U.S. patent application number 15/488743 was filed with the patent office on 2017-08-24 for biomarkers of therapeutic responsiveness.
This patent application is currently assigned to Meso Scale Technologies, LLC.. The applicant listed for this patent is Meso Scale Technologies, LLC.. Invention is credited to Simone Barbero, Eli N. Glezer, Anu Mathew.
Application Number | 20170242012 15/488743 |
Document ID | / |
Family ID | 51259726 |
Filed Date | 2017-08-24 |
United States Patent
Application |
20170242012 |
Kind Code |
A1 |
Barbero; Simone ; et
al. |
August 24, 2017 |
BIOMARKERS OF THERAPEUTIC RESPONSIVENESS
Abstract
The present invention relates to methods of diagnosing breast
cancer in a patient, as well as methods of monitoring the
progression of breast cancer 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: |
Barbero; Simone;
(Germantown, MD) ; Glezer; Eli N.; (Del Mar,
CA) ; Mathew; Anu; (North Potomac, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Meso Scale Technologies, LLC. |
Rockville |
MD |
US |
|
|
Assignee: |
Meso Scale Technologies,
LLC.
Rockville
MD
|
Family ID: |
51259726 |
Appl. No.: |
15/488743 |
Filed: |
April 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14168629 |
Jan 30, 2014 |
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15488743 |
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61759431 |
Feb 1, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/52 20130101;
G01N 2333/916 20130101; C12Q 1/42 20130101; G01N 33/57415 20130101;
A61K 31/5377 20130101; G01N 2333/912 20130101; C12Q 1/485
20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61K 31/5377 20060101 A61K031/5377 |
Goverment Interests
STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH
[0002] This invention was made with federal support under
HHSN261201000075C 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 breast cancer, said method comprising (a)
obtaining a test sample from a patient undergoing said treatment
regimen for breast cancer; (b) measuring a level of a biomarker in
said test sample, wherein said biomarker comprises (i) total and
phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof; (c) comparing said level to a normal control
level of said biomarker; and (d) evaluating from said comparing
step (c) whether said patient is responsive to said treatment
regimen.
2. A method for evaluating the efficacy of a treatment regimen in a
patient diagnosed with breast cancer, said method comprising (a)
ordering a test comprising a measurement of a level of a biomarker
in a test sample obtained from a patient undergoing said treatment
regimen for breast cancer, wherein said biomarker comprises (i)
total and phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof; (b) comparing said level to a normal control
level of said biomarker; and (c) evaluating from said comparing
step (b) whether said patient is responsive to said treatment
regimen.
3. A method of administering a treatment regimen to a patient in
need thereof for treating breast cancer, comprising: (a) obtaining
a test sample from a patient undergoing said treatment regimen for
breast cancer; (b) measuring a level of a biomarker in said test
sample, wherein said biomarker comprises (i) total and
phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof; (c) comparing said level to a normal control
level of said biomarker; (d) evaluating from said comparing step
(c) whether said patient is responsive to said treatment regimen;
and (e) adjusting said treatment regimen based on said evaluating
step (d).
4. A method of administering a treatment regimen to a patient in
need thereof for treating breast cancer, comprising: (a) obtaining
a test sample from a patient prior to the commencement of said
treatment regimen for breast cancer; (b) measuring a level of a
biomarker in said test sample, wherein said biomarker comprises (i)
total and phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof; (c) comparing said level to a normal control
level of said biomarker; (d) evaluating from said comparing step
(c) whether said patient will be responsive to said treatment
regimen; and (e) administering said treatment regimen based on said
evaluating step (d).
5. A method of administering a treatment regimen to a patient in
need thereof for treating breast cancer, comprising: (a) evaluating
a level of a biomarker in a test sample obtained from a patient
undergoing said treatment regimen for breast cancer relative to a
normal control level of said biomarker, wherein said biomarker
comprises (i) total and phosphorylated isoforms of Akt, MEK, mTOR,
GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,
and (iii) combinations thereof; and (b) adjusting said treatment
regimen based on said evaluating step (a).
6. A method of administering a treatment regimen to a patient in
need thereof for treating breast cancer, comprising: (a) evaluating
a level of a biomarker in a test sample obtained from a patient
prior to the commencement of said treatment regimen for breast
cancer relative to a normal control level of said biomarker,
wherein said biomarker comprises (i) total and phosphorylated
isoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof,
(ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof; and
(b) administering said treatment regimen based on said evaluating
step (a).
7. A method of treating breast cancer, comprising administering a
VEGF-inhibitor to a patient whose levels of a biomarker for RCC or
metastatic melanoma were determined to be at least 1.5-fold
greater/less than those values in a normal control, wherein said
biomarker of breast cancer is selected from (i) total and
phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof.
8. The method of claim 1 wherein said measuring step comprises
conducting a multiplexed assay measurement of a plurality of said
biomarkers in said test sample, wherein said multiplexed assay
measurement is conducted using one reaction volume comprising said
test sample.
9. The method of claim 1 wherein said method comprises measuring
levels of two or more biomarkers.
