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Cancer Patent Abstract
The invention is directed to diagnostic and monitoring methods (assays)
for cancer and kits that may be used in such methods. More particularly,
an aspect of the invention relates to the use of activated Stat5
for diagnosing and monitoring breast cancer and predicting the effectiveness
of cancer treatment. The invention also relates to the use of screening
assays for discovering compounds that effect levels of activated
Stat5.
Cancer Patent Claims
What is claimed is:
1. A method of determining reduced risk of breast cancer progression,
comprising: a) obtaining a sample of breast tissue from an individual
in need of breast cancer prognosis, wherein said sample comprises
cancer cells; b) determining positive or negative Stat5 activation
status of said sample by detecting tyrosine phosphorylated Stat5
localized in cancer cell nuclei of said sample; and c) determining
from said status a reduced risk of breast cancer progression associated
with said cancer cells if the status is positive.
2. The method of claim 1 wherein said sample of breast tissue is
a breast tumor sample from an individual with node-negative breast
cancer.
3. The method of claim 1 wherein, in step b), said detecting comprises
contacting said sample with an antibody that detects tyrosine phosphorylated
Stat5.
4. The method of claim 1 wherein said detecting comprises a method
selected from the group consisting of immunoblotting, immunohistochemistry
and immunocytochemistry.
5. The method of claim 1 wherein said detecting comprises Fluorescence-Activated
Cell Sorting (FACS).
6. The method of claim 3, wherein said antibody is an antibody
to tyrosine-phosphorylated Stat5.
7. The method of claim 1 wherein said sample is a tissue section
sample.
8. The method of claim 1 further comprising analyzing the levels
of activated Stat5 in conjunction with additional breast cancer
markers.
9. The method of claim 1 wherein, in step b), said detecting comprises
treating said sample in a microwave oven or by other heat-based
methods of antigen retrieval and then detecting tyrosine phosphorylated
Stat5 localized in cancer cell nuclei of the resulting treated sample.
10. The method of claim 1 wherein, in step b), said detecting comprises
treating said sample in a microwave oven or by another heat-based
method of antigen retrieval in an appropriate antigen retrieval
solution and then detecting tyrosine phosphorylated Stat5 localized
in cancer cell nuclei of the resulting treated sample.
11. The method of claim 10, wherein said appropriate antigen retrieval
solution is an aqueous buffer with a pH of about 7-10.
12. The method of claim 11 wherein said antigen-retrieval buffer
is 1 mM Tris at pH 10.
13. The method of claim 8, wherein said analyzing comprises univariate
or multivariate analysis.
14. The method of claim 3, wherein said antibody is a monoclonal
antibody recognizing the phosphopeptide KAVDG(phospho Y)VKPQIK (SEQ
ID NO: 1), and which specifically recognizes tyrosine phosphorylated
isoforms of Stat5, but not unphosphorylated isoforms, and does not
recognize Stat5 mutants in which the tyrosine residue has been substituted
with phenylalanine.
15. The method of claim 1, wherein said activated Stat5 comprises
Stat5 a phosphorylated on amino acid residue Tyr694.
16. The method of claim 1, wherein said activated Stat5 comprises
Stat5b phosphorylated on amino acid residue Tyr699.
17. The method of claim 1 further comprising, after step c): d)
obtaining a subsequent tissue sample from a recurrent tumor in said
individual, wherein said subsequent tissue sample contains breast
cancer cells; and e) determining positive or negative Stat5 activation
status of said subsequent sample by detecting tyrosine phosphorylated
Stat5 localized in cancer cell nuclei of said subsequent sample;
and f) determining from said status of step e) a reduced risk of
breast cancer progression associated with said cancer cells of said
subsequent tissue sample if the status of said subsequent sample
is positive.
18. The method of claim 17, wherein step f) comprises determining
from said positive status that radical mastectomy is not necessary.
19. The method of claim 1, wherein said breast tissue sample is
a biopsy sample or a pathological archived material.
20. The method of claim 6, wherein in step b) detecting of tyrosine
phosphorylated Stat5 localized in cancer cell nuclei comprised detecting
binding of said antibody in said cancer cell nuclei in said breast
tissue sample.
