(s) are conducted in a single assay chamber.
26. The method of claim 25 wherein said assay chamber is a single
well of an assay plate.
27. The method of claim 25 wherein said assay chamber is a
cartridge.
28. The method of any one of the preceding claims wherein said
sample is selected from the group consisting of blood, peripheral
blood mononuclear cells (PBMC), isolated blood cells, serum and
plasma.
29. The method of any one of the preceding claims wherein said
sample is selected from a group consisting of biopsy tissue,
intestinal mucosa, saliva, cerebral spinal fluid, and urine.
30. The method of any one of the preceding claims wherein said
level(s) are measured using an immunoassay.
Description
FIELD OF THE INVENTION
[0002] This application relates to assay methods useful in the
detection and treatment of renal cell carcinoma (RCC).
BACKGROUND OF THE INVENTION
[0003] Challenges in the field of oncology include the lack of
efficient means for early cancer detection and for specific cancer
subtyping and for measuring and/or predicting responsiveness to
therapy. There is a need for new cancer biomarkers that can provide
early and specific diagnosis of cancer and enable targeted therapy
and prognosis. The need for new diagnostics has been the impetus
behind many initiatives targeting the discovery and development of
new biomarkers for cancer. The hope is that the identification of
suitable biomarkers will allow for the development of early cancer
detection screening tests and will lead to improved cancer therapy
and a reduction in the mortality associated with many cancers.
[0004] Kinases are specialized proteins that function within
intracellular communication networks known as signal transduction
pathways. Preclinical studies have shown that these pathways are
important in the development of tumor vasculature and in the
proliferation of tumor cells, leading to tumor growth and
metastases. Therefore, by blocking the kinases involved in these
signaling pathways, tumor growth and proliferation may be
controlled. Kinases are located on multiple levels of signaling
pathways. Receptor tyrosine kinases are located upstream in the
signaling pathway of tumor vasculature (e.g., VEGFR and PDGFR) and
tumor cells (e.g., Kit and FLT-3). Serine/threonine kinases are
located downstream in the signaling pathway within the cells of
tumors and tumor vasculature (e.g., RAF/MEK/ERK).
[0005] Multiple kinase inhibition targets various signaling
pathways in tumor cells and tumor vasculature. For example,
preclinical studies have shown that by providing upstream blockade
of VEGF and PDGF receptors in the VEGF-signaling pathway as well as
downstream blockade of the RAF/MEK/ERK pathway, sorafenib
(Nexavar.RTM., Bayer Healthcare Pharmaceuticals) simultaneously
decreases both angiogenesis and tumor cell proliferation, which
blocks tumor cell growth. Moreover, sorafenib treatment causes a
reduction in tumor vasculature in xenograft studies (Chang et al.
2007, Wilhelm et al, 2004). Further, an inhibitor of VEGFR-2 and 3,
and PDGFR-13 signaling pathways, sorafenib is one of a few
VEGF-targeted therapies that have demonstrated clinical utility in
RCC treatment (reviewed by Garcia and Rini 2007). In addition,
sunitinib malate (Sutent.RTM., Pfizer, Inc.), has been approved for
the treatment of advanced RCC. Sunitinib blocks the tyrosine kinase
activities of VEGFR2, PDGFR.beta., and c-Kit, thereby inhibiting
angiogenesis and cell proliferation. This agent also inhibits the
phosphorylation of Fms-related tyrosine kinase 3 (FLT3), another
receptor tyrosine kinase expressed by some leukemic cells. Finally,
cediranib (AZD2171, AstraZeneca) is a potent inhibitor of VEGFR-1,
VEGFR-2, VEGFR-3, and in particular VEGFR-2, the predominant
receptor through which VEGF exerts its effect on angiogenesis.
Cediranib is in clinical development for the treatment of advanced.
RCC, among other cancers.
[0006] Elevated levels of phospho-Akt have been correlated with
poor prognosis in RCC (Horiguchi et al. 2003). One of the
downstream targets of Akt is the p21 protein, which becomes more
stable after phosphorylation (Li et al. 2002). p21 is a prognostic
marker in clear cell RCC, in which elevated nuclear and cytosolic
p21 levels are indicative of poor prognosis (Weiss et al.
2007).
[0007] Another tyrosine kinase receptor associated with RCC is
c-Met. Miyata et al. (2006) suggested that phospho c-Met is a
predictor of metastasis and survival in RCC patients.
[0008] AMPK is a stress-responsive protein kinase that is activated
under conditions such as hypoxia. Cross-talk between AMPK and Akt
is essential for angiogenesis under hypoxic conditions (Nagata et
al. 2003), which would occur during anti-angiogenic therapy. It has
been suggested that tumor AMPK activity may stabilize VEGF mRNA
under conditions of nutrient deprivation, which suggests that AMPK
activation is a pro-angiogenic signal. The activity of another
factor, STAT3, is also reportedly induced with hypoxia in RCC
leading to VEGF expression (Jung et al. 2005).
[0009] There is a complex relationship between GSK3.beta. activity
and sorafenib in melanoma cells (Panka et al. 2008). Sorafenib
activates GSK3.beta. in these cells, which in turn undermines the
lethality of the drug in the cells by acting as an anti-apoptotic
agent.