10. The method of claim 9 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.
11. The method of claim 1, wherein said treatment regimen comprises
administration of a therapeutic agent comprising gefitinib.
12. The method of claim 1 further comprising one or more additional
measuring steps including: (x) measuring a baseline level(s) of
said biomarker before said treatment regimen is initiated, and said
evaluating step further comprises comparing said level and said
baseline level; and (y) measuring an interim level of said
biomarker during said treatment regimen and said evaluating step
further comprises comparing said level, said interim level and said
baseline level.
13. 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 breast cancer.
14. 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 breast cancer.
15. The method of claim 1 further comprising determining from said
level of said biomarker the disease progression of breast
cancer.
16.-22. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of co-pending
application Ser. No. 14/168,629, filed Jan. 30, 2014, which claims
priority of U.S. Provisional Application No. 61/759,431 filed on
Feb. 1, 2013, the entire contents of which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0003] This application relates to assay methods useful in the
detection and treatment of breast cancer.
BACKGROUND OF THE INVENTION
[0004] 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.
SUMMARY OF THE INVENTION
[0005] The invention provides a method for evaluating the efficacy
of a treatment regimen in a patient diagnosed with breast cancer,
said method comprising
[0006] (a) obtaining a test sample from a patient undergoing said
treatment regimen for breast cancer;
[0007] (b) measuring a level of a biomarker in said test sample,
wherein said biomarker comprises (i) total and phosphorylated
isoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof,
(ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof;
[0008] (c) comparing said level to a normal control level of said
biomarker; and
[0009] (d) evaluating from said comparing step (c) whether said
patient is responsive to said treatment regimen.
[0010] The invention further provides a method for evaluating the
efficacy of a treatment regimen in a patient diagnosed with breast
cancer, said method comprising
[0011] (a) ordering a test comprising a measurement of a level of a
biomarker in a test sample obtained from a patient undergoing said
treatment regimen for breast cancer, wherein said biomarker
comprises (i) total and phosphorylated isoforms of Akt, MEK, mTOR,
GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,
and (iii) combinations thereof;
[0012] (b) comparing said level to a normal control level of said
biomarker; and
[0013] (c) evaluating from said comparing step (b) whether said
patient is responsive to said treatment regimen.
[0014] Another embodiment of the invention is a method of
administering a treatment regimen to a patient in need thereof for
treating breast cancer, comprising:
[0015] (a) obtaining a test sample from a patient undergoing said
treatment regimen for breast cancer;
[0016] (b) measuring a level of a biomarker in said test sample,
wherein said biomarker comprises (i) total and phosphorylated
isoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof,
(ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof;
[0017] (c) comparing said level to a normal control level of said
biomarker;
[0018] (d) evaluating from said comparing step (c) whether said
patient is responsive to said treatment regimen; and
[0019] (e) adjusting said treatment regimen based on said
evaluating step (d).
[0020] Still further, the invention contemplates a method of
administering a treatment regimen to a patient in need thereof for
treating breast cancer, comprising:
[0021] (a) obtaining a test sample from a patient prior to the
commencement of said treatment regimen for breast cancer;
[0022] (b) measuring a level of a biomarker in said test sample,
wherein said biomarker comprises (i) total and phosphorylated
isoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof,
(ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof;
[0023] (c) comparing said level to a normal control level of said
biomarker;
[0024] (d) evaluating from said comparing step (c) whether said
patient will be responsive to said treatment regimen; and
[0025] (e) administering said treatment regimen based on said
evaluating step (d).
[0026] An alternative or additional embodiment of the invention is
a method of administering a treatment regimen to a patient in need
thereof for treating breast cancer, comprising:
[0027] (a) evaluating a level of a biomarker in a test sample
obtained from a patient undergoing said treatment regimen for
breast cancer relative to a normal control level of said biomarker,
wherein said biomarker comprises (i) total and phosphorylated
isoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof,
(ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof;
and
[0028] (b) adjusting said treatment regimen based on said
evaluating step (a).
[0029] Moreover, the invention includes a method of administering a
treatment regimen to a patient in need thereof for treating breast
cancer, comprising:
[0030] (a) evaluating a level of a biomarker in a test sample
obtained from a patient prior to the commencement of said treatment
regimen for breast cancer relative to a normal control level of
said biomarker, wherein said biomarker comprises (i) total and
phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof; and
[0031] (b) administering said treatment regimen based on said
evaluating step (a).
[0032] The invention also provides a kit for the analysis of a
breast cancer panel comprising
[0033] (a) a multi-well assay plate comprising a plurality of
wells, each well comprising at least four discrete binding domains
to which capture antibodies to the following human analytes are
bound: (i) total and phosphorylated isoforms of Akt, MEK, mTOR,
GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,
and (iii) combinations thereof;
[0034] (b) in one or more vials, containers, or compartments, a set
of labeled detection antibodies specific for said human analytes;
and
[0035] (c) in one or more vials, containers, or compartments, a set
of calibrator proteins.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 shows the fractional marker levels (to 0 h time
point) for a representative set of biomarkers.
[0037] FIGS. 2(a)-(d) show the relative levels of optimal
biomarkers as a function of IC50 values for the 1 hour time point
(cut off for sensitivity is 1 uM). Each point on the graphs
represents a cell line.
