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Cancer Patent Abstract
The invention provides methods for prognosis, diagnosis, staging
and disease progression in human cancer patients related to expression
levels of a variety of immunohistochemical and genetic markers associated
with poor cancer prognosis, and in particular those markers related
to tumor invasiveness, metastasis and spread. The invention also
provides methods using a predictive index for prognosis of cancer
patients for metastasis, recurrence and relapse of neoplastic disease.
The methods of the invention are useful for making clinical decisions
on cancer treatment, surveillance and surgical intervention.
Cancer Patent Claims
I claim:
1. A method for making a prognosis of disease course in a human
breast or prostate cancer patient, the method comprising the step
of: (a) obtaining a sample of a tumor from the human cancer patient;
(b) determining a level of nuclear localization of p53 protein in
the tumor sample and comparing the level of nuclear localization
of p53 protein in the tumor sample with the level of nuclear localization
of p53 protein in a non-invasive, non-metastatic tumor sample; (c)
determining a level of thrombospondin 1 expression in the tumor
sample and comparing the level of thrombospondin 1 expression in
the tumor sample with the level of thromobospoin 1 expression in
a non-invasive, non-metastatic tumor sample; (d) determining by
immunohistochemistry an extent of microvascularization in the tumor
sample and comparing the extent of microvascularization in the tumor
sample with the extent of microvascularization in a non-invasive,
non-metastatic tumor sample; and (e) preparing a prognostic index
comprising the results of the determination of the levels of nuclear
localization of p53, thrombospondin 1 expression, and the extent
of microvascularization in the tumor sample, wherein said prognosis
is predicted from considering a likelihood of further neoplastic
disease which is made when the level of nuclear localization of
p53 protein in the tumor sample is greater than the level of nuclear
localization of p53 protein in the non-invasive, non-metastatic
tumor sample; the level of thrombospondin 1 expression in the tumor
sample is less than the level of thromobospondin 1 expression in
the non-invasive, non-metastatic tumor sample; and the extent of
microvascularization in the tumor sample is greater than the extent
of microvascularization in the non-invasive, non-metastatic tumor
sample.
2. The method of claim 1, wherein the level of nuclear localization
of p53 protein in the tumor sample is from about twofold to about
tenfold greater than the level of nuclear localization of p53 protein
in the non-invasive, non-metastatic tumor sample.
3. The method of claim 1, wherein the level of thrombospondin 1
expression in the tumor sample is from about twofold to about tenfold
less than the level of thrombospondin 1 expression in the non-invasive,
non-metastatic tumor sample.
4. The method of claim 1, wherein the extent of microvascularization
in the tumor sample is from about twofold to about tenfold greater
than the extent of microvascularization in the non-invasive, non-metastatic
tumor sample.
5. The method of claim 1, wherein the level of nuclear localization
of p53 protein in the tumor sample is from about twofold to about
tenfold greater than the level of nuclear localization of p53 protein
in the non-invasive, non-metastatic tumor sample, and wherein the
level of thrombospondin 1 expression in the tumor sample is from
about twofold to about tenfold less than the level of thrombospondin
1 expression in the non-invasive, non-metastatic tumor sample and
wherein the extent of microvascularization in the tumor sample is
from about twofold to about tenfold greater than the extent of microvascularization
in the non-invasive, non-metastatic tumor sample.
6. The method of claim 1, wherein the level of nuclear localization
of p53 protein in the tumor sample is from about fivefold greater
than the level of nuclear localization of p53 protein in the non-invasive,
non-metastatic tumor sample, and wherein the level of thrombospondin
1 expression in the tumor sample is from about fivefold less than
the level of thrombospondin 1 expression in the non-invasive, non-metastatic
tumor sample and wherein the extent of microvascularization in the
tumor sample is from about sixfold greater than the extent of microvascularization
in the non-invasive, non-metastatic tumor sample.
7. The method of claim 1, wherein the level of nuclear localization
of p53, the level of thrombospondin 1 expression and the extent
of microvascularization are determined by immunohistochemical staining.
8. The method of claim 1 wherein the cancer is breast cancer.
9. The method of claim 1 wherein the cancer is prostrate cancer.
Cancer Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to cancer diagnosis and treatment, and specifically
to the determination of a predictive index for prognosis of cancer
patients for metastasis, recurrence and relapse of neoplastic disease.