SUMMARY OF THE INVENTION
[0010] The present invention provides a method for evaluating the
efficacy of a treatment regimen in a patient diagnosed with renal
cell carcinoma (RCC), wherein the method includes the following
steps:
[0011] (a) measuring a level of a biomarker in a test sample
obtained from a patient undergoing a treatment regimen for RCC,
wherein the biomarker is selected from the group consisting of
total Akt, total Erk1/2, total Met, total GSK3b, total Hif1a, total
p21, total AMPKa1, total VEGF, total PlGF, total VEGFR-1/Flt-1,
phosphorylated Akt, phosphorylated Erk1/2, phosphorylated Met,
phosphorylated STAT3, phosphorylated GSK3b, and phosphorylated
AMPKa1; and
[0012] (b) evaluating from that level whether the patient is
responsive to the treatment regimen.
[0013] The method may include measuring a level of two or more
biomarkers.
[0014] In one embodiment, the biomarkers are selected from the
group consisting of total Akt, phosphorylated STAT3, total p21,
total VEGF, total PlGF, total VEGFR-1/Flt-1, phosphorylated Erk1/2,
p21 and phosphorylated AMPKa1. And in a further embodiment, the
phosphorylated AMPKa1 is phosphorylated at amino acid 174 (AMPKa1
p174).
[0015] Still further, the measuring step may include measuring
levels of a first biomarker and an additional biomarker, wherein
the first biomarker is a total form of a biomarker and the
additional biomarker is a phosphorylated form of that biomarker.
For example, the measuring step may include measuring levels of a
pair of a first and an additional biomarker selected from the group
consisting of (a) total Akt and phosphorylated Akt; (b) total
Erk1/2 and phosphorylated Erk1/2; (c) total Met and phosphorylated
Met; (d) total GSK3b and phosphorylated GSK3b; and (e) total AMPKa1
and phosphorylated AMPKa1.
[0016] The method of the invention may be used to evaluate the
responsiveness or non-responsiveness of a patient to a treatment
regimen, e.g., the biomarker level(s) reflect responsiveness or
non-responsiveness to a treatment regimen.
[0017] The treatment regimen may comprise administration of a
therapeutic agent that modulates one or more biological activity
and/or one or more signaling pathway and the level(s) of the
biomarker indicates modulation of the biological activity and/or
signaling pathway by the therapeutic agent. In one embodiment, the
signaling pathway is selected from the group consisting of the
VEGF-signaling pathway and Raf/Ras/Erk/Mek signaling pathway. Still
further, the therapeutic agent is an agonist of a given signaling
pathway, or alternatively, the therapeutic agent is an antagonist
of a signaling pathway.
[0018] In one embodiment, the therapeutic agent is a multi-kinase
inhibitor, for example, sorafenib, sunitinib, and cediranib. In a
specific embodiment, the therapeutic agent is sorafenib. Still
further, the therapeutic agent may cause a reduction in tumor
vasculature.
[0019] Moreover, the invention provides a method in which the
measuring and evaluating steps are carried out by a point of care
device and the method further includes providing a report to an
end-user, by the device, concerning whether the patient is
responsive to the therapeutic regimen.
[0020] The method of the present invention may include measuring a
baseline level(s) of the biomarker before the therapeutic regimen
is initiated, and the evaluating step further comprises comparing
the level and the baseline level. Moreover, the method may further
comprise measuring an interim level of the biomarker during the
therapeutic regimen and the evaluating step further comprises
comparing the level, the interim level and the baseline level.
[0021] Still further, the evaluating step of the instant method may
include comparing the level of the biomarker to a detection cut-off
level, wherein the level above the detection cut-off level is
indicative of RCC. Alternatively, the evaluating step comprises
comparing the level of the biomarker to a detection cut-off level,
wherein the level below the detection cut-off level is indicative
of RCC.
[0022] The measuring step of the method of the instant invention
may be conducted on a single sample. Moreover, the measuring step
may employ a multiplexed assay. The measuring step(s) may be
conducted in a single assay chamber, and the assay chamber may be a
single well of an assay plate or the assay chamber may be a
cartridge.
[0023] The sample used in the method of the present invention may
be selected from the group consisting of blood, peripheral blood
mononuclear cells (PBMC), isolated blood cells, serum and plasma.
Still further, the sample may be biopsy tissue, intestinal mucosa,
saliva, cerebral spinal fluid, or urine.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. The articles "a" and "an" are used herein to refer to one
or to more than one (i.e., to at least one) of the grammatical
object of the article. By way of example, "an element" means one
element or more than one element.
[0025] The present invention includes the use of biomarkers for
cancer diagnostics including the use of biomarkers for predicting
(prior to treatment) and/or determining (after commencement of
treatment) whether a cancer is resistant to a specific course of
treatment. Suitable biomarkers including total and/or
phosphorylated Akt, Erk1/2, Met, STAT3, GSK3.beta., Hif1.alpha.,
p21, AMPKa1, VEGF, PlGF, VEGFR-1/Flt-1. The invention includes the
use of these biomarkers (or other proteins associated with the VEGF
and/or RAF/MEK/ERK signaling pathways) to indicate if treatment
with a therapeutic regimen targeting the VEGF signaling pathways
and/or the RAF/MEK/ERK pathway results in responsive or
non-responsive outcomes. Analysis of human tumor xenograft samples
from mice in assays for total and/or phosphorylated Akt, Erk1/2,
Met, STAT3, GSK3.beta., Hif1.alpha., p21, AMPKalpha1, VEGF, PlGF,
VEGFR-1/Flt-1, and other pharmacodynamic factors shows that these
proteins are biomarkers for RCC and that the levels of these
proteins can be used to determine the responsiveness or
non-responsiveness of cancers (including RCC) to such treatment.
