U.S. patent application number 14/725862 was filed with the patent office on 2016-05-26 for compositions and methods for prognosis and treatment of cancer.
The applicant listed for this patent is NOVAZOI THERANOSTICS, INC.. Invention is credited to Ritu Aneja, Padmashree C.G. Rida.
Application Number | 20160146781 14/725862 |
Document ID | / |
Family ID | 54701431 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160146781 |
Kind Code |
A2 |
Aneja; Ritu ; et
al. |
May 26, 2016 |
COMPOSITIONS AND METHODS FOR PROGNOSIS AND TREATMENT OF CANCER
Abstract
A protocol for assessing the prognosis for a patient diagnosed
with a neoplasm or suspected of having a neoplasm is provided
herein. The protocol involves the steps of determining a mitotic
cells to proliferating cells ratio (M:P ratio) in a neoplastic
tissue sample obtained from the patient and producing a prognosis
for the neoplasm based on the M:P ratio.
Inventors: |
Aneja; Ritu; (Lilburn,
GA) ; Rida; Padmashree C.G.; (Plano, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVAZOI THERANOSTICS, INC. |
Plano |
TX |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150346191 A1 |
December 3, 2015 |
|
|
Family ID: |
54701431 |
Appl. No.: |
14/725862 |
Filed: |
May 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62006242 |
Jun 1, 2014 |
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Current U.S.
Class: |
424/94.5 ;
435/29; 435/6.14; 702/19 |
Current CPC
Class: |
G16H 50/30 20180101;
G01N 33/5011 20130101; G01N 2800/52 20130101; G16H 15/00 20180101;
G01N 33/5005 20130101; G16H 50/20 20180101; G16B 99/00 20190201;
G01N 2800/7028 20130101; G01N 33/57496 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G06F 19/00 20060101 G06F019/00 |
Goverment Interests
[0002] This invention was made with government support under grant
number RO1 CA 169127 awarded by the National Cancer Institute (NCI)
at the National Institutes of Health (NIH). The government has
certain rights in the invention.
Claims
1. A method of assessing the prognosis for a patient who has been
diagnosed with, or is suspected to have, a neoplasm, the method
comprising: (a) exposing at least a portion of a neoplastic tissue
sample from the patient to two binding agents under conditions
sufficient to allow the binding agents to bind pre-selected markers
within the sample portion, wherein a first binding agent
specifically targets mitotic cells in the sample portion and a
second agent specifically targets proliferating cells in the sample
portion; (b) exposing the sample portion in step (a) to detection
reagents suitable for visualizing and discriminating between
proliferating cells that are mitotic and proliferating cells that
are non-mitotic; and (c) determining the ratio of mitotic cells to
proliferating cells (M:P ratio) within the sample portion, and (d)
providing a prognosis based on the M:P ratio and the type of
neoplasm.
2. The method of claim 1, wherein the neoplasm is Luminal B subtype
breast cancer or triple negative breast cancer (TNBC), and wherein
an M:P ratio above a predetermined threshold indicates a worse
prognosis.
3. The method of claim 1, wherein the neoplasm is cancer and
wherein the method further comprises the step of determining a
grade of the cancer, based on a conventional grading system, and
further adjusting the grade of the cancer based on the
stratification provided by the M:P ratio.
4. The method of claim 1, wherein step (c) comprises flow cytometry
to determine the percentages of mitotic cells and proliferating
cells.
5. The method of claim 1, wherein the first binding agent targets a
phosphorylated form of histone H3 and wherein the second binding
agent targets Ki-67.
6. The method of claim 1, further comprising the steps of: (d)
processing a sample of tumor tissue or cancer cells from the
patient in a form suitable for visualization and demarcation of
cell nuclei, individually distinguishable centrosomes (iCTRs) and
megacentrosomes (mCTRs) in a region of interest (ROI) defined by a
plurality of cell nuclei; (e) determining the numbers of iCTRs and
mCTRs associated with each cell nucleus in the ROI; (f) determining
the volume of each iCTR and mCTR in the ROI; and (g) calculating
one or more centrosome amplification scores (CASs) values for the
sample based on steps (e) and (f), wherein M:P ratio and the one or
more CASs provide a measure of a level of risk and/or a prognosis
associated with the cancer and indicate the severity of the cancer,
the degree of intratumoral heterogeneity, or both.
7. The method of claim 1, wherein the patient is treated with at
least one antineoplastic agent based on the results from step
(b).
8. The method of claim 7, wherein the at least one antineoplastic
agent is selected from the group consisting of anti-mitotic agents,
anti-interphase agents, anti-microtubule agents,
anthracycline-based agents, and aromatase inhibitor agents.
9. A method of identifying a chemotherapeutic agent for a neoplasm,
the method comprising: (a) exposing a first group of neoplastic
cells to two binding agents under conditions sufficient to allow
the binding agents to bind pre-selected markers within the first
group of neoplastic cells, wherein a first binding agent
specifically targets mitotic cells in the group of neoplastic cells
and a second agent specifically targets proliferating cells in the
group of neoplastic cells; (b) exposing the group of neoplastic
cells to detection reagents suitable for visualizing and
discriminating between proliferating cells that are mitotic and
proliferating cells that are non-mitotic; (c) determining a first
ratio of percent mitotic cells to percent proliferating cells
within the group of neoplastic cells; (d) treating a second group
of neoplastic cells with a candidate chemotherapeutic agent; (e)
exposing the treated cells in step (d) to the two binding agents in
step (a) under conditions sufficient to allow the binding agents to
bind pre-selected markers within the treated cells; (f) exposing
the treated cancer cells in step (e) to the detection reagents in
step (b); (g) determining a second ratio of percent mitotic cells
to percent proliferating cells within the treated neoplastic cells;
and (h) determining whether the second ratio is reduced in
comparison to the first ratio, wherein a candidate chemotherapeutic
agent that reduces the first ratio is a chemotherapeutic agent for
neoplasm.
10. The method of claim 9, wherein the neoplasm is breast
cancer.
11. A method of identifying a chemotherapeutic agent for a
neoplasm, the method comprising: (a) exposing at least a portion of
a neoplastic tissue sample to two binding agents for a time and
under conditions sufficient to allow the agents to bind
pre-selected markers within the sample portion, wherein a first
binding agent specifically targets mitotic cells in the sample
portion and a second binding agent specifically targets
proliferating cells in the sample portion; (b) exposing the sample
portion in step (a) to detection reagents suitable for visualizing
and discriminating between proliferating cells that are mitotic and
proliferating cells that are non-mitotic; (c) determining a first
ratio of percent mitotic cells to percent proliferating cells
within the sample portion; (d) treating the patient with a
candidate chemotherapeutic agent; (e) providing a tissue sample
from the treated patient, wherein the tissue sample is suspected of
including neoplastic cells; (f) exposing at least a portion of the
sample in step (e) to the two binding agents in step (a); (g)
determining a second ratio of percent mitotic cells to percent
proliferating cells within the sample portion from the treated
patient; and (h) determining whether the second ratio is reduced in
comparison to the first ratio, wherein a candidate chemotherapeutic
agent that reduces the first ratio is a neoadjuvant
chemotherapeutic agent.
12. The method of claim 11, wherein the neoplasm is cancer.
13. The method of claim 12, wherein the cancer is breast
cancer.
14. A method of improving a conventional grading system for cancer,
wherein the conventional grading system comprises analysis of
cellular mitosis, cellular proliferation, or both, and wherein the
method comprises substituting the conventional analysis of cellular
mitosis, cellular proliferation, or both, in the grading system
with an analysis of M:P ratio, wherein the M:P ratio is defined by
the percent mitotic cells and percent proliferating cells in a
common tissue sample.
15. The method of claim 14, wherein the grading system is the
Nottingham grading system for breast cancer or the Gleason grading
system for prostate cancer.
16. A composition comprising a cocktail of two cell cycle specific
binding agents, wherein: a first binding agent specifically targets
mitotic cells in a sample, and a second binding agent specifically
targets proliferating cells in the sample.
17. The composition of claim 16, wherein the first binding agent
binds to a phosphorylated form of histone H3 and wherein the second
binding agent binds Ki-67.
18. A kit comprising: a first binding agent specifically targeting
mitotic cells in a sample; a second binding agent specifically
targeting proliferating cells in the sample; one or more detection
reagents for visualizing bound complexes indicative of mitotic and
proliferative cells; and instructions for use.
19. The kit of claim 18, wherein the first binding agent binds to a
phosphorylated form of histone H3 and wherein the second binding
agent binds Ki-67.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 62/006,242, filed Jun. 1, 2014. The entirety
of the aforementioned application is incorporated herein by
reference.
FIELD
[0003] The present invention relates to compositions and methods
for predicting clinical outcomes, selecting cancer therapies, and
assessing a cancer patient's response to a cancer therapy. More
specifically, the methods involve compositions and methods for
determining the ratio of mitotic cells to proliferating cells to
aid in making these determinations.
BACKGROUND
[0004] Therapeutic planning for individualized management of breast
cancer relies on patient stratification based on risk conferred by
clinicopathologic factors. Prognostic and predictive markers
commonly used for assessing risk associated with a breast tumor or
its "aggressive potential", include expression status of cell
proliferation marker Ki67, estrogen receptor (ER), progesterone
receptor (PR), extent of amplification of Human Epidermal Growth
Factor Receptor 2 gene, and mitotic index (MI) of the tumor.
[0005] The mitotic index (MI) is determined by counting the number
of mitotic cells per 10 HPFs (high-power fields) in a section of
tumor tissue and has been shown to carry a strong prognostic value.
Literature reports indicate that error-prone divisions of tumor
cells lead to chromosomal instability to enable generation of
genetic diversity out of which superlative karyotypes can be
eventually selected. Thus, the higher the mitotic frequency within
the proliferative population of tumor cells, the higher the
probability of aggressive clones emerging to fuel tumor
progression. The mitotic score within a tumor is therefore a
crucial indicator of the risk of acquiring an aggressive phenotype.
However, mitosis (M-phase) is only a part of the whole
proliferative cycle and is relatively infrequent, as reflected in a
mean tumor doubling time of 45-325 days. Infrequent mitoses
underlie the failure of drugs that specifically target the M-phase
in neoplastic cells.
[0006] Another prognostic factor, the Ki67 index (KI) is defined as
the percentage of Ki67-positive neoplastic cells. Ki67 protein is
present during all cell cycle phases (G1, S, G2 and M)
characteristic of cell proliferation. As an adjunct to
tumor-grading, pathologists have long been using Ki67
immunohistochemical staining to quantify the proliferating cell
population within tumors. The percentage of Ki67-positive nuclei
(referred to as Ki67 Index or KI) yields crucial information about
disease prognosis, predicts relative responsiveness to
chemotherapy, estimates residual risk in patients on standard
therapy, and serves as a dynamic biomarker for neoadjuvant
treatment efficacy.
[0007] Although KI is a universally accepted prognostic marker for
cell proliferation, there is tremendous ambiguity in the
nomenclature of proliferation cells in diagnostic pathology. In
particular, the terms "actively proliferating", "actively dividing"
and "mitotically active" cells are often used synonymously.
However, a cell in the "proliferation cycle" may not be actually
"dividing", whereas an "actively dividing" cell is indeed
"proliferating."
[0008] Among the above-described markers, MI is an integral
component of the Nottingham Grading System (NGS), which is a
modification of the Scarff-Bloom-Richardson breast tumor-grading
system. KI measurement is not routinely mandated according to ASCO
guidelines and KI has never been integrated into NGS. Extensive
research has focused on evaluating KI and MI either separately or
comparatively as markers of prognosis, yet surprisingly the two
indices have never been studied integratively.
[0009] Tumor-grading in NGS involves microscopically evaluating
three histological parameters, including tubule formation, nuclear
pleomorphism, and mitotic activity/10 high-power fields (HPF), and
assigning a score of 1 to 3 for each of them: tubule formation
(>75%=1, 10% to 75%=2, and <10%=3), nuclear pleomorphism
(none=1, moderate=2, and marked =3), and mitotic activity found in
10 HPF, based on a HPF size of 0.274 mm.sup.2 (<7 mitoses=1, 7
to 14 mitoses=2, and >14 mitoses=3). Summation of the three
scores thus obtained (ranging from 3 to 9) determines the placement
of the tumor into one of three Nottingham Grades. A combined score
of 3, 4, or 5=Nottingham Grade (NG) I; a combined score of 6 or
7=NG II; and a combined score of 8 or 9=NG III. Multivariate
analyses in large cohorts of breast cancer patients have
consistently demonstrated that histologic grade of a tumor is a
powerful prognostic indicator of disease recurrence and patient
death independent of lymph node status and tumor size.
[0010] Despite widespread use of NGS by clinicians for patient
stratification, prognostic heterogeneity persists within each
Nottingham Grade. One drawback of the NGS is that about 30-60% of
breast tumors are categorized as Nottingham grade (NG) II (the
intermediate between the lowest grade of NG I and the highest grade
of NG III), a classification that is not too informative for
therapeutic decision-making. Gene expression studies suggest that
many of these tumors are much more similar to NG I or NG III tumors
in terms of their expression profiles, implying that many NG II
patients may be either overtreated or undertreated. Also, the
recommendation of cytotoxic chemotherapy for all invasive lesions
is far from ideal when one considers that node-negative tumors
smaller than 10 mm have survival rates of >90% without
chemotherapy. Hence there is a need to refine the NGS and enhance
its prognostic accuracy by identifying quantifiable biomarkers for
breast tumors that (i) can discriminate more sharply the risk posed
by breast tumors, (ii) can be accurately and reliably determined
via a clinically-facile method, (iii) are robust and applicable in
some, if not all, of the subtypes of breast carcinomas, and (iv)
yield more accurate patient stratification.
[0011] The accuracy of NGS cannot be improved unless the precision
in determining its constituent parameters is enhanced. One source
of error pertains to mis-estimation of mitotic cells due to visual
recognition from hematoxylin-eosin (H&E)-stained slides (an
inherently error-prone process) and subjectivity (both intra- and
inter-observer) arising from different choices of regions to be
assessed.