Cancer Patent Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser.
No. 09/760,899, filed on Jan. 17, 2001, now abandoned.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to diagnostic and monitoring methods
and assays for cancer and kits that may be used in such methods.
More particularly, the application relates to the use of activated
Stat5 for diagnosing and monitoring cancer and predicting the prognosis
of (breast) cancer patients and the outcome of cancer therapies,
especially breast cancer. The invention also relates to screening
assays for discovering compounds that affect levels of activated
Stat5.
2. Related Art
One of the most pressing health issues today is diagnosing, monitoring
and treating cancer and particularly breast cancer. Breast cancer
is the leading form of cancer in women, and the second leading cause
after lung cancer of cancer death among this population in the United
States. In the industrialized world, about one woman in every nine
can expect to develop breast cancer in her lifetime. In the United
States, the annual incidence breast cancer is about 180,000 new
cases and approximately 48,000 deaths each year (Parkin 1998; Apantaku
2000). Approximately two million women living in the United States
alone have been diagnosed with breast cancer at some point in their
lives. Breast cancer also occurs among men, though far more rarely
(approximately 1,600 new cases diagnosed in the U.S. 1998). Treatment
for male breast cancer is guided by our understanding of the disease
in women.
Despite ongoing improvements in understanding the disease, breast
cancer has remained to a large extent resistant to medical intervention.
Most clinical initiatives are focused on early diagnosis, followed
by conventional forms of intervention, particularly surgery, radiation,
hormone suppression, and chemotherapy. Such interventions are of
limited success, particularly in patients where the tumor has undergone
metastasis. In patients with breast cancer without detectable lymph
node metastases, socalled node negative breast cancer, the risk
of death from breast cancer recurrence within 10 years is also high,
approximately 30% (McGuire, Tandon et al. 1992). There is a pressing
need to improve the arsenal of diagnostic tools and methods available
to provide more precise and more effective information that will
allow successful treatment in the least invasive way possible. Specifically,
markers that can identify patients with very low risk of disease
recurrence and death after initial surgery would reduce the extent
of overtreatment with expensive and potentially toxic supplementary
regimes. The invention meets that need by providing new methods
and markers for monitoring breast cancer.
Breast Cancer
Development of cancer is a multistep process of genetic alterations
that transform normal cells into highly malignant derivatives (Kinzler
and Vogelstein 1996; Lengauer, Kinzler et al. 1998). Tumors within
the breast may arise from any of its component tissues (e.g. connective
tissue and epithelial structures). However, it is the epithelial
tissue compartment that gives rise to most common malignant breast
neoplasms.
A number of risk factors for carcinoma of the breast have been
identified. These include: geographic influences, genetic predisposition,
increasing age, length of reproductive life, parity, age at birth
of first child, obesity, exogenous estrogens, fibrocystic changes
with a typical epithelial hyperplasia and carcinoma of the contralateral
breast or endometrium (Cole 1980; Stoll 1998). The chief forms of
carcinoma of the breast are classified as infiltrating or noninfiltrating
arising in the ducts. These include intraductal carcinoma, comedocarcinoma,
simple or usual type (including scirrhous carcinoma), medullary
carcinoma, colloid carcinoma, Paget's disease of the breast and
tubular carcinoma. Infiltrating and noninfiltrating carcinomas also
arise in the lobules and are referred to as in situ lobular carcinoma
and infiltrating lobular carcinoma (Simmons and Osborne 1999; Styblo
and Wood 1999).
Among the large group of breast cancer patients with localized
tumors and without detectable metastases to nearby lymph nodes,
many will be cured by surgery because the tumors have not spread
to surrounding tissues and lymph nodes. However, others have occult
micrometastatic disease and could benefit from supplementary radiation
or adjuvant anti-hormone therapy or chemotherapy. There is a need
for diagnostic markers to discriminate between tumors with low risk
for micrometastatic spread and those with higher risk. Tumor markers
that signify low risk of micrometastatic disease may directly affect
the therapeutic decision of whether to use supplementary radiation
or adjuvant hormone or chemotherapy. Furthermore, such tumor markers
may also affect the surgeon's recommendation of whether to choose
breast conserving surgery or mastectomy.