[0038] FIG. 3 shows the relative levels of the combined markers
phospho-Akt, phospho-GSK3beta, phospho-MEK, and phospho-mTOR as a
function of IC50 values for the 1 hour time point. Each point on
the graph represents the sum of the relative levels of each marker
shown in FIG. 2(a)-(d) for one cell line as a function of its 1050
value for gefitinib.
DETAILED DESCRIPTION OF THE INVENTION
[0039] 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.
[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] 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
breast cancer or a patient having a predisposition to breast
cancer. The patient may or may not exhibit symptoms associated with
one or more of these conditions.
[0042] "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.
[0043] 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. Aside from non-melanoma skin cancer, breast cancer
is the most common cancer among women in the United States. It is
also one of the leading causes of cancer death among women of all
races and Hispanic origin populations. About 1 in 8 (12%) women in
the US will develop invasive breast cancer during their lifetime.
The American Cancer Society estimates that in 2013 about 232,340
new cases of invasive breast cancer will be diagnosed in women,
about 64,640 new cases of carcinoma in situ (CIS) will be diagnosed
(CIS is non-invasive and is the earliest form of breast cancer),
and about 39,620 women will die from breast cancer. Breast cancer
is the second leading cause of cancer death in women, second only
to lung cancer. The chance that breast cancer will be responsible
for a woman's death is about 1 in 36 (about 3%). Death rates from
breast cancer have been declining since about 1989, with larger
decreases in women younger than 50. These decreases are believed to
be the result of earlier detection through screening and increased
awareness, as well as improved treatment.
[0044] The levels of pharmacodynamic markers can be assessed to
determine the effects of investigational agents, assaying tumors
directly or surrogate tissues such as plasma. The ultimate goal is
to incorporate predictive pharmacodynamic markers in early clinical
studies of new oncology drugs. This would allow early evaluation of
investigational agents based on human pharmacology data in a `real
time` setting. The biomarkers identified herein can be used for
cancer diagnostics, e.g., to predict (prior to treatment) and/or
determine (after commencement of treatment) whether a cancer is
resistant to a specific course of treatment. The following
biomarkers were identified as valuable in the diagnosis and
prediction of responsiveness to treatment for breast cancer: (i)
total and phosphorylated forms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof. The invention includes the use of these
biomarkers to indicate if treatment with a therapeutic regimen
targeting EGFR tyrosine kinase activity results in responsive or
non-responsive outcomes.
[0045] Accordingly, the present invention includes a method and kit
configured to measure the levels of one or more of (i) total and
phosphorylated isoforms of Akt, MEK, mTOR, GSK3beta, and
combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN, and (iii)
combinations thereof to determine if a tumor is responsive and/or
non-responsive to treatment with drugs targeting EGFR tyrosine
kinase activity (e.g., gefitinib). In addition, the invention also
relates to methods and kits to detect abnormal EGFR tyrosine kinase
activity in a patient and/or to detect an alteration in EGFR
tyrosine kinase activity by measuring one or more of the biomarkers
identified above and comparing those levels to a normal control
and/or a baseline level to determine if an abnormality or
alteration is present in a patient sample.
[0046] Therefore, the invention provides a method for evaluating
the efficacy of a treatment regimen in a patient diagnosed with
breast cancer, said method comprising (a) measuring a level of a
biomarker in a test sample obtained from a patient undergoing said
treatment regimen for breast cancer, wherein said biomarker is
selected from the group consisting of (i) total and phosphorylated
isoforms of Akt, MEK, mTOR, GSK3beta, and combinations thereof,
(ii) p70S6K, eIF4EBP1, PTEN, and (iii) combinations thereof; and
(b) evaluating from said level whether said patient is responsive
to said treatment regimen.
[0047] The methods 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 HER2/neu, ER, PR, Ki67, and combinations thereof. Additional
diagnostic methods can be used in combination with the panels
described herein, as referenced in Misek et al, Int J. Proteomics,
Vol. 2011, Article ID 343582, pages 1-9, the disclosure of which is
incorporated herein by reference in its entirety.
[0048] 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 MEK and phosphorylated MEK; total
GSK3.beta. and phosphorylated GSK3.beta.; and total mTOR and
phosphorylated mTOR.
[0049] The level(s) of the various biomarkers identified herein may
reflect the responsiveness or non-responsiveness of breast cancer
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 breast cancer, 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 Breast cancer. 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.
[0050] 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 regimens used in cancer treatment. In one embodiment,
the therapeutic regimen comprises administration of a therapeutic
agent that modulates one or more biological activities and the
level(s) of said one or more biomarkers indicate modulation of said
biological activities by said therapeutic agent. In particular, the
biological activity includes EGFR tyrosine kinase activity and the
therapeutic agent may be an agonist or an antagonist of such
signaling pathway(s). In a preferred embodiment, the therapeutic
agent is gefitinib (Iressa), and it can be administered alone or in
combination with one or more additional therapeutic agents, e.g.,
trastuzumab, aromasin, anastrazole, taxotere (or another taxane),
doxorubicin, adriamycin, Cytoxan, methotrexate, fluorouracil, and
combinations thereof.