[0012] A second source of error pertains to current diagnostic
practices that take MI and KI into consideration as independent
entities, while in reality, mitosis is a cell-cycle phase snugly
nested within the proliferative cycle. In the absence of a unified
view of mitosis and proliferation, the kinetic information on how
fast the proliferative tumor cell population is actually cycling is
lost. There is a need to improve the accuracy of tumor grading and
more optimal selection of therapies.
SUMMARY
[0013] In one aspect, method of assessing the prognosis for a
patient diagnosed with a neoplasm or suspected of having a neoplasm
includes the steps of: (a) exposing at least a portion of a
neoplastic tissue sample from the patient to two binding agents
under conditions sufficient to allow binding to pre-selected
markers within the sample portion, where a first binding agent
specifically targets mitotic cells in the sample portion and a
second agent specifically targets proliferating cells in the sample
portion; (b) exposing the sample portion in step (a) to detection
reagents suitable for visualizing and discriminating between
proliferating cells that are mitotic and proliferating cells that
are non-mitotic; (c) determining the ratio of mitotic cells to
proliferating cells (M:P ratio) within the sample portion; and (d)
providing a prognosis based on the M:P ratio and the type of
neoplasm.
[0014] In certain embodiments, this method can aid in determining
whether a patient will benefit from an anti-mitotic or
anti-microtubule therapy or chemotherapy. In other embodiments,
this method can aid in determining the extent of intratumoral
heterogeneity.
[0015] In some embodiments, the method further includes the step of
determining the histological grade of the cancer, based on a
conventional grading system, and further adjusting the histological
grade of the cancer based on the M:P ratio.
[0016] The patient may be suspected of having a neoplasm or may
have already been diagnosed with a neoplasm. In some embodiments,
the patient has been diagnosed with malignant neoplasm, such as
cancer. In one embodiment, the patient has a carcinoma. In certain
preferred embodiments, the patient has been diagnosed with a breast
neoplasm or breast cancer.
[0017] In one embodiment, an M:P ratio above a predetermined
threshold level differentiates patients having a Luminal A subtype
of breast cancer from patients having a Luminal B subtype of breast
cancer.
[0018] In some embodiments, an M:P ratio above a predetermined
threshold indicates a worse prognosis for a patient with Luminal B
subtype breast cancer or triple negative breast cancer (TNBC). In
general, among Nottingham Grade-matched tumors of luminal B or TNBC
subtypes, the higher the M:P ratio or the equivalent thereto, the
greater the intratumoral heterogeneity and the poorer the patient's
prognosis.
[0019] In another embodiment, the patient has been diagnosed with a
prostate neoplasm or prostate cancer.
[0020] In some embodiments, the determination step includes flow
cytometry to determine the percentages of mitotic cells and
proliferating cells. In one embodiment, additional markers are
labeled to ensure that only tumor/neoplastic cells are analyzed. In
other embodiments, the determination step employs an image analysis
step employing a computer readable medium, wherein a nuclear
segmentation step aids in the identification of cells that are
mitotic or proliferative and the computer readable medium
determines the percentages of mitotic cells and proliferating
cells. Alternatively, serial sections are stained with
hematoxylin-eosin (H&E) and/or are stained for a neoplastic
markers and/or markers specific for non-neoplastic cells, whereby
multi-stained images of mitotic neoplastic cells, proliferative
neoplastic cells and non-neoplastic cells are overlaid over one
another to identify cancer cells and tumor regions unequivocally.
This "virtual multiple staining" strategy ensures that stromal
cells, immune cells and other non-tumor cells are not quantitated
in the analysis. In another embodiment, in addition to staining
mitotic and proliferative cells, multiplexed immunohistochemistry
(IHC) may be used to stain for tumor-specific antigens or non-tumor
antigens in the same sample sections to distinguish tumor cells
from non-tumor cells surrounding or infiltrating the tissue.
[0021] In some embodiments, the first binding agent and the second
binding agent is an antibody or a biologically active fragment
thereof. In certain preferred embodiments, the first binding agent
targets a phosphorylated form of histone H3 and the second binding
agent targets Ki-67. The sample may be exposed to the first and
second binding agents separately or simultaneously. In some
embodiments, the method may further include the step of exposing
the sample to a DNA binding agent.
[0022] The sample may be collected from a variety of different
tissues. In some embodiments, the sample is a histological tissue
section. In other embodiments, the sample includes whole blood,
leukocytes or a cell suspension prepared from a tissue sample.
[0023] In some embodiments, the patient is treated with at least
one antineoplastic agent based on the results from the M:P ratio
determination. In certain embodiments, the antineoplastic agent is
an anti-mitotic agent, an anti-interphase agent, an
anti-microtubule agent, an anthracycline-based agents or an
aromatase inhibitor agent. In other embodiments, the antineoplastic
agent is a centrosome declustering agent.
[0024] In another aspect, a method of identifying a potential
chemotherapeutic agent for cancer includes the steps of: (a)
exposing a first group of the neoplastic cells to two binding
agents under conditions sufficient to allow the binding agents to
bind pre-selected markers within the first group of neoplastic
cells, wherein a first binding agent specifically targets mitotic
cells in the first group of neoplastic cells and a second agent
specifically targets proliferating cells in the first group of
neoplastic cells; (b) exposing the first portion of neoplastic
cells to detection reagents suitable for visualizing and
discriminating between proliferating cells that are mitotic and
proliferating cells that are non-mitotic; (c) determining a first
ratio of percent mitotic cells to percent proliferating cells
within the first group of neoplastic cells; (d) treating a second
group of the neoplastic cells with a candidate chemotherapeutic
agent; (e) exposing the treated cells in step (d) to the two
binding agents in step (a) under conditions sufficient to allow the
binding agents to bind pre-selected markers within the treated
neoplastic cells; (f) exposing the treated neoplastic cells in step
(e) to the detection reagents in step (b); (g) determining a second
ratio of percent mitotic cells to percent proliferating cells
within the treated neoplastic cells; and (h) determining whether
the second ratio is reduced in comparison to the first ratio,
wherein a candidate chemotherapeutic agent that reduces the first
ratio is a potential chemotherapeutic agent.
[0025] In some embodiments, the first group and the second group of
neoplastic cells are cultured neoplastic cells. In other
embodiments, the first group and the second group of neoplastic
cells are cells located in vivo, such as cells in an in vivo tumor
model. For example, a plurality of neoplastic cells can be injected
into mice to form tumors in vivo, whereby the mice can be subjected
to various treatment modalities and regimens comprising one or more
candidate therapeutic drugs to determine the responsiveness of the
mice to these drugs.
[0026] In other embodiments, a method of identifying a
chemotherapeutic agent for neoplastic tissues includes the steps
of: (a) exposing at least a portion of a neoplastic tissue sample
to two binding agents for a time and under conditions sufficient to
allow the agents to bind pre-selected markers within the sample
portion, wherein a first binding agent specifically targets mitotic
cells in the sample portion and a second binding agent specifically
targets proliferating cells in the sample portion; (b) exposing the
sample portion in step (a) to detection reagents suitable for
visualizing and discriminating between proliferating cells that are
mitotic and proliferating cells that are non-mitotic; (c)
determining a first ratio of percent mitotic cells to percent
proliferating cells within the sample portion; (d) treating the
patient with a candidate chemotherapeutic agent; (e) providing a
tissue sample from the treated patient, wherein the tissue sample
is suspected of including neoplastic cells; (f) exposing at least a
portion of the sample in step (e) to the two binding agents in step
(a); (g) exposing the sample portion in step (f) to the detection
reagents in step (b); (h) determining a second ratio of percent
mitotic cells to percent proliferating cells within the sample
portion from the treated patient; and (i) determining whether the
second ratio is reduced in comparison to the first ratio, wherein a
candidate chemotherapeutic agent that reduces the first ratio is a
potential neoadjuvant chemotherapeutic agent.
[0027] In another aspect, a method of improving a grading system
for neoplastic tissue is provided where the revised grading system
comprises generating a new score (Ki67-Adjusted Mitotic Score, or
KAMS) derived by using MI and KI scores determined by conventional
methods (e.g., in different fields of view from different slides)
and then using these numbers to approximate the quotient of percent
mitotic cells divided by percent Ki67-positive cells as further
described below.
[0028] In another aspect, a method of improving a grading system
for neoplastic tissue is provided where the revised grading system
includes analysis of cellular mitosis, and cellular proliferation
in which the method substitutes for conventional analyses of
cellular mitosis and/or cellular proliferation in the grading
system, whereby an analysis of the M:P ratio is determined based on
the percent mitotic cells to percent proliferating cells in a
common tissue sample.
[0029] In another aspect, a composition includes a cocktail of two
cell cycle specific binding agents, including or consisting of a
first binding agent specifically targeting mitotic cells in a
sample, and a second binding agent specifically targeting
proliferating cells in the sample. In certain preferred
embodiments, the first binding agent binds to a phosphorylated form
of histone H3 and the second binding agent binds Ki-67. In another
embodiment, the cocktail further includes a third binding agent
that binds DNA.
[0030] In another aspect, a kit for assessing the prognosis for a
patient who has been diagnosed with a neoplasm includes a first
binding agent specifically targeting mitotic cells in a sample; a
second binding agent specifically targeting proliferating cells in
a sample; one or more detection reagents for visualizing bound
complexes indicative of mitotic and proliferative cells; and
instructions for use. In certain embodiments, the first and second
binding agents are combined in the same container.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1, Panel A schematically depicts the divergent
perspectives of a pathologist and a researcher regarding an
actively dividing cell. Whereas a pathologist views Ki67-positivity
and mitosis as two mutually-exclusive events in cell-cycle, a
researcher views mitosis as a subset of the full cycle of a
proliferating or Ki67-positive cell. Panel B shows micrographs
depicting various stages of mitosis in an H&E-stained
paraffin-embedded breast tumor tissue section. Scale bar is 20
.mu.m. Panel C compares mitotic count determinations from
H&E-stained and p-H3-stained slides. Panel Ci depicts
bar-graphs representing average mitotic counts determined by
counting mitotic figures from H&E-stained or p-H3-stained
slides, by each of the three pathologists. Panel Cii depicts box
and whisker plots representing the average time taken by the three
pathologists to score H&E-stained or p-H3-stained slides. Panel
Ciii depicts a mean and standard deviation plot showing the
difference in the ICC of p-H3-based versus H&E-based counting,
with the confidence intervals. (t-test p<0.05).
[0032] FIG. 2 depicts Kaplan-Meier survival plots (Breast
cancer-specific survival) showing stratification of Lum B and TNBC
patients (n=495) from the Nottingham University dataset.
[0033] FIG. 3 depicts Kaplan-Meier survival plots (Progression-free
survival) showing stratification of a combined set of Lum B and
TNBC patients (n=1070) from the Emory University dataset.
[0034] FIG. 4 depicts Kaplan-Meier survival plots (Overall
survival) showing stratification of a combined set of Lum B and
TNBC patients (n=880) from the Northside Hospital dataset.
[0035] FIG. 5 depicts a patient grade-adjustment model, which
creates an adjusted Nottingham Grade based on KAMS values of Lum B
and TNBC patients.
[0036] FIG. 6 shows the histological grades of 1455 patients from
the Nottingham University dataset (for whom progression-free
survival data was available) and grades adjusted according to the
grade adjustment model depicted in FIG. 5.
[0037] FIG. 7 shows the histological grades of 1460 patients from
the Nottingham University dataset (for whom breast cancer-specific
survival data was available) and grades adjusted according to the
grade adjustment model depicted in FIG. 5.
[0038] FIG. 8 depicts the mean KAMS values of Lum A and Lum B
patients in NG I, NG II and NG III, respectively, in a combined
dataset comprising patients from Northside Hospital, Atlanta, Emory
University Hospital, Atlanta and Nottingham University Hospital,
UK.
[0039] FIG. 9 depicts the extraction and integration of KI and MI
from the same microscopic field using immunofluorescence
microscopy.
[0040] FIG. 10 depicts the extraction and integration of KI and MI
to derive M:P ratio from the same microscopic field using dual
antibody immunohistochemistry.
[0041] FIG. 11 depicts how the extent of centrosome amplification
and inherent mitotic propensity (i.e., the M:P ratio) determine the
rate at which intratumoral heterogeneity (ITH) is generated.
DETAILED DESCRIPTION
[0042] A protocol for assessing the prognosis for a patient
diagnosed with a neoplasm or suspected of having a neoplasm (such
as cancer or benign tumor) and in treating cancer patients is
provided herein. Any patient (e.g., a human of any age, gender, or
ethnicity) diagnosed with a neoplasm or suspected of having a
neoplasm may be selected as a subject for the present methods. The
accompanying descriptions serve to illustrate, but do not limit,
the invention.
Definitions.
[0043] As used herein, the term "neoplastic tissue," "neoplastic
cells," or "neoplasms" refers to an abnormal mass of tissue or a
proliferation of cells. The growth of neoplastic cells exceeds that
of normal tissue around it and it is not coordinated with that of
the normal tissue around it. Neoplasms may be benign (e.g., benign
tumor and atypical hyperplasia), pre-malignant (e.g., carcinoma in
situ and pre-cancer) or malignant (e.g., cancer). The term "cancer"
refers to any of the various malignant neoplasms characterized by
the proliferation of cells that have the capability to invade
surrounding tissue and/or metastasize to new colonization sites,
including but not limited to leukemias, lymphomas, carcinomas,
melanomas, sarcomas, germ cell tumors and blastomas. Exemplary
cancers include cancers of the brain, bladder, breast, cervix,
colon, head and neck, kidney, lung, non-small cell lung,
mesothelioma, ovary, prostate, stomach and uterus, leukemia and
medulloblastoma.
[0044] Neoplastic tissues can originate from any cell type or
tissue found in a mammal, including, but not limited to hepatic,
skin, breast, prostate, neural, optic, intestinal, cardiac,
vasculature, lymph, spleen, renal, bladder, lung, muscle,
connective, tissue, pancreatic, pituitary, endocrine, reproductive
organs, bone, and blood. The neoplastic tissue for analysis may
include any type of solid tumor or hematological cancer. In some
embodiments, the neoplastic tissue is a breast cancer tissue. In
other embodiments, the neoplastic tissue is a breast tissue with
atypical hyperplasia.