The invention relates to the determination of a variety of immunohistochemical
and genetic markers associated with poor cancer prognosis, and in
particular those markers related to tumor invasiveness, metastasis
and spread. The invention particularly relates to the use of certain
markers associated with tumor invasiveness, metastasis and spread
to provide a prognostic index for making clinical decisions on cancer
treatment, surveillance and surgical intervention.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United
States. Clinically, a broad variety of medical approaches, including
surgery, radiation therapy and chemotherapeutic drug therapy are
currently being used in the treatment of human cancer (see the textbook
CANCER: Principles & Practice of Oncology, 2d Edition, De Vita
et al., eds., J. B. Lippincott Company, Philadelphia, Pa., 1985).
However, it is recognized that such approaches continue to be limited
by a fundamental inability to accurately predict the likelihood
of metastasis and tumor recurrence or the most efficacious treatment
regime for minimizing the occurrence of these negative outcomes.
The discovery and clinical validation of markers for cancer of
all types which can predict prognosis, likelihood of invasive or
metastatic spread is one of the major challenges facing oncology
today. In breast cancer, for example, 70% of the approximately 186,00
annual cases present as node lumph negative; however, 30% of these
cases will recur after local therapy (mastectomy or "lumpectomy")
(Boring et al., 1992, Clin. J. Cancer 42: 19-38). Although adjuvant
chemotherapy has been demonstrated to improve survival in node negative
breast cancer patients (Mansour et al., 1989, N. Engl. J. Med. 320:
485-490), it remains uncertain how to best identify patients whose
risk of disease recurrence exceeds their risk of significant therapeutic
toxicity (Osbourne, 1992, J. Clin. Oncol. 10: 679-682).
Current approaches to answer these questions stratify node negative
breast cancer on the basis of primary tumor size, pathological grade,
DNA S-phase fraction (SPF) and steroid hormone receptor status (Allegra
et al., 1979, Cancer Treat. Rep. 63: 1271-1277; Von Rosen et al.,
1989, Breast Cancer Res. Treat. 13: 23-32; Fischer et al., 1992,
J. Natl. Cancer Inst. 11: 152-258; Clark et al., 1994, N. Engl.
J. Med. 320: 627-633). For example, moderately and well-differentiated
tumors <1 cm in size are thought to require only local excision
regardless of receptor status, while such tumors from 1 to 3 cm
in size that express normal levels of hormone receptor are treated
with hormone therapy (Fischer et al., 1993, in Cancer Medicine,
3d ed., Holland et al., eds., Philadelphia: Lea & Febiger, pp.
1706-1774). On the other hand, patients with tumors larger than
2 cm that are poorly differentiated and/or hormone receptor negative
are treated with adjuvant chemotherapy (Early Breast Cancer Trialistic
Collaborative Group 1992, Lancet 339: 1-15; The Ludwig Breast Cancer
Study Group 1989, N. Engl. J. Med. 320: 491-496). However, therapeutic
indications are much less clearly defined for patients having moderately
differentiated tumors of 1 to 3 cm in size where the hormone receptor
status is borderline or unknown (Gasparini et al., 1993, J. Natl.
Cancer Inst. 85: 1206-1219). Deciding the most appropriate therapy
for this group of patients, comprising about 70,000 women annually,
would benefit from the development of validated prognostic analysis.
Similar prognostic tools are needed in most other forms of cancer.
Thus, there is a need in this art for developing methods for making
clinical decisions on adjuvant therapy, tumor surveillance and the
likelihood of disease progression based on validated tumor markers
statistically correlated with tumor invasiveness, metastasis and
recurrence.
SUMMARY OF THE INVENTION
The present invention provides methods for predicting a disease
course in a human cancer patient. The invention also provides a
prognostic (risk) index for making predictions about disease progression
and prognosis, and for determining the proper course of treatment
for an individual patient using the index to grade the patient's
tumor and estimate their chances for survival.
In a first aspect the invention provides a method for making a
prognosis of disease course in a human cancer patient. The method
comprises the following steps. First, a sample of a tumor from the
human cancer patient is obtained. Then, the levels of three tumor
markers in the tumor sample are determined, and compared with levels
of these markers in a control, non-invasive, non-metastatic tumor
sample of the same type. The tumor markers tested are nuclear localization
of p53 protein (which is used as an indicator of p53 mutation),
thrombospondin 1 expression, and the extent of microvascularization
in the tumor sample (as a measure of angiogenesis in the sample).
