U.S. patent application number 17/035607 was filed with the patent office on 2021-01-14 for apparatus and method for absolute quantification of biomarkers for solid tumor diagnosis.
The applicant listed for this patent is QUANTICISION DIAGNOSTICS INC.. Invention is credited to Jiandi Zhang.
Application Number | 20210011010 17/035607 |
Document ID | / |
Family ID | 1000005117558 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210011010 |
Kind Code |
A1 |
Zhang; Jiandi |
January 14, 2021 |
APPARATUS AND METHOD FOR ABSOLUTE QUANTIFICATION OF BIOMARKERS FOR
SOLID TUMOR DIAGNOSIS
Abstract
Provided is a method for quantitative analysis of a sample. The
method includes steps of (a) providing a singular marker
representative of one or more features of the sample, the sample
comprising a population of individual units of the marker; (b)
measuring the marker with dot blot analysis, wherein the
quantitation result is an absolute amount of the marker's
population of individual units in the sample, normalized by the
sample volume or by the sample weight; and (c) obtaining an
objective determination of the one or more features of the sample
based on the quantitation result of the marker. Also disclosed is a
reference database and a method to use the reference database for
diagnosing cancer in a patient.
Inventors: |
Zhang; Jiandi; (Chapel Hill,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
QUANTICISION DIAGNOSTICS INC. |
Chapel Hill |
NC |
US |
|
|
Family ID: |
1000005117558 |
Appl. No.: |
17/035607 |
Filed: |
September 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16022259 |
Jun 28, 2018 |
10788484 |
|
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17035607 |
|
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62526425 |
Jun 29, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G16H 50/20 20180101;
G01N 33/54306 20130101; G01N 33/57484 20130101; G01N 33/6893
20130101; G01N 33/574 20130101; G01N 2800/52 20130101 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/68 20060101 G01N033/68; G01N 33/574 20060101
G01N033/574 |
Claims
1. A method for quantitative analysis of a sample, comprising: (a)
providing a singular marker representative of one or more features
of the sample, the sample being a formalin-fixed paraffin-embedded
(FFPE) tissue sample and comprising a population of individual
units of the marker; (b) quantifying the marker extracted from the
FFPE tissue sample with dot blot analysis, wherein the quantitation
result is an absolute amount of the marker's population of
individual units in the sample, normalized by the protein weight;
and (c) obtaining a determination of the one or more features of
the sample based on the quantitation result of the marker.
2. The method of claim 1, wherein the step (b) comprises measuring
the marker with quantitative dot blot (QDB) analysis.
3. The method of claim 2, wherein the step (b) comprises: (b1)
incubating the sample with a solution comprising a binding agent,
wherein: the binding agent is capable of specifically binding to an
individual unit of the marker, and the binding agent is capable of
being quantified by the dot blot analysis; and (b2) measuring the
marker through QDB analysis of the binding agent.
4. The method of claim 3, wherein the marker is a protein marker
and the binding agent is an antibody.
5. The method of claim 4, wherein the antibody is an analyte
specific regent (ASR) antibody.
6. The method of claim 4, wherein the antibody is an in vitro
diagnostics (IVD) antibody.
7. The method of claim 4, wherein the antibody is capable of being
assayed in immunohistochemistry.
8. The method of claim 1, wherein the sample is a biopsy tissue
from a subject.
9. The method of claim 8, wherein the subject is a cancer
patient.
10. The method of claim 9, further comprising (d) evaluating the
patient based on the quantitation result of the marker.
11. The method of claim 10, wherein, in step (d), evaluating the
patient comprises diagnosis and prognosis of a cancer for the
patient.
12. The method of claim 11, wherein the diagnosis and prognosis of
a cancer comprises disease-free survival, overall survival, hazard
ratio, or treatment prediction for the cancer.
13. The method of claim 10, wherein, in step (d), evaluating the
patient is conducted by a commercial kit.
14. A reference database for diagnosing cancer in a patient based
on antibody-based quantitative analysis of a marker in a
formalin-fixed paraffin-embedded_(FFPE) biopsy sample from the
patient, comprising a plurality of reference profiles, each of the
plurality of the reference profile prepared by: (a) providing a
formalin-fixed paraffin-embedded (FFPE)_biopsy sample from a cancer
patient with a known diagnosis; (b) measuring said marker with dot
blot analysis for the biopsy sample, wherein the quantitation
result is an absolute amount of said marker in the biopsy sample,
normalized by the protein weight thereof; and (c) associating the
quantitation result with the known diagnosis of the cancer patient
thereby obtaining a reference profile.
15. The reference database of claim 14, wherein the step (b)
comprises: (b1) incubating the biopsy sample with a solution
comprising a binding agent that specifically binding to said
marker; and (b2) measuring said marker through quantitative dot
blot (QDB) analysis of the binding agent.
16. The reference database of claim 15, wherein the marker is a
protein marker and the binding agent is an antibody.
17. A method for diagnosing cancer in a patient, comprising:
providing a reference database of claim 14; obtaining a biopsy
sample from the patient; measuring the marker used in the reference
database in the biopsy sample with QDB analysis, wherein the
measured result is an absolute amount of the marker in the biopsy
sample; comparing the quantitation result of the marker with that
of each reference profile stored in the reference database; and
identifying a reference profile in the reference database that has
the best match and outputting the known diagnosis associated with
the identified reference profile.
18. The method of claim 17, wherein the cancer is a solid tumor
cancer.
19. A kit for diagnosing cancer in a patient, the kit comprising
the reference database of claim 14.
20. A kit for diagnosing cancer in a patient, the kit comprising a
unit configured to access online the reference database of claim
14.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/022,259, filed Jun. 28, 2018, which claims
the benefit of provisional application No. 62/526,425, filed on
Jun. 29, 2017, the disclosure of which is hereby incorporated by
reference. Further, U.S. application Ser. No. 14/721,205, filed on
May 26, 2015, and U.S. application Ser. No. 15/433,586, filed on
Feb. 15, 2017 (now U.S. Pat. No. 10,295,544), are also hereby
incorporated by reference in their entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present disclosure relates to methods and apparatus for
quantification of biomarker at tissue level for diagnostics purpose
using immunoblot method. Specifically, this disclosure provides
methods and apparatus to measure the expression level of a
biomarker using quantitative dot blot analysis (QDB), and when
combined with a reference database, to provide diagnosis and
prognosis for solid tumors.
BACKGROUND
[0003] Protein analysis is the basis of modern biological research.
It centers on antigen-antibody interaction to measure levels of
antigen of interest under various medical or experimental
conditions. An antigen by definition is a foreign molecule that
triggers the production of an antibody by the immune system when
introduced into the body. The high specificity of the antibody
against a specific antigen makes it a powerful tool in clinical,
pharmaceutical and biomedical research.
[0004] An antigen includes, but not limited to a chemical compound,
a peptide, a protein, an RNA, a DNA, a cell (proteins released in
situ), or a virus particle (proteins released in situ). The
molecule of antigen, as a whole or in part, may be introduced into
a host animal, such as a donkey, a goat, or a rabbit to generate a
large quantity of antibody against the introduced antigen of
interest. Furthermore, the introduced antigen, or part of the
antigen, may have more than one epitopes, thus may generate a
corresponding number of antibodies against the antigen of
interest.