[0045] The term "leukemia" refers to broadly progressive, malignant
diseases of the blood-forming organs and is generally characterized
by a distorted proliferation and development of leukocytes and
their precursors in the blood and bone marrow. Leukemia diseases
include, for example, acute nonlymphocytic leukemia, chronic
lymphocytic leukemia, acute granulocytic leukemia, chronic
granulocytic leukemia, acute promyelocytic leukemia, adult T-cell
leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic
leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic
leukemia, leukemia cutis, embryonal leukemia, eosinophilic
leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic
leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell
leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic
leukemia, lymphoblastic leukemia, lymphocytic leukemia,
lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell
leukemia, mast cell leukemia, megakaryocytic leukemia,
micromyeloblastic leukemia, monocytic leukemia, myeloblastic
leukemia, myelocytic leukemia, myeloid granulocytic leukemia,
myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia,
plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia,
Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and
undifferentiated cell leukemia.
[0046] The term "carcinoma" refers to a malignant new growth made
up of epithelial cells tending to infiltrate the surrounding
tissues and give rise to metastases. Exemplary carcinomas include,
for example, acinar carcinoma, acinous carcinoma, adenocystic
carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,
carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell
carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid
carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,
bronchiolar carcinoma, bronchogenic carcinoma, cerebriform
carcinoma, cholangiocellular carcinoma, chorionic carcinoma,
colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform
carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical
carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma
durum, embryonal carcinoma, encephaloid carcinoma, epiennoid
carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,
carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,
gelatinous carcinoma, giant cell carcinoma, carcinoma
gigantocellulare, glandular carcinoma, granulosa cell carcinoma,
hair-matrix carcinoma, hematoid carcinoma, hepatocellular
carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid
carcinoma, infantile embryonal carcinoma, carcinoma in situ,
intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's
carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma,
lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma,
lymphoepithelial carcinoma, carcinoma medullare, medullary
carcinoma, melanotic carcinoma, carcinoma molle, mucinous
carcinoma, carcinoma muciparum, carcinoma mucocellulare,
mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma,
carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma,
carcinoma ossificans, osteoid carcinoma, papillary carcinoma,
periportal carcinoma, preinvasive carcinoma, prickle cell
carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney,
reserve cell carcinoma, carcinoma sarcomatodes, schneiderian
carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell
carcinoma, carcinoma simplex, small-cell carcinoma, solanoid
carcinoma, spheroidal cell carcinoma, spindle cell carcinoma,
carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma,
string carcinoma, carcinoma telangiectaticum, carcinoma
telangiectodes, transitional cell carcinoma, carcinoma tuberosum,
tuberous carcinoma, verrucous carcinoma, and carcinoma
villosum.
[0047] The term "sarcoma" generally refers to a tumor which arises
from transformed cells of mesenchymal origin. Sarcomas are
malignant tumors of the connective tissue and are generally
composed of closely packed cells embedded in a fibrillar or
homogeneous substance. Sarcomas include, for example,
chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma,
myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma,
liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,
botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal
sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal
sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,
giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,
idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic
sarcoma of B cells, lymphomas (e.g., Non-Hodgkin Lymphoma),
immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's
sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,
malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic
sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and
telangiectaltic sarcoma.
[0048] The term "melanoma" is taken to mean a tumor arising from
the melanocytic system of the skin and other organs. Melanomas
include, for example, acral-lentiginous melanoma, amelanotic
melanoma, benign juvenile melanoma, Cloudman's melanoma, S91
melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo
maligna melanoma, malignant melanoma, nodular melanoma subungal
melanoma, and superficial spreading melanoma.
Methods for Prognosis of Patients Diagnosed with a Neoplastic
Condition
[0049] Independent determinations of KI and MI to date, coupled
with their disjointed consideration in decision-making (which
disregards the fact that mitosis is an integral part of the
proliferative cell-cycle), fail to harness their full prognostic
potential. Notwithstanding differences of opinion concerning the
relative merits of KI and MI, it is indisputable that faster tumor
growth is a sign of more aggressive disease. Faster tumor growth
can result from two possible scenarios: (i) KI increases and MI
increases proportionally with KI, or (ii), both KI and MI increase
but MI does not increase proportionally to the increase in KI.
Moreover, recent studies have clearly divulged that majority of
cells within proliferative cell population in a tumor, are not
actually dividing (i.e., are not in M-phase of cell-cycle) but are
instead, populating interphase. The more speedily cells transit
through the cell-cycle, the higher will be the proportion of
mitotic cells observed in the proliferating population.
[0050] The present application utilizes a novel metric that
rationally integrates KI and MI into a ratio for prognosis and
treatment of neoplastic conditions. This new metric, the M-to-P
ratio (or M:P ratio), reveals the cycling kinetics of the
proliferative cells in a tumor. These kinetics change as the agenda
of a tumor evolves. By capturing this "kinetics" element, the
M-to-P ratio directly measures the proportion of proliferative
cells that pose an immediate threat of engendering highly
aggressive progeny cells due to erroneous mitoses that could drive
chromosomal instability and intratumoral heterogeneity. The M-to-P
ratio of a sample thus illuminates a fundamental aspect of that
tumor's biology and its quantitation measures the risk of a tumor
being or rapidly become metastatic. This metric also enables deeper
risk-segmentation of patients (based on their cell cycling kinetics
of their neoplastic tissues) into prognostically meaningful
subgroups to improve selection of a more appropriate treatment
regimen for patients.
[0051] The inventors of the present application have discovered
that independent determinations of the Ki67 index (KI) and the
mitosis index (MI) in different fields of view from different
slides fail to harness their full prognostic potential. Further, it
was discovered that the previous prognostic accuracy of MI was
compromised by subjectivity and errors in visual determination. To
establish this, the inventors first brought KI and MI on the same
measurement scale. Specifically, the inventors obtained the
clinicopathologic data for a large cohort of breast carcinoma cases
from three different hospitals (Nottingham University Hospital, UK;
Emory University Hospital, Atlanta, US; and Northside Hospital,
Atlanta, US).
[0052] In all three hospitals, only mitotic score information was
available for all patients. Therefore, the inventors first
converted MI (categorical-variable) into a mitotic cell percentage.
Briefly, 10 HPFs were evaluated in at least 5 patient samples and
on average had .about.500 cells. Average mitotic cell counts were
determined for each mitotic score category by counting the number
of mitotic cells in 10 HPFs for a total of 267 cases (140 cases
from Emory University Hospital and 127 cases from Northside
Hospital) spanning all three Nottingham grades. It was then
determined that the average mitotic counts for Mitotic Scores 1, 2,
and 3 are 2.94, 11.12 and 32.62, respectively. These mitotic cell
counts provided an estimate of number of mitotic cells per 500
cells (10 HPFs), thus providing the mitotic cell percentage.
[0053] A Ki67-Adjusted mitotic score (KAMS) for each patient was
calculated simply as quotient of percent mitotic cells divided by
percent Ki67-positive cells. A KAMS determination utilizes the KI
and MI information of a given sample as these indices are currently
derived, namely, from different slides (and therefore different
microscopic fields of view), and from totally different scales of
measurement (MI is determined as total mitotic cells in 10 HPFs
while KI is the percentage of nuclei that stain positive for Ki67).
Based on the concept that the more speedily cells transit through
the cell-cycle, the higher will be the proportion of mitotic cells
observed in the proliferating population, the inventors reasoned
that the ratio of the number of mitotic cells in a field of view to
the number of proliferating cells in the same microscopic field of
view (or the mitosis-to-proliferation ratio; or M:P ratio) would
provide the most direct and accurate measurement of cell cycling
kinetics within a sample.
[0054] KAMS analysis of the Nottingham University dataset revealed
that NG II and NG III can be further segmented into "high cycling
kinetics" and "low cycling kinetics" subclasses with prognosis (as
assessed by the patients' clinical outcomes such as breast
cancer-specific survival, progression-free survival,
metastasis-free survival and overall survival) that were
statistically different from each other. These results show that
KAMS can uncover a new layer of information about the cell cycle
kinetics in tumor samples; this information was previously
overlooked in the Nottingham Breast Tumor Grading System which does
not integrate the information provided by MI and KI.
[0055] As shown below, KAMS has the ability to stratify a combined
cohort comprising Luminal B (defined as samples that are ER+ and/or
PR+, Her2+ and ER+ and/or PR+, Her2- with KI of 15% or above) and
triple-negative breast cancers (TNBCs) drawn from all three
Nottingham Grades into two subclasses with different cell cycling
kinetics, in all three datasets assessed by the inventors (FIGS.
2-4). The low KAMS (low cycling kinetics, lower-risk) subclass had
better outcomes that were very similar to that of NG II patients;
the high KAMS (high cycling kinetics, high-risk) subclass had
poorer outcomes that were very close to that of NG III patients.
These data show that KAMS and cell cycling kinetics have prognostic
value in Luminal B and TNBC patients. In particular, these data
also show that a KAMS determination in Luminal B and TNBC patients
can aid in discerning subgroups of patients that possess greater
ITH and bear a higher risk of metastasis and therapy resistance
than others.
[0056] The inventors then evaluated the effectiveness of using KAMS
as a classifier for improving patient risk-stratification in the
Nottingham University dataset. The threshold KAMS value that best
stratifies Luminal B and TNBC patients into a high cycling kinetics
(higher-risk) and low cycling kinetics (lower-risk) subclasses with
significantly different survival probabilities (p<0.05) was
determined (FIG. 5) and log-rank tests were performed for breast
cancer specific survival (BCSS) and progression-free survival (PFS)
(FIGS. 6 and 7).
[0057] Low cycling kinetics Luminal B and TNBC patients were found
to have outcomes that closely match those of patients originally in
NGII; this subclass of Luminal B and TNBC patients were therefore
moved into the "Adjusted NG II" category. Similarly, high cycling
kinetics Luminal B and TNBC patients were found to have outcomes
that closely match those of patients originally in NG III; the high
cycling kinetics subclass of Luminal B and TNBC patients were
therefore moved into the "Adjusted NG III" category. Following this
grade adjustment and re-stratification of patients in the
Nottingham University dataset, the inventors' grade adjustment
model was found to have much better model fit statistics and hazard
ratios (FIGS. 6 and 7) than the original Nottingham Grading System
indicating the strong risk-predictive value of KAMS above and
beyond that provided by NG variables.
[0058] Further, as shown below, KAMS can be used to distinguish
Luminal A and Luminal B subtypes in the clinic since they have very
significantly different and characteristic cell cycling kinetics
(FIG. 8).
[0059] Immunofluorescence-based (FIG. 9) and
immunohistochemistry-based (FIG. 10) methods may be used to
determine the M:P ratio in clinical samples. Accordingly, the M:P
ratio metric can provide a measure of mitotic propensity of a
proliferative population and a measure of the risk posed by the
proliferative population due to erroneous mitoses that could drive
chromosomal instability and intratumoral heterogeneity. The present
methods reduce inter-observer variability, enhance the
reproducibility and accuracy of MI determinations, and may be used
to risk stratify NG and KI-based groups, which has profound
clinical implications as further elaborated below.
[0060] In one aspect, a method of assessing the prognosis for a
patient who has been diagnosed with a neoplastic condition,
includes the step of providing a tissue sample suspected of
containing neoplastic cells from the patient and exposing at least
a portion of the sample to at least two binding agents under
conditions sufficient for binding the binding agents to the
neoplastic cells. Whereas the first binding agent specifically
targets all mitotic cells in the sample portion, the second binding
agent specifically targets all proliferating cells in the sample
portion. Upon binding of the binding agents to the cells, the
sample is further exposed to detection reagents suitable for
visualizing proliferating cells and discriminating between
proliferating cells that are mitotic and proliferating cells that
are non-mitotic. Following this step, the M:P ratio within a common
portion of the sample is determined.
[0061] A serial section of the sample that has been stained with
hematoxylin-eosin, or stained with a cancer-specific marker, or a
marker that labels only non-cancer cells, is then processed for
either immunohistochemical visualization, immunofluorescence
visualization, or visualization using quantum dots to ensure that
the region of the sample being profiled for M:P ratio comprises
tumor cells only. The determination of M:P ratio in a clinical
tissue sample could be combined with methods that allow the
visualization of apoptotic cells (marked by a marker for apoptosis)
that would allow quantification of net tumor growth kinetics
(arising from the addition of new cells via mitoses in the
proliferative population within a tumor, minus the loss of cells
due to apoptosis).
[0062] In certain embodiments, determination of M:P scores in
tissue samples or tissue sections may include staining and imaging
of a tissue sample (e.g., whole-slide imaging or imaging of
specific regions of interest), whereby the image analysis is
carried out to determine mitotic and proliferative cells. From
these images, an M:P ratio can be quantitated in areas deemed as
Ki67 "hot-spots" exclusively, or from both "Ki67 Hotspots" and
"Ki67 non-hotspot regions" after giving appropriate weights to
these types of regions. In addition, M:P ratios may be derived from
"Mitotic hotspots" and regions that are not "mitotic hotspots"
after giving these types of regions appropriate weights.
[0063] An M:P ratio may be derived from any region of interest or
from multiple regions after giving them appropriate weights (e.g.,
M:P ratios in regions that show high expression of certain
biomarkers or high CAS (see below) may have special prognostic
significance). In each case, an optimal scoring method may be
determined via retrospective studies of samples with known clinical
outcomes, whereby the weighted model providing the best concordance
with clinical outcomes would be selected as the M:P ratio scoring
method. Preferably, these image analysis steps are computer-aided
with the use of appropriate software.