[0051] 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 gefitinib, one suitable therapeutic
regimen comprises administering the drug once 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. 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.
[0052] 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.
[0053] Alternatively, the measuring step may comprise measuring a
level(s) of a biomarker before a therapeutic regimen is initiated
to predict whether breast cancer 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.
[0054] 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 breast cancer.
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 breast cancer.
[0055] 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
breast cancer. 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 breast cancer; 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 breast cancer; or iii) an algorithm based on the
levels of the multiple markers is used to determine if breast
cancer is present.
[0056] As described herein, the measured levels of one or more
biomarkers may be used to detect or monitor cancer (e.g., breast
cancer) 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.
[0057] In addition, the methods of the present invention may be
used in combination with other methods of diagnosing breast cancer
in a patient. In one embodiment, the patient may also be subjected
to one or more diagnostic tools designed to detect breast cancer.
For example, imaging methods may be used to provide images of the
breast to look for tumors. In addition, a biopsy may be performed.
Imaging methods that may be performed include ultrasound, computed
tomography (CT) scan and magnetic resonance imaging (MRI).
[0058] 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.
[0059] 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 breast cancer. 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.
[0060] 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, .sigma..sub.D 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.
[0061] 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.
[0062] 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.
[0063] 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 0.9.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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 al., "A Latex Bead-Based Flow
Cytometric Immunoassay Capable of Simultaneous Typing of Multiple
Pneumococcal Serotypes (Multibead Assay)" Olin. 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, L E. et al., "Simultaneous Quantification of Six
Human Cytokines in a Single Sample Using Microparticle-based Flow
Cytometric Technology," Olin. Chem (1999) 45:1693-1694).
[0070] 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
(MULTI-SPOT.RTM. and MULTI-ARRAY.RTM. plates and SECTOR.RTM.
instruments, Meso Scale Discovery, a division of Meso Scale
Diagnostics, LLC, Rockville, Md.).
[0071] The present invention relates to a kit for the analysis of a
panel of target analytes. The kit is preferably configured to
conduct a multiplexed assay of two or more of the following
analytes: (i) total and phosphorylatedisoforms of Akt, MEK, mTOR,
GSK3beta, and combinations thereof, (ii) p70S6K, eIF4EBP1, PTEN,
and (iii) combinations thereof. The kit can include (a) a single
panel arrayed on a multi-well plate which is configured to be used
in an electrochemiluminescence assay, as well as (b) associated
consumables, e.g., detection antibodies, calibrators, and optional
diluents and/or buffers. Alternatively, the multi-well plates and
associated consumables can be provided separately.
[0072] The panel is preferably configured in a multi-well assay
plate including a plurality of wells, each well having an array
with "spots" or discrete binding domains. Preferably, the array
includes one, four, seven, ten, sixteen, or twenty-five binding
domains, and most preferably, the array includes one, four, seven,
or ten binding domains. A capture antibody to each analyte is
immobilized on a binding domain in the well and that capture
antibody is used to detect the presence of the target analyte in an
immunoassay. Briefly, a sample suspected of containing that analyte
is added to the well and if present, the analyte binds to the
capture antibody at the designated binding domain. The presence of
bound analyte on the binding domain is detected by adding labeled
detection antibody. The detection antibody also binds to the
analyte forming a "sandwich" complex (capture
antibody-analyte-detection antibody) on the binding domain.
[0073] The multiplexed immunoassay kits described herein allow a
user to simultaneously quantify multiple biomarkers. The panels are
selected and optimized such that the individual assays function
well together. The sample may require dilution prior to being
assayed. Sample dilutions for specific sample matrices of interest
are optimized for a given panel to minimize sample matrix effects
and to maximize the likelihood that all the analytes in the panel
will be within the dynamic range of the assay. In a preferred
embodiment, all of the analytes in the panel are analyzed with the
same sample dilution in at least one sample type. In another
preferred embodiment, all of the analytes in a panel are measured
using the same dilution for most sample types.
[0074] For a given panel, the detection antibody concentration and
the number of labels per protein (UP ratio) for the detection
antibody are adjusted to bring the expected levels of all analytes
into a quantifiable range at the same sample dilution. If one wants
to increase the high end of the quantifiable range for a given
analyte, then the L/P can be decreased and/or the detection
antibody concentration is decreased. On the other hand, if one
wants to increase the lower end of the quantifiable range, the L/P
can be increased, the detection antibody concentration can be
increased if it is not at the saturation level, and/or the
background signal can be lowered.
[0075] Calibration standards for use with the assay panels are
selected to provide the appropriate quantifiable range with the
recommended sample dilution for the panel. The calibration
standards have known concentrations of one of more of the analytes
in the panel. Concentrations of the analytes in unknown samples are
determined by comparison to these standards. In one embodiment,
calibration standards comprise mixtures of the different analytes
measured by an assay panel. Preferably, the analyte levels in a
combined calibrator are selected such that the assay signals for
each analyte are comparable, e.g., within a factor of two, a factor
of five or a factor of 10. In another embodiment, calibration
standards include mixtures of analytes from multiple different
assay panels.