The molecular basis of cancer is still being determined. Underlying
genome instability facilitates progressive accumulation of growth-promoting
traits in premalignant cells under selective pressure from various
growth barriers (Cahill et al 1999). Growth-promoting characteristics
of cancer include self-sufficiency in growth signals, insensitivity
to anti-growth signals, evasion of apoptosis, limitless replicative
potential, sustained angiogenesis, tissue invasion and metastasis
(Hanahan and Weinberg 2000). Associated with this stepwise progression
of tumor cells toward increasing malignancy is a gradual loss of
tissue-specific cell differentiation.
Loss of tumor cell differentiation appears to be particularly prominent
at the transition from localized, surgically curable cancer to metastatic
disease (Hart and Easty 1991; Freije, MacDonald et al. 1998; Rivadeneira,
Simmons et al. 2000). This transition also is the single most critical
determinant of prognosis for patients with solid tumors (McGuire
1991; Tubiana 1999). Assessment of the activity of transcriptional
regulators that maintain cell and tissue-specific differentiation
in primary tumors may therefore be useful for predicting the risk
of occult micrometastases and tumor recurrence. Such informative
tumor markers may directly influence treatment decisions by either
providing prognostic distinction between low- and high-risk malignancies,
or by predicting tumor response to specific adjuvant therapies or
tumor response to specific modes of surgery (breast conservation
surgery vs. mastetomy).
In breast cancer, receptors for estrogen and progesterone are related
to the state of mammary epithelial cell differentiation and have
prognostic value for disease outcome in certain cases. Estrogen
and progesterone receptor (ER/PR) status is particularly useful
as a predictive marker of positive response to adjuvant anti-estrogen
therapy in node-positive breast cancer. However, the ER/PR status
is not clinically useful to predict prognosis in node-negative cancer
(Fitzgibbons, Page et al. 2000). This may be due to the high proportion
of ER/PR positive, localized tumors. There is a need to identify
low-risk breast cancer patients who may be spared from costly and
potentially toxic adjuvant antiestrogen treatment or chemotherapy.
There is also a need to identify low-risk breast cancer patients
who may benefit from less invasive procedures such as breast conserving
surgery, or lumpectomy, with or without post-surgical radiation
therapy, instead of mastectomy. The benefits of less extensive and
less invasive therapeutic regimes to patients with good prognosis
may include avoidance of side-effects, improved mental and physical
health, improved quality of life, and lower financial burden. The
benefits to society are particularly the cost-saving aspects of
avoiding unneccessary overtreatment. One means of accomplishing
this is to obtain better prognostic markers for node-negative, as
well as other types of breast cancer. These needs are met by the
invention.
Diagnosis of Breast Cancer
The definitive diagnosis of all types of breast disease is based
on histologic evaluation of tissue samples using the light microscope.
The histologic criteria used to define most breast lesions are historic
but nonetheless quite reproducible for identifying fully invasive
breast cancers.
Improved detection and screening routines, and the development
and increasing utilization of fine needle aspirates (FNAs) and core
needle biopsies for obtaining tissue samples have been major advances
in both detection and diagnosis. Stereotactic image guidance of
needle biopsies has tremendously improved our ability to sample
suspicious lesions, particularly non-palpable masses, as small as
a few millimeters in diameter nearly anywhere in the breast. This
has dramatically increased the detection of small, more treatable
breast cancers and decreased unnecessary surgery in an enormous
number of patients with insignificant benign disease. Recent accomplishments
include the identification of a small number of tissue-based biomarkers
that are helpful in predicting clinical outcome and response to
therapy (e.g., S-phase fraction, estrogen and progesterone receptors,
c-erbB-2) and the discovery of genes (BRCA-1 and BRCA-2) associated
with familial risk for breast cancers (Dahiya and Deng 1998; Fitzgibbons,
Page et al. 2000).
However, diagnosing breast cancer still requires some type of biopsy
procedure. In addition, current diagnostic and prognostic methods
cannot absolutely distinguish breast cancers that are treatable
by surgery alone from those that are likely to recur or have already
spread through micrometastases. As a result, at least 50 percent
of breast cancer patients with node negative disease are treated
with some form of adjuvant therapy. Moreover, available methods
are inadequate for predicting the response of breast cancers to
specific types of adjuvant therapies.