[0064] In contrast to determining the mitotic index (MI) and
nuclear Ki67 positivity as two independent variables, the present
method determines the proportion of mitotic cells within all of the
proliferating cell pool in a common field. Mitotic cell positivity
may be scored as the total number of cells expressing an M-phase
specific marker that can be labeled with a suitable M-phase
specific binding agent (e.g., "first binding agent"). Proliferative
cell positivity may be scored as the total number of cells
selectively expressing a marker during all proliferative phases of
the cell cycle (i.e., G1, S, G2 and M). Nuclear Ki67 antigen is an
exemplary proliferative cell marker that can be tracked using a
suitable "second binding agent," such as an anti-Ki67 antibody.
Within the population of proliferating cells in a portion of the
tissue sample, a subpopulation of mitotic (M phase) cells exists,
where the proportion of mitotic cells to proliferating cells
defines the M:P ratio.
[0065] The tissue sample can be manipulated by, for example,
sectioning or dissociation, and exposed to the first and second
binding agents, either sequentially or simultaneously, for a time
and under conditions sufficient to allow the agents to detectably
label cells within the sample.
[0066] M:P ratios reveal the dynamic agenda of an evolving tumor
and provides highly actionable information that can aid risk
stratification of unselected cohorts of operable early-stage breast
cancer patients, especially those with Luminal B and TNBC subtypes.
Incorporation of a M:P ratio thus maximizes the use of available
biomarker information to facilitate personalized medicine for
breast cancer management.
[0067] In certain embodiments, a portion of the patient's tissue
may be further analyzed to quantitate the numeric degree and
structural degree of centrosomal amplification. Centrosome
amplification is a key driver of chromosomal instability that
underlies the generation of karyotypic diversity and the evolution
of more aggressive and malevolent phenotypes such as metastases and
therapeutic resistance. Quantitation of centrosome amplification
can include the steps of: (a) processing a sample of tumor tissue
or neoplastic cells from the patient in a form suitable for
visualization and demarcation of cell nuclei, individually
distinguishable centrosomes (iCTRs) and megacentrosomes (mCTRs) in
a region of interest (ROI) defined by a plurality of cell nuclei;
(b) determining the numbers of iCTRs and mCTRs associated with each
cell nucleus in the ROI; (c) determining the volume of each mCTR in
the ROI; and (d) calculating one or more centrosome amplification
scores (CASs) values for the sample based on steps (b) and (c),
wherein the one or more CASs indicate the severity of centrosome
amplification, the frequency of centrosome amplification, or both,
and wherein the one or more scores provide a measure of a level of
risk and/or a prognosis associated with the neoplastic tissue.
[0068] Intratumoral heterogeneity (ITH) in cancers is crucial for
orchestrating the growth, survival, invasion and spread of cancer
cells in a patient's body. The generation of ITH and metastatic
clones relies on frequent passage of cancer cells through
error-prone mitoses.
[0069] Information from M:P scores and CAS scores can provide an
important measure of the rate at which intratumoral heterogeneity
is being generated, thereby providing a better prediction of
metastatic risk and therapy resistance. The higher the M:P ratio
and CAS scores, the greater the intratumoral heterogeneity and the
poorer the patient's prognosis. Further integrating CAS scores into
this analysis can further improve the assessment of intratumoral
heterogeneity to more accurately predict a patient's prognosis,
including metastatic risk, and provide a more rational, efficacious
basis for treatment.
[0070] For example, in some embodiments images of tissue samples
that have been stained for mitotic and proliferative cells may be
overlaid with images of serial sections stained for a variety of
biomarkers, including amplified centrosomes. This can provide new
cumulative measures of risk that combine M:P ratio with different
biomarkers so as to provide more appropriate weightage to the M:P
ratio. Thus, these other biomarkers can be employed for more
accurate risk prognostication and selection of more optimal
therapies.
[0071] The M:P ratios or the cumulative measures of risk described
above could also be used in conjunction with clinicopathological
variables routinely determined in the clinic, including the results
of other risk-predictive tests (such as Oncotype Dx) or
gene-expression information to yield new risk models for improved
patient stratification and personalization of cancer treatment.
Compositions and Kits
[0072] In another aspect, the present application provides
compositions and kits for the prognosis methods described herein.
In one embodiment, the composition includes a cocktail of two cell
cycle specific binding agents. The first binding agent specifically
targets all mitotic cells in a sample. The second binding agent
specifically targets all proliferating cells in the sample. In
another embodiment, the cocktail consists of the first binding
agent specifically targeting all mitotic cells in a sample, and the
second binding agent specifically targeting all proliferating cells
in the sample.
[0073] Each of the first and second binding agents may be an
antibody or a biologically active fragment thereof. In certain
preferred embodiments, the first binding agent targets a
phosphorylated form of histone H3 and the second binding agent
targets Ki-67.
[0074] In another embodiment, a kit for neoplasm prognosis includes
a first cell cycle specific binding agent specifically targeting
all mitotic cells in a sample; a second cell cycle specific binding
agent specifically targeting all proliferating cells in a sample;
one or more detection reagents for visualizing bound complexes
indicative of mitotic and proliferative cells; and instructions for
use. The first and second binding agents may be included in the
same container or in separate containers.
[0075] The above described cocktail may be used in performing the
various methods described herein. For example, one can use a
cocktail/composition including both an antibody that specifically
binds mitotic cells (e.g., an anti-PH3 antibody) and an antibody
that specifically binds proliferating cells (e.g., an anti-Ki67
antibody), thus enabling the simultaneous detection of both types
of target cells in the same defined portion of a sample (e.g., in
the same field of view in paraffin-embedded tissue sections).
Co-immunostaining the same sample or same portion of a sample
(e.g., the same tissue) with both agents enables the observer to
score both Ki67-positive and mitotic cell simultaneously. It also
ensures that the same scale is used for scoring both the
parameters. To facilitate simultaneous detection of mitotic and
proliferating cells within the same portion of a sample, the agents
targeting these cell types may be labeled to allow for distinct
recognition (e.g., with two distinct colors etc.). For example, an
alkaline phosphatase reaction produces a pink color when labeling
Ki67, and a horseradish peroxidase reaction produces a brown color
when labeling PHI Preferably, the antibodies within a cocktail
exhibit little cross-reactivity.
Processing of Tissue Samples and Methodology for Analysis
[0076] 1. Patient Selection.
[0077] A wide variety of patients diagnosed with a neoplastic
condition or suspected of having a neoplastic condition can benefit
from the present methods.
[0078] 2. Cell and Tissue Sources.
[0079] As noted above, the present application includes methods of
assessing the prognosis for a patient who has been diagnosed with a
neoplasm or cancer. These methods and others described herein may
commence by providing a biological sample that is suspected of
including neoplastic cells. The biological sample can be a cell
sample, a tissue sample or a sample of biological fluids carrying
cells, such as blood, urine, tears, lymph, bile, cerebrospinal
fluid, interstitial fluid, aqueous or vitreous humor, colostrum,
sputum, amniotic fluid, saliva, anal and vaginal secretions,
perspiration, semen, transudate, exudate, and synovial fluid.
[0080] Any cell or tumor cell type can serve as a cell or tissue
sample for the inventive method, including those described above.
Cells can originate from a variety of different sources, including
the breast, prostrate, lung, brain, colon, bladder, kidney, cervix,
testis, ovary, liver, pancreas, head and neck, anogenital tissue,
adrenal gland, and blood. The present methods may be applied to any
patient (e.g., a human of any age, gender, or ethnicity) who has
been diagnosed with a neoplasm or cancer. This includes patients
diagnosed with a breast cancer; a biliary tract cancer; a bladder
cancer; a brain cancer (e.g., a glioblastomas or medulloblastomas);
a cervical cancer; a choriocarcinoma; a colon cancer; an
endometrial cancer; an esophageal cancer; a gastric cancer; a
hematological neoplasm (e.g., acute lymphocytic leukemia or
lymphoma, leukemia/lymphoma, hairy cell leukemia, chronic
lymphocytic leukemia, chronic myelogenous leukemia, multiple
myeloma, or an adult T-cell leukemia/lymphoma); an intraepithelial
neoplasm including Bowen's disease and Paget's disease; a liver
cancer; a lung cancer; a neuroblastoma; a melanoma, an oral cancer
including squamous cell carcinoma; an ovarian cancer including
ovarian cancer arising from epithelial cells, stromal cells, germ
cells and mesenchymal cells; a pancreatic cancer; a prostate
cancer; a rectal cancer; a sarcoma, including angiosarcoma,
gastrointestinal stromal tumors, leiomyosarcoma, rhabdomyosarcoma,
liposarcoma, fibrosarcoma, and osteosarcoma; a renal cancer
including renal cell carcinoma and Wilms tumor; a skin cancer
including basal cell carcinoma and squamous cell cancer; a
testicular cancer including germinal tumors such as seminoma,
non-seminoma (teratomas, choriocarcinomas), stromal tumors, and
germ cell tumors; and a thyroid cancer including thyroid
adenocarcinoma and medullary carcinoma. Typically, the cell sample
is derived from tumor tissue that was surgically removed from a
human patient or other mammal.
[0081] It should be appreciated that a variety of different tumor
types can arise in certain organs, which may differ with regard to,
for example, their clinical and/or pathological features and/or the
agents expressed. Tumors arising in a variety of different organs
are discussed, for example, in the WHO Classification of Tumours
series, 4th ed, or 3rd ed (Pathology and Genetics of Tumours
series), by the International Agency for Research on Cancer (IARC),
WHO Press, Geneva, Switzerland. In certain embodiments, the
neoplasm or cancer may be one that is considered resistant to
treatment (e.g., hormone resistant or chemotherapeutic
resistant).
[0082] 3. Biopsy Techniques.
[0083] A variety of biopsy techniques may be used to obtain a cell
or tissue sample such as, but not limited to excisional (i.e.,
removal of an entire lesion) or incisional (i.e., where a portion
or wedge of tissue is removed). In some cases, a fine-needle may be
required to withdraw cellular material from a tissue mass using
aspiration techniques (e.g., aspiration biopsy). Further, cell or
tissue samples may be cells isolated from any cell suspension, body
fluid samples, or cells dislodged from tumor by any other
means.
[0084] 4. Preparation of Tissue Samples.
[0085] In the present methods, normal, neoplastic and/or cancer
tissue samples can be formalin-fixed paraffin-embedded or may be
fresh-frozen in an OCT compound (such compounds are well-known in
the art) and sectioned or fixed with methanol or any other
appropriate fixative (such fixatives, processes and types are
well-known in the art). Formalin-fixed, paraffin-embedded tissue
must be subjected to de-paraffinization, peroxide quenching and
antigen retrieval (e.g., heating under pressure in a citrate
buffer) prior to the staining steps that allow visualization of
centrosomes and nuclei within the sample.
[0086] 5. Labeling and Detection of Target Cell Antigens or Target
Cell Subpopulations.
[0087] When assessing the number of mitotic cells and/or the number
of proliferating cells, one can use any method known in the art,
including immunohistochemistry (by bright field or fluorescence)
and flow cytometry (see, e.g., Vignon et al., PLoS ONE 8(7):
e68425. doi:10.1371/journal.pone.0068425). Both fluorescence
(direct and indirect) and immunohistochemical (IHC) staining
methods may be employed for the purpose of staining mitotic cells,
proliferating cells, centrosomes and/or DNA for visualization
purposes.
[0088] For immunohistochemistry or immunofluorescence, the sample
can be fresh frozen or "fixed" with a fixative such as formaldehyde
or glutaraldehyde as described above. The fluorescence-based and/or
immunohistochemical-based staining methods may employ any one of
the variety of antibodies directed against mitosis specific
markers, such as MPM-2 and phospho Histone H3 (PHH3); proliferation
specific markers, such as Ki67; and centrosomal markers, such as
.gamma.-tubulin. For viewing the cells by immunofluorescence, the
sample may be exposed to an agent (e.g., a primary or secondary
antibody) that is conjugated to a chromophor (e.g., a fluorochrome
or fluorophore). Thus, the secondary antibodies for detecting Ki67,
MPM-2, PHH3 and .gamma.-tubulin, may be conjugated to suitable
chromophors, such as Alexa Fluor 555, Alexa Fluor 488,
TRITC-conjugated, FITC-conjugated etc. Many of the steps of
preparing and analyzing a sample can be automated and/or
computer-aided.
[0089] In certain preferred embodiments, determination of M-to-P
ratios employs the use of clinically-facile multi-color
immunohistochemistry methods, where different colored detection
labels distinguish between different target markers. This improves
simplifies the detection process and improves the accuracy of
patient risk-stratification.
[0090] In certain embodiments, an anti-pericentrin antibody may be
used as the primary antibody for labelling centrosomes instead of
anti-.gamma.-tubulin antibody. In other embodiments, the primary
antibody itself is conjugated to a fluorophore or quantum dots or
an enzyme for enabling visualization. When using quantum dots,
visualization of centrosomes and quantitation of CASs may be
multiplexed with (or carried out simultaneously along with)
visualization of other proteins in the same sample.
[0091] In certain embodiments, as an alternative to
fluorescence-based detection of centrosomes, immunohistochemical
(IHC) staining may be employed for imaging centrosomes. For
example, an HRP-based detection system employing hematoxylin
counterstain may be used for imaging centrosomes (as brown colored
dots) using a brightfield imaging system with optical sections
(i.e., z-stacks) followed by image deconvolution to enable
software-assisted 3D volume rendering as further described below.
Centrosome volume ranges may be determined from
immunohistochemically stained normal tissues to aid in analysis of
iCTRs and mCTRs in tumor tissues. Alternatively, an alkaline
phosphatase-based detection system (producing red color instead of
brown) may be used in place of the HRP-based system for IHC. In
other embodiments, there could be variation in the primary antibody
used for labelling centrosomes. For example, instead of using
.gamma.-tubulin, pericentrin may be used for labelling whole
centrosomes.
[0092] 6. Binding Agents and Antibodies.