In the practice of the invention, a poor prognosis, that is, a prognosis
of the likelihood of further neoplastic, particularly metastatic,
disease, is made when the level of nuclear localization of p53 in
the tumor sample is greater than the level of nuclear localization
of p53 protein in the non-invasive, non-metastatic tumor sample;
the level of thrombospondin 1 expression in the tumor sample is
less than the level of thrombospondin 1 expression in the non-invasive,
non-metastatic tumor sample; and the extent of microvascularization
in the tumor sample is greater than the extent of microvascularization
in the non-invasive, non-metastatic tumor sample.
In a preferred embodiment, the determination of a poor prognosis
is made when the level of nuclear localization in the tumor sample
is from about twofold to about tenfold, more preferably about fivefold,
greater than the level of nuclear localization of p53 protein in
the non-invasive, non-metastatic tumor sample.
In a preferred embodiment, the determination of a poor prognosis
is made when the level of thrombospondin 1 expression in the tumor
sample is from about twofold to about tenfold, more preferably about
fivefold, less than the level of thrombospondin 1 expression in
the non-invasive, non-metastatic tumor sample.
In a preferred embodiment, the determination of a poor prognosis
is made when the extent of microvascularization in the tumor sample
is from about twofold to about tenfold, more preferably about sixfold,
greater than the extent of microvascularization in the non-invasive,
non-metastatic tumor sample.
In a more preferred embodiment, the determination of a poor prognosis
is made when the level of nuclear localization of p53 in the tumor
sample is from about twofold to about tenfold greater than the level
of nuclear localization of p53 protein in the non-invasive, non-metastatic
tumor sample, and the level of thrombospondin 1 expression in the
tumor sample is from about twofold to about tenfold less than the
level of thrombospondin 1 expression in the non-invasive, non-metastatic
tumor sample and the extent of microvascularization in the tumor
sample is from about twofold to about tenfold greater than the extent
of microvascularization in the non-invasive, non-metastatic tumor
sample. Most preferably, the level of nuclear localization of in
the tumor sample is from about fivefold greater than the level of
nuclear localization of p53 protein in the non-invasive, non-metastatic
tumor sample, the level of thrombospondin 1 expression in the tumor
sample is from about fivefold less than the level of thrombospondin
1 expression in the non-invasive, non-metastatic tumor sample and
the extent of microvascularization in the tumor sample is from about
sixfold greater than the extent of microvascularization in the non-invasive,
non-metastatic tumor sample in determining a poor prognosis for
a cancer patient.
In preferred embodiments, the levels of nuclear localization of
p53, thrombospondin 1 expression and the extent of microvascularization
are determined by immunohistochemical staining and detected by microscopy.
The invention also provides methods wherein the results of the
determination of the levels of nuclear localization of p53, thrombospondin
1 expression, and the extent of microvascularization are used to
prepare a prognostic or "risk" index for making a prognostic
determination. In this aspect of the invention, a prognostic index
is prepared comprising the product of the percentage of cells in
the tumor sample that are positive for nuclear localization of p53
protein and one plus the intensity of immunohistochemical staining;
the product of the percentage of cells in the tumor sample that
are positive for microvascularization and one plus the intensity
of immunohistochemical staining; and the product of the percentage
of cells in the tumor sample that are positive for thrombospondin
1 expression and one plus the intensity of immunohistochemical staining.
In calculating these products, the intensity of staining is assigned
a value of 0 for staining equal to a negative control, a value of
1 for weak staining greater than the negative control, a value of
2 for moderate staining intensity, a value of 3 for staining intensity
equal to a positive control, and a value of 4 for staining intensity
greater than the positive control. The calculated products of each
of the tumor marker determinations are then weighted on a scale
of from +1 to -4, and the index is produced as the sum of the weighted
products for nuclear localization of p53, thrombospondin 1 expression
and microvascularization. In the practice of the invention, a prognosis
of a likelihood of further neoplastic, particularly metastatic,
disease is made when this sum is less than about -5.
In additional embodiments, the prognostic index is produced by
preparing a weighted scale of expression levels of the tumor markers
related to progression observed in a representative sample of a
particular tumor type, wherein the different values in the weighted
scale are related to increased invasiveness or metastatic spread
in the representative sample.
The methods of the invention are also provided for identifying
a human cancer patient at risk for additional neoplastic disease,
for staging malignant disease in a human cancer patient and assessing
the relative risk of metastatic disease versus the risk of toxicity
(such as leukocytopenia, for example) from chemotherapeutic treatment.