Treatment decisions for individual breast cancer patients are frequently
based on the number of axillary lymph nodes involved with disease,
estrogen receptor and progesterone receptor status, size of the
primary tumor, and stage of disease at diagnosis (Tandon, Clark
et al. 1989). However, even with this variety of factors, it is
currently not possible to predict accurately the course of disease
for all breast cancer patients. There is clearly a need to identify
new markers in order to separate patients with good prognosis, who
might need no supplementary therapy beyond surgical removal of the
malignant breast tumor, from those whose cancer is more likely to
recur and who might benefit from additional and more exhaustive
treatment forms.
Despite extensive efforts over several decades, only a limited
number of immunohistochemical breast tumor markers have been identified.
Among immunohistochemical markers, hormone receptor status remains
the only to have gained standard clinical use for evaluating node-negative
breast tumors (Fitzgibbons, Page et al. 2000). With improving methods
for screening and detection of early breast cancer the proportion
of node-negative cases is expected to continue to rise (Elledge
and McGuire 1993). Parameters that have been established to be important
for the prognosis of patients with breast malignancies in general
and that are used by clinicians include: size of primary tumor,
stage of disease at diagnosis, number of axillary lymph nodes involved
with disease, and hormonal receptor status (ER/PR) (Fitzgibbons,
Page et al. 2000). Abnormal status of ErbB-2 or p53, as well as
other histological and genetic markers, also are associated with
poor prognosis especially in node-positive tumors (Slamon, Clark
et al. 1987; Fresno, Molina et al. 1997; Pharoah, Day et al. 1999).
In this regard, U.S. Pat. No. 5,599,681 has suggested the use of
an antibody that specifically binds to a reversible phosphorylation
site of the c-erbB2 oncoprotein in its active form to screen for
the metastatic potential of tumors in patients with node-negative
breast cancer. Nowhere, however, was it suggested that screening
for activated Stat5 could be used to predict the metastatic potential
of breast cancer.
There remain deficiencies in the art with respect to the identification
of markers linked with the progression of breast cancer, the development
of diagnostic methods to monitor disease progression and the development
of therapeutic methods and compositions; to treat breast diseases
and cancers. The identification of markers which are differentially
expressed or activated in breast cancer would be of considerable
importance in the development of a rapid, inexpensive method to
improve diagnosing of breast cancer and to predict tumor behavior
with respect to patient prognosis and responsiveness to individual
therapeutic options. The identified marker(s) would also be useful
as a target of therapeutic compositions, of in screening assays
for therapeutic compounds.
The diagnostic and monitoring methods of the invention meet many
needs in this area.
Therapeutic Regimes for Treating Breast Cancer
Treatment of breast cancer is multifaceted and complex. The choice
of therapeutic approach is guided by a series of criteria based
on a limited set of tumor characteristics. Nearly all patients with
breast cancer will have some type of surgery. This may be supplemented
by local therapy with radiation, or by systemic therapy including
hormone suppression or chemotherapy. To kill cancer cells that may
have spread beyond the breast and nearby tissues, physicians employ
oral or intravenous systemic therapy. Examples of systemic treatments
for breast cancer are chemotherapy and antiestrogen therapy. Systemic
therapy given to patients after surgery is often referred to as
adjuvant therapy. The goal of adjuvant therapy is to kill hidden
cancer cells. Even in the early stages of the disease cancer cells
can break away from the primary breast tumor and spread through
the bloodstream. These cells usually cause no detectable symptoms
and usually do not show up on an x-ray and cannot be felt during
a physical examination. But they can establish new tumors in other
locations in the body. Furthermore, oncologists sometimes give patients
neo-adjuvant therapy--that is, systemic therapy before surgery,
typically to shrink the tumor.
The following summarizes the main principles of treatment of breast
cancer according to current guidelines endorsed by the U.S. National
Cancer Consortium Network and the American Cancer Society (1999).
The text below maintains an emphasis on treatment of node-negative
breast cancer, as it relates to the present invention.