[0093] In certain embodiments, cell samples may be stained with one
or more antibodies, biologically active fragments thereof, and/or
binding agents directed against mitosis-specific or
proliferation-specific cell markers. Although the invention is not
so limited, either the first and/or the second binding agent can be
an antibody. As used herein, the term "antibody" encompasses
monoclonal antibodies, polyclonal antibodies, multivalent
antibodies, multispecific antibodies, single chain antibodies,
human or humanized antibodies, and antibody fragments or other
variants that retain the ability to specifically bind a target
antigen. Antibodies used in the present application can be
purchased commercially or, if necessary or desired, can be
generated using techniques well known in the art. The same is true
for any antibody useful in the context of the present methods
(e.g., for antibodies that stain, label, or target mitotic cells,
proliferating cells, pericentriolar matrix (PCM) etc.). (See, e.g.,
Antibodies: A Laboratory Manual (Second Edition), Edited by Edward
A. Greenfield. 2014, Cold Spring Harbor Laboratory Press).
[0094] In certain preferred embodiments, the first binding agent
binds to a mitotic specific marker. Exemplary mitosis specific
markers include MPM-2 and phospho Histone H3 (PHH3). Exemplary
mitosis specific binding agents include MPM-2 monoclonal antibody,
anti-phospho Histone H3 antibody, Phospho-Histone H3 Ser28
(PHH3).
[0095] In other preferred embodiments, the second binding agent
binds a proliferation-specific cell marker. Exemplary
proliferation-specific markers include Ki-67, proliferating cell
nuclear antigen (PCNA), Ki-S2, Ki-S5, MCM2, MCM3, MCM4, MCM5, MCM6,
MCM7, MCM10, CAF-1 p60, CAF-1 p150, Pomfil2, Unc-53, CDC6, CDC7,
CDC7 protein kinase, Dbf4, CDC14, CDC14 protein phosphatase, CDC45,
topoisomerase 2 alpha, DNA polymerase delta, replication protein A
(RPA), replication factor C (RFC) and FEN1. Anti-Ki-67 antibodies
used in the present invention can be purchased commercially or, if
necessary or desired, can be generated using techniques well known
in the art.
[0096] As noted above, antibodies for use in the present invention
can also be produced by injecting an antigen into laboratory or
farm animals to evoke high expression levels of antigen-specific
antibodies in the serum, which can then be removed from the animal.
Polyclonal antibodies can be recovered directly from serum.
Monoclonal antibodies can be produced by fusing antibody-secreting
spleen cells from immunized mice with immortal myeloma cell to
create monoclonal hybridoma cell lines that express the specific
antibody in cell culture supernatant.
[0097] In certain embodiments, cell samples may be stained with one
or more antibodies, biologically active fragments thereof, and/or
binding agents directed against pericentriolar matrix components.
Preferably, the primary antibody or binding agent specifically
binds an antigen, protein or component of the pericentriolar matrix
(PCM) that shows substantial localization to centrosomes at all
stages of the cell cycle (i.e., interphase, mitosis (including
prophase, metaphase, anaphase, telophase) and cytokinesis). In some
embodiments, the primary antibody or binding agent is conjugated to
a fluorophore or quantum dot or enzyme, etc. to facilitate
visualization of signal. When using quantum dots, visualization of
centrosomes and quantitation of CASs may be multiplexed with (or
carried out simultaneously along with) visualization of other
proteins in the same sample. In other embodiments, a secondary
antibody or binding agent that binds to the primary antibody or
binding agent is used to facilitate visualization. By colocalizing
with centrosomes, the PCM binding agents produce a detectable
signal above background so as to provide reliable image acquisition
and 3D volume rendering. Volume rendering creates a binary image
for volume determination.
[0098] Components of the PCM that localize to the PCM throughout
the cell cycle include proteins include .gamma.-tubulin,
pericentrin, centromere protein J (CPAP/Sas-4) and ninein.
Accordingly, these PCM components may be targeted using e.g.,
anti-.gamma.-tubulin antibodies, including e.g., T3320, T-3195,
T-3559, and C7604 (Sigma-Aldrich); ab11317, ab16504, ab27074
(Abcam); and sc-7396 (Santa Cruz Biotechnology); anti-pericentrin
antibodies, including e.g., A301-348A, A301-349A and IHC-00264
(Bethyl Laboratories); ABT59 (EMD Millipore); ab4448, ab28144,
ab99342, ab84542, ABIN968665, ABIN253211, ABIN253210, ABIN910327
(Abcam); CPBT-42894R1I, CPBT-42892RH, CPBT-42891RN (Creative
BioMart); sc-28145, sc-28143, sc-28144, sc-68928 (Santa Cruz
Biotechnology), HPA016820, HPA019887 (Sigma-Aldrich); NB 100-61071,
NBP100-61072, NBP1-87771 and NBP1-87772 and (Novus Biologicals);
anti-centromere protein J antibodies, including e.g., ABIN527721,
ABIN527722 and ABIN527723 (Abcam); 101-10278 (Ray-Biotech); and
CABT-22656MH (Creative BioMart); and anti-ninein antibodies,
including e.g., ab52473, ab4447 (Abcam); 41-3400 (Life
Technologies); orb100831 (Biorbyt); HPA005939 (Atlas Antibodies);
sc-376420 and sc-292089 (Santa Cruz Biotechnology).
[0099] Alternatively, or in addition, the antibodies or binding
agents may target one or more of the following: the nucleus of a
cell, comprised of key structural components such as the nuclear
envelope, nucleoplasm, nucleoskeleton, nuclear lamina (including
lamin proteins, such as LEM3), RNA molecules, chromosomes,
chromatin, including euchromatin and heterochromatin, nucleolus,
and other subnuclear bodies (e.g., Cajal bodies, Gemini of coiled
bodies or gems, RAFA domains, polymorphic interphase karyosomal
association (PIK), promyelocytic leukaemia (PML) bodies,
paraspeckles, splicing speckles and perichromatin fibrils). In
other embodiments, the antibody or binding agent is an antibody or
binding agent that is capable of binding to any subcellular
organelle that is present as one copy per cell or whose number of
copies per cell is constant for a given cell cycle phase and is
well-established.
[0100] In other embodiments, centriolar markers are used to stain
centrioles and provide 3-dimentional information about centriolar
volumes and structural aberrations.
[0101] Although the invention is not so limited, when any nuclear
component or nuclear membrane component is targeted, the stain may
be a fluorescent protein-based marker for the nucleus. Exemplary
fluorescent protein-based nuclear markers include, but are not
limited to CellLight Nucleus-Green Fluorescent Protein (C 10602),
CellLight.RTM.Nucleus-RFG (Red Fluorescent Protein; 10603),
CellLight.RTM.Nucleus-Cyan Fluorescent Protein and Alexa Fluor 488
conjugate of Histone Hl (H13188)); nuclear counterstains for live
cells and unfixed tissues, such as Hoechst 33342 dye and SYTO dyes
40 (S11351), 11 (S7573), 13 (S7575), 12 (S7574), 14 (S7576), 16
(S7578), 17 (S7579) and 59 (511341)); nucleic acid stains,
including dimeric cyanine dyes, and fluorescein-12-dUTP (C7604);
4',6-diamindino-2-phenylindole (DAPI; D1306, D3571, D21490);
Hoechst stains, such as Hoechst 33258, Hoescht 34580, Hoechst
S769121 (N21485) and Hoeshst 33342 (H1399, H3570 and H21492);
BOBO-1 (B3582), BOBO-3 (B3586), SYTOX (S7020), SYTOX (Si 1368),
SYTOX Blue (511348, S34857), YO-PRO-1 dye (Y3603), TOTO-1 (T3600),
TOTO-3 (T3604), TO-PRO-3 (T3605), YOYQ-1 (Y3601), propidium iodide
(P1304MP, P3566, P21493); and other chromosome banding dyes,
including 7-aminoactinomycin D (7-AAD, A1310) and
9-amino-6chloro-2-methoxyacridine (ACMA, A1324).
Quantitating the Numeric and Structural Degree of Centrosome
Amplification (CA)
[0102] As described above, the method may additionally include
quantitating the numeric and structural degree of centrosome
amplification (CA) in tumor samples as further described in U.S.
patent application Ser. No. 14/632,778, filed Feb. 26, 2015, the
disclosure of which is expressly incorporated by reference herein.
A protocol for determining one or more CA scores (CAS) in normal,
neoplastic and/or cancer cells using a standardized, quantitative
method may be utilized in the methods described herein. This
methodology involves a key transformative step of classifying
centrosomes into individually distinguishable centrosomes (iCTRs)
and megacentrosomes (mCTRs). Although these types of aberrations
often occur together, their biological origin and clinical
consequences may be different. These two different types of
aberrations can make different contributions to the development and
progression of neoplasms or cancer, hence the classification scheme
herein facilitates quantitation of these types of aberrations
separately.
[0103] iCTRs are centrosomes that stain positive for
.gamma.-tubulin, with centrosomes numbers and boundaries clearly
distinguishable and volumes that lie within the range of centrosome
volumes found in normal tissue (e.g., 0.23-0.76 cubic microns for
breast tissue immunostained for .gamma.-tubulin). mCTRs are
centrosomes in a neoplastic region that stain positive for
.gamma.-tubulin and whose volume is greater than the upper limit of
the centromere volume range found in corresponding normal tissue
(e.g., 0.76 cubic micron for breast tissue immunostained for
.gamma.-tubulin). mCTRs could either be centrosomes with aberrantly
large volumes or could represent a situation wherein multiple
centrosomes are clumped together so closely that their precise
numbers and boundaries cannot be discerned or resolved.
[0104] For each cell in a sample, a measure of the severity of
centrosome amplification (numerical or structural) with reference
to a normal centrosome numbers and volumes may be determined In
addition, for each sample, the frequency of numerical and
structural amplification may be quantitated through calculation of
CA score for iCTRs (CAS.sub.i) and CA score for mCTRs (CAS.sub.m),
respectively. Scaling factors may be included in algorithms to
ensure that CAS.sub.i and CAS.sub.m have the same weight in the
cumulative CA score (CAS.sub.total).
[0105] As used herein, the term "normal centrosomes" refers to
centrosomes found in normal tissue (including adjacent non-involved
tissue in a tumor core biopsy or resected tumor tissue) and stain
positive for .gamma.-tubulin, with numbers and boundaries clearly
distinguishable and volume not exceeding the normal range of
centrosomes of the corresponding tissue or cell type. For each
tissue type, the normal range of centrosome volumes is determined
from a large cohort of normal tissue samples. For example,
centrosome volumes (as determined by immunostaining for
.gamma.-tubulin) in normal breast tissue range from 0.23 to 076
cubic microns; in normal pancreatic cell tissues from 0.20 to 0.56
cubic microns; and in normal bladder cell tissues from 0.35 to 0.74
cubic microns.
[0106] Generally, most normal somatic tissues average between 1-2
normal centrosomes per nucleus and no mCTRs. By contrast, cancer
cells may have >2 iCTRs and several mCTRs per nucleus.
Three-dimensional analysis of iCTRs and mCTRs in cancer cells can
provide a useful tool for optimizing a risk profile of cancer in a
patient to facilitate a more risk-adapted and optimal course of
treatment.
[0107] In one embodiment, the method includes the step of
processing a sample of neoplastic tissue from the patient to
facilitate three dimensional visualization and demarcation of cell
nuclei, iCTRs and mCTRs in a region of interest (ROI) defined by a
plurality of cell nuclei. Three dimensional image data is generated
so as to provide volume rendering of the iCTRs and mCTRs. In some
embodiments, the 3D image is produced by confocal imaging of
immunofluorescently stained centrosomes. In other embodiments,
immunohistochemical (IHC) staining methods (e.g., HRP-based
detection with hemotoxylin counterstain) are used to produce 3D
image of centrosomes. Imaging of the centrosomes (brown colored
dots) will be done using a bright field imaging system with optical
sections (i.e., z-stacks) followed by image deconvolution, to
enable software-assisted 3D volume rendering. Centrosome volume
range as determined in the immunohistochemically stained normal
tissues will be used to determine iCTRs and mCTRs in the tumor
tissues. Images could either be obtained from 10-15 microscopic
fields of view for each sample or by whole-slide imaging as long as
optical sections are acquired for 3D volume rendering. For slides
stained immunofluorescently for centrosomes, imaging is carried out
in areas determined to be "tumor areas" based on comparison with a
serial section stained with hematoxylin eosin (wherein tumor areas
are pre-marked). In slides stained immunohistochemically for
centrosomes, only iCTRs and mCTRs in tumor areas will be analyzed
for CAS determination.
[0108] From this image data, the following are determined: [0109]
(i) the number of iCTRs and mCTRs associated with each cell nucleus
in the ROI, [0110] (ii) the volume of each iCTR and mCTR associated
with each cell nucleus in the ROI, [0111] (iii) the average number
of excess iCTRs (i.e., iCTRs in excess of 2) amongst cells that
have >2 centrosomes; this gives a measure of the "severity" of
numerical amplification present in the cells that bear numerically
amplified centrosomes, [0112] (iv) the percentage of cell nuclei
that have excess iCTRs (i.e., iCTRs in excess of 2); this gives a
measure of the "frequency" or "prevalence" of numerical centrosome
amplification, [0113] (v) the average volume deviation (compared to
the upper limit of the volume of normal centrosomes) of mCTRs among
the cells that bear mCTRs; this gives a measure of the "severity"
of structural amplification present in cells that bear structurally
amplified centrosomes or mCTRs, [0114] (vi) the percentage of cell
nuclei that have mCTRs associated with them; this gives a measure
of the "frequency" or "prevalence" of structural amplification of
centrosomes.
[0115] Based on these numerical and structural determinations, one
or more CASs are determined as further described below. The scores
indicate the severity of centrosome amplification, the frequency of
centrosome amplification, or both, in the sample and provide a
measure of the level of risk associated with the neoplastic
tissue.
Treatment with Antineoplastic Agents
[0116] The presently described methods provide a more accurate
predictions of a patient's prognosis, including metastatic risk,
and provide a more rational, efficacious basis for treatment. For
example, in some embodiments, M:P ratio profiling of patients may
help to decide which Luminal B patients should be given hormone
therapy and chemotherapy (i.e., perhaps the higher-risk high
cycling kinetics subclass) and who should receive hormone therapy
alone. Further, when combined with determination of CAS scores, M:P
ratios might help clinicians to decide who might benefit from
declustering drugs and anti-mitotic therapeutics, including
chemotherapeutic agents.