The methods of the invention are provided for prognosis of disease
course in a cancer patient suffering from any specific cancer of
any tissue of origin. In preferred embodiments, the cancer is breast
cancer, prostate cancer or melanoma.
Specific preferred embodiments of the present invention will become
evident from the following more detailed description of certain
preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B and 1C are histograms of the intensity of tumor marker
staining versus tumor histology for four histological subsets described
in Example 1. The p values indicate significance of the observed
differences between samples of different histologies, determined
using paired, one-tail t-test analysis.
FIG. 2 is a graph showing the increase in p53 nuclear accumulation
and microvascularization and decrease in TSP-1 expression with tumor
progression for a cohort of breast cancer samples as described in
Example 1.
FIGS. 3A and 3B is a graph of a retrospective study of patient
survival of 40 breast cancer patients as described in Example 2,
comparing patients having a prognostic risk index of greater than
or equal to -5 (GE-5) with patients having a prognostic risk index
of less than -6 (LE-6).
FIG. 4 is a graph of a retrospective study patient survival of
104 prostate cancer patients as described in Example 3, comparing
patients having a prognostic risk index of greater than or equal
to -7 (GE-7) FIG. 3A with patients having a prognostic risk index
of less than -8 (LE-8).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a method for making a prognosis
about disease course in a human cancer patient. For the purposes
of this invention, the term "prognosis" is intended to
encompass predictions and likelihood analysis of disease progression,
particularly tumor recurrence, metastatic spread and disease relapse.
The prognostic methods of the invention are intended to be used
clinically in making decisions concerning treatment modalities,
including therapeutic intervention, diagnostic criteria such as
disease staging, and disease monitoring and surveillance for metastasis
or recurrence of neoplastic disease.
The methods of the invention are preferably performed using human
cancer patient tumor samples, most preferably samples preserved,
for example in paraffin, and prepared for histological and immunohistochemical
analysis.
The invention also provides an index for use with the methods of
the invention to relate three tumor markers (p53 nuclear accumulation,
thrombospondin-1 expression and microvascularization) with disease
progression, particularly invasiveness and metastatic spread. The
indices of the invention can be prepared as described herein for
any tumor type, provided that there is available a representative
cohort of samples of the tumor type having varying degrees of tumor
invasiveness and metastatic spread, to enable the production of
a weighted scale of expression levels of the three tumor markers.
Preferably, the size of the cohort is sufficiently large to enable
statistical analyses to verify the significance of differences in
tumor marker expression for disease progression.
Indices as provided by the invention can also be constructed using
any relevant tumor marker associated with disease progression, again
provided that there is available a representative cohort of samples
of the tumor type having varying degrees of tumor invasiveness and
metastatic spread, to enable the production of a weighted scale
of expression levels of the three tumor markers. Preferably, the
size of the cohort is sufficiently large to enable statistical analyses
to verify the significance of differences in tumor marker expression
for disease progression. Additional tumor markers can be added to
the three tumor markers used in the practice of this invention,
or other tumor markers can replace any of the markers described
herein, if such markers meet the proviso discussed above.
The methods of the invention are practiced by determining expression
levels of the three preferred tumor markers (p53 nuclear accumulation,
thrombospondin-1 expression and microvascularization) in a human
cancer patient sample. In preferred embodiments, expression levels
are determine immunohistochemical. However, expression levels can
be determined using any appropriate and convenient method. For example,
in situ polymerase chain reaction and in situ nucleic acid hybridization
methods for determining expression levels of TSP-1 fall within the
methods of the invention. Additionally, site-specific mutation analysis,
including sequence analysis or mutant allele-specific amplification
of mutant p53, can be used for determining expression levels of
mutant p53 in a tumor sample. Similarly, any method for detecting
microvascularization, including any method of specific staining,
fall within the ambit of the methods of the present invention. Detection
methods are chosen appropriate for the labeling or identification
of any of the three tumor markers used in the practice of the invention.