Breast conserving surgery--"Lumpectomy" removes only
the breast lump and the surrounding area, or margin, of normal tissue.
If cancer cells are present at the margin (the edge of the excisional
biopsy or lumpectomy specimen), a re-excision can usually be done
to remove the remaining cancer. In most cases, lumpectomy is combined
with 6 to 7 weeks of supplementary radiation therapy following surgery.
This combination of lumpectomy and radiation is often referred to
as "breast conserving therapy".
Mastectomy--In a "simple (total) mastectomy" procedure
surgeons remove the entire breast but do not remove any lymph nodes
from under the arm, or muscle tissue from beneath the breast. In
a "modified radical mastectomy", surgeons remove the entire
breast and some of the axillary (underarm) lymph nodes. Modified
radical mastectomy is the most common surgery for patients with
breast cancer in whom doctors remove the whole breast. "Radical
mastectomy" removes not only the entire breast, but axillary
lymph nodes and the chest wall muscles under the breast as well.
The less extensive modified radical mastectomy has proved as effective
as radical mastectomy, which is nowadays rarely performed due to
disfiguration and frequent side-effects.
Lymph node surgery--Regardless of whether a breast cancer patient
has a mastectomy, or a lumpectomy for invasive cancer, the physicians
need to determine whether the cancer has spread. The regional lymph
nodes in the underarm drain lymph from the breast, and are typically
the first sites of spread. Furthermore, lymph node involvement increases
the likelihood that cancer cells have spread through the blood-stream
to other parts of the body.
While lymph node surgery itself does not improve the chance for
a cure, this is the only way to accurately determine if the cancer
has spread to the lymph nodes. This usually means removing some
or all of the lymph nodes in the armpit. Typically 10 to 20 lymph
nodes in the armpit are examined by an operation called "axillary
lymph node dissection". Although axillary lymph node dissection
is a safe procedure with low rates of serious side effects, efforts
are ongoing to develop new ways of detecting the spread of cancer
to lymph nodes that are less invasive and do not involve a full
lymph node dissection. Such alternative methods include the "sentinel
lymph node biopsy" (Orr, Hoehn et al. 1999; Sugg, Ferguson
et al. 2000), and new detection methods for breast cancer cells
in bone marrow and blood (Berois, Varangot et al. 2000; Braun, Pantel
et al. 2000; Fetsch, Cowan et al. 2000; Ikeda, Miyoshi et al. 2000;
Kraeft, Sutherland et al. 2000; Zhong, Kaul et al. 2000). It is
possible that these newer methods in the future may replace lymph
node dissection as a means of determining micrometastatic spread
of cancer.
Sentinel lymph node biopsy--In the sentinel lymph node biopsy procedure
the surgeon finds and removes the `sentinel node`--the first lymph
node into which a tumor drains, and therefore the one most likely
to contain cancer cells. Many doctors recommend it for most women
with breast cancer, but others still consider it investigational.
In a sentinel lymph node biopsy the surgeon injects a radioactive
substance and/or a blue dye into the area around the tumor. Lymphatic
vessels carry these materials into the sentinel node. The doctor
can either see the blue dye or detect the radioactivity with a geiger
counter, and then cuts out the node for examination. If the sentinel
node contains cancer, the surgeon will have to perform an axillary
dissection--removal of more lymph nodes in the axilla (armpit).
If the sentinel node is cancer-free, the patient and her physicians
may consider avoiding more lymph node surgery and its potential
side effects. Although the sentinel node procedure is relatively
new and its long-term effectiveness is uncertain (Orr, Hoehn et
al. 1999; Sugg, Ferguson et al. 2000), it may turn out to be equally
as effective in determining lymph node spread as full lymph node
dissection.
Detection of disseminated cancer cells in blood and bone marrow--Recent
methods for detecting metastatic breast cancer cells in blood (Berois,
Varangot et al. 2000; Fetsch, Cowan et al. 2000; Kraeft, Sutherland
et al 2000) or in bone marrow (Braun, Pantel et al. 2000; Ikeda,
Miyoshi et al. 2000; Zhong, Kaul et al. 2000) are typically based
on the detection of cytokeratin markers characteristic to breast
cancer cells by immunological methods or by gene-based testing.