Accordingly, the present invention includes a method comprising
measuring the levels of one or more of total and/or phosphorylated
Akt, Erk1/2, Met, STAT3, GSK3.beta., Hif1.alpha., p21, AMPKa1,
VEGF, PlGF, VEGFR-1/Flt-1 to determine if a tumor is responsive
and/or non-responsive to treatment with drugs targeting the VEGF or
RAF/MEK/ERK pathways (e.g., sorafenib).
[0026] Therefore, the invention provides a method for evaluating
the efficacy of a treatment regimen in a patient diagnosed with
renal cell carcinoma (RCC), said method comprising (a) measuring a
level of a biomarker in a test sample obtained from a patient
undergoing said treatment regimen for RCC, wherein said biomarker
is selected from the group consisting of total Akt, total Erk1/2,
total Met, total GSK3b, total Hif1a, total p21, total AMPKa1, total
VEGF, total PlGF, total VEGFR-1/Flt-1, phosphorylated Akt,
phosphorylated Erk1/2, phosphorylated Met, phosphorylated STAT3,
phosphorylated GSK3b, and phosphorylated AMPKa1; and (b) evaluating
from said level whether said patient is responsive to said
treatment regimen. The method may include measuring a level of two
or more biomarkers, or a panel of three or more such biomarkers.
The panel may further comprise one or more additional biomarkers
selected from the group consisting of carbonic anhydrase IX (CAIX),
X-linked inhibitor of apoptosis protein (MAP), minichromosome
maintenance 2 (Mcm2), 5100, S100A10, brain fatty acid binding
protein (B-FABP), p53, COX-2, B7-H1, B7-H4, osteopontin (OPN),
Ki-67, miR-28, miR-185, miR-27 and let-7f-2 (four human
micro-RNAs), beta-subunit of human chorionic gonadotropin (b-hCG),
thymidine phosphorylase, IMPS, pro-MMP-7, MDR-1/P-glycoprotein,
VCAM-1, IFN-.alpha., (Gimenez et al., Future Oncol. (2009) 5(2):
197-205), sVEGFR-3, VEGF-C (Garcia et al., Curr. Opin. Oncol.
(2009) 21: 266-71), CA-XII, CXCR3, CXCR4, IGF-1, B7-H3, PD-1,
Bcl-2, PTEN, Cyclin A, p27, Skp2, EpCAM/KSA, EMA, E-Cad,
alpha-catenin, Cad-6, surviving, EphA2, Smac/DIABLO, PCNA,
Caveolin-1, AR, CD44, Annexin II, Gelsolin, Vimentin, CA-125,
aberrant DNA methylation, Na-K ATPase .alpha. 1 subunit, vitamin D
receptor, retinoid X receptor (Lane et al., Urol. Clin. N. Am.
(2008) 35: 613-25), gamma-enolase, pyruvate kinase type M2, iNOS,
TS, CD10, CD154, AgNOR, Gp200, markers of the mTOR pathway (e.g.,
phosphorylated protein kinase B (AKT) and phosphorylated S6 kinase)
(Tununguntla et al., J. Urol. (2008) 179: 2096-2102), C-reactive
protein, CD57+, Fas, APAF1, and DAPK1 (Ljungberg, Curr. Op. Urol.
(2007), 17: 303-08.
[0027] In one embodiment, the method includes measuring a level of
a first biomarker and an additional biomarker, wherein the first
biomarker is a total form of a biomarker and the additional
biomarker is a phosphorylated form of that biomarker. Diagnosis of
the presence or state of a cancer could be based on the absolute
levels of one or both of these forms. Alternatively, the diagnosis
could be based on the ratio of phosphorylated to total forms (i.e,
based on the fraction of a specific biomarker that is present in a
phosphorylated form). In one example, the method may include
measuring a level of total Akt as the first biomarker and measuring
a level of phosphorylated Akt as the additional biomarker.
Similarly, the method may comprise measuring one or more of the
following pairs of first and additional biomarkers, i.e., total and
phosphorylated biomarkers: total Erk1/2 and phosphorylated Erk1/2;
total Met and phosphorylated Met; total GSK3b and phosphorylated
GSK3b; and total AMPKa1 and phosphorylated AMPKa1.
[0028] In one embodiment, the biomarker measured in the method of
the present invention is selected from total Akt, phosphorylated
STATS, total p21, total VEGF, total PlGF, total VEGFR-1/Flt-1,
phosphorylated Erk1/2, p21 and phosphorylated AMPKa1. In one
specific embodiment, the measured biomarker is AMPKa1
phosphorylated at amino acid 174.
[0029] The level(s) of the various biomarkers identified herein may
reflect the responsiveness or non-responsiveness of a renal cell
carcinoma to a given treatment regimen. A response to a therapeutic
regimen includes a detectable reduction to some extent of one or
more of the symptoms of RCC, including, but not limited to: (1)
reduction in the number of cancer cells; (2) reduction in tumor
size; (3) inhibition (i.e., slowing to some extent, preferably
stopping) of cancer cell infiltration into peripheral organs; (4)
inhibition (i.e., slowing to some extent, preferably stopping) of
tumor metastasis; (5) inhibition, to some extent, of tumor growth;
(6) relieving or reducing to some extent one or more of the
symptoms associated with the disorder; and/or (7) increasing, to
some extent, the overall survival of a patient relative to that
observed for the standard of care for RCC. A response to a
therapeutic regimen may also comprise maintenance of a therapeutic
benefit, including, but not limited to (1) inhibiting an increase
in the number of cancer cells; (2) inhibiting an increase in tumor
size; (3) inhibiting cancer cell infiltration into peripheral
organs; (4) inhibiting tumor metastases; (5) relieving or reducing
to some extent one or more of the symptoms associated with the
disorder; and/or (6) inhibiting a recurrence or onset of one or
more of the symptoms associated with the disorder.