[0117] In some embodiments, M:P ratios can be used to predict who
is more likely to benefit from specific types of neoadjuvant
chemotherapy. In other embodiments, the M:P ratios can be used to
stratify TNBCs into high cycling kinetics (higher-risk) who might
require aggressive treatment and low cycling kinetics subclasses
for whom less aggressive therapy may suffice.
[0118] In view of the foregoing, in certain embodiments, the
patient is treated with at least one antineoplastic agent based on
the results from the M:P determination. Exemplary antineoplastic
agents include anti-mitotic agents, anti-interphase agents,
anti-microtubule agents, anthracycline-based agents and aromatase
inhibitor agents.
[0119] In certain embodiments, the patient is administered one or
more centrosome declustering agents, including but not limited to
griseofulvin; noscapine, noscapine derivatives, such as brominated
noscapine (e.g., 9-bromonoscapine), reduced bromonoscapine (RBN),
N-(3-brormobenzyl) noscapine, aminonoscapine and water-soluble
derivatives thereof; CW069; the phenanthridene-derived
poly(ADP-ribose) polymerase inhibitor, PJ-34;
N2-(3-pyridylmethyl)-5-nitro-2-furamide,
N2-(2-thienylmethyl)-5-nitro-2-furamide,
N2-benzyl-5-nitro-2-furamide, an anthracine compound as described
in U.S. Patent Application Publication 2008/0051463; a
5-nitrofuran-2-carboxamide derivative as described in U.S.
Provisional Application 61/619,780; and derivatives and analogs
therefrom.
[0120] In another embodiment, the patient is administered an
inhibitor of HSET, a key mediator of centrosome clustering. The
inhibitor of HSET can be a small molecule drug or a nucleic
acid-based therapeutic, such as an siRNA, an shRNA-encoded
expression vector or an antisense oligonucleotide, whereby the
inhibitor inhibits the activity and/or expression of HSET in the
targeted cell. Alternatively, or in addition, the patient may be
administered an inhibitor of a protein that is upregulated with
HSET or inhibitors of other proteins implicated in centrosome
clustering. HSET co-regulated product targets include, but are not
limited to Npap60L, CAS, Prc1, Ki67, survivin, phospho-survivin,
Hif1.alpha., aurora kinase B, p-Bc12, Mad1, Plk1, FoxM1, KPNA2,
Aurora A and combinations thereof. In other embodiments, the
patient is administered one or more agents that block the nuclear
accumulation of HSET during interphase.
[0121] siRNAs are double-stranded RNAs that can be engineered to
induce sequence-specific post-transcriptional gene silencing of
mRNAs. Synthetically produced siRNAs structurally mimic the types
of siRNAs normally processed in cells by the enzyme Dicer. siRNAs
may be administered directly in their double-stranded form or they
may be expressed from an expression vector is engineered to
transcribe a short double-stranded hairpin-like RNA (shRNA) that is
processed into a targeted siRNA inside the cell. Suitable
expression vectors include viral vectors, plasmid vectors and the
like and may be delivered to cells using two primary delivery
schemes: viral-based delivery systems using viral vectors and
non-viral based delivery systems using, for example, plasmid
vectors. Exemplary viral vectors may include or be derived from an
adenovirus, adeno-associated virus, herpesvirus, retrovirus,
vaccinia virus, poliovirus, poxvirus, HIV virus, lentivirus,
retrovirus, Sindbis and other RNA viruses and the like.
[0122] As used herein, the term "oligonucleotide" refers to a
single stranded nucleic acid containing between about 15 to about
100 nucleotides. An antisense oligonucleotide comprises comprise a
DNA backbone, RNA backbone, or chemical derivative thereof, which
is designed to bind via complementary binding to an mRNA sense
strand of a target gene (such as HSET) so as to promote RNase H
activity, thereby leading to degradation of the mRNA. Preferably,
the antisense oligonucleotide is chemically or structurally
modified to promote nuclease stability and/or increased binding.
The single stranded antisense oligonucleotide may be synthetically
produced or it may be expressed from a suitable expression vector.
In addition, the antisense oligonucleotide may be modified with
nonconventional chemical or backbone additions or substitutions,
including but not limited to peptide nucleic acids (PNAs), locked
nucleic acids (LNAs), morpholino backboned nucleic acids,
methylphosphonates, duplex stabilizing stilbene or pyrenyl caps,
phosphorothioates, phosphoroamidates, phosphotriesters, and the
like.
[0123] In certain embodiments, the small molecule drug targets the
motor domain of HSET and/or specifically binds to the
HSET/microtubule binary complex so as to inhibit HSET's
microtubule-stimulated and/or microtubule-independent ATPase
activities. In a specific embodiment, the small molecule drug is
AZ82 or CW069 or a therapeutically effective derivative, salt,
enantiomer, or analog thereof.
[0124] AZ82 binds specifically to the KIFC1/microtubule (MT) binary
complex and inhibits the MT-stimulated KIFC1 enzymatic activity in
an ATP-competitive and MT-noncompetitive manner with a Ki of 0.043
.mu.M. Treatment with AZ82 causes centrosome declustering in BT-549
breast cancer cells with amplified centrosomes.
[0125] Alternatively, or in addition, the patient may be
administered a poly(ADP-ribose) polymerase (PARP) inhibitor, an
inhibitor of the Ras/MAPK pathway, an inhibitor of the
PI3K/AKT/mTOR pathway, an inhibitor of FoxM1, Hif1.alpha.,
survivin, Aurora, Plk1 or a combination thereof. Exemplary PARP
inhibitors include, but are not limited to olaparib, iniparib,
velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827,
INO-1001, CEP-9722, PJ-34, Tiq-A, Phen, PF-01367338 and
combinations thereof. Exemplary Ras/MAPK pathway agents include,
but are not limited to MAP/ERK kinase (MEK) inhibitors, such as
trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026,
RO4987655, RO5068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162,
GDC-0973 and combinations thereof. Exemplary PI3K/AKT/mTOR pathway
inhibitors include, but are not limited to everolimus,
temsirolimus, GSK2126458, BEZ235, PIK90, PI103 and combinations
thereof.
[0126] Other prescribed therapies. Alternatively, or in addition to
administering centrosome declustering drugs, HSET-targeted drugs,
or others described above, a patient exhibiting high CA scores may
be additionally treated with adjuvant chemotherapeutic agents to
further reduce the risk of adverse events, such as metastasis,
disease relapse, and poor survival. Adjuvant chemotherapies may
include administration of cyclophosphamide, taxanes, such as
docetaxel and paclitaxel; anthracyclines, such as epirubicin and
doxorubicin; gemcitabine, cisplatin, fluorouracil, ixabepilone,
capecitabine, epidermal growth factor receptor-targeting agents,
and combinations thereof.
[0127] The appropriate dosage ("therapeutically effective amount")
of the therapeutic agent(s) will depend, for example, on the
severity and course of the breast cancer, the mode of
administration, the bioavailability of the therapeutic agent(s),
previous therap(ies), the age and weight of the patient, the
patient's clinical history and response to the therapeutic
agent(s), the type of the therapeutic agent used, discretion of the
attending physician, etc. The therapeutic agent(s) are suitably
administered to the patent at one time or over a series of
treatments and may be administered to the patient at any time from
diagnosis onwards. The therapeutic agent(s) may be administered as
the sole treatment or in combination with other drugs or therapies
useful in treating the breast cancer. When used with other drugs,
the therapeutic agent(s) may be used at a lower dose to reduce
toxicities and/or side effects.
[0128] The therapeutic agent(s) may be administered to the patient
with known methods, such as intravenous administration as a bolus
or by continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical and/or inhalation routes.
As a general proposition, the therapeutically effective amount(s)
of the above described therapeutic agent(s) will be in the range of
about 1 ng/kg body weight/day to about 100 mg/kg body weight/day
whether by one or more administrations. In a particular
embodiments, each therapeutic agent is administered in the range of
from about 1 ng/kg body weight/day to about 10 mg/kg body
weight/day, about 1 ng/kg body weight/day to about 1 mg/kg body
weight/day, about 1 ng/kg body weight/day to about 100 .mu.g/kg
body weight/day, about 1 ng/kg body weight/day to about 10 .mu.g/kg
body weight/day, about 1 ng/kg body weight/day to about 1 .mu.g/kg
body weight/day, about 1 ng/kg body weight/day to about 100 ng/kg
body weight/day, about 1 ng/kg body weight/day to about 10 ng/kg
body weight/day, about 10 ng/kg body weight/day to about 100 mg/kg
body weight/day, about 10 ng/kg body weight/day to about 10 mg/kg
body weight/day, about 10 ng/kg body weight/day to about 1 mg/kg
body weight/day, about 10 ng/kg body weight/day to about 100
.mu.g/kg body weight/day, about 10 ng/kg body weight/day to about
10 .mu.g/kg body weight/day, about 10 ng/kg body weight/day to
about 1 .mu.g/kg body weight/day, 10 ng/kg body weight/day to about
100 ng/kg body weight/day, about 100 ng/kg body weight/day to about
100 mg/kg body weight/day, about 100 ng/kg body weight/day to about
10 mg/kg body weight/day, about 100 ng/kg body weight/day to about
1 mg/kg body weight/day, about 100 ng/kg body weight/day to about
100 .mu.g/kg body weight/day, about 100 ng/kg body weight/day to
about 10 .mu.g/kg body weight/day, about 100 ng/kg body weight/day
to about 1 .mu.g/kg body weight/day, about 1 .mu.g/kg body
weight/day to about 100 mg/kg body weight/day, about 1 .mu.g/kg
body weight/day to about 10 mg/kg body weight/day, about 1 .mu.g/kg
body weight/day to about 1 mg/kg body weight/day, about 1 .mu.g/kg
body weight/day to about 100 .mu.g/kg body weight/day, about 1
.mu.g/kg body weight/day to about 10 .mu.g/kg body weight/day,
about 10 .mu.g/kg body weight/day to about 100 mg/kg body
weight/day, about 10 .mu.g/kg body weight/day to about 10 mg/kg
body weight/day, about 10 .mu.g/kg body weight/day to about 1 mg/kg
body weight/day, about 10 .mu.g/kg body weight/day to about 100
.mu.g/kg body weight/day, about 100 .mu.g/kg body weight/day to
about 100 mg/kg body weight/day, about 100 .mu.g/kg body weight/day
to about 10 mg/kg body weight/day, about 100 .mu.g/kg body
weight/day to about 1 mg/kg body weight/day, about 1 mg/kg body
weight/day to about 100 mg/kg body weight/day, about 1 mg/kg body
weight/day to about 10 mg/kg body weight/day, about 10 mg/kg body
weight/day to about 100 mg/kg body weight/day.
[0129] In certain embodiments, the therapeutic agent(s) are
administered at a dose of 500 .mu.g to 20 g every three days, or 10
.mu.g to 400 mg/kg body weight every three days. In other
embodiments, each therapeutic agent is administered in the range of
about 10 ng to about 100 ng per individual administration, about 10
ng to about 1 .mu.g per individual administration, about 10 ng to
about 10 .mu.g per individual administration, about 10 ng to about
100 .mu.g per individual administration, about 10 ng to about 1 mg
per individual administration, about 10 ng to about 10 mg per
individual administration, about 10 ng to about 100 mg per
individual administration, about 10 ng to about 1000 mg per
injection, about 10 ng to about 10,000 mg per individual
administration, about 100 ng to about 1 .mu.g per individual
administration, about 100 ng to about 10 .mu.g per individual
administration, about 100 ng to about 100 .mu.g per individual
administration, about 100 ng to about 1 mg per individual
administration, about 100 ng to about 10 mg per individual
administration, about 100 ng to about 100 mg per individual
administration, about 100 ng to about 1000 mg per injection, about
100 ng to about 10,000 mg per individual administration, about 1
.mu.g to about 10 .mu.g per individual administration, about 1
.mu.g to about 100 .mu.g per individual administration, about 1
.mu.g to about 1 mg per individual administration, about 1 .mu.g to
about 10 mg per individual administration, about 1 .mu.g to about
100 mg per individual administration, about 1 .mu.g to about 1000
mg per injection, about 1 .mu.g to about 10,000 mg per individual
administration, about 10 .mu.g to about 100 .mu.g per individual
administration, about 10 .mu.g to about 1 mg per individual
administration, about 10 .mu.g to about 10 mg per individual
administration, about 10 .mu.g to about 100 mg per individual
administration, about 10 .mu.g to about 1000 mg per injection,
about 10 .mu.g to about 10,000 mg per individual administration,
about 100 .mu.g to about 1 mg per individual administration, about
100 .mu.g to about 10 mg per individual administration, about 100
.mu.g to about 100 mg per individual administration, about 100
.mu.g to about 1000 mg per injection, about 100 .mu.g to about
10,000 mg per individual administration, about 1 mg to about 10 mg
per individual administration, about 1 mg to about 100 mg per
individual administration, about 1 mg to about 1000 mg per
injection, about 1 mg to about 10,000 mg per individual
administration, about 10 mg to about 100 mg per individual
administration, about 10 mg to about 1000 mg per injection, about
10 mg to about 10,000 mg per individual administration, about 100
mg to about 1000 mg per injection, about 100 mg to about 10,000 mg
per individual administration and about 1000 mg to about 10,000 mg
per individual administration. The therapeutic agent(s) may be
administered daily, or every 2, 3, 4, 5, 6 and 7 days, or every 1,
2, 3 or 4 weeks.
[0130] In other particular embodiments, the therapeutic agent(s)
are administered at a dose of about 0.0006 mg/day, 0.001 mg/day,
0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day, 0.06 mg/day,
0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day,
10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600
mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day or 10,000 mg/day. As
expected, the dosage(s) will be dependent on the condition, size,
age and condition of the patient.
[0131] Various alternatives (e.g., different types of cancers and
types of reagents) may be utilized. It is to be understood that
various combinations can be employed and any one or more of the
listed alternatives can be excluded from the compositions of the
invention.