[0005] A typical immunodetection process has three major steps. The
first step is sample application, in which prepared samples
containing an antigen of interest is first bound to a solid phase,
such as nitrocellulose or PVDF membrane, multi-well plate with
protein binding capacity, glass slide in immunohistochemistry
analysis (IHC) or membrane at QDB plate, Zhang et al. "Quantitative
dot blot analysis (QDB), a versatile high throughput immunoblot
method" Oncotarget 2017 Vol. 8, page 58553-58562, which is
incorporated by reference herein in its entirety. The second step
is to form and label the antigen-antibody complex (i.e.,
immunocomplex) of interest. This step involves the sub-steps of
blocking, incubation and washing. In the blocking sub-step,
non-specific protein binding sites on the membrane are blocked
using a blocking buffer to shield them from non-specific protein.
In some case of IHC, slice on the glass slide needs to be further
processed (de-paraffined and antigen retrieval) before the blocking
buffer is applied. After blocking, the membrane is incubated in the
incubation step with antibody against the antigen of interest to
form membrane-bound antigen-antibody complex. The unbound
antibodies are washed away. The antibody used herein is often
commercially available as a pre-labelled antibody. One may also
perform the labeling sub-step on site. In either case, the antibody
shall be labeled, either directly with a reporter, e.g., a reporter
enzyme, or indirectly labelled using a secondary antibody
conjugated with a reporter.
[0006] The third step is detection. Signals emitted by the reporter
enzyme are detected and recorded, which yield information related
to the quantity or quality of the immunocomplex bound on the
membrane.
[0007] Different methods of labeling the antibody in turn
necessitate different corresponding detection methods. For example,
the third step of detection may be a color reaction by visual
inspection or chemiluminescence signals detectable through scanner,
X-ray film or microplate reader, etc. The antibody may also be
fluorescence-labeled and detected through scanner
[0008] Multiple techniques of protein analysis are variations of
this typical immunodetection process. These techniques include, but
not limited to, Western blot analysis, Dot blot analysis,
immunohistochemistry (IHC), Enzyme-linked immunosorbent assay
(ELISA), reverse phase protein microarray (RPPM) and newly
developed Quantitative Dot Blot analysis (QDB) and Zestern
analysis.
[0009] In immunohistochemical analysis, the tissue is first
processed either as paraffin-embedded block or as a frozen tissue,
and is further sliced at certain thickness before the tissue slice
is placed on a slide. Next, the slide is going through a typical
immunodetection process to form immunocomplex between antigen of
interest and detection antibody on the slide. The pre-labeled
antibody is used to allow detection of antigen of interest on the
slide by a pathologist with the aid of microscope. For slice from
paraffin-embedded block, the slice needs to go through further
steps to facilitate antigen-antibody interaction. These steps may
include de-paraffin of the slice, and antigen retrieval step to
expose the antigen for antigen-antibody interaction.
[0010] In clinical practice, IHC is widely used to detect any
changes at protein level of a biomarker for diagnostic or
prognostic purpose. A typical IHC report of a biomarker either
expresses as "+" or "-", or it is further categorized as "0, 1+,
2+, 3+". For example, the expression level of one commonly used
biomarker for breast cancer diagnosis, Human epidermal growth
factor receptor 2 (Her2), is assessed using IHC to determine if
Her2-dependent therapy should be included in the treatment plan.
The IHC results are categorized into four groups: 0 and 1+, 2+, and
3+. 0 and 1+ group is considered as negative, 3+ is considered as
positive while 2+ is considered as equivocal.
[0011] Attempts trying to unite the intensity of staining with the
percentage of cells stained lead to scoring system including H
score (or H index) developed to provide more informative detail
about IHC analysis. In other cases, the intensity of the staining,
for example, strong or weak and the percentages of the cells
stained are both provided. For example, the results can be reported
as (percentage, intensity). However, all these results are
semi-quantitative in nature.
[0012] Although IHC provides valuable morphological information,
and numerous efforts have been invested to standardize the
analytical procedure, its result is still qualitative, affected by
various factors including the process of treatment, the field of
view and inter-observer variability. It is also rather challenging
to adapt this technique into a high throughput format.
[0013] The uncertainty associated with IHC analysis consequently
leads to significantly increased error in diagnosis. In fact, in
Her2's case, there are as much as 30% inconsistency between IHC
with Fluorescence In Situ Hybridization technique (FISH).
[0014] The categorized results of IHC analysis also makes it
difficult for further data analysis. For example, while there are
significant differences among individual patients with positive
results, they are all considered in the same category in clinical
practice. Thus, results from IHC analysis are unable to be used for
extensive data analysis to provide more accurate, more predictive
diagnosis or prognosis as we expected.
[0015] There have been several attempts to measure quantitatively
the biomarker at tissue level with success. For example, Hermark
assay, based on the proximity assay, was able to quantify Her2
levels at breast cancer tissues.sup.4. However, this technique
measures the relative Her2 level among breast cancer patients.
[0016] In the meantime, the absolute Her2 level was achieved with
SRM-MS technique. Using an isotope labeled protein standard, the
absolute Her2 levels were measured, and a cutoff of 740 amole/.mu.g
was proposed based on the Receiving Operative Characteristics (ROC)
analysis.
[0017] Nonetheless, these methods are complicated and labor
intensive, not suitable for high throughput analysis of a large
number of samples. The complex process also leads to significantly
increased operating cost and efforts.
[0018] The recently developed QDB method, on the other hand, has
been demonstrated suitable to process complex lysates prepared
either from cells or tissues in a high throughput format.
Introduction of a protein standard, either in the form of a
recombinant protein, or a purified protein, conveniently translates
this method into an absolute quantitative assay to measure the
absolute content of a specific protein at cellular or tissue
level.
[0019] In this invention, the effort was made to adopt QDB assay in
measuring the expression level of a biomarker at tissue level for
clinical use. In one application of current invention, the IHC
assays currently available for diagnostics purpose can be converted
into absolute quantitation assays with clear advantages of
accurate, quantitative, objective, high throughput, and more
comprehensive to aid clinical diagnosis or prognosis. The absolute
values from QDB analyses can be further processed in large scale
for data analysis to provide more predictive and accurate diagnosis
and prognosis.
SUMMARY OF THE INVENTION
[0020] The present invention provides method to quantify the
expression levels of biomarkers at tissue levels. The evaluation of
biomarkers is quantitatively measured instead of categorized as in
current prevailing methods.
[0021] Consequently, the present invention provides a continuous
result to replace the current prevailing discrete result. Thus, the
present invention opens more opportunities than the current
prevailing method of IHC and FISH.
[0022] One aspect of the present invention relates to a method for
quantitative analysis of a sample. The method includes steps of (a)
providing a singular marker representative of one or more features
of the sample, which contains a population of individual units of
the marker; (b) measuring the marker with dot blot analysis,
wherein the quantitation result is an absolute amount of the
marker's population of individual units in the sample, normalized
by the sample volume or by the sample weight; and (c) obtaining an
objective determination of the one or more features of the sample
based on the quantitation result of the marker.
[0023] The sample can be a tissue from a subject. In one embodiment
of current invention, the tissue refers to a biopsy tissue. In
another embodiment of current invention, the tissue refers to a
frozen tissue. Yet in another embodiment of current invention, the
tissue refers to a Formalin Fixed specimen, and yet in another
embodiment of current invention, the tissue refers to a Formalin
Fixed Paraffin Embedded specimen (FFPE specimen).
[0024] The subject can be a patient. Specifically, the subject can
be a cancer patient.