[0030] The therapeutic regimen used in the method of the present
invention may include radiation treatment, chemotherapy, treatment
with therapeutic drugs, immune system modulation or other
therapeutic regimes used in cancer treatment. In one embodiment,
the therapeutic regimen comprises administration of a therapeutic
agent that modulates one or more biological activities and/or one
or more signaling pathways and the level(s) of said one or more
biomarkers indicate modulation of said biological activities and/or
said signaling pathways by said therapeutic agent. In particular,
the signaling pathways may include the VEGF-signaling pathway and
Raf/Ras/Erk/Mek signaling pathway and the therapeutic agent may be
an agonist or an antagonist of such signaling pathway(s).
[0031] The therapeutic regimen may include administration of a
therapeutic agent or a combination of therapeutic agents to a
patient one or more times over a given time period. For example, if
the therapeutic agent is sorafenib, one suitable therapeutic
regimen comprises administering the drug twice daily until the
patient is no longer clinically benefiting from treatment or until
unacceptable toxicity occurs. This treatment regimen may be
accompanied by the administration of one or more additional
chemotherapeutic agents or palliative agents. The level(s) of
biomarkers may be measured before treatment, one or more times
during the administration period, and/or after treatment is
suspended. If sunitinib is the selected therapeutic agent, one
example of a suitable therapeutic regimen comprises administration
of the drug once daily for four weeks, followed by a two week
period in which sunitinib is not administered to the patient. This
cycle may be repeated one or more times. This treatment regimen may
also be accompanied by the administration of one or more additional
chemotherapeutic agents or palliative agents. The level(s) of
biomarkers may be measured at one or more time points in the
treatment regimen, e.g., before treatment, one or more times during
the four week administration period, and/or after the four week
administration period. Therefore, the method may include measuring
an interim level of a biomarker during the therapeutic regimen and
the evaluating step further comprises comparing that level, the
interim level and the baseline level.
[0032] In addition, the level of a biomarker may be determined at
any time point before and/or after initiation of treatment. In one
embodiment, the biomarker is used to gauge the efficacy of a
therapeutic regimen. Therefore, the method of the present invention
may include measuring a baseline level(s) of a biomarker before a
therapeutic regimen is initiated, and the evaluating step further
comprises comparing the level and the baseline level. Moreover, the
method may further comprise measuring an interim level of the
biomarker during the therapeutic regimen and the evaluating step
further comprises comparing the level, the interim level and the
baseline level.
[0033] Alternatively, the measuring step may comprise measuring a
level(s) of a biomarker before a therapeutic regimen is initiated
to predict whether a RCC will be responsive or non-responsive to a
given therapeutic regimen. The method may further comprise
modifying the therapeutic regimen based on the level(s) of a
biomarker observed during the measuring step, e.g., increasing or
decreasing the dosage, frequency, or route of administration of a
therapeutic agent, adding an additional therapeutic agent and/or
palliative agent to a treatment regimen, or if the therapeutic
regimen includes the administration of two or more therapeutic
and/or palliative agents, the treatment regimen may be modified to
eliminate one or more of the therapeutic and/or palliative agents
used in the combination therapy.
[0034] Still further, the evaluating step may include comparing the
level of a biomarker to a detection cut-off level, wherein a level
above the detection cut-off level is indicative of RCC.
Alternatively, the evaluating step comprises comparing a level of a
biomarker to a detection cut-off level, wherein a level below the
detection cut-off level is indicative of RCC.
[0035] In one embodiment of the present invention, the level of a
biomarker is compared to a detection cut-off level or range,
wherein the biomarker level above or below the detection cut-off
level (or within the detection cut-off range) is indicative of RCC.
Furthermore, the levels of two or more biomarkers may both be used
to make a determination. For example, i) having a level of at least
one of the markers above or below a detection cut-off level (or
within a detection cut-off range) for that marker is indicative of
RCC; ii) having the level of two or more (or all) of the markers
above or below a detection cut-off level (or within a detection
cut-off range) for each of the markers is indicative of RCC; or
iii) an algorithm based on the levels of the multiple markers is
used to determine if RCC is present.
[0036] As described herein, the measured levels of one or more
biomarkers may be used to detect or monitor cancer (e.g., RCC)
and/or to determine the responsiveness of a cancer to a specific
treatment regimen. The specific methods/algorithms for using
biomarker levels to make these determinations, as described herein,
may optionally be implemented by software running on a computer
that accepts the biomarker levels as input and returns a report
with the determinations to the user. This software may run on a
standalone computer or it may be integrated into the
software/computing system of the analytical device used to measure
the biomarker levels or, alternatively, into a laboratory
information management system (LIMS) into which crude or processed
analytical data is entered. In one embodiment, biomarkers are
measured in a point-of-care clinical device which carries out the
appropriate methods/algorithms for detecting, monitoring or
determining the responsiveness of a cancer and which reports such
determination(s) back to the user.
[0037] In addition, the methods of the present invention may be
used in combination with other methods of diagnosing RCC in a
patient. In one embodiment, the patient may also be subjected to
one or more diagnostic tools designed to detect RCC. For example,
imaging methods may be used to provide images of the kidney to look
for tumors. In addition, a kidney biopsy may be performed. Imaging
methods that may be performed include ultrasound, computed
tomography (CT) scan and magnetic resonance imaging (MRI).