In a preferred embodiment, the present invention uses immunohistochemical
methods for detecting expression levels of the tumor markers of
the invention. In the practice of the invention, antibodies or antisera,
preferably polyclonal antisera, and most preferably monoclonal antibodies
specific for each marker are used to detect expression levels, using
anti-p53, anti-TSP-1 and anti-CD31 antibody immunostaining. Detection
of these antibodies can be realized by direct labeling of the antibodies
themselves, with labels including a radioactive label such as .sup.3H,
.sup.14C, .sup.35S, .sup.125I or .sup.131I, a fluorescent label,
a hapten label such as biotin, or an enzyme such as horse radish
peroxidase or alkaline phosphatase. Alternatively, unlabeled primary
antibody is used in conjunction with labeled secondary antibody,
comprising antisera, polyclonal antisera or a monoclonal antibody
specific for the primary antibody. In a preferred embodiment, the
primary antibody or antisera is unlabeled, the secondary antisera
or antibody is conjugated with biotin and enzyme-linked streptavidin
is used to produce visible staining for histochemical analysis.
Detection and quantitation of the tumor markers is provided using
methods appropriate for the staining or other detection method used.
In preferred embodiments, immunohistochemically stained sections
of a tumor sample are analyzed microscopically, most preferably
by light microscopy of a sample stained with a stain that is detected
in the visible spectrum, using any of a variety of such staining
methods and reagents known to those with skill in the art. Most
preferably the methods of the invention are practiced by those with
skill in the histological arts, but embodiments of the invention
provided to permit relatively unskilled technicians to properly
interpret tumor marker results are also within the scope of the
methods provided.
The following Examples are intended to further illustrate certain
preferred embodiments of the invention and are not limiting in nature.
EXAMPLE 1
Tumor Progression/Prognosis Analysis for Breast Cancer
Nuclear localization of p53 protein, thrombospondin 1 expression
levels and extent of microvascularization were determined immunohistochemically
as follows.
Tumor blocks from breast cancer patients were obtained from Western
Medical Center and H. Lee Moffitt Cancer Center and examined independently
by two pathologists to confirm the diagnosis for tumor type and
stage. Representative sections of each tumor sample were chosen
on the basis of pathological examination for immunohistochemical
staining. Tissue sections 5 microns in thickness were cut and prepared
on slides using standard histological preparation techniques. Since
paraffin sections were used, slides were first deparaffinized using
Histoclear (Biogenics, Calif.). Antigens were exposed for immunohistochemical
staining by pronase digestion (for CD31 detection) and by microwave
boiling (for p53 and thrombospondin 1 (TSP-1) detection) using antigen
recovery solution (Biogenics). Slides were then incubated in a solution
of 3% hydrogen peroxide in distilled water at room temperature for
10 min, then rinsed briefly with water. Slides were then incubated
for 10 min at room temperature using 100 .mu.L goat serum as blocking
buffer. Excess blocking buffer was removed from the slides by shaking,
and the slides then incubated with primary antibody at room temperature
for 30 min. The primary antibodies used in these assays were: antibody
DO1 for p53 (obtained from Santa Cruz Biotech, Santa Cruz, Calif.);
antibody clone 12 for TSP-1 (Immunotech, Inc., Westbrook, ME); and
an endothelial cell-specific antibody reactive with the cell surface
antigen CD31 for microvascularization (Dako, Carpenteria, Calif.).
Slides were rinsed twice with phosphate buffered saline (PBS) for
5 min after primary antibody incubation.
For detection of primary antibody binding, tissue sections were
then incubated with biotinylated goat antimouse immunoglobulin for
20 min at room temperature in a humidified chamber (70-100% relative
humidity). Slides were rinsed twice with PBS after this incubation,
and then treated with a solution of peroxidase-conjugated streptavidin
for 20 min. at room temperature. After being rinsed again with PBS,
the slides were incubated in a solution of 3,3'-diaminobenzidine
for 3 min at room temperature. Slides were rinsed with PBS for 5
min, exposed to hematoxylin for 1 min, rinsed with water for 10
min, dehydrated in an ascending ethanol series, cleared with xylene,
mounted and viewed by light microscopy.
Microscopic analyses were performed at 200.times. magnification
as follows. The malignant cells on the slide were counted, and the
number of stained cells and staining intensity determined. Each
slide was scored independently by two pathologists. Scoring of staining
intensity was relative to the following scale: 0=staining intensity
equal to the negative control 1=staining intensity weak but greater
than negative control 2=staining intensity moderate (more than negative
control, but less than positive control) 3=staining intensity strong,
equal to positive control 4=staining intensity greater than positive
control.
Control slides used for comparison were paraffin-embedded MCF-7
40F cells (ATCC #HTB-22) for p53 and TSP-1. Microvascularization
controls were paraffin-embedded tumor specimens showing high reactivity
with anti-CD31 antibody.