[0076] A calibration curve may be fit to the assay signals measured
with calibration standards using, e.g., curve fits known in the art
such as linear fits, 4-parameter logistic (4-PL) and 5-parameter
(5-PL) fits. Using such fits, the concentration of analytes in an
unknown sample may be determined by backfitting the measured assay
signals to the calculated fits. Measurements with calibration
standards may also be used to determine assay characteristics such
as the limit of detection (LOD), limit of quantification (LOQ),
dynamic range, and limit of linearity (LOL).
[0077] A kit can include the following assay components: a
multi-well assay plate configured to conduct an immunoassay for one
of the panels described herein, a set of detection antibodies for
the analytes in the panel (wherein the set comprises individual
detection antibodies and/or a composition comprising a blend of one
or more individual detection antibodies), and a set of calibrators
for the analytes in the panel (wherein the set comprises individual
calibrator protein compositions and/or a composition comprising a
blend of one or more individual calibrator proteins). The kit can
also include one of more of the following additional components: a
blocking buffer (used to block assay plates prior to addition of
sample), an antibody diluent (used to dilute stock detection
antibody concentrations to the working concentration), an assay
diluent (used to dilute samples), a calibrator diluent (used to
dilute or reconstitute calibration standards) and a read buffer
(used to provide the appropriate environment for detection of assay
labels, e.g., by an ECL measurement). The antibody and assay
diluents are selected to reduce background, optimize specific
signal, and reduce assay interference and matrix effect. The
calibrator diluent is optimized to yield the longest shelf life and
retention of calibrator activity. The blocking buffer should be
optimized to reduce background. The read buffer is selected to
yield the appropriate sensitivity, quantifiable range, and slowest
off-rate.
[0078] The reagent components of the kit can be provided as liquid
reagents, lyophilized, or combinations thereof, diluted or
undiluted, and the kit includes instructions for appropriate
preparation of reagents prior to use. In a preferred embodiment, a
set of detection antibodies are included in the kit comprising a
plurality of individual detection antibody compositions in liquid
form. Moreover, the set of calibrators provided in the kit
preferably comprise a lyophilized blend of calibrator proteins.
Still further, the kit includes a multi-well assay plate that has
been pre-coated with capture antibodies and exposed to a
stabilizing treatment to ensure the integrity and stability of the
immobilized antibodies.
[0079] As part of a multiplexed panel development, assays are
optimized to reduce calibrator and detection antibody non-specific
binding. In sandwich immunoassays, specificity mainly comes from
capture antibody binding. Some considerations for evaluating
multiplexed panels include: (a) detection antibody non-specific
binding to capture antibodies is reduced to lower background of
assays in the panel, and this can be achieved by adjusting the
concentrations and LIP of the detection antibodies; (b)
non-specific binding of detection antibodies to other calibrators
in the panel is also undesirable and should be minimized; (c)
non-specific binding of other calibrators in the panel and other
related analytes should be minimized; if there is calibrator
non-specific binding, it can reduce the overall specificity of the
assays in the panel and it can also yield unreliable results as
there will be calibrator competition to bind the capture
antibody.
[0080] Different assays in the panel may require different
incubation times and sample handling requirements for optimal
performance. Therefore, the goal is to select a protocol that's
optimized for most assays in the panel. Optimization of the assay
protocol includes, but is not limited to, adjusting one or more of
the following protocol parameters: timing (incubation time of each
step), preparation procedure (calibrators, samples, controls,
etc.), and number of wash steps.
[0081] The reagents used in the kits, e.g., the detection and
capture antibodies and calibrator proteins, are preferably
subjected to analytical testing and meet or exceed the
specifications for those tests. The analytical tests that can be
used to characterize kit materials include but are not limited to,
CIEF, DLS, reducing and/or non-reducing EXPERION, denaturing
SOS-PAGE, non-denaturing SOS-PAGE, SEC-MALS, and combinations
thereof. In a preferred embodiment, the materials are characterized
by CIEF, DLS, and reducing and non-reducing EXPERION. One or more
additional tests, including but not limited to denaturing SDS-PAGE,
non-denaturing SOS-PAGE, SEC-MALS, and combinations thereof, can
also be used to characterize the materials. In a preferred
embodiment, the materials are also subjected to functional testing,
i.e., a binding assay for the target analyte, as well as one or
more characterization tests, such as those listed above. If the
materials do not meet or exceed the specifications for the
functional and/or characterization tests, they can be subjected to
additional purification steps and re-tested. Each of these tests
and the metrics applied to the analysis of raw materials subjected
to these tests are described below:
[0082] Capillary Isoelectric Focusing (CIEF) is a technique
commonly used to separate peptides and proteins, and it is useful
in the detection of aggregates. During a CIEF separation, a
capillary is filled with the sample in solution and when voltage is
applied, the ions migrate to a region where they become neutral
(pH=pl). The anodic end of the capillary sits in acidic solution
(low pH), while the cathodic end sits in basic solution (high pH).