[0038] As used herein, the term "cancer" is intended to mean a
class of diseases characterized by the uncontrolled growth of
aberrant cells, including all known cancers, and neoplastic
conditions, whether characterized as malignant, benign, soft tissue
or solid tumor. In one embodiment, the cancerous condition is
metastatic RCC. An estimated 54,390 new cases and an estimated more
than 13,000 deaths in the United States of RCC were reported in
2008. Of all kidney tumors, 85% are RCC, and of those patients
diagnosed with RCC, 25% present with advanced disease. RCC is
frequently an incidental finding via ultrasonography and CT scan.
Approximately 15% to 48% of new cases are discovered incidentally
and 25% to 30% of patients have metastases at initial
presentation.
[0039] The assays of the present invention may be conducted by any
suitable method. In one embodiment, the measuring step is conducted
on a single sample, and it may be conducted in a single assay
chamber or assay device, including but not limited to a single well
of an assay plate, a single assay cartridge, a single lateral flow
device, a single assay tube, etc.
[0040] As used herein, the term "sample" is intended to mean any
biological fluid, cell, tissue, organ or combinations or portions
thereof, which includes or potentially includes a biomarker of a
disease of interest. For example, a sample can be a histologic
section of a specimen obtained by biopsy, or cells that are placed
in or adapted to tissue culture. A sample further can be a
subcellular fraction or extract, or a crude or substantially pure
nucleic acid molecule or protein preparation. In one embodiment,
the samples that are analyzed in the assays of the present
invention are blood, peripheral blood mononuclear cells (PBMC),
isolated blood cells, serum and plasma. Other suitable samples
include biopsy tissue, intestinal mucosa, saliva, cerebral spinal
fluid, and urine.
[0041] As used herein, a "biomarker" is a substance that is
associated with a particular disease. A change in the levels of a
biomarker may correlate with the risk or progression of a disease
or with the susceptibility of the disease to a given treatment. A
biomarker may be useful in the diagnosis of disease risk or the
presence of disease in an individual, or to tailor treatments for
the disease in an individual (choices of drug treatment or
administration regimes and/or to predict responsiveness or
non-responsiveness to a particular therapeutic regimen). In
evaluating potential drug therapies, a biomarker may be used as a
surrogate for a natural endpoint such as survival or irreversible
morbidity. If a treatment alters a biomarker that has a direct
connection to improved health, the biomarker serves as a "surrogate
endpoint" for evaluating clinical benefit A sample that is assayed
in the diagnostic methods of the present invention may be obtained
from any suitable patient, including but not limited to a patient
suspected of having RCC or a patient having a predisposition to
RCC. The patient may or may not exhibit symptoms associated with
one or more of these conditions.
[0042] As used herein, the term "level" refers to the amount,
concentration, or activity of a biomarker. The term "level" may
also refer to the rate of change of the amount, concentration or
activity of a biomarker. A level can be represented, for example,
by the amount or synthesis rate of messenger RNA (mRNA) encoded by
a gene, the amount or synthesis rate of polypeptide corresponding
to a given amino acid sequence encoded by a gene, or the amount or
synthesis rate of a biochemical form of a biomarker accumulated in
a cell, including, for example, the amount of particular
post-synthetic modifications of a biomarker such as a polypeptide,
nucleic acid or small molecule. The term can be used to refer to an
absolute amount of a biomarker in a sample or to a relative amount
of the biomarker, including amount or concentration determined
under steady-state or non-steady-state conditions. Level may also
refer to an assay signal that correlates with the amount,
concentration, activity or rate of change of a biomarker. The level
of a biomarker can be determined relative to a control marker or an
additional biomarker in a sample. For example, the level of a
phosphorylated protein can be presented as the absolute amount of
the phosphorylated protein or as the ratio of the amount of the
phosphorylated form of the protein to the total amount of the
protein.
[0043] According to one aspect of the invention, the level(s) of
biomarker(s) are measured in samples collected from individuals
clinically diagnosed with, suspected of having or at risk of
developing RCC. Initial diagnosis may have been carried out using
conventional methods, e.g., biopsy or other conventional diagnostic
methods. The level(s) of biomarker(s) are also measured in healthy
individuals. Specific biomarkers valuable in distinguishing between
normal and diseased patients are identified by visual inspection of
the data, for example, by visual classification of data plotted on
a one-dimensional or multidimensional graph, or by using
statistical methods such as characterizing the statistically
weighted difference between control individuals and diseased
patients and/or by using Receiver Operating Characteristic (ROC)
curve analysis. A variety of suitable methods for identifying
useful biomarkers and setting detection thresholds/algorithms are
known in the art and will be apparent to the skilled artisan.
[0044] For example and without limitation, diagnostically valuable
biomarkers may be first identified using a statistically weighted
difference between control individuals and diseased patients,
calculated as
D - N .sigma. D * .sigma. N ##EQU00001##
wherein D is the median level of a biomarker in patients diagnosed
as having, for example, kidney cancer, N is the median (or average)
of the control individuals, op is the standard deviation of D and
.sigma..sub.N is the standard deviation of N. The larger the
magnitude, the greater the statistical difference between the
diseased and normal populations.
[0045] According to one embodiment of the invention, biomarkers
resulting in a statistically weighted difference between control
individuals and diseased patients of greater than, e.g., 1, 1.5, 2,
2.5 or 3 could be identified as diagnostically valuable
markers.
[0046] Another method of statistical analysis for identifying
biomarkers is the use of z-scores, e.g., as described in Skates et
al. (2007) Cancer Epidemiol. Biomarkers Prev. 16(2):334-341.