[0025] On the other hand, the step (b) of the above method can
include measuring the marker with quantitative dot blot (QDB)
analysis. More specifically, the step (b) can include: (b1)
incubating the sample with a solution containing a binding agent,
which is capable of specifically binding to an individual unit of
the marker and is capable of being quantified by the dot blot
analysis; and (b2) measuring the marker through QDB analysis of the
binding agent.
[0026] The binding agent can be capable of being assayed based on
antibody-antigen interaction including immunohistochemistry.
Moreover, the binding agent can be capable of being assayed in flow
cytometry.
[0027] Preferably, the marker is a protein marker and the binding
agent is an antibody. Further, the antibody can be an analyte
specific regent (ASR) antibody, and it can also be an in vitro
diagnostics (IVD) antibody.
[0028] Additionally, the above-described method further includes
step (d) of evaluating the patient based on the quantitative result
of the marker. Particularly, step (d) includes diagnosis and
prognosis of a cancer for the patient. Examples of diagnosis and
prognosis of a cancer for the patient include disease-free
survival, overall survival, Hazard Ratio (HR), or treatment
prediction for the cancer.
[0029] The quantitatively measured biomarker level disclosed in
this invention can be categorized based on mathematical analysis of
available information to facilitate the comparison from this
invention to the current prevailing method, and to help better
explore the clinical significance of the absolute level of a
biomarker.
[0030] One embodiment of current invention is through immunoblot
analysis. The expression level of a biomarker can be measured using
an antibody-based method.
[0031] By defining the linear range of the antibody-based method,
the absolute level of a biomarker can be measured with a protein
standard in the same measurement.
[0032] In one embodiment of current invention, dot blot method can
be used to achieve the absolute level of a biomarker at tissue.
[0033] Purified or recombinant protein is used to establish a
protein standard curve using image-based dot blot analysis. The
image is converted into number through image capture and
analysis.
[0034] The captured signal is used to establish a protein standard
curve.
[0035] Total protein extracted from FFPE block can be used in dot
blot analysis, and the captured signal can be converted into
absolute biomarker level using the referenced standard curved
described above.
[0036] In one embodiment of current invention, the Quantitative Dot
Blot analysis (QDB) method can be used to achieve the absolute
level of a biomarker at tissue level.
[0037] The protein expression level of a biomarker can be measured
relatively through immunoblot assay including QDB method in the
absence of a protein standard. As long as the process of analysis
is well controlled (e.g., with the positive and negative controls),
it provides informative result for reference.
[0038] In QDB analysis, the absolute level of a biomarker can be
measured using a validated detection antibody. There is no
limitation of methods to validate the detection antibody. The
antibody can be validated through Western blot analysis, or it can
be validated based on prior experience. In one embodiment of
current invention, a well-accepted antibody for
immunohistochemistry can be used directly in QDB analysis. For
example, ASR or IVD antibodies can be used in QDB analysis. These
antibodies include, but not limited to, EP3 and 4B5 clones for HER2
protein, MIB1 for ki67 and SP1 for Estrogen Receptor (ER).
Likewise, a detection antibody for other antibody-based assay,
including, but not limiting, flow cytometry, can be used in QDB
analysis.
[0039] A detection antibody (antibody A) can be validated through
comparative study with an existing detection antibody (antibody B).
When more than one sample are analyzed with both antibody A and
antibody B with similar result, antibody A can be considered
validated
[0040] Sample for QDB analysis can be prepared from frozen tissue
or from a formalin-fixed tissue or from a
formalin-fixed-paraffin-embedded tissue block (FFPE block). It can
also be prepared from tissues preserved in other format as long as
the antigen-antibody interaction is allows to happen with the
sample.
[0041] The absolute level of a biomarker from more than one sample
can be combined with relevant clinical information to setup a
protein database (AKA, QDB database). Mathematical analysis of QDB
database provides information for medical use. For example, the
putative association between absolute level of a biomarker and the
disease free survival (DFS) can be explored to provide predictive
clinical prognosis for a patient.
[0042] These and other embodiments of the present invention rely at
least in part upon the finding that the absolute nature of the QDB
analysis allows for combining results from more than one source for
analysis. The continued addition of new information from QDB
analysis allows growing of a QDB database. Information obtained
through analysis of a QDB database provides diagnosis or prognosis
for a patient.
[0043] Protein can be extracted in non-denatured form, or it can be
further denatured through heating and/or reducing reagent. The
non-denatured form should be taken in their broadest context. It
can be in native form; or it may refer a state where the antigen is
either not exposed to reducing agents (e.g., Dithiothreitol (DTT)
or (.beta.-mercaptoethanol) or not exposed to a sufficient amount
of reducing reagent to completely denature the antigen.
[0044] In one embodiment of the current invention, an antigen with
predetermined amount can be included as operational control to
ensure the consistency of experiment. The absolute level of the
antigen needs to be measured each time, and only when the measured
result is within the certain range of the pre-determined amount
before the overall experiment can be accepted as valid.
[0045] The QDB analysis can also be performed using the same lysate
with more than one antibody against the same biomarker. These
antibodies can direct against the same epitope, or against
different epitopes of the biomarker. A cutoff reference can be
developed for these antibodies respectively, and the sample can be
determined either as positive or negative in reference to these
cutoff reference values. Samples agreed by all antibodies can be
accepted as positive or negative, and those with disagreeable
results can be assigned as equivocal.
[0046] In one embodiment of current invention, the absolute levels
of more than one biomarker can be quantified using the same sample,
and the combination of these information together with relevant
clinical information, including, but not limited to, the disease
free survival, the overall survival, the hazard ratio, the side
response, the age, the progression stage of the disease, can be
used to find a pattern, and this pattern can be used for diagnosis
and prognosis purpose.
[0047] In one embodiment of current invention, the absolute level
of a biomarker can be measured with more than one antibody, and the
absolute levels of a biomarker based on each antibody respectively
over relevant clinical information, including, but not limited to,
the disease free survival, the overall survival, the side response,
the hazard ratio, the age, the progression stage of the disease,
can be used to find a pattern respectively, and individual pattern
from different antibodies can be used to provide antibody-specific
information for diagnosis and prognosis purpose.
[0048] Another aspect of the present invention relates to a
reference database for diagnosing cancer in a patient based on
quantitative analysis of a marker in a biopsy sample from the
patient. The reference database includes a plurality of reference
profiles. each of the plurality of the reference profile is
prepared by steps of: (a) providing a biopsy sample from a cancer
patient with a known diagnosis; (b) measuring said marker with dot
blot analysis for the biopsy sample, wherein the quantitation
result is an absolute amount of said marker in the biopsy sample,
normalized by the biopsy sample volume or by the biopsy sample
weight; and (c) associating the quantitation result with the known
diagnosis of the cancer patient thereby obtaining a reference
profile.
[0049] The step (b) described above can include (b1) incubating the
biopsy sample with a solution containing a binding agent that
specifically binding to said marker; and (b2) measuring said marker
through QDB analysis of the binding agent.
[0050] In the above reference database, preferably the marker is a
protein marker and the binding agent is an antibody.
Advantageously, the reference database is stored in a computational
memory chip.
[0051] Yet another aspect of the present invention is directed at a
method for diagnosing cancer in a patient. The method includes
steps of (i) providing a reference database described above, (ii)
obtaining a biopsy sample from the patient; (iii) measuring the
marker used in the reference database in the biopsy sample from
patient with dot blot analysis, the measured result being an
absolute amount of the marker in the biopsy sample; (iv) comparing
the measured result of the marker with that of each reference
profile stored in the reference database; and (v) identifying a
reference profile in the reference database that has the best match
and outputting the known diagnosis associated with the identified
reference profile.