EXAMPLES
Example 1
Materials and Methods for Analyzing Mitotic Cells and Proliferative
Cells in the Same Field
[0132] 1. Data-Mining.
[0133] Clinical records of 4342 breast cancer patients diagnosed
between 2005 and 2009 were obtained from Northside Hospital,
Atlanta. Histologic grading was performed by pathologists at
Northside Hospital in accordance with Table 1. 2731 patients out of
the total were excluded from this analysis due to missing
information regarding KI, MI, hormone-receptor status or OS.
Clinical records of the remaining 1611 patients who met all
inclusion criteria were used for the analyses described below.
Table 1 depicts the clinicopathologic characteristics of the breast
cancer patient cohort analyzed in the Northside Hospital study.
[0134] The data mining was further extended to cover deidentified
clinical records of 1492 breast cancer patients obtained from
Nottingham University Hospital, UK (Table 2) and 1597 breast cancer
patients obtained from Emory University Hospital, Atlanta (Table
3). The patients' clinicopathologic characteristics are described
in Tables 2 and 3.
TABLE-US-00001 TABLE 1 Clinicopathologic characteristics of 1611
breast cancer patients from Northside Hospital. Number of patients
(Total n = % out Factor Status 1611) of total ER Positve 1259 78.15
Negative 339 21.04 Unknown 13 0.81 PR Positive 1086 67.41 Negative
511 31.72 Unknown 14 0.87 HER2 Postive 1088 67.54 Negative 520
32.28 Unknown 3 0.19 Glandular differentiation 1 171 10.61 Score 2
451 28.00 3 989 61.39 Nuclear Grade 1 435 27.00 2 728 45.19 3 448
27.81 Mitotic Score 1 819 50.84 2 472 29.30 3 448 27.81 Nottingham
Grade I 539 33.46 II 638 39.60 III 434 26.94 Subtype ER/PR- Her2+
131 8.13 Luminal A 584 36.25 Luminal B Her2+ 384 23.84 Luminal B
Her2- 299 18.56 Triple Neg 197 12.23 Unknown 16 0.99 Ki67 <=15
848 52.64 <=30 317 19.68 >30 446 27.68 Metastasis Status
Local Relapse 27 1.68 Distant 23 1.43 None 1064 66.05 Unknown 547
33.95 Race European- 1290 80.07 American African-American 210 13.04
Other 111 6.89 Sex Female 1606 99.69 Male 4 0.25 Unknown 1 0.06 Age
<50 539 33.46 50-69 837 51.96 70-75 219 13.59 >75 16 0.99
Median Follow up time 1783 days Median Age 56
TABLE-US-00002 TABLE 2 Clinicopathologic characteristics of 1492
breast cancer patients from Nottingham University Hospital, UK.
Number of patients Factor Status (Total n = 1492) % out of total ER
Negative 376 25.3 Positive 1109 74.7 Missing 7 -- PR Negative 582
40.4 Positive 857 59.6 Missing 53 -- HER2 Negative 1249 87.2
Positive 182 12.7 Unknown 1 0.1 Missing 60 -- Mitotic Score 1 543
36.4 2 278 18.6 3 671 45.0 Nuclear Grade 1 36 2.4 2 575 38.5 3 881
59.0 Nottingham Grade 1 225 17.1 2 490 32.8 3 747 50.1 Subtype
HER2+ 85 6.0 Luminal A 577 40.8 Luminal B 499 35.3 Triple Negative
254 18.0 Missing 77 -- K167 <=15 741 49.7 16-30 205 13.7 >30
548 36.6 Metastasis Status None 979 66.0 Distant 5051 34.0 Unknown
8 -- Age <50 497 33.3 50-69 955 64.0 70-75 40 2.7 Tubule
Formation 1 93 6.2 2 493 33.0 3 906 60.7
TABLE-US-00003 TABLE 3 Clinicopathological characteristics of 1597
breast cancer patients from Emory University Hospital, Atlanta.
Number of patients Factor Status (Total n = 1597) % out of total ER
Negative 336 21.0 Positive 1261 79.0 PR Negative 563 35.3 Positive
1030 64.7 Missing 4 -- HER2 0 1384 86.7 Positive 213 13.3 Mitotic
Score 1 925 58.0 2 398 25.0 3 272 17.1 Missing 2 -- Nuclear Grade 1
175 11.0 2 731 45.9 3 687 43.1 Missing 4 -- Nottingham Grade 1 411
26.0 2 725 45.9 3 444 28.1 Missing 17 -- Subtype HER2+ 64 4.0
Luminal A 463 29.0 Luminal B 828 51.8 Triple Negative 242 15.2 Ki67
<=15 575 36.0 16-30 301 18.8 >30 721 45.1 Metastasis Status
Distant 4 0.30 None 679 42.52 Unknown 914 57.2 Race Black 758 48.2
White 758 48.2 Others 56 3.51 Missing 25 -- Sex Female 707 98.5
Male 11 1.5 Unknown 879 -- Age <50 431 27.0 50-69 883 55.3 70-75
130 8.1 >75 153 9.6 Tubule Formation 1 158 9.9 2 463 29.0 3 973
61.0 Missing 3 --
[0135] 2. KAMS Ratio Determination.
[0136] To compare MI, a categorical variable, based on mitotic
score (i.e., 1, 2 or 3) to KI, a continuous variable expressed as a
percentage, MI was converted to a percentage as follows. Briefly,
10 HPFs were evaluated in at least 5 patient samples, where on
average, 10 HPFs were found to have .about.500 cells. For patients
with mitotic scores 1 and 2, mitotic cell counts were assumed to
have values of 3.5 and 11 (average cell count value of those score
ranges), respectively. For patients with a mitotic score of 3, a
mitotic cell count of 15 (which is the floor value for mitotic
score 3 category) was assumed. These mitotic cell counts provided
an estimate of the number of mitotic cells per 500 cells (10 HPFs),
thus providing the percentage of cells undergoing mitosis. The KAMS
ratio for each patient was calculated simply as a quotient of
percent mitotic cells divided by percent Ki67-positive cells.
[0137] 3. Statistical Analysis.
[0138] Differences among baseline results were established using a
one-way analysis of variance (ANOVA) alongside a post-hoc Tukeys
range test. Survival curves were obtained via the Kaplan-Meier
method with significance determined using the log-rank test.
Survival time was measured from the initial diagnosis to either an
event (death) or to the final follow up (censor) and was thus an
indicator of overall survival (OS). Progression-free survival (PFS)
is calculated as the time interval from first diagnosis to date of
first local recurrence in the absence of metastasis or metastasis
in the absence of local recurrence or death (if that occurred
without recurrence or metastasis). Breast cancer-specific survival
(BCSS) was calculated as the time from first diagnosis to death
from breast cancer. To obtain hazard ratios and the fit statistics,
a Cox proportional hazard model was employed. For categorical
variables, the lowest risk-group was used as the reference to that
parameter's hazard. For ideal thresholds, the FINDCUT macro
developed by Jayawant N. Mandrekar et al. from Mayo Clinic
(http://www2.sas.com/proceedings/sugi28/261-28.pdf) was used, which
identifies the optimal cut-off point for continuous variables
(i.e., M:P ratio) that predicts time to event outcomes. Using a
macro by Mithat Gonen (% c-index), a concordance index (c-index)
was determined for these models with censored outcomes. In order to
determine if c-indices of multiple models were significantly
different, a 100.times. bootstrap method was utilized where the
model was trained on 60% of the samples and validated on the
remaining 40% of the samples. The mean c-indices could then be
compared using a student t-test.
[0139] To assess degree of agreement between pathologists for
mitosis identification using either phoshpho-histone H3 (p-H3) or
H&E staining, the intraclass correlation test (ICC) was
employed using a macro developed by Li Lu and Nawar Shara
(http://www.lexjansen.com/nesug/nesug07/sa/sa13.pdf). The average
change in mitotic count determined using the two methods was
compared via a t-test. All relevant tests were two-sided and used a
significance level alpha of 0.05. All statistical analysis was done
in either SAS or in Microsoft Excel.
[0140] 4. Immunofluorescent Staining on Tissue Sections.
[0141] Formalin-fixed, paraffin-embedded tissue sections were
incubated at 60.degree. C.-70.degree. C. for 2 h, followed by 2
xylene washes (5 min each) and sequential ethanol washes (100%,
95%, 70% and 50%). Antigen retrieval was carried out in citric acid
buffer (pH 6.0) at 98.degree. C. for 20 min. Slides were allowed to
cool down and blocked in 5% BSA/PBS (30 min). Tissue sections were
then incubated for 1 h with a cocktail of rat anti-human p-H3
antibody (1:500) (Abcam, Cambridge, UK) and mouse anti-human DM1A
(.alpha.-tubulin, 1:1000) (Sigma-Aldrich, MO, USA) followed by
donkey anti-rat Alexa 488 (1:2000) and goat anti-mouse Cy5 (1:2000)
(Invitrogen, Grand Island, N.Y.) secondary antibody incubation for
1 h. Slides were then washed 3 times in PBS followed by 1 h
incubation with rabbit anti-human Ki67 (1:1000) (Abcam, Cambridge,
UK) and goat anti-rabbit Alexa 405 (Invitrogen, 1:2000) secondary
antibody incubation for 1 h. Slides were again washed 3 times in
PBS followed by incubation with Propidium Iodide (0.1 ug/ml) for 15
min and washing in PBS. Finally, coverslips were mounted on the
slides using Prolong Antifade mounting medium (Invitrogen, Grand
Island, N.Y.). Ziess LSM 700 confocal microscope was used to
capture immunofluorescence images at 63.times. objective
magnification.
[0142] Paraffin-embedded slides were processed as described in the
immunofluorescent staining section. Following antigen retrieval,
peroxide quenching was done in 5% H2O2 solution for 30 min and the
slides were blocked in 5% BSA/PBS (30 min). Tissue sections were
then incubated for 1 h with rabbit-human p-H3 antibody (1:500)
(Abcam, Cambridge, UK). Peroxide-based antibody detection kit was
used (Universal LSAB.TM.+ Kit/HRP) (Dako, Golstrup, Denmark) to
develop brown color. Slides were then counterstained with
Haematoxylin (Fisher Scientific, Waltham, Mass.) for 5 min followed
by 3 H.sub.2O washes. For H&E staining, slides were directly
stained with Hematoxylin (5 min incubation) after tissue
rehydration. Slides were then dipped in acid alcohol followed by
ammonia water and then stained with Eosin Y (1 min incubation)
(Fisher Scientific, Waltham, Mass.). After 3 H.sub.2O washes, all
tissues were then dehydrated in sequential ethanol washes (50%,
70%, 95%, 100%) followed by 3 xylene washes. Coverslips were
mounted using toluene-based mounting medium (Secure Mount, Fisher
Scientific, Waltham, Mass.).
[0143] 5. Representative Protocol for Double-Color
Immunohistochemistry [0144] 1. Put slides in the oven at 67.degree.
C. for 2 hours. [0145] 2. Put slides in a slide holder and perform
each step in the following order. Put in Xylene three times five
minutes each. [0146] 3. Quench endogenous hydrogen peroxide
activity for 45 minutes by placing slides in: 95 ml MeOH, 5 ml
hydrogen peroxide (30% by vol) [0147] 4. Put slides in a sequential
order in 100%, 95%, 70%, and 50% EtOH, 5 minutes each. [0148] 5.
Prepare citrate buffer: 210 mg citrate in 100 ml pure water and add
two drops Triton X-100. [0149] 6. Preheat pressure cooker. Put
slides in citric acid solution and pressure cook for 10 minutes at
125.degree. C. Let it cool down (will take another 20-30 minutes).
[0150] 7. Put slides in ice for 30 minutes to cool down to room
temperature. [0151] 8. Put slides in TBST 2.times.5 minutes. [0152]
9. Block slides using antibody diluents and blocker (BioGenex,
#QA900-91, LOT QA9000807) for one hour at room temperature. [0153]
10. Gently dry area around the sections and draw a line with a
liquid blocker pen to prevent spillage of antibody. Prepare rat
anti-human phospho-Histone H3 (PH3) antibody [Abeam, #abl0543, LOT
GR1054868] at a dilution of 1:1000 with BioGenex blocker. [0154]
11. Incubate slides with primary antibody overnight at 4.degree. C.
[0155] 12. Tap off antibody and wash three times with TBST 5
minutes for each wash. [0156] 13. Put Polyclonal Dako Rabbit
anti-Rat Immunoglubulins/HRP (#P0450, LOT 00082902) for one hour.
[0157] 14. Incubate with substrate -chromogen solution until brown
color develops with Dako Kit (1-2 minutes). 1 ml DAB substrate [LOT
10081846]+1 drop chromogen [LOT 10081846], use immediately. [0158]
15. Rinse gently with water and let the slides stay in water for
two minutes. [0159] 16. Block the slides with 5% BSA in PBS+ 0.05%
Triton X-100. [0160] 17. Prepare rabbit anti-human Ki67 antibody
(Abeam, #ab16667, LOT GR 1054868) at a dilution of 1:1000. [0161]
18. Incubate slides with Ki67 primary antibody overnight 4.degree.
C. [0162] 19. Tap off antibody and wash three times with TBST for 5
minutes each. [0163] 20. Put Linker for Alkaline Phosphatase
(Biogenex, QA900-91, LOT HK3310307) for 35 minutes. [0164] 21. Wash
slides with TBST three times for 5 minutes each. [0165] 22. From
SIGMA Alkaline Phosphatase Magenta kit (#AM0100-1KT), mix 20 ul
liquid substrate initiator from the [LOT 026K1143] and 20 ul SIGMA
Alkaline Phosphatase Magenta liquid for two minutes and put SIGMA 1
ml Alkaline Phosphatase Magenta liquid substrate buffer [LOT
026K1144]. [0166] 23. Incubate with substrate-chromogen solution
until red color develops (-10 minutes). [0167] 24. Rinse with
water. While slide is still wet, counterstain with hematoxylin
(EMD, #65067-75) for 1.5 min. [0168] 25. Rinse until slide runs
clear. [0169] 26. Put slides sequentially in 50%, 70%, 95% and 100%
EtOH for 5 minutes each. [0170] 27. Put slides in Xylene three
times for 5 minutes each in the fume hood. [0171] 28. Add mounting
medium (Secure Mount, Protocol, #022-208) to slide while still
moist from Xylene, and add coverslip. Let dry in the fume hood and
put transparent nail polish around the cover slip.