[0047] Another method of statistical analysis that can be useful in
the inventive methods of the invention for determining the efficacy
of particular candidate analytes, such as particular biomarkers,
for acting as diagnostic marker(s) is ROC curve analysis. An ROC
curve is a graphical approach to looking at the effect of a cut-off
criterion, e.g., a cut-off value for a diagnostic indicator such as
an assay signal or the level of an analyte in a sample, on the
ability of a diagnostic to correctly identify positive or negative
samples or subjects. One axis of the ROC curve is the true positive
rate (TPR, i.e., the probability that a true positive
sample/subject will be correctly identified as positive, or
alternatively, the false negative rate (FNR=1-TPR, the probability
that a true positive sample/subject will be incorrectly identified
as a negative). The other axis is the true negative rate, i.e.,
TNR, the probability that a true negative sample will be correctly
identified as a negative, or alternatively, the false positive rate
(FPR=1-TNR, the probability that a true negative sample will be
incorrectly identified as positive). The ROC curve is generated
using assay results for a population of samples/subjects by varying
the diagnostic cut-off value used to identify samples/subjects as
positive or negative and plotting calculated values of TPR or FNR
and TNR or FPR for each cut-off value. The area under the ROC curve
(referred to herein as the AUC) is one indication of the ability of
the diagnostic to separate positive and negative samples/subjects.
In one embodiment, a biomarker provides an AUC.gtoreq.0.7. In
another embodiment, a biomarker provides an AUC.gtoreq.0.8. In
another embodiment, a biomarker provides an AUC.gtoreq.0.9.
[0048] Diagnostic indicators analyzed by ROC curve analysis may be
a level of an analyte, e.g., a biomarker, or an assay signal.
Alternatively, the diagnostic indicator may be a function of
multiple measured values, for example, a function of the
level/assay signal of a plurality of analytes, e.g., a plurality of
biomarkers, or a function that combines the level or assay signal
of one or more analytes with a patient's scoring value that is
determined based on visual, radiological and/or histological
evaluation of a patient. The multi-parameter analysis may provide
more accurate diagnosis relative to analysis of a single
marker.
[0049] Candidates for a multi-analyte panel could be selected by
using criteria such as individual analyte ROC areas, median
difference between groups normalized by geometric interquartile
range (IQR) etc. The objective is to partition the analyte space to
improve separation between groups (for example, normal and disease
populations) or to minimize the misclassification rate.
[0050] One approach is to define a panel response as a weighted
combination of individual analytes and then compute an objective
function like ROC area, product of sensitivity and specificity,
etc. See e.g., WO 2004/058055, as well as US2006/0205012, the
disclosures of which are incorporated herein by reference in their
entireties.
[0051] Biomarker levels may be measured using any of a number of
techniques available to the person of ordinary skill in the art,
e.g., direct physical measurements (e.g., mass spectrometry) or
binding assays (e.g., immunoassays, agglutination assays and
immunochromatographic assays). The method may also comprise
measuring a signal that results from a chemical reactions, e.g., a
change in optical absorbance, a change in fluorescence, the
generation of chemiluminescence or electrochemiluminescence, a
change in reflectivity, refractive index or light scattering, the
accumulation or release of detectable labels from the surface, the
oxidation or reduction or redox species, an electrical current or
potential, changes in magnetic fields, etc. Suitable detection
techniques may detect binding events by measuring the participation
of labeled binding reagents through the measurement of the labels
via their photoluminescence (e.g., via measurement of fluorescence,
time-resolved fluorescence, evanescent wave fluorescence,
up-converting phosphors, multi-photon fluorescence, etc.),
chemiluminescence, electrochemiluminescence, light scattering,
optical absorbance, radioactivity, magnetic fields, enzymatic
activity (e.g., by measuring enzyme activity through enzymatic
reactions that cause changes in optical absorbance or fluorescence
or cause the emission of chemiluminescence). Alternatively,
detection techniques may be used that do not require the use of
labels, e.g., techniques based on measuring mass (e.g., surface
acoustic wave measurements), refractive index (e.g., surface
plasmon resonance measurements), or the inherent luminescence of an
analyte.
[0052] Binding assays for measuring biomarker levels may use solid
phase or homogenous formats. Suitable assay methods include
sandwich or competitive binding assays. Examples of sandwich
immunoassays are described in U.S. Pat. No. 4,168,146 and U.S. Pat.
No. 4,366,241, both of which are incorporated herein by reference
in their entireties. Examples of competitive immunoassays include
those disclosed in U.S. Pat. No. 4,235,601, U.S. Pat. No. 4,442,204
and U.S. Pat. No. 5,208,535, each of which are incorporated herein
by reference in their entireties.
[0053] Multiple biomarkers may be measured using a multiplexed
assay format, e.g., multiplexing through the use of binding reagent
arrays, multiplexing using spectral discrimination of labels,
multiplexing of flow cytometric analysis of binding assays carried
out on particles, e.g., using the Luminex.RTM. system. Suitable
multiplexing methods include array based binding assays using
patterned arrays of immobilized antibodies directed against the
biomarkers of interest. Various approaches for conducting
multiplexed assays have been described (See e.g., US 20040022677;
US 20050052646; US 20030207290; US 20030113713; US 20050142033; and
US 20040189311, each of which is incorporated herein by reference
in their entireties. One approach to multiplexing binding assays
involves the use of patterned arrays of binding reagents, e.g.,
U.S. Pat. Nos. 5,807,522 and 6,110,426; Delehanty J-B, Printing
functional protein microarrays using piezoelectric capillaries,
Methods Mol. Bio. (2004) 278: 135-44; Lue R Y et al., Site-specific
immobilization of biotinylated proteins for protein microarray
analysis, Methods Mol. Biol. (2004) 278: 85-100; Lovett,
Toxicogenomics: Toxicologists Brace for Genomics Revolution,
Science (2000) 289: 536-537; Berns A, Cancer: Gene expression in
diagnosis, nature (2000), 403, 491-92; Walt, Molecular Biology:
Bead-based Fiber-Optic Arrays, Science (2000) 287: 451-52 for more
details). Another approach involves the use of binding reagents
coated on beads that can be individually identified and
interrogated. See e.g., WO 9926067, which describes the use of
magnetic particles that vary in size to assay multiple analytes;
particles belonging to different distinct size ranges are used to
assay different analytes. The particles are designed to be
distinguished and individually interrogated by flow cytometry.