[0052] In the above method, the cancer being diagnosed is solid
tumor.
[0053] Still another aspect of the present invention relates to an
apparatus for diagnosing cancer in a patient. The apparatus
contains the reference database just described.
[0054] Yet another aspect of the present invention relates to a kit
for diagnosing cancer in a patient. The kit contains the above
reference database. Alternatively, the kit contains a unit
configured to access online the above reference database.
[0055] The details of the invention are set forth in the drawing
and the description below. Other features, objects, and advantages
of the invention will be apparent to those persons skilled in the
art upon reading the drawing and the description, as well as from
the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIGS. 1A-1D: Measuring the absolute her2 level with EP3
antibody using dot blot analysis. Purified her2 protein was used to
define a dose curve, and the captured image was converted into
number using ImageQuant from Li-Cor. A dose curve was established,
and used to convert the captured dot blot image from patient
samples into absolute Her2 level among these patients.
[0057] FIGS. 2A-2H defining the linear range of three known
antibodies for IHC analyses of Her2, Ki67 and ER in QDB analysis.
Recombinant proteins of Her2, Ki67 and ER were used as protein
standard. The dose response curves were established using QDB
analyses with these antibodies respectively.
[0058] FIGS. 3A-3D show the absolute levels of Her2, Ki67, ER in
breast cancer tissues extracted from FFPE specimens, measured with
QDB analysis, and the absolute Her2 levels in frozen stomach
tissues. The result was further grouped based on their IHC results
respectively.
[0059] FIGS. 4A-4D show how to define a cutoff value to convert
continuous data into categorized results based on Her2 absolute
levels measured with QDB analysis, and the corresponding IHC
results, obtained from local hospital.
[0060] FIG. 5 shows how to analyze the clinical data using absolute
value of a biomarker. In this case, the potential link between Her2
absolute level and the histologic grade of breast cancer patients
was investigated.
DETAILED DESCRIPTION
[0061] Before the present methods are described, it is to be
understood that this invention is not limited to particular method
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0062] Unless otherwise defined in this disclosure, all technical
and scientific terms used herein have the same meaning as is
commonly understood by one of skills in the art to which this
disclosure belongs.
[0063] The subject methods are useful primarily for diagnostic
purposes. Thus, as used herein, the terms "determining,"
"measuring," and "assessing," and "assaying" are used
interchangeably and include both quantitative and qualitative
determinations. These terms can also refer to both quantitative and
semi-quantitative determinations and as such, the term
"determining" is used interchangeably herein with "assaying,"
"measuring," and the like. Where a quantitative determination is
intended, the phrase "determining an amount" of an analyte and the
like is used. Where either a quantitative and semi-quantitative
determination is intended, the phrase "determining a level" of an
analyte or "detecting" an analyte is used.
[0064] Whenever applicable, "quantitative" assays in general
provide information on the amount of an analyte in a sample
relative to a reference (control), and are usually reported
continuously numerical, where a "zero" value can be assigned where
the analyte is below the limit of detection. "Semi-quantitative"
assays yielding an approximation of the quantity or amount of a
substance; between a qualitative and a quantitative result, where a
"zero" value can be assigned where the analyte is below the limit
of detection.
[0065] The term "diagnosis" is used herein to refer to the
identification of a molecular or pathological state, disease or
condition, such as the identification of a molecular subtype of
breast cancer or other type of cancer.
[0066] The term "prognosis" is used herein to refer to the
prediction of the likelihood of cancer-attributable death or
progression, including recurrence, metastatic spread, and drug
resistance, of a neoplastic disease, such as breast cancer. The
term "predicative" is used here to refer to the prediction of the
outcome of therapeutic intervention.
[0067] The terms "subject," "host," "patient," and "individual" are
used interchangeably herein to refer to any mammalian subject for
whom diagnosis or therapy is desired, particularly humans.
[0068] As used herein, the term "unit," in the context of an
antibody-based essay, refers to an epitope of a target protein. In
a more general setting, "unit" could represent a hybridizing region
recognized by a binding agent.
Quantitation by QDB
[0069] The quantitatively measurement of the protein expression
level of a biomarker can be achieved at tissue level using
immunoblot method. The immunoblot method is to be considered to its
broadest context. As long as the analytical process is
antibody-based, and a solid phase is involved in the process, it
can be considered as immunoblot analysis. The solid phase includes,
but not limited to, a membrane, the ELISA plate, the QDB plate
(AKA, the plate used in QDB analysis).
[0070] There is no limitation to how the antibody is validated. The
antibody can be validated using Western blot analysis when linear
epitope is used; it can also be validated based on prior
experience. For example, a lot of antibodies have been used in
clinical diagnosis and prognosis. These antibodies include, but not
limited to ASR or IVD antibodies, and other clinically accepted
antibody for immunohistochemistry or flow cytometry analysis. All
these antibodies can be used in QDB analysis without further
validation.
[0071] A detection antibody can also be validated in reference to a
known detection antibody. In one embodiment of current invention, a
detection antibody is used to analyze more than one sample, and the
results are compared with those from a known antibody. For example,
4B5 clone from Roche for HER2 protein. When the concordance is
beyond certain value, this antibody can be considered as a
validated antibody.
[0072] In one embodiment of current invention, the expression level
of a biomarker is measured with a typical immunoblot method. This
method includes, but not limited to two features, including 1) a
validated antibody and 2), A dose curve can be established using a
protein standard.
[0073] In one embodiment of current invention, the expression level
of a biomarker is measured with a typical immunoblot method. This
method incudes, but not limited to three features, including 1) a
validated antibody and 2) a dose curve can be established using a
protein standard, and 3) the immunocomplex on the membrane is
individual quantified, without the interference from neighboring
unit during the detection process.
[0074] In one embodiment of current invention, a reference sample
can be included in the experiment. The reference sample is
processed with a sample of interest in the same immunoblot
analysis. The result of the reference sample is compared with that
of sample of interest to determine if the sample of interest is
positive when the result of sample of interest is above that of
reference sample, or vice versa.
[0075] In another embodiment of current invention, more than one
reference sample are included in the experiment. The reference
samples are processed together with sample of interest in the same
immunoblot analysis. The results of reference samples are used to
define a ranked order, and the result of sample of interest is
determined in reference to those of reference samples. For example,
if reference sample 1 and reference sample 2 are included to define
the upper limit of 1+ and 2+, and the result from sample of
interest is between those of reference sample 1+ and reference
sample 2+, the sample of interest is assigned as 2+.
[0076] In one embodiment of current invention, the expression level
of a biomarker is measured quantitatively using dot blot method.
The Dot blot analysis includes, but not limited to, features
including a) a validated antibody; b), defined linear range when
the antibody is used to analyze one type of sample, and c), the
immunocomplex on the membrane is detected as an image, and d), the
conversion of image into a number.
[0077] In another embodiment of current invention, the expression
level of a biomarker is measured quantitatively using QDB method.
The QDB analysis incudes, but not limited to, features including a)
a validated antibody; b), defined linear range when the antibody is
used to analyze one type of sample; and c), the immunocomplex on
the membrane is individual quantified, without the interference
from neighboring unit during the detection process.