Compounds of equal isoelectric points (pl) are "focused" into sharp
segments and remain in their specific zone, which allows for their
distinct detection based on molecular charge and isoelectric point.
Each specific antibody solution will have a fingerprint CIEF that
can change over time. When a protein solution deteriorates, the
nature of the protein and the charge distribution can change.
Therefore, CIEF is a particularly useful tool to assess the
relative purity of a protein solution and it is a preferred method
of characterizing the antibodies and calibrators in the plates and
kits described herein. The metrics used in CIEF include pl of the
main peak, the pl range of the solution, and the profile shape, and
each of these measurements are compared to that of a reference
standard.
[0083] Dynamic Light Scattering (DLS) is used to probe the
diffusion of particulate materials either in solution or in
suspension. By determining the rate of diffusion (the diffusion
coefficient), information regarding the size of particles, the
conformation of macromolecular chains, various interactions among
the constituents in the solution or suspension, and even the
kinetics of the scatterers can be obtained without the need for
calibration. In a DLS experiment, the fluctuations (temporal
variation, typically in a .mu.s to ms time scale) of the scattered
light from scatterers in a medium are recorded and analyzed in
correlation delay time domain. Like CIEF, each protein solution
will generate a fingerprint DLS for the particle size and it's
ideally suited to detect aggregation. All IgGs, regardless of
binding specificity, will exhibit the same DLS particle size. The
metrics used to analyze a protein solution using DLS include
percentage polydispersity, percentage intensity, percentage mass,
and the radius of the protein peak. In a preferred embodiment, an
antibody solution meets or exceeds one or more of the following DLS
specifications: (a) radius of the antibody peak: 4-8 nm (antibody
molecule size); (b) polydispersity of the antibody peak: <40%
(measure of size heterogeneity of antibody molecules); (c)
intensity of the antibody peak: >50% (if other peaks are
present, then the antibody peak is the predominant peak); and (d)
mass in the antibody peak: >50%.
[0084] Reducing and non-reducing gel electrophoresis are techniques
well known in the art. The EXPERION.TM. (Bio-Rad Laboratories,
Inc., www.bio-rad.com) automated electrophoresis station performs
all of the steps of gel-based electrophoresis in one unit by
automating and combining electrophoresis, staining, destaining,
band detection, and imaging into a single step. It can be used to
measure purity. Preferably, an antibody preparation is greater 50%
pure by Experion, more preferably, greater than 75% pure, and most
preferably greater than 80% pure. Metrics that are applied to
protein analysis using non-reducing Experion include percentage
total mass of protein, and for reducing Experion they include
percentage total mass of the heavy and light chains in an antibody
solution, and the heavy to light chain ratio.
[0085] Multi-Angle Light Scattering (MALS) detection can be used in
the stand-alone (batch) mode to measure specific or non-specific
protein interactions, as well as in conjunction with a separation
system such as flow field flow fractionation (FFF) or size
exclusion chromatography (SEC). The combined SEC-MALS method has
many applications, such as the confirmation of the oligomeric state
of a protein, quantification of protein aggregation, and
determination of protein conjugate stoichiometry. Preferably, this
method is used to detect molecular weight of the components of a
sample.
[0086] As used herein, a lot of kits comprise a group of kits
comprising kit components that meet a set of kit release
specifications. A lot can include at least 10, at least 100, at
least 500, at least 1,000, at least 5,000, or at least 10,000 kits
and a subset of kits from that lot are subjected to analytical
testing to ensure that the lot meets or exceeds the release
specifications. In one embodiment, the release specifications
include but are not limited to kit processing, reagent stability,
and kit component storage condition specifications. Kit processing
specifications include the maximum total sample incubation time and
the maximum total time to complete an assay using the kit. Reagent
stability specifications include the minimum stability of each
reagent component of the kit at a specified storage temperature.
Kit storage condition specifications include the range of storage
temperatures for all components of the kit, the maximum storage
temperature for frozen components of the kit, and the maximum
storage temperature for non-frozen components of the kit. A subset
of kits in a lot is reviewed in relation to these specifications
and the size of the subset depends on the lot size. In a preferred
embodiment, for a lot of up to 300 kits, a sampling of 4-7 kits are
tested; for a lot of 300-950 kits, a sampling of 8-10 kits are
tested; and for a lot of greater than 950 kits, a sampling of 10-12
kits are tested. Alternatively or additionally, a sampling of up to
1-5% preferably up to 1-3%, and most preferably up to 2% is
tested.
[0087] In addition, each lot of multi-well assay plates is
preferably subjected to uniformity and functional testing. A subset
of plates in a lot is subjected to these testing methods and the
size of the subset depends on the lot size. In a preferred
embodiment, for a lot of up to 300 plates, a sampling of 4-7 plates
are tested; for a lot of 300-950 plates, a sampling of 8-10 plates
are tested; and for a lot of greater than 950 plates, a sampling of
10-12 plates are tested. Alternatively or additionally, a sampling
of up to 1-5% preferably up to 1-3%, and most preferably up to 2%
is tested. The uniformity and functional testing specifications are
expressed in terms of % CV, Coefficient of Variability, which is a
dimensionless number defined as the standard deviation of a set of
measurements, in this case, the relative signal detected from
binding domains across a plate, divided by the mean of the set.