These new methods may also lead to an alternative approach to lymph
node dissection for determining whether a breast cancer has spread
beyond the local tumor area.
Radiation therapy--Radiation is used to destroy cancer cells left
behind in the breast, chest wall, or lymph nodes after surgery.
Radiation treatments usually take place 5 days a week over a period
of 6 to 8 weeks. Side effects most likely to occur include swelling
and heaviness in the breast, sunburn-like skin changes in the treated
area, and fatigue. Changes to the breast tissue and skin usually
go away in 6 to 12 months. In some women, the breast becomes smaller
and firmer after radiation therapy. Radiation therapy of axillary
(armpit area) lymph nodes can also cause lymphedema. Although generally
safe, it is evident that radiation therapy comes at a considerable
expense and with potentially serious side-effects. Radiation therapy
also involves a major risk for abnormal fetal development, and cannot
be used to treat pregnant women with breast cancer.
New tumor markers that signify good prognosis may reduce the need
for supplementary radiation therapy.
Chemotherapy--Patients receive this treatment of anti-cancer drugs
intravenously (injected into a vein) or by mouth. Either way, the
drugs travel in the bloodstream and move throughout the entire body.
Doctors who prescribe these drugs (medical oncologists) generally
use a combination of medicines proven more effective than a single
drug. For women with node-negative breast cancer the most frequently
used chemotherapy options are CMF (cyclophosphamide, methotrexate,
and fluorouracil), CAF (cyclophosphamide, doxorubicin), and AC (doxorubicin
(Adriamycin) and cyclophosphamide) (1999). Doctors give chemotherapy
in cycles, with each period of treatment followed by a recovery
period. The total course of chemotherapy usually lasts 3 to 6 months
depending on the combinations used. This is significant both in
terms of cost and reduced well-being. The side effects of chemotherapy
are many and potentially severe, and depend on the type of drugs
used, the amount taken, and the length of treatment. Doxorubicin
and epirubicin may cause heart damage, although doctors limit the
dose and perform periodic tests to check heart function in order
to prevent this side effect. Other side effects include loss of
appetite, nausea and vomiting, mouth sores, hair loss, and changes
in the menstrual cycle. Because chemotherapy can damage the blood-producing
cells of the bone marrow, a drop in white blood cells can raise
a patient's risk of infection, a shortage of blood platelets can
cause bleeding or bruising after minor cuts or injuries; and a decline
in red blood cells can lead to fatigue due to anemia.
New tumor markers that identify patients with excellent prognosis
may eliminate the need for adjuvant chemotherapy among these patients.
Hormone therapy--Estrogen, a female sex hormone produced by the
ovaries, promotes growth of some breast cancers. Doctors use several
approaches to block the effect of estrogen or to lower estrogen
levels. The most commonly used antiestrogen drug is tamoxifen, taken
daily in pill form, usually for 5 years. Studies show that tamoxifen
can reduce the chances of breast cancer coming back after surgery
if the breast cancer cells contain receptors for estrogen or progesterone.
Tamoxifen may be used to treat metastatic breast cancer, but also
a significant number of patients with node-negative cancer receive
tamoxifen treatment.
Adjuvant Herceptin therapy--A new form of adjuvant breast cancer
treatment involves the use of Herceptin, a drug that antagonizes
activity of the Her2/neu oncogene reecently introduced for select
patients with node-positive breast cancer (Stebbing, Copson et al.
2000). Herceptin therapy will not be discussed in more detail here.
Therapeutic considerations in node-negative breast cancer--Decisions
about types of surgery (breast conserving lumpectomy, radical mastectomy),
radiation therapy, adjuvant chemotherapy or hormonal therapy are
currently based on the status of axillary lymph nodes, the size
of the malignant tumor and its histologic type (appearance under
a microscope), and hormone receptor status. For example, if regional
lymph nodes are negative (do not contain any cancer cells) and the
tumor measures half a centimeter or smaller, the patient needs no
adjuvant (post-surgery) therapy. In current practice, a substantial
number of patients with node-negative breast cancer with larger
tumors receive adjuvant therapies with questionable benefit in terms
of relatively limited improvement in prognosis considering the associated
increased morbidity and serious side-effects (McGuire, Tandon et
al. 1992). These adjuvant therapies also come at high cost as described
above. Furthermore, the choice of the less invasive breast conserving
surgery (lumpectomy) is generally preferred by doctors and patients
over mastectomy, but more specific guidelines and better prognostic
tumor markers are needed to guide this selection. There is therefore
a strong need for new markers to identify breast cancer patients
with low risk for disease recurrence and death.