[0078] The categorized results in current prevailing form of IHC
should be considered in its broadest context. These results can be
binary (positive or negative), or ranked (0, 1+, 2+, 3+ or strong,
medium or weak), or by percentage, or in combination of all the
above. Several scoring systems for IHC, including H score, is still
considered as categorized results, as the result is discrete in
nature
[0079] The result from the method disclosed in this invention is a
continuous data. Although this data can be converted into a
categorized result as in prevailing IHC system to facilitate the
comparison and understanding of the clinical significance of the
result from this invention, this conversion is based on a cutoff
reference derived from mathematical analysis, and the category
where it belongs to can vary in reference to difference cutoff
reference. In one embodiment of current invention, the measured
Her2 levels in breast cancer tissue can be used for statistical
analysis, and a cutoff value can be derived from Receiver Operative
Characteristic analysis (ROC) based on IHC results, and the
absolute level in breast cancer tissue can be assigned as either
"positive" or "negative" in reference to the derived cutoff value
from ROC analysis, with absolute Her2 level above the cutoff value
as positive, and verse versa.
Absolute Measurement
[0080] The result from current invention can be relative, or, in
combination with a protein standard, to be absolute. The terms
"relative" and "absolute," referring to two ways to take a
measurement, should be taken into their broadest context. While
relative measurement is measuring one thing compared to another
thing, absolute measurement is measuring things in known amounts
with standard units. Perhaps, the most significant difference
between these two measurements lies in each's applicable scope. A
relative result is only meaningful under the same experimental
setting, while an absolute result should be comparable across a
number of different analyses, even analyses taken at vastly
separate places or times.
[0081] Accordingly, the absolute measurement used herein is
achieved with the inclusion of a protein standard. This protein
standard should be taken into its broadest context. It refers to an
antigen of pre-determined amount, or one antigen of more than one
pre-determined amount. It includes, but not limited to, the amount
pre-determined by weight, by the percentage, by the volume, by the
number of molecules (for example, the mole), or a combination of
any two or more of above. It can be in pure form, or it can be in a
mixture. It can be in native form, or it can be in non-denatured
form, or it is in denatured form. In this disclosure, the
non-denatured form can be considered as any form between native
form and denatured form.
Biomarkers
[0082] A "sample" or "patient sample" or "biological sample," which
is used interchangeably herein, generally refers to a sample which
may be tested for a particular molecule, preferably a specific
marker molecule associated with a biological signature, such as a
biomarker shown in the paragraph below. Samples may include, but
are not limited to, peripheral blood cells, CNS fluids, serum,
plasma, buccal swabs, urine, saliva, tears, pleural fluid and the
like. A sample used in the present invention generally refers to a
tissue.
[0083] The term "marker" or "biomarker" here is to be defined at
its broadest context. A "marker" or "biomarker," which is used
interchangeably herein, generally refer to an organic biomolecule
(e.g., a polypeptide) which is differentially present in a sample
taken from a subject of one phenotypic status (e.g., having a
disease) as compared with that from another phenotypic status
(e.g., not having the disease or having a different disease). A
biomarker is thus often established if differentially present
between two different phenotypic statuses, when the mean or median
level of the biomarker in a first phenotypic status relative to a
second phenotypic status is calculated to represent statistically
significant differences.
[0084] In the present invention, the biomarker is protein molecule
measurable related with a biological or a disease state. It can be
well established diagnostic biomarkers (for example, a diagnostic
biomarker for IHC), or it can be biomarkers newly identified for in
vitro diagnostics.
[0085] In one embodiment of present invention, the biomarker is
used for diagnosis of solid tumor.
[0086] The known biomarkers for diagnostics include, but not
limited to ACTH, ACTIN, ADENOVIRUS, AFP, ALK-1, AMYLOID A, ANDROGEN
RECEPTOR, ANNEXIN, ARGINASE-1, BAP1, B-AMYLOID, BCL-1,BCL-2, BCL-6,
BEREP4, Beta-Catenin, BOB1, BRACHYURY, BRST-2, C3d, C4d,
CALCITONIN, CALDESMON, CALPONIN, CALRETININ, CD14, CD117, CD117,
BM, CD123, CD138, CD15, CD163, CD1a, CD2, CD20, CD21, CD23, CD25,
CD3, CD30, CD31, CD33, CD34, CD34, BM, CD38, CD4, CD43, CD45RA,
CD45RB, CD45RO, CD5, CD56, CD57, CD61, CD68, CD7, CD79a, CD8, CD99,
CDX2, CEAm, CEAp, Chromogranin, Chymotrypsin, Claudin 3, Claudin 4,
CK MIX, CK20, CK34BE12, CK5/6, CK7, CK19, CKAE1/AE3, CKCAM5.2, CMV,
c-Myc, CXCL13, CYCLIN D3, D2-40, DBA-44, Desmin, DOG-1, EBV, LMP,
E-Cadherin, EGFR, EMA, EMA-Perineurioma, ER, ERG, Factor 13a,
Factor 8, FOXP1, FSH, GALECTIN-3, Gastrin, GATA-3, GFAP, GH,
Glucagon, Glut1, Glutamine synthetase, Glypican-3, GPC, GRANZYME,
H. pylori, HBC, HBS, hCG, HepPar 1, Her2neu, HGAL, HHV-8, HMB-45,
HPL, HSV, IDH1, IgA, IgD, IgG , IgG4, IgM, Inhibin, INI, Insulin,
ISH EBV, ISH KAPPA, ISH LAMBDA, KAPPA, KBA62, KI67, Lambda,
LANGERIN, LAT, LEF1, LH, LM02, LYSOZYME, MAP-2, MCT, MELAN A, MITF,
MLH1, MNDA, MOC-31, MPO, MSH2, MSH6, MUC2, MUC5AC, MUM1, Myogenin,
Napsin A, NB84, NEU N, Neurofilament, NKI/C3, NKX3.1, NPM, NSE,
NUT, 2-Oct, Oct-3/4, p16, p53, p57, p63, Parvovirus, PAX-2, PAX-5,
PAX8, PCSK9, PD-1, Perforin, PHH3, PHLDA1, PIN4, PLAP, PMS2, PR,
PRAP, Prolactin, Prox1, PSA, RNA, S100, S100P, SALL4, SF-1, SMA,
SMMS, Somatostatin, SOX-10, SOX11, SCAP, S1P, SREBP, Spirochetes,
STATE, SV40, SYNAP, Tamm-Horsfall, T-bet, TCL-1, TCR, TCR, Gamma,
TdT, THYRO, TIA-1, TOXO, Transthyretin, TRAP, TSH, TTF1,
Tyrosinase, Vimentin, WT1, WT1 (C-19), ZAP70.
[0087] This method can be adopted to analyze the antigen in fresh
tissue, or it could be used to analyze antigen in formalin fixed
paraffin embedded block (FFPE). The antigen can be extracted from
the tissue or FFPE by any method as long as the antigen from
extraction process allows the process of antibody-antigen
interaction to happen. For antigen in FFPE block, the slice needs
to be de-paraffined with and without the antigen retrieval step to
facilitate antigen-antibody interaction.
Reference Database
[0088] The QDB results from more than one sample of interest,
together with their matching clinical information, can be used to
setup a database for mathematical analysis (AKA, QDB database). A
reference database can thus be built for diagnostic purposes.
[0089] The terms "reference" and "control" are used interchangeably
to refer to a known value or set of known values against which an
observed value may be compared. The known value represents an
understood correlation between two parameters, e.g., a level of
expression of a marker and its associated phenotype. As used
herein, the known value constitute a reference profile in a
reference database.