[0088] One type of uniformity testing is protein A/G testing.
Protein A/G binding is used to confirm that all binding domains
within a plate are coupled to capture antibody. Protein A/G is a
recombinant fusion protein that combines IgG binding domains of
Protein A and protein G and it binds to all subclasses of human
IgG, as well as IgA, IgE, IgM and, to a lesser extent, IgD. Protein
A/G also binds to all subclasses of mouse IgG but not mouse IgA,
IgM, or serum albumin, making it particularly well suited to detect
mouse monoclonal IgG antibodies without interference from IgA, IgM,
and serum albumin that might be present in the sample matrix.
Protein A/G can be labeled with a detectable moiety, e.g., a
fluorescent, chemiluminescent, or electrochemiluminescent label,
preferably an ECL label, to facilitate detection. Therefore, if
capture antibody is adhered to a binding domain of a well, it will
bind to labeled protein A/G, and the relative amount of capture
antibody bound to the surface across a plate can be measured.
[0089] In addition to the uniformity testing described above, a
uniformity metric for a subset of plates within a lot can be
calculated to assess within-plate trending. A uniformity metric is
calculated using a matrix of normalized signals from protein A/G
and/or other uniformity or functional tests. The raw signal data is
smoothed by techniques known in the art, thereby subtracting noise
from the raw data, and the uniformity metric is calculated by
subtracting the minimum signal in the adjusted data set from the
maximum signal.
[0090] In a preferred embodiment, a subset of plates in a lot is
subjected to protein A/G and functional testing and that subset
meet or exceed the following specifications:
TABLE-US-00001 TABLE 3(a) Plate Metrics Preferred Specification for
a subset of 96 well Metric multi-well plates Average intraplate CV
.ltoreq.10% Maximum intraplate CV .ltoreq.13% Average Uniformity
.ltoreq.25% Maximum Uniformity .ltoreq.37% CV of intraplate
averages .ltoreq.18% Signal, lower boundary >1500 Signal, upper
boundary <10.sup.(6)
[0091] As disclosed in U.S. Pat. No. 7,842,246 to Wohlstadter et
al., the disclosure of which is incorporated herein by reference in
its entirety, each plate consists of several elements, e.g., a
plate top, a plate bottom, wells, working electrodes, counter
electrodes, reference electrodes, dielectric materials, electrical
connects, and assay reagents. The wells of the plate are defined by
holes/openings in the plate top. The plate bottom can be affixed,
manually or by automated means, to the plate top, and the plate
bottom can serve as the bottom of the well. Plates may have any
number of wells of any size or shape, arranged in any pattern or
configuration, and they can be composed of a variety of different
materials. Preferred embodiments of the invention use industry
standard formats for the number, size, shape, and configuration of
the plate and wells. Examples of standard formats include 96, 384,
1536, and 9600 well plates, with the wells configured in
two-dimensional arrays. Other formats may include single well
plates (preferably having a plurality of assay domains that form
spot patterns within each well), 2 well plates, 6 well plates, 24
well plates, and 6144 well plates. Each well of the plate includes
a spot pattern of varying density, ranging from one spot within a
well to 2, 4, 7, 9, 10, 16, 25, etc., as described hereinabove.
[0092] Each plate is assembled according to a set of preferred
specifications. In a preferred embodiment, a plate bottom meets or
exceeds the following specifications:
TABLE-US-00002 TABLE 3(b) Plate bottom specifications 96-well
(round well) specifications in Parameter inches Length range (C to
C)* 3.8904-3.9004 (A1-A12 and H1-H12)** Width range (C to C)
2.4736-2.4836 (A1-A12 and H1-H12) Well to well spacing
0.3513-0.3573 *C to C well distance is the center of spot to center
of spot distance between the outermost wells of a plate.
[0093] In a further preferred embodiment, the plate also meets or
exceeds defined specifications for alignment of a spot pattern
within a well of the plate. These specifications include three
parameters: (a) .DELTA.x, the difference between the center of the
spot pattern and the center of the well along the x axis of the
plate (column-wise, long axis); (b) .DELTA.y, the difference
between the center of the spot pattern and the center of the well
along the y axis of the plate (row-wise, short axis); and (c)
.alpha., the counter-clockwise angle between the long axis of the
plate bottom and the long axis of the plate top of a 96-well plate.
In a preferred embodiment, the plate meets or exceeds the following
specifications: .DELTA.x.ltoreq.0.2 mm, .DELTA.y.ltoreq.0.2 mm, and
.alpha..ltoreq.0.1.degree..
[0094] The following non-limiting examples serve to illustrate
rather than limit the present invention.
Examples
Measurement of Biomarkers Indicative of Gefitinib Sensitivity in
the Treatment of Breast Cancer
[0095] Multiplex immunoassay kits were used for detection of total
and/or phosphorylated biomarkers (supplied by Meso Scale
Diagnostics, LLC., Rockville, 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. (or arbitrary
units) 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.