Markers for low-risk cancer and patient follow-up--Better prognostic
tumor markers may also have the benefit of reducing the frequency
of follow-up visits among patients with low-risk cancer. Tumor markers
identifying low-risk breast cancer patients may also allow reduced
frequency and lighten the extensive requirements for patient follow-up.
While this is primarily a cost issue, it also positively impacts
the patient's quality of life. Routine surveillance and follow-up
for all patients who have had invasive breast cancer curently includes
the following: a history and physical exam every 4-6 months for
2 years, then every 6 months for 3 years, and then, once every year
(1999). Women who have had a lumpectomy and radiation (breast conservation
therapy) should undergo mammography of the treated breast at 6 months
after radiation therapy, and then mammography of both breasts on
an annual basis. Women who have had a mastectomy should get a mammogram
of the remaining breast annually after the surgery. Because tamoxifen
increases a postmenopausal woman's risk of developing cancer of
the endometrium (lining of the upper part of the uterus), postmenopausal
patients taking this drug also should have an annual pelvic exam.
Markers indicating low-risk for tumor recurrence therefor may benefit
both patients and society by reduced costs associated with fewer
and less extensive follow-up examinations.
Monitoring of recurrent breast cancer--Work-up for a suspected
recurrence of breast cancer includes a biopsy to confirm the first
recurrence whenever possible. A recurrence may be local, meaning
that cancer has returned to the breast, underarm lymph nodes, or
nearby tissues, or systemic, which means that cancer has spread
to distant organs. There exist a series of guidelines to treat locally
recurring breast cancer. The current recommendations for treatment
of the locally recurring tumor depend in large part on what mode
of treatment was used for the original tumor (1999). New markers
that predict the biological behavior of breast cancer may affect
the choice of follow-up therapy, depending on whether the recurrent
tumor is deemed low or high risk. For instance, local recurrence
of a tumor positive for a marker indicating low risk of distant
spread may allow the use of less intensive therapeutic approaches
than if the tumor is negative for this same marker. For example,
reexcision and possibly local radiation may suffice instead of radical
mastectecomy with or without adjuvant chemotherapy or anti-hormone
therapy.
Stat5
The Signal Transducer and Activator of Transcription (STAT) family
of transcription factors provide a signaling link between cell surface
hormone and cytokine receptors and specific response elements in
the promoters of selective genes. Seven mammalian STAT genes have
been identified. The Stat5 transcription factor is involved in regulation
of cell growth, differentiation, and cell survival (Wakao, Gouilleux
et al. 1994). It exists as two highly homologous isoforms, Stat5a
and 5b, which have more than 95% amino acid homology and are encoded
by separate genes (Liu, Robinson et al. 1995; Grimley, Dong et al.
1999). Stat5 is required for normal mammary epithelial cell development
and differentiation (Liu, Robinson et al. 1997; Udy, Towers et al.
1997; Moriggl, Topham et al. 1999).
Stat5 polypeptides typically are cytoplasmic and quiescent under
homeostatic conditions. Their activation results from phosphorylation
of the highly conserved C-terminal tyrosine at Tyr694 in Stat5a
or the corresponding Tyr699 in Stat5b by certain intracellular tyrosine
kinases. This phosphorylation permits dimer pair formation which
is needed for Stat5 to bind to DNA.
This initial phosphotyrosyl "on-switch" is a generic
Stat feature (Darnell 1997; Darnell 1998) and is triggered when
cells with cognate receptors are exposed to a variety of stimuli
including cytokines, immune complexes, microbiologic agents or non-peptidyl
compounds. Although the spectrum of agonists thus is heterogeneous,
the bulk implicated in triggering Stat5 activation belong to the
class I and class II cytokine superfamilies. (See Table 4 of (Grimley,
Dong et al. 1999). These cytokines utilize receptors lacking a catalytic
domain (Liu, Gaffen et al. 1998), so that the Stat activation is
most often dependent upon an auxiliary protein kinase (Leonard and
O'Shea 1998).