Alternatively, TSP-1 expression was determined using image analysis
(IA) techniques. Slides immunohistochemically stained for detection
of TSP-1 expression as described above were analyzed using a CAS
200 image analysis system (Cell Analysis Systems, Lombard, Ill.)
to quantitate the staining intensity of TSP-1 marker positive cells
as described (see Figge et al., 1991, Amer. J. Pathol. 139: 1213-1221
and Esteban et al., 1993, Amer. J. Clin. Pathol. 22: 32-38). This
analytical method uses a two-color system to sample image data using
2 solid-state video cameras, each with its own optical filter, mounted
on a light microscope. Video signals are sent to an image capture
board, which samples and digitizes the analog signal. The digital
value of the signal sample is proportional to the amplitude of the
video signal and is stored in an interactive computer. Measurements
are obtained from calibrated conversion of pixel information from
the video image.
For IA of TSP-1 expression in breast cancer tumor samples, the
instrument was set at threshold values optimized to distinguish
between cell membrane, nuclear and cytosolic portions of the stained
image, and the zero pixel set-point adjusted using a tumor section
stained with an isotype-matched irrelevant (i.e., unrelated) antibody.
At least 10 fields of positive area on the slide were scanned for
each tumor specimen. Video values were converted to the product
of the positive areas and positively-stained areas, expressed as
optical density (O.D.) Units using instrument software. Antigen
preservation control was evaluated using vimentin staining (1:200
antibody dilution, obtained from Dako, Carpenteria, Calif.). The
results of IA were consistent with results obtained by visual analysis
of the stained tumor sections.
The values of staining intensity related to "positive"
or "negative" predicted outcomes were determined based
on univariate analysis of the markers on survival, using a training
subset (n=42) for which survival data were known. Immunohistochemistry
(IHC) scores were assigned based on the product of the percentage
of cells positive in the sample times (1+ intensity of staining),
using the staining intensity scale described above. Tissue sections
with immunodetectably nuclear p53 observed in more than 5% of the
cells with 2+ staining intensity (corresponding to an IHC value
>15) were considered positive. (It is noted that the presence
of nuclear-located p53 is used as a marker for mutant p53, consistent
with the difference in cellular location of mutant p53 versus wildtype
known in the prior art (Hall & Lane, 1994, J. Pathol. 172: 1-4).
For IA of TSP-1 expression, positive sections were determined to
have a value of >30 O.D. For angiogenesis, microvessels were
counted in the region of greatest vessel density over at least 10
fields; samples designated as positive had >70 vessels per field.
Statistics, including Fischer's exact test and unpaired one-tailed
t test were performed using a software program (GraphPad Software,
v2.05, San Diego, Calif.) to compare values for each markers' incidence
and intensity of expression as a function of histological progression.
The results of these assays are shown in Tables I and II. Table
I presents the results for the tested markers based on a dichotomy
of invasive versus non-invasive ductal breast carcinoma (as determined
by pathological examination of breast tumor samples and registry
information provided for each sample), while Table II shows the
difference in staining patterns observed for the 4 histological
subsets studied. These results show that highly significant changes
in all three of the tested markers were observed in the transition
from non-invasive to invasive disease. The frequency of nuclear
p53 localization and microvascularization were found to be increased
(>5-fold) significantly (p <0.0001) in invasive tumor tissue,
while the frequency of thrombospondin 1 expression decreased (>5-fold)
significantly (p<0.0001) in these tumor samples.
When the tumor samples are further distinguished based on four
(rather than two) subsets of morphological and histochemical criteria,
additional differences were detected. As shown in Table II, frequency
of nuclear localization of p53 increased significantly (p=0.006)
in a comparison between low-grade and high-grade ductal carcinoma
in situ (DCIS), even though both subsets are non-invasive. In these
assays, nuclear p53 staining was not detected in any of the low-grade
DCIS samples, while 31% (6/22) of the high-grade DCIS samples showed
positive staining.
For the transition between high-grade (but non-invasive) DCIS to
frankly invasive ductal carcinoma with negative lymph nodes, only
the decline in TSP-1 expression was significant (p <0.002), with
the frequency of TSP-1 expression declining from 82% (18/22 samples)
to 32% (6/19 samples). In addition, the transition from invasive
ductal carcinoma without lymph node metastasis to invasive disease
accompanied by lymph node metastasis showed significant changes
in p53 nuclear localization, TSP-1 expression and microvascularization.