[0090] Accordingly, a reference database can be prepared storing a
number of reference profiles for diagnostic purpose, each recording
a marker expression level of a sample obtained from a subject with
either a known diagnosis or known clinical outcome after
therapy.
[0091] In one embodiment, the present invention also includes a
method of determining a patient profile that best matches one or
more reference profiles in a reference database. The method
includes steps of (a) comparing, on a suitably programmed computer,
the level of expression of a marker in a sample from a patient with
reference profiles in a reference database to determine a measure
of similarity between the patient profile and each of the reference
profiles; (b) identifying, on a suitably programmed computer, a
reference profile in a reference database that best matches the
patient profile based on a maximum similarity among the measures of
similarity determined in step (a); and (c) outputting to a user
interface device, a computer readable storage medium, or a local or
remote computer system; or displaying, the maximum similarity or
the associated phenotype of the reference profile in the reference
database that best matches the patient profile.
Absolute Value-Based Diagnostic Method
[0092] The putative association between the expression level of a
biomarker and a clinical trait is explored using a mathematical
method to analyze the information from a QDB database. The examples
include, but not limited to, the relative expression level of HER2
with the disease free survival of the patient. The predicative
value of Ki67 in treating stomach cancer patients. This information
may provide prognosis for other patient in the same analysis.
[0093] As pointed out above, diagnostic tests used in clinical
practice often rely on a single analyte, and therefore do not
capture the value of its potentially intrinsic correlations with
other parameters, including some clinical readings. Moreover,
diagnostic tests are frequently not quantitative, relying on
immunohistochemistry, which often yields different results in
different laboratories, in part because the reagents are not
standardized, and in part because the interpretations are
subjective and cannot be easily quantified.
[0094] When the protein standard is included in the QDB analysis,
the absolute level of a biomarker can be achieved, greatly
facilitating benchmarking across different clinical laboratories.
The QDB results from more than one sample of interest, together
with their matching clinical information, can be used to setup a
QDB database for mathematical analysis. The putative association
between the absolute level of a biomarker and a clinical trait is
explored using a mathematical method. The examples include, but not
limited to, the absolute expression level of HER2 with the disease
free survival of the patient. This information may provide
diagnosis and prognosis for a patient in the future when the
absolute level of the biomarker is measured.
[0095] The absolute results from more than one QDB analysis can be
combined together to increase the size of the database for
mathematical analysis. The putative association between the
absolute level of a biomarker and a clinical trait is explored
using a mathematical method. The examples include, but not limited
to, the absolute expression level of HER2 with the disease free
survival of the patient, or the predicative value of Ki67 in
treating stomach cancer patient. This information may provide
prognosis and diagnosis for a patient with known absolute level of
a biomarker.
[0096] The absolute level of more than one biomarker can be
measured using a sample of interest. These results, together with
the clinical information of the patient of interest, can be used to
setup a QDB reference database for mathematical analysis to provide
diagnosis and prognosis to a patient when the absolute levels of
these biomarkers are measured. Reference profiles can be obtained,
each carrying the absolute level of biomarker for a patience with
either a known diagnosis or known clinical outcome after therapy.
Later for a new patient, his or her absolute level of the
biomarkers can be determined and compared with those in the
reference database. A match with the absolute level of a stored
reference profile can be indicative of the associated diagnosis or
prognosis for therapy for the new patient.
[0097] The clinical trait is to be considered in its broadest
context. The trait may include, but not limited to, age, sex, blood
pressure, glucose level, cancer stage, hazard ratio, disease free
survival, or any information relevant to the diagnosis, prevention,
treatment of the patient.
[0098] Another substantial advantage comes from the diagnostic
method of the present invention. Specifically, the method can also
be utilized for screening patients prior to entry into a clinical
trial. A major impediment to cancer therapeutic trials aimed at
personalized treatment is the lack of biomarkers available for
stratifying the patients. The validation of an absolute value-based
screening tool for cancer patients could significantly reduce the
costs of such trials by using a single standard and refining the
study entry process. With reduced costs for identification and
screening of patients, the new methods for screening of the present
invention facilitate recruitment, screening, and/or selection of
patients from a broader range of populations and/or clinic
settings, thereby offering underserved patient populations the
opportunity to engage in clinical trials, which has been a major
limitation to the majority of previously conducted trials in
oncology.
[0099] Moreover, today's oncologists frequently have a number of
treatment options available to them, including those labeled as
"standard of care," and other drugs that do not carry such a label
yet exhibited efficacy in certain types of cancer patients.
Likelihood of good treatment outcome can be maximized if patients
could be assigned to their optimal cancer treatment options as
quickly as possible following diagnosis. The method of the present
invention fulfills these needs by providing an enabling tool.
[0100] The absolute level of a biomarker can be measured with QDB
analysis, or it may be measured with other method in addition to
QDB analysis. In one embodiment of current invention, the absolute
level of a sample of interest is verified with QDB analysis.
[0101] The absolute level of a biomarker for setting up a QDB
database can be measured with QDB analysis, or it can be verified
with QDB analysis. A QDB database should be considered in its
broadest context. A QDB database includes at least one sample of
interest with the absolute level of one biomarker measured or
verified with QDB analysis.
[0102] The absolute level of a biomarker can be measured using more
than one antibody. These antibodies can be against the same
epitope, or they can be against different epitope of the biomarker.
The absolute levels from these antibodies may be the same, or they
are different from each other. When the absolute levels are
different from each other using different antibodies, they should
be specified with the antibody used, and be processed individually
for diagnosis and prognosis purpose.
[0103] In one embodiment of current invention, when more than one
detection antibody are used to measure the absolute level of a
biomarker in a sample, If the diagnosis or prognosis is the same
from these antibodies, the diagnosis or prognosis is considered as
valid. If the diagnosis or prognosis is in disagreement, the
diagnosis or prognosis is considered as "equivocal", which requires
further verification.
[0104] Any antibody used for antigen-antibody interaction can be
used for QDB analysis as long as it is validated as specific to the
antigen of interest. In one embodiment of current invention, the
antibody is monoclonal. In yet another embodiment of current
invention, the antibody is polyclonal.
[0105] In one embodiment of current invention, an antibody for
immunohistochemistry can be used for QDB analysis using tissue or a
cell from a human or an animal.
[0106] In one embodiment of current invention, an antibody for flow
cytometry can be used for QDB analysis using tissue or a cell from
a human or an animal.
[0107] In yet another embodiment of current invention, an antibody
for antigen-antibody based assay can be used for QDB analysis using
tissue or a cell from a human or an animal.
[0108] In one embodiment of current invention, an antibody for a
biomarker for QDB analysis of a sample can be labeled with
fluorescence dye detectable at one wavelength, while another
antibody for another biomarker for QDB analysis of the same sample
can be labeled with fluorescence day detectable at another
wavelength can be used simultaneously to analyze both biomarkers in
the same sample.
[0109] In another embodiment of current invention, an antibody for
a biomarker for QDB analysis of a sample can be labeled with one
isotope, while another antibody for another biomarker for QDB
analysis of the same sample can be labeled with another isotope can
be used simultaneously to analyze both biomarkers in the same
sample. In yet another embodiment of current invention, an antibody
for a biomarker for QDB analysis of a sample can be labeled in one
method, for example, with Horseradish peroxide (HRP); while another
antibody for another biomarker for QDB analysis of the same sample
can be labeled with a florescent dye to allow simultaneously
measurement of two biomarkers in the same sample.