[0096] The following cell lines were used, representing different
breast cancer subtypes and having known differences in sensitivity
to gefitinib as indicated by their IC50 values. Cells were treated
with 1 uM gefitinib.
TABLE-US-00003 TABLE 4 Cell Line Sub-type IC50 (.mu.M) HER2 ER
MDA-MB-175 Luminal 0.03 - + BT-474 HER2 amplified 0.13 + - HCC1954
HER2 amplified 0.23 + - SKBR3 HER2 amplified 0.34 + - SUM190 HER2
amplified 0.38 + - MDA-MB-468 Basal/EGFR amplified 0.78 - - UACC812
HER2 amplified 1.25 + - T47D Luminal 1.7 - + CAL 51 Basal/vimentin+
3.3 - - MDA-MB-453 HER2 amplified 5.7 + - ZR75-1 Luminal 7 - +
HCC1937 Basal 10 - - EFM19 Luminal 10 - + BT20 Basal >10 - -
MCF7 Luminal >10 - + MDA-MB-231 Basal/vimentin+ >10 - - CAMA1
Luminal >10 - +
[0097] 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 pl MSD read buffer to
each well; (8) read plate immediately on MSD 516000 Reader
(supplied by Meso Scale Diagnostics, LLC).
[0098] Fractional marker levels (to 0 h time point) for a
representative set of biomarkers is shown in FIG. 1. The levels of
several markers (e.g., phospho-Akt, phospho-GSK3beta, phospho-MEK,
and phospho-mTOR, were decreased correlating with IC50 values.
[0099] The fractional levels of each biomarker (relative to the t=0
h values) in each cell line were correlated to the known gefitinib
IC50 values for the cells lines. Table 5 summarizes the correlation
coefficients for each of the markers at each of the time points
analyzed across all of the cell lines' IC50 values. The sums of the
correlation coefficients for each marker suggest which markers
change in levels over multiple time points indicating a persistent
response to drug. The top four markers were found to be
phospho-Akt, phospho-GSK3beta, phospho-MEK, and phospho-mTOR.
TABLE-US-00004 TABLE 5 Correlation coefficients for changes in
levels of each marker versus IC50 values for the 17 breast cancer
cell lines evaluated at individual time points. The sums of the
correlation coefficients for each marker are shown. p- p- p- p- p-
p- p-Akt MEK GSK3? mTOR mTOR p70S6K MEK 4EBP1 Akt EGFR IGF1R p70S6K
VEGF 10 min 0.70 0.59 0.49 0.24 -0.16 0.36 -0.14 0.31 -0.05 -0.18
0.01 -0.10 0.19 0.5 h 0.75 0.71 0.54 0.48 0.35 0.50 0.31 0.29 0.43
0.41 0.20 0.18 0.60 1 h 0.79 0.62 0.63 0.66 0.59 0.56 0.47 0.58
0.17 0.63 0.50 0.20 0.37 8 h 0.79 0.57 0.69 0.65 0.44 0.55 0.55
0.34 0.32 0.50 0.25 0.14 0.24 24 h 0.71 0.56 0.56 0.51 0.42 0.21
0.41 0.04 0.47 -0.19 0.24 0.38 0.17 48 h 0.64 0.62 0.58 0.61 0.78
0.16 0.57 0.51 0.69 0.57 0.47 0.75 -0.01 SUM 4.38 3.68 3.49 3.16
2.43 2.35 2.17 2.07 2.04 1.73 1.67 1.55 1.55 p- VEGFR2 PTEN Raptor
GSK3? Rictor PTEN EGFR ERK PI3K p-IGF1R 4EBP1 cMet 10 min 0.23 0.02
0.06 -0.41 -0.19 -0.20 -0.23 0.16 -0.37 0.07 -0.13 -0.30 0.5 h 0.48
0.17 -0.01 -0.37 0.18 -0.02 -0.01 0.03 -0.45 0.13 -0.02 0.07 1 h
0.04 0.41 -0.05 0.29 0.10 0.38 0.24 0.13 0.06 0.07 0.04 8 h 0.22
0.25 0.07 0.15 -0.12 0.04 -0.06 -0.26 0.25 -0.20 -0.36 0.07 24 h
0.21 0.23 0.00 0.27 0.01 0.00 -0.18 -0.05 -0.20 -0.09 -0.41 48 h
0.12 0.17 0.62 0.52 0.31 0.00 0.21 -0.17 0.25 -0.09 0.25 0.15 SUM
1.30 1.24 0.69 0.47 0.30 0.21 -0.02 -0.16 -0.26 -0.29 -0.29
-0.38
[0100] The relative levels of optimal biomarkers as a function of
IC50 values for the 1 hour time point are illustrated in FIGS.
2(a)-(d) (cut off for sensitivity is 1 uM). Each point on the
graphs represents a cell line. The relative levels of the combined
markers phospho-Akt, phospho-GSK3beta, phospho-MEK, and
phospho-mTOR as a function of IC50 values for the 1 hour time point
is illustrated in FIG. 3. Each point on the graph represents the
sum of the relative levels of each marker shown in FIG. 2(a)-(d)
for one cell line as a function of its 1050 value for
gefitinib.
[0101] 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.
* * * * *
References