The incidence of samples with p53 nuclear localization in tumor
samples comprising metastatic cancer increased from 47% (9/19 samples)
to 82% (14/17 samples) (p=0.041), the incidence of samples with
high microvessel counts increased from 53% (10/19 samples) to 100%
(17/17 samples) (p=0.001), and the incidence of samples with pronounced
TSP-1 staining decreased from 32% (6/19 samples) in tumor without
lymph node involvement to 0% (0/17 samples) in tumors associated
with metastasis-positive lymph nodes (p=0.02).
TABLE-US-00001 TABLE I Marker Profile: Invasive versus Non-invasive
Ductal Breast Carcinoma Percent Positive Markers Studied: Tumor
Type p53 TSP-1 Microvasc. Non-invasive (n = 48) 12 83 12 Invasive
(n = 36) 64 17 75 P value* <0.0001 <0.0001 <0.0001 *P value
determined by unpaired one-tailed t-test.
TABLE-US-00002 TABLE II Marker Profile of Breast Carcinoma Progression
Percent Positive Markers Studied: Tumor Type p53 TSP-1 Microvasc.
Low-grade DCIS.sup.1 (n = 26) 0 89 4 High-grade DCIS (n = 36) 31*
82 23 Invasive - LN(-).sup.2 (n = 19) 47 32* 53 Invasive - LN(+).sup.3
(n = 17) 82* 0 100* * = p < 0.05 for paired one-tailed t-test,
comparing the designated group with the group immediately above
it in the Table .sup.1ductal carcinoma in situ .sup.2lymph node
negative .sup.3lymph node positive
These results demonstrated that nuclear localization of p53, decreased
thrombospondin 1 expression, and increased microvascularization
were significantly correlated with increased invasiveness of primary
breast cancer, increased metastasis, and poorer prognosis for breast
cancer patients whose tumors had these markers. To determine whether
disease progression was linked not only to the incidence, but also
the degree of marker expression as well, the intensity of staining
of the markers as determined above by immunohistochemistry or image
analysis was plotted versus tumor histology for the four histological
subsets described above. These results are shown in FIGS. 1A through
1C. These results demonstrate a distinct pattern of differences
in intensity and degree of expression of the three tumor markers
assayed above. These results show that nuclear p53 accumulation
and the number of tumor microvessels increased in both the transition
from low-grade DCIS to high-grade DCIS, and also in the transition
from invasive tumors without evidence of metastatic spread to invasive
tumors having metastasis-positive lymph node involvement FIGS. 1A
and 1B). TSP-1 expression showed a significant decline in intensity
between high-grade DCIS and invasive cancer prior to metastatic
spread.
These results are also shown graphically in FIG. 2, where the increase
in p53 nuclear accumulation and microvascularization and decrease
in TSP-1 expression with tumor progression is shown. These data
suggest that a coordinated relationship existed between nuclear
p53 accumulation and angiogenesis, while TSP-1 expression was inversely
correlated with these two factors. Invasion and metastasis in breast
cancer were associated in a statistically-significant way with acquisition
of dysfunctional p53 (as evidenced by nuclear accumulation), decreased
TSP-1 expression, and increased angiogenesis.
EXAMPLE 2
Tumor Prognostic Index
The results obtained in the assays described in Example 1 above
were used to construct a prognostic (risk) index relating tumor
progression and increasingly poorer disease prognosis with positive
marker results, graded by intensity of immunohistochemical staining
of each of the tumor markers.
The IHC scores obtained in Example 1 were used to construct a tumor
progression/prognosis (risk) index as follows. Scores for each of
the markers were associated with a integer index scale from +1 to
-4. This index scale was constructed for each marker based on the
following IHC staining results as follows. The p53 nuclear accumulation
score was derived from the percentage of cell staining with anti-p53
antibody multiplied by (1 +intensity of staining), using the intensity
of staining and positive control cells described in Example 1. The
TSP-1 score was derived from IA data obtained as described in Example
1, as weighted based on the percentage of cells staining positively
for TSP-1. Angiogenesis was scored as greatest number of microvessels
per field stained with anti CD31 antibody, after a minimum scan
of 10 fields. These scores and their associated weighted index scores
are described below in Table III.