[0110] As used herein "reporter enzyme" is to be taken in its
broadest context. A reporter enzyme can be any modification of the
antibody in immunodetection assay with the purpose to aid the
detection of the antibody. For example, a report enzyme can be, but
not limited to, antibody directly labeled with radioactive isotope
like Iodide 125, or reporter enzymes like alkaline phosphatase or
horseradish peroxidase, or with a fluorescence day. The detection
of the amount of reporter enzymes associated with antibody is
through the formation of a detectable product as the readout of the
amount of reporter enzymes in the detection reaction. The product
can be radioactive, luminescent, fluorescent, or a product with
characteristic absorbance or reflection spectrum in the visible or
outside the visible range. When a complement is used to detect the
bound antigen-antibody complex, it may either be labeled in any one
of the above ways, or be detected in turn by a specific
anti-complement antibody.
[0111] A report enzyme can be, but not limited to, antibody
indirectly labeled with radioactive isotope like Iodide 125, or
reporter enzymes like alkaline phosphatase or horseradish peroxide.
Antibody can be, but not limited to, indirectly labeled through a
secondary antibody, and the secondary antibody is directly or
indirectly labeled with radioactive isotope like Iodide 125, or
reporter enzymes like alkaline phosphatase or horseradish peroxide.
In one embodiment, the secondary antibody is labeled with biotin,
and indirectly further labeled with a horseradish peroxide through
a streptavidin molecule.
[0112] As used herein "antigen" and an "antibody" are to be taken
in their broadest context. An "antigen" can be a molecule, a cell,
a virus, or a particle. The term "antigen" may be used to refer to
a chemical compound, a peptide, a protein, an RNA, a DNA, a cell
(proteins released in situ), or a virus particle (proteins released
in situ) or any molecules that may evoke the production of one or
more antibodies by a host animal, including human. An antigen may
also be a product comprising any two or more of the molecules or
moieties crosslinked together. An antigen can exists either in a
pure form, or it can exist in a mixture. An antigen can be in a
modified form (e.g., modified by a chemicals) or be in an
unmodified form.
[0113] Reference herein to an "antibody" is to be taken in its
broadest context. An "antibody" is a polypeptide that binds to "an
antigen". An antibody includes, but is not limited to, a
traditional antibody, a fragment of a traditional antibody
containing an antigen binding site, a recombinant antibody
containing an antigen binding site, a protein which binds to an
antigen, and a product that comprises of crosslinking any two or
more of the above. An antibody can exist either in a pure form, or
in a mixture. An antibody can be in a modified form (e.g., modified
by a chemical) or be an unmodified form.
[0114] "Solid tumor" is to be considered in its broadest term. A
solid tumor refers to any tissue, cancer or non-cancer, which is
connected at cellular level, and not existing as a single cell. It
includes, but not limited to, breast cancer, prostate cancer,
bladder cancer, stomach cancer, kidney cancer, Melanoma of skin,
Cervix uteri cancer, head and neck cancer, brain cancer, thyroid
cancer lung cancer, liver cancer, pancreatic cancer, and colorectal
cancer.
[0115] It is to be understood that the exemplary embodiments
described herein are that for presently preferred embodiments and
thus should be considered in a descriptive sense only and not for
purposes of limitation. Descriptions of features or aspects within
each embodiment should typically be considered as available for
other similar features or aspects in other embodiments.
[0116] Further, Examples put forth are not intended to represent
that the experiments below are all or the only experiments
performed. Efforts have been made to ensure accuracy with respect
to numbers used (e.g., amounts, temperature, etc.) but some
experimental errors and deviations should be accounted for.
MATERIALS AND METHODS
[0117] Human subjects and human cell lines Both Frozen tissues and
formalin fixed paraffin embedded (FFPE) slices were obtained from
local hospitals together with their clinical information including
IHC scores (0, 1+, 2+or 3+). Some of them were with FISH results.
MCF-7 and BT-474 cell lines were purchased from the Cell Bank of
Chinese Academy of Sciences (Shanghai, China), and lysates prepared
from these two cell lines were used as HER2 negative and positive
controls.
[0118] General reagents. All general reagents used for cell culture
were purchased from Thermo Fisher Scientifics (Waltham, Mass., USA)
including cell culture medium and culture dishes. The protease
inhibitors were purchased from Sigma Aldrich (St. Louis, Mo., USA).
All other chemicals were purchased from Sinopharm Chemicals
(Beijing, P. R. China). Recombinant human HER2/ErbB2/CD340
(676-1255) protein was purchased from Sino Biological Inc.
(Beijing, China). QDB plate was manufactured by Yantai Zestern
Biotechnique Co. Ltd at Yantai, China.
[0119] Ventana anti-HER2/neu(4B5) rabbit monoclonal primary
antibody and anti-Human estrogen receptor (clone Sp1) were
purchased from Roche Diagnostics GmbH. Rabbit anti-HER2 antibody
(clone EP3) and Mouse anti-human Ki67 (clone MIB1) were purchased
from ZSGB-BIO (Beijing, China). HRP labeled Donkey Anti-Rabbit IgG
secondary antibody was purchased from Jackson Immunoresearch lab
(Pike West Grove, Pa. USA).
[0120] Preparation of Cell and Tissue Lysates
[0121] For frozen tissues, about 150 mg tissues were cut from
tissue biopsies, and processed in 300 .mu.l lysis buffer (50 mM
HEPES, 137 mM NaCl, 5 mM EDTA, 1 mM MgCl, 10 mM
Na.sub.2P.sub.2O.sub.7, 1% TritonX-100, 10% glycerol) with protease
inhibitors (2 .mu.g/ml Leupeptin, 2 .mu.g/ml Aprotinin, 1 .mu.g/ml
pepstatin, 2 mM PMSF, 2 mM NaF) with a handheld tissue homogenizer
for 30 s before they were centrifuged at 12000.times.g for 5 mins.
The supernatants were collected for immunoblot analysis. The total
protein concentration was measured using Pierce BCA protein assay
kit in accordance to the manufacturer's instructions. To prepare
lysates from MCF-7 and BT-474 cells, cells were lysed in lysis
buffer with protease inhibitors by pipetting up and down for 50
times. Supernatants were collected after centrifugation and the
total protein concentration was determined by the BCA protein assay
kit.
[0122] For Formalin fixed paraffin embedded (FFPE) blocks, two
2.times.15 .mu.m slices were collected , de-paraffined and
processed in 300 .mu.l lysis buffer (50 mM HEPES, 137 mM NaCl, 5 mM
EDTA, 1 mM MgCl, 10 mM Na.sub.2P.sub.2O.sub.7, 1%TritonX-100, 10%
glycerol) with protease inhibitors (2 .mu.g/ml Leupeptin, 2
.mu.g/ml Aprotinin, 1 .mu.g/ml pepstatin, 2 mM PMSF, 2 mM NaF)
before they were centrifuged at 12000.times.g for 5 mins. The
supernatants were collected for immunoblot analysis. The total
protein concentration was measured using Pierce BCA protein assay
kit in accordance to the manufacturer's instructions. To prepare
lysates from MCF-7 and BT-474 cells, cells were lysed in lysis
buffer with protease inhibitors by pipetting up and down for 50
times. Supernatants were collected after centrifugation and the
total protein concentration was determined by the BCA protein assay
kit.