Vignali has described a multiplex binding assay in which 64
different bead sets of microparticles are employed, each having a
uniform and distinct proportion of two dyes (Vignali, D. A A,
"Multiplexed Particle-Based Flow Cytometric Assays" J. ImmunoL
Meth. (2000) 243: 243-55). A similar approach involving a set of 15
different beads of differing size and fluorescence has been
disclosed as useful for simultaneous typing of multiple
pneumococcal serotypes (Park, M. K et A, "A Latex Bead-Based Flow
Cytometric Immunoassay Capable of Simultaneous Typing of Multiple
Pneumococcal Serotypes (Multibead Assay)" Clin. Diag. Lab ImmunoL
(2000) 7: 4869). Bishop, J E et al. have described a multiplex
sandwich assay for simultaneous quantification of six human
cytokines (Bishop, LE. et al., "Simultaneous Quantification of Six
Human Cytokines in a Single Sample Using Microparticle-based Flow
Cytometric Technology," Clin. Chem (1999) 45:1693-1694).
[0054] A diagnostic test may be conducted in a single assay
chamber, such as a single well of an assay plate or an assay
chamber that is an assay chamber of a cartridge. The assay modules,
e.g., assay plates or cartridges or multi-well assay plates),
methods and apparatuses for conducting assay measurements suitable
for the present invention are described for example, in US
20040022677; US 20050052646; US 20050142033; US 20040189311, each
of which is incorporated herein by reference in their entireties.
Assay plates and plate readers are now commercially available
(MULTISPOT.RTM. and MULTI-ARRAY.RTM. plates and SECTOR.RTM.
instruments, Meso Scale Discovery.RTM., a division of Meso Scale
Diagnostics, LLC, Gaithersburg, Md.).
[0055] The following non-limiting examples serve to illustrate
rather than limit the present invention.
EXAMPLES
Measurement of Biomarkers Indicative of Sorafenib Resistance in the
Treatment of RCC
[0056] A panel of biomarkers was measured in RCC xenograft tissue
extracts from mice. The mice were implanted with human 786-0 RCC
cells and the cells were allowed to grow until a tumor of
approximately 12 mm diameter was formed. The "Set A" group of mice
received no drug treatment prior to removal of the xenograft
tissue. The other two groups of mice--"Set C" and "Set D"--were
treated with sorafenib prior to removal of the xenograft tissue.
The "Set C" group showed sensitivity to the drug, i.e., the tumor
size decreased. The "Set D" developed resistance to the drug and
the tumor size increased to 16 mm in diameter during treatment.
[0057] Multiplex immunoassay kits were used for detection of total
and/or phosphorylated biomarkers (supplied by Meso Scale
Diagnostics, LLC, Gaithersburg, Md.). Levels of each biomarker were
determined by calibration of the assays with were either purified
calibrator proteins or using control cell lysates from
appropriately treated cultured cell lines (e.g., cells subjected to
conditions known to induce or reduce levels of a specific
biomarker). Calibration curves were derived by testing serial
dilutions of the calibrator lysates or purified proteins. Levels of
biomarkers in test samples were back-calculated from the
calibration curves and were expressed in terms of wt. of protein
per weight of tissue extract (for purified calibrators) or in terms
of weight of crude control lysate protein per well (for lysate
calibrators). Titrations of tumor extracts were carried out to
determine the linearity of the assay response to sample dilution
and to select the sample dilution that would be appropriate to use
for each assay panel.
[0058] In general, the assay format was as follows, with minor
alterations for specific assay panels as indicated in the assay
protocols provided with each assay kit (supplied by Meso Scale
Diagnostics, LLC): (1) block MSD MULTI-SPOT.RTM. plate for 1 hour
with appropriate MSD.RTM. blocking solution and wash; (2) add 25
.mu.l assay diluent to each well, if specified; (3) add 25 .mu.l
calibrator, or sample (diluted as appropriate) to each well; (4)
incubate with shaking for 1-3 hours (time as specified) and wash
the well; (5) add 25 .mu.l labeled detection antibody solution to
each well; (6) incubate with shaking for 1-2 hours (time as
specified) and wash the well; (7) add 150 .mu.l MSD read buffer to
each well; (8) read plate immediately on MSD SI6000 Reader
(supplied by Mesa Scale Diagnostics, LLC). The results of these
studies are summarized in Tables 1 and 2 below.
[0059] A significant difference in the level of a biomarker in
sorafenib sensitive and resistant tumors (see the far-right column
in Table 2) indicates that the marker can be used to characterize
the RCC tumor as responsive or non-responsive. The use of specific
profiles of the levels of multiple markers (as shown in Tables 1
and 2) provides a signature pattern of changes in the levels of
specific biomarkers that further strengthens the characterization
of RCC tumors as sorafenib-responsive or non-responsive. The
measurement of this set of markers (or individual markers or
subsets of markets selected from the set of markers) could greatly
improve the effectiveness of treatment with this therapeutic
agent.