TABLE-US-00003 TABLE III p53, TSP-1 and Angiogenesis Indices Index
p53 Score Thrombospondin Score Microvascularization Score 1 0 30
>30 0 30 0 31 60 25 29 31 70 -1 61 90 20 24 71 85 -2 91 120 15
19 86 100 -3 121 450 10 14 101 123 -4 >150 0 9 >123
The tumor prognosis (risk) index is then prepared by the sum of
the index scores for p53 accumulation, TSP-1 expression and angiogenesis
(microvascularization), with poor prognosis being determined for
tumors having an summed index score of -5 or less.
The efficacy the prognostic (risk) index was assessed using Log
rank tests on survival versus index score. The significance of the
index scores on survival are shown in Table IV. In this table, it
can be seen that a statistically-significant difference was observed
in survival between patients having tumors with a summed index score
.gtoreq.-5 when compared with patients having tumors with a summed
index score .ltoreq.-6. Similarly, a statistically-significant difference
was observed in survival between patients having tumors with a summed
index score .gtoreq.-6 when compared with patients having tumors
with a summed index score <-7. Finally, there was a statistically-significant
difference was observed in survival between patients having tumors
with a summed index score >-7 when compared with patients having
tumors with a summed index score <-8.
TABLE-US-00004 TABLE IV Log Rank Tests to Compare Survival Index
Score Log Rank Test P Value .gtoreq.-5 vs. <-6 p < 0.0001
.gtoreq.-6 vs. <-7 p < 0.0007 .gtoreq.-7 vs. <-8 p <
0.0548
These results are shown graphically in FIG. 3 in a retrospective
analysis of 40 breast cancer patients having a summed prognostic
(risk) index score greater than -5 (GE -5) or less than -6 (LE -6).
In this study, survival was correlated with risk index scores greater
than or equal to -5 (about 80% survival at 60 months post-diagnosis)
versus about 20% survival for patient having tumors with index scores
less than -6.
These results were also analyzed using multivariate analysis including
angiogenesis, tumor size and lymph node status. These results showed
that the index was more predictive of patients' prognosis for survival
than the commonly-used indices of tumor size or lymph node status.
These results demonstrate that the tumor progression index is a
statistically reliable predictor of tumor prognosis and disease
progression for breast cancer.
EXAMPLE 3
Tumor Progression/Prognosis Analysis for Prostate Cancer
The immunohistochemical analyses described above in Example 1 were
applied to prostate cancer samples. In addition, androgen receptor
expression was assayed for these tumors, due to the recognized correlation
between androgen receptor expression and poor prognosis/survival
in these patients.
In this study, 104 prostate cancer patient tumor samples were assayed
for nuclear accumulation of p53, TSP-1 expression, androgen receptor
(AR) gene expression and microvascularization. These assays were
performed immunohistochemically as described above in Example 1,
except that androgen receptor expression was determined using an
anti-AR antibody (Biogenics, used at 1:20 dilutions).
The results of these studies closely paralleled the results obtained
with breast carcinoma, and the indices derived from the p53, TSP-1
and angiogenesis/microvascularization data were identical to those
shown in Table III. In addition, AR expression was found to be negatively
correlated with prognosis and survival using multivariate analysis
(Cox regression analysis), which showed statistical significance
(p=0.0077). Interestingly, the presence of nuclear accumulation
of p53 was correlated with AR expression (p<0.0041).
The risk index incorporating levels of nuclear p53, TSP-1 expression
and angiogenesis was found to be significantly associated with survival.
Survival in patients with a risk index of -8 or less was significantly
lower than that in patients with a prognostic (risk) index of -7
or greater (p<0.0001). The risk index was also associated with
survival (p<0.0061) even after adjustment for age and stage.
These results are shown graphically in FIG. 4. This Figure illustrates
the results of a retrospective analysis of 104 prostate cancer patients
having a summed prognostic (risk) index score greater than -7 (GE
-7) or less than -8 (LE -8). In this study, survival was correlated
with risk index scores greater than or equal to -7 (about 95 months
to 20% survival post-diagnosis) versus about 30 months to 20% survival
post-diagnosis for patients having tumors with index scores less
than -8. These results demonstrated that the prognosis (risk) index
was reliable for predicting poor prognosis/increased disease progression
based on the tested tumor markers.
It should be understood that the foregoing disclosure emphasizes
certain specific embodiments of the invention and that all modifications
or alternatives equivalent thereto are within the spirit and scope
of the invention as set forth in the appended claims.
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