[0123] Dot blot analysis Purified her2 protein was serially diluted
as indicated in the figure, and spotted on the a membrane together
with total proteins extracted from FFPE slices. These samples were
processed in a typical dot blot process until the image was
captured using C-Digit Western blot scanner from Li-Cor. The image
was converted into a number using ImageQuanti program from Li-Cor,
and a protein standard curve was established to convert the images
captures from patient samples into absolute biomarker levels.
[0124] QDB analysis The linear range of a specific antibody (EP3 or
4B5 clone for Her2; MIB1 for Ki67, SP1 for Estrogen receptor (ER))
was determined by using a pooled lysate from patients testing
positive respectively for these biomarkers. The lysates were
prepared first by mixing in equal amount tissue lysates prepared
from 3 to 4 breast or stomach cancer tissues. The pooled lysates
were serially diluted from 0-2 .mu.g to define the linear range of
QDB analysis. A protein standard either obtained commercially or
expressed and purified in the company was also serially diluted
from 0-500 .mu.g, and used to define the linear range of QDB
analysis.
[0125] The samples were applied onto the QDB plates at 2 .mu.l/unit
in triplicate, and were processed as described previous.sup.15. A
primary antibody was used for primary antibody incubation at 100
.mu.l/well overnight at 4.degree. C. and a donkey anti-rabbit or
donkey anti-mouse secondary antibody was incubated with the plate
for 4 hours at room temperature. The plates were briefly rinsed
twice with TBST, and washed 5.times.10 mins before they were
inserted into a white 96-well plate pre-filled with ECL solution
prepared according to the manufacturer's instruction at 100
.mu.l/well for 3 mins. The chemiluminescence signal from individual
well of the plate was quantified by using the Tecan Infiniti 200
pro Microplate reader with the option "plate with cover".
[0126] A linear regression formula was established using serially
diluted protein standard respectively. The formula was used to
calculate the total HER2 level in each breast cancer tissue
samples, and the results were corrected by total protein amount
measured by the BCA protein determination kit. To ensure the
consistency of the experiments, two cell lysates prepared from
BT474 and MCF7 were used as positive and negative controls. The
lysates were prepared in small aliquots. The absolute biomarker
levels from two cell lysates were measured with QDB analysis and
documented prior to being included and processed as regular samples
in all the QDB experiments performed. Samples with
chemiluminescence reading less than 2 fold over blank were regarded
as non-detectable, and entered as 0 for data analysis. The final
result was further corrected by the percentage of the purity of the
recombinant HER2 protein.
[0127] Statistical analysis Common tests for statistical
significance include, among others, t-test, ANOVA, Kruskal-Wallis,
Wilcoxon, Mann-Whitney and odds ratio. Biomarkers, alone or in
combination, provide measures of relative likelihood that a subject
belongs to a phenotypic status of interest. As such, biomarkers can
find use as markers for, for example, disease, therapeutic
effectiveness of a drug, and the like. Biomarkers are thus analytes
in assays that facilitate diagnosis and the like.
[0128] All the data were presented as Mean.+-.Standard Deviation
(SD). The difference between individual groups was calculated using
Microsoft Excel using unpaired two-tailed Student's t tests. P
value <0.05 was considered statistically significant. Receiver
operating characteristic (ROC) curves were generated using the
GraphPad Prism software version 7.0 (GraphPad Software Inc., USA),
as indicated in the figure legend.
EXAMPLES
[0129] Example 1 teaches how to measure absolute Her2 level in FFPE
specimen using Dot blot analysis.
[0130] In 1A, recombinant Her2 protein was serially diluted and
spotted onto a membrane together with four patient samples
extracted from FFPE blocks.
[0131] The spotted protein standards and patient samples on the
membrane was processed in dot blot analysis using EP3 clone for
Her2 protein, and the image was captured using C-Digit Western blot
scanner from Li-Cor, as shown in FIGS. 1A and 1B.
[0132] The image in FIG. 1A was converted into numbers using
ImageQuant software from Li-Cor, and these numbers were used to
establish a protein standard curve, as shown in FIG. 1C.
[0133] The images from 4 patient samples were also converted into
number using the same computer software, and the numbers were
converted into absolute Her2 level based on the protein standard
curve established in section [0098].
[0134] FIGS. 2A-2H teach how to define the linear range of the QDB
analysis using a specific antibody. FIG. 2A showed the linear range
of QDB analysis of frozen breast cancer tissues using Her2-EP3
antibody; FIG. 2B showed the linear range of QDB analysis of FFPE
slices using Her2-EP3 antibody; FIG. 2C showed the linear range of
QDB analysis of a recombinant Her2 protein, FIG. 2D showed the
linear range of QDB analysis of frozen stomach cancer tissue using
MIB1 antibody, FIG. 2E showed the linear range of QDB analysis of
FFPE slices using MIB1 antibody against Ki67, 2F showed the linear
range of QDB analysis of a recombinant fragment of Ki67 protein
using MIB1 antibody, FIG. 2G showed the linear range of QDB
analysis of a recombinant fragment of Estrogen Receptor (ER)
protein using SP1 antibody, and FIG. 2H showed the linear range of
QDB analysis of FFPE slices using SP1 antibody against ER.
[0135] FIGS. 3A-3D show the absolute level of Her2 (A), Ki67 (B)
and ER (C) in breast cancer tissues using Her2-4B5, MIB1 and SP2
antibodies respectively. In FIG. 3D, the Her2 absolute levels in
frozen stomach cancer were measured using Her2-EP3 antibody. The
results were average of three independent experiments in
triplicate, and were grouped based on their IHC scores.
[0136] FIGS. 4A-4D show how to convert a QDB result into a
categorized result using Receiving Operative Characteristic (ROC)
analysis. The results from QDB analysis were separated into
negative and positive groups based on their IHC results, and these
two groups of numbers were used to calculate the cutoff value with
promising specificity and sensitivity. FIGS. 4A and 4B showed ROC
curves of QDB results from HER2-EP3 and HER2-4B5 antibodies
respectively. FIGS. 4C and 4D showed how to separate QDB results
into negative and positive groups based on the suggested cutoff
value from ROC analysis.
[0137] In FIG. 5, the absolute Her2 levels from 263 FFPE specimens
were measured, and plotted as a group based on the provided
histologic grade judged by local hospital based on Nottingham
histologic scores. There were 34 samples in group I, 125 samples
with grade II, and 104 samples in grade III group. The averages of
three grades were 0.08, 0.73, and 3.15 nmole/g. The her2 level were
significantly higher with patients in Grade III group than those
from Grade I and Grade II group, and absolute her2 level was
significantly higher among patients in Grade II group than those in
Grade I group. This result cannot be achieved with the current
prevailing categorized system with both IHC and FISH analyses.
[0138] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed. To the extent a definition of a term set
out in a document incorporated herein by reference conflicts with
the definition of a term explicitly defined herein, the definition
set out herein controls.
[0139] It is believed that the following claims particularly point
out certain embodiments or combined thereof that are directed to
one of the disclosed inventions and are novel and non-obvious.
Inventions embodied in other combinations or subcombinations of
features, functions, elements and/or properties may be claimed
through amendment of the present claims or presentation of new
claims in this or a related application. Such amended or new
claims, whether they are directed to a different invention or
directed to the same invention, whether different, broader,
narrower, or equal in scope to the original claims, are also
regarded as included within the subject matter of the inventions of
the present disclosure.
* * * * *