TABLE-US-00001 TABLE 1 The average and standard deviations for the
levels of each tested biomarker for tumor tissue extracts from mice
in groups A, C and D. The extracts were diluted to the following
concentrations (in terms of weight of extract protein per well)
prior to analysis: 0.5 ug total protein/well for phospho/total Akt,
phospho/total Erk1/2, total p38 phospho/total Met, and p21; 1 ug
protein/well for phospho/total MEK1/2, phospho/total GSK3.beta.,
and phospho/total AMPK.alpha.1; 2 ug protein/well for phosphoSTAT3;
2.5 ug protein/well for phospho/total Jnk, VEGF, bFGF, PlGF, and
VEGFR-1; and 4 ug protein/well for Hif1.alpha., The biomarker
levels are expressed in the units shown in the leftmost column.
Marker Concentrations +/- SD Tumor Tumor Tumor Group A Group C
Group D (untreated) (treated) (resistant) Akt (ng calibrator lysate
Total 143 +/- 25 142 +/- 43 93 +/- 25 protein/well) Phospho 312 +/-
58 348 +/- 85 354 +/- 84 Erk1/2 (ng calibrator lysate Total 462 +/-
81 577 +/- 212 486 +/- 306 protein/well) Phospho 106 +/- 27 102 +/-
45 206 +/- 103 Jnk (ng calibrator lysate Total 730 +/- 193 983 +/-
356 878 +/- 231 protein/well) Phospho 91 +/- 27 74 +/- 29 110 +/-
22 Met (ng calibrator lysate Total 406 +/- 166 850 +/- 375 595 +/-
137 protein/well) Phospho 15 +/- 7 34 +/- 7 41 +/- 6 Gsk3b (ng
calibrator lysate Total 4360 +/- 55 3771 +/- 1491 3829 +/- 1058
protein/well) Phospho 935 +/- 261 1188 +/- 446 1474 +/- 294 Mek1/2
(ng calibrator lysate Total 332 +/- 59 350 +/- 90 359 +/- 103
protein/well) Phospho 90 +/- 8 116 +/- 21 113 +/- 24 AMPKa1 (pg/ug
tumor Total 20 +/- 2 29 +/- 13 26 +/- 10 protein) p174 5.5 +/- 0.6
7.3 +/- 4.4 3.8 +/- 0.9 p285 17 +/- 2 29 +/- 15 19 +/- 5 STAT3 (ng
calibrator lysate Phospho 60 +/- 32 65 +/- 16 41 +/- 10
protein/well) Hif1a (ng calibrator lysate Total 14 +/- 3 19 +/- 3
14 +/- 3 protein/well) p38 (ng calibrator lysate Total 529 +/- 174
449 +/- 266 428 +/- 227 protein/well) bFGF (pg/ug tumor protein)
Total 2.7 +/- 0.8 2.1 +/- 0.6 2.6 +/- 1.3 VEGF (pg/ug tumor
protein) Total 0.68 +/- 0.51 11.4 +/- 3.7 26 +/- 15 PlGF (pg/ug
tumor protein) Total 0.015 +/- 0.006 0.083 +/- 0.031 0.144 +/-
0.073 VEGFR-1 (pg/ug tumor Total 0.05 +/- 0.03 0.26 +/- 0.08 0.39
+/- 0.14 protein) p21 (pg/ug tumor protein) Total 1.6 +/- 0.4 1.1
+/- 0.3 2.2 +/- 0.9
TABLE-US-00002 TABLE 2 Comparison of the relative level of each
biomarker in the three groups of test mice: untreated (A), treated
and sensitive to sorafenib (C) and treated and resistant to
sorafenib (D). The presence of a significantly different level of a
biomarker in one group relative to another is shown through the use
of an arrow in the appropriate table cell. For example, an arrow
pointing upward in the "Resistant vs. Sensitive" column for a
specific marker, indicates that the sorafenib-resistant mice had
higher levels of that marker than the sensitive mice. The number of
arrows is indicative of the magnitude of the change. Sensitive (C)
Resistant (D) Resistant (D) vs UnTreated vs Untreated vs Sensitive
(A) (A) (C) Akt Total no diff .dwnarw. .dwnarw. Phospho no diff no
diff no diff Erk1/2 Total no diff no diff no diff Phospho no diff
.uparw. .uparw. Jnk Total no diff no diff no diff Phospho no diff
no diff no diff Met Total .uparw. no diff no diff Phospho
.uparw..uparw. .uparw..uparw. no diff Gsk3b Total no diff no diff
no diff Phospho no diff .uparw. no diff Mek1/2 Total no diff no
diff no diff Phospho no diff no diff no diff AMPKa1 Total no diff
no diff no diff p174 no diff .dwnarw. .dwnarw. p285 no diff no diff
no diff STAT Phospho no diff no diff .dwnarw. Hif1a Total no diff
no diff no diff p38 Total no diff no diff no diff bFGF Total no
diff no diff no diff VEGF Total .uparw..uparw. .uparw..uparw.
.uparw. PlGF Total .uparw..uparw. .uparw..uparw. .uparw. VEGFR-1
Total .uparw..uparw. .uparw..uparw. .uparw. p21 Total no diff no
diff .uparw.
[0060] Various publications and test methods are cited herein, the
disclosures of which are incorporated herein by reference in their
entireties. In cases where the present specification and a document
incorporated by reference and/or referred to herein include
conflicting disclosure, and/or inconsistent use of terminology,
and/or the incorporated/referenced documents use or define terms
differently than they are used or defined in the present
specification, the present specification shall control.
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