Example 2
Quantification of Mitotic Figures from H&E-Stained Slides
Underestimates Mitotic Population
[0172] KI and MI are normally determined by pathologists in
different tissue sections and evaluated on disparate scales, which:
(i) overlook the fact that mitotic cells comprise a subset of
cycling cells; (ii) make a direct cell-matched comparison of KI and
MI impossible (FIG. 1, Panel A) and (iii) preclude evaluation of
mitotic propensity and cell-cycling kinetics of the proliferative
population in a tumor. Evidence provided herein suggests that the
proportion of mitotic cells amongst the proliferative population
within a tumor provides a measure of the risk associated with the
tumor due to erroneous mitoses. This "dangerous" fraction of
proliferating cells can be quantitated with a high degree of
accuracy by simultaneous visualization of both mitotic and
Ki67-positive cells in the same field (FIG. 1, Panel A).
[0173] A commonly used marker to identify M-phase cells is p-H3. In
order to assess the value of p-H3 for determining mitotic score by
immunocytochemistry, 45 paraffin-embedded breast tumor sections
were stained with either H&E or anti-p-H3 antibody. Three
pathologists determined mitotic scores based on H&E and p-H3
for the same pre-marked areas in the two sets of slides in a
blinded manner (FIG. 1, Panel B). Higher and more reproducible
mitotic scores resulted when mitotic cells were counted in
p-H3-stained slides vs. H&E stained sections (FIG. 1, Panel
Ci). Average mitotic scores via p-H3 staining were higher by an
average of 46.6% (p<0.0001) (FIG. 1, Panel Ci). The better
contrast in p-H3 and easier recognition of mitotic figures enabled
more rapid determination of mitotic scores in p-H3-stained slides
(FIG., Panel Cii), as the average time spent scoring p-H3 slides
was .about.37% lower than for H&E slides.
[0174] Next, an Intraclass correlation coefficient (ICC) was used
to assess consistency of measurements made by the three
pathologists. There was a significant increase in agreement among
the three pathologists when evaluating mitotic scores using p-H3
staining (ICC=0.57) compared to mitotic scores using H&E
staining (ICC=0.38) (p<0.05). These data underscore that p-H3
stain significantly increases inter-observer reproducibility than
H&E in evaluation of MI.
Example 3
Integration of KI and MI Enhances Patient Risk-Stratification
[0175] FIG. 2. Panel Ai depicts Kaplan-Meier survival plots (Breast
cancer-specific survival) showing stratification of Lum B patients
(n=495) from the Nottingham University dataset based on an ideal
threshold (KAMS threshold=0.375). Panel Aii shows the tests of
equality of the survival function over strata, which indicate that
the survival difference between the groups is statistically
significant. Panel Aiii shows the summary of the number of censored
and uncensored values in the survival analysis of Lum B patients.
Panel Bi depicts Kaplan-Meier survival plots showing stratification
of TNBC patients (n=250) from the Nottingham University dataset
based on an ideal threshold (KAMS threshold=0.413). Panel Bii shows
the tests of equality of the survival function over strata, which
indicate that the survival difference between the groups is
statistically significant. Panel Biii shows the summary of the
number of censored and uncensored values in the survival analysis
of TNBC patients. Note that above threshold Lum B and TNBC patients
show poorer prognosis.
[0176] FIG. 3. Panel Ai depicts Kaplan-Meier survival plots
(Progression-free survival) showing stratification of a combined
set of Lum B and TNBC patients (n=1070) from the Emory University
dataset, based on an ideal threshold (KAMS threshold=0.111). Panel
Aii shows the tests of equality of the survival function over
strata, which indicate that the survival difference between the
groups is statistically significant. Panel Aiii shows the summary
of the number of censored and uncensored values in the survival
analysis of the patient cohort. Note that above threshold Lum B and
TNBC patients show poorer prognosis.
[0177] FIG. 4. Panel Ai depicts Kaplan-Meier survival plots
(Overall survival) showing stratification of a combined set of Lum
B and TNBC patients (n=880) from the Northside Hospital dataset,
based on an ideal threshold (KAMS threshold=0.318). Panel Aii shows
the tests of equality of the survival function over strata, which
indicate that the survival difference between the groups is
statistically significant. Panel Aiii shows the summary of the
number of censored and uncensored values in the survival analysis
of the Northside Hospital Lum B and TNBC patients. Note that above
threshold Lum B and TNBC patients show poorer prognosis.
[0178] FIG. 5 depicts a patient grade-adjustment model, which
creates an adjusted Nottingham Grade based on KAMS values of Lum B
and TNBC patients. This histological grade-adjustment model was
then tested to see if incorporation of KAMS-classifier subsequent
to conventional Nottingham classification would improve
stratification of patients. In this model, all Lum A patients
originally in NG III were adjusted into the "Adjusted NGII"
category owing to their relatively good prognosis (this adjustment
is not depicted in FIG. 5 since these patients represent <5% of
the total cohort). All the Lum B and TNBC patients were then
categorized by the KAMS classifier into a low-KAMS (low cell
cycling kinetics) subclass and a high-KAMS (high cell cycling
kinetics) subclass based on an ideal threshold. The "low cycling
kinetics" subclass was then combined with the remainder of patients
from the original NG II to generate the "Adjusted NG II" cohort.
The "high cycling kinetics" subclass was then combined with the
remainder of patients from the original NG III to generate the
"Adjusted NG III" cohort.
[0179] FIG. 6 shows the histological grades of 1455 patients from
the Nottingham University dataset (for whom progression-free
survival data was available) were adjusted according to the grade
adjustment model depicted in FIG. 5. Panel Ai depicts the
Kaplan-Meier survival plot (Progression-free survival) of patients
stratified by the original Nottingham Grading System. Panel Aii
shows the tests of equality of the survival functions over strata,
which indicate that the survival differences between the groups are
statistically significant. Panel Aiii shows the summary of the
number of censored and uncensored values for each Nottingham Grade
in the survival analysis of the Nottingham University patients
classified by the original Nottingham Grading System. Panel Bi
depicts the Kaplan-Meier survival plot (Progression-free survival)
of the Nottingham University patients after they were stratified
and re-classified using the KAMS-classifier, thus yielding the
"Adjusted NG I, II and III". The adjusted grading system boasts a
better separation between PFS of the adjusted grades. Panel Bii
shows the tests of equality of the survival functions over strata,
which indicate that the survival differences between the Adjusted
Nottingham Grades are statistically significant. Panel Biii shows
the summary of the number of censored and uncensored values for
each Adjusted Nottingham Grade in the survival analysis of the
Nottingham University patients. Panel C shows a comparison of the
model fit statistics, hazard ratios and concordance indices for the
original and KAMS classifier-adjusted Nottingham Grades. Panel C
shows a decrease of all 3 model-fit statistics (-2 log L, Akaike
Information Criterion or AIC, and Schwarz Bayesian Criterion or
SBC) for the adjusted model alongside an increase in hazard ratios
(using NG I as the reference point for both models) both indicating
the superior fit of the adjusted model, improved patient
stratification and more accurate risk-segmentation of patients
using the KAMS-classifier. A comparison of mean c-index (c-index is
a measure of concordance for time-to-event data, in which
increasing values between 0.5 and 1.0 indicate improved concordance
between predicted and actual outcomes) of 100 bootstraps of the
dataset (using 60% cases as training set and 40% cases as
validation set), between the original NGS and KAMS-adjusted system
shows comparable c-index for original and adjusted grading systems.
Analysis of distribution of various breast cancer subtypes among
the patient-cohort prior to (Panel Di) and after the KAMS-based
grade reassignment (Panel Dii) shows the ability of this metric to
distinguish between high- and low-risk breast cancer subtypes as
the proportion of Lum B patients in the adjusted NG III is higher
than in the original NG III. Moreover, this metric allowed the
identification of lower-risk TNBC patients who moved from the
original NG III into the adjusted NG II.
[0180] FIG. 7 shows the histological grades of 1460 patients from
the Nottingham University dataset (for whom breast cancer-specific
survival data was available) were adjusted according to the grade
adjustment model depicted in FIG. 5. Panel Ai depicts the
Kaplan-Meier survival plot (breast cancer-specific survival) of
patients stratified by the original Nottingham Grading System.
Panel Aii shows the tests of equality of the survival functions
over strata, which indicate that the survival differences between
the groups are statistically significant. Panel Aiii shows the
summary of the number of censored and uncensored values for each
Nottingham Grade in the survival analysis of the Nottingham
University patients classified by the original Nottingham Grading
System. Panel Bi depicts the Kaplan-Meier survival plot (breast
cancer-specific survival) of the Nottingham University patients
after they were stratified and re-classified using the
KAMS-classifier, thus yielding the "Adjusted NG I, II and III". The
adjusted grading system boasts a better separation between BCSS of
the adjusted grades. Panel Bii shows the tests of equality of the
survival functions over strata, which indicate that the survival
differences between the Adjusted Nottingham Grades are
statistically significant. Panel Biii shows the summary of the
number of censored and uncensored values for each Adjusted
Nottingham Grade in the survival analysis of the Nottingham
University patients. Panel C shows a comparison of the model fit
statistics, hazard ratios and concordance indices for the original
and KAMS classifier-adjusted Nottingham Grades. Panel C shows a
decrease of all 3 model-fit statistics (-2 log L, Akaike
Information Criterion or AIC, and Schwarz Bayesian Criterion or
SBC) for the adjusted model, alongside an increase in hazard ratios
(using NG I as the reference point for both models) both indicating
the superior fit of the adjusted model, improved patient
stratification and more accurate risk-segmentation of patients
using the KAMS-classifier. A comparison of mean c-index (c-index is
a measure of concordance for time-to-event data, in which
increasing values between 0.5 and 1.0 indicate improved concordance
between predicted and actual outcomes) of 100 bootstraps of the
dataset (using 60% cases as training set and 40% cases as
validation set), between the original NGS and KAMS-adjusted system
shows comparable c-index for original and adjusted grading systems.
Analysis of distribution of various breast cancer subtypes among
the patient-cohort prior to (Panel Di) and after the KAMS-based
grade reassignment (Panel Dii) clearly shows the ability of this
metric to distinguish between high- and low-risk breast cancer
subtypes as the proportion of Lum B patients in the adjusted NG III
is higher than in the original NG III. Moreover, this metric
allowed the identification of lower-risk TNBC patients who moved
from the original NG III into the adjusted NG II.
[0181] FIG. 8, Panels A, B and C depict the mean KAMS values of Lum
A and Lum B patients in NG I, NG II and NG III, respectively, in a
combined dataset comprising patients from Northside Hospital,
Atlanta, Emory University Hospital, Atlanta and Nottingham
University Hospital, UK. Within each Nottingham Grade, the
difference in the mean KAMS of Lum A and Lum B patients is
statistically significant (p<0.0001).
[0182] FIG. 9 depicts the extraction and integration of KI and MI
from the same microscopic field using immunofluorescence
microscopy. Field 1 and 2 show different fields depicting mitotic
propensities observed in two breast tumors immunostained for Ki67,
p-H3, .alpha.-tubulin and DNA (Propidium Iodide). Sample in top row
has 13 Ki67-positive cells, 1 p-H3-positive cells in a field,
M-to-P ratio for field 1=1/13.times.100=7.69. Sample in bottom row
has 13 Ki67-positive cells, 2 p-H3-positive cells, M-to-P ratio for
field 2=2/13.times.100=15.3.
[0183] FIG. 10 depicts the extraction and integration of KI and MI
to derive M-to-P Ratio from the same microscopic field using dual
antibody immunohistochemistry. The antibodies used were directed
against Ki67 and p-H3 and nuclei were visualized using
hematoxylin.
[0184] FIG. 11 depicts how the extent of centrosome amplification
and inherent mitotic propensity (i.e., the M-to-P Ratio) determine
the rate at which intratumoral heterogeneity (ITH) is generated.
Centrosomes are depicted as small circles within the cell. This
schematic describes how a tumor cell population (with different
degrees of centrosome amplification and mitotic propensity) in the
vicinity of a blood vessel evolves over time. This tumor cell
population could represent either an entire primary tumor, an
individual clone within a primary tumor, or a metastatic tumor
evolving in parallel with a primary tumor in another location. Four
scenarios are illustrated for this population which is at a very
early stage in its lifetime: Rows 1 and 2--evolution of ITH when
two cell populations with similarly high mitotic propensity start
off with either low or high levels of centrosome amplification,
respectively. Rows 3 and 4--evolution of ITH when two cell
populations with similarly low mitotic propensity start off with
either low or high levels of centrosome amplification,
respectively. Each row has 4 panels depicting sequential snapshots
of tumor population over time. ITH level of the tumor population is
represented by the histogram in the background, the color of the
histogram representing the level/degree of ITH at a particular
time/stage in tumor evolution. The height of the histogram depicts
the maximum level of ITH attained in each of the four scenarios.
Variety of clones produced is represented by the number of
differently colored cells. The rate of ITH is depicted by the
variety of clones produced, tumor size and time it took the tumor
to reach ITH peak which demarcates the switch in tumor agenda from
mitosis to metastasis. Metastasis (if any) is depicted by black
arrows pointing towards the blood vessel in the final panel in each
row. Highest risk of metastasis occurs when both centrosome
amplification levels and inherent mitotic propensity are high (Row
2).
[0185] The above description is for the purpose of teaching the
person of ordinary skill in the art how to practice the present
invention, and it is not intended to detail all those obvious
modifications and variations of it which will become apparent to
the skilled worker upon reading the description. It is intended,
however, that all such obvious modifications and variations be
included within the scope of the present invention, which is
defined by the following claims. The claims are intended to cover
the claimed components and steps in any sequence which is effective
to meet the objectives there intended, unless the context
specifically indicates the contrary.
* * * * *
References