The Janus tyrosine kinases (Jaks) form biochemically stable associations
with class I and class II cytokine receptors. A non-covalent linkage
facilitates Jak phosphorylations during receptor ligation and increases
the odds of interactions between Jaks and Stat5 recruited to receptor-Jak
complexes (Leonard and O'Shea 1998). This critical and conserved
mutual relationship has engendered the scientific vernacular of
"Jak-Stat pathway" (Liu, Gaffen et al. 1998). However,
Jaks are not the sole means of Stat activation.
Stat5a and Stat5b can also be tyrosine phosphorylated by a number
of cytokines commonly designated as "growth factors" which
bind to receptor tyrosine kinases (RTKs). The RTKs possess intrinsic
catalytic properties, and may trigger Stat5 signals absent a direct
linkage to the Jak enzyme system (Chen, Sadowski et al. 1997). In
addition, Stat5 tyrosine phosphorylation might be effected by cytosolic
protein kinases in the Src or Tec families. As "nonreceptor
tyrosine kinases" (NTKs), the latter enzymes can function without
extrinsic stimulation due to receptor ligation. The Src-family kinase
Lck has been implicated in Stat5 phosphorylation during T cell proliferation
(Welte, Leitenberg et al. 1999) and constitutively active NTKs,
RTKs or analogous oncoproteins may be particularly significant in
maintaining a constitutive phosphorylation of Stat5 in autonomously
proliferating neoplastic cells (For example, See (Lacronique, Boureux
et al. 1997; Wellbrock, Geissinger et al. 1998)).
In addition to the initial activation switch of Stat5, which involves
phosphorylation of a tyrosine residue within a conserved C-terminal
segment and causes dimerization of Stat5 molecules (Gouilleux, Wakao
et al. 1994), a second coordinated activation event is required
for functional activation. This involves translocation of dimerized
Stat5 from the cytoplasm into the cell nucleus, which permits Stat5
to come in proximity of and bind to gene regulatory promoter elements,
and thus regulate transcription of specific genes (Gouilleux, Wakao
et al. 1994; Kazansky, Kabotyanski et al. 1999). Because Stat5 not
only requires phosphorylation of a specific tyrosine residue, but
also needs to translocate into the cell nucleus in order to function
as an active DNA-binding transcription factor, amounts of tyrosine
phosphorylated Stat5 located within the cell nucleus will reflect
the levels of activated Stat5 more accurately than overall cellular
levels of tyrosine phosphorylated Stat5. For instance, tyrosine
phosphorylation of Stat5a by the Src tyrosine kinase has been shown
not to be accompanied by nuclear translocation (Kazansky, Kabotyanski
et al. 1999), illustrating that quantitation of tyrosine phosphorylation
status alone without assessing nuclear localization is not sufficient
for accurate determination of levels of activated Stat5. Correspondingly,
Stat transcription factors may become dephosphorylated within the
cell nucleus and loose the ability to bind to DNA (Haspel and Darnell
1999), making assays that detect nuclear Stat5 protein levels alone
also not sufficient for accurate determination of levels of activated
Stat5. In the present description, the term `levels of activated
Stat5` refers to levels of tyrosine phosphorylated Stat5 within
the cell nucleus.
Antibodies that bind exclusively to tyrosine phosphorylated Stat5
can be used to detect activated Stat5 in the nuclei of cells by
immunocytochemistry or immunohistochemistry, provided that proper
steps are taken to achieve antigen retrieval of this cryptic antigenic
site. This antigenic site is cryptic, or unavailable, unless the
phosphorylated tyrosine bound to the SH2 domain of the partner molecule
in the dimer is dissociated by specific treatment.
Detection of active, tyrosine phosphorylated Stat5 by immunohistochemistry
in tissue sections has been reported (Jones, Welte et al. 1999).
Stat5 activation in normal mouse mammary gland tissue in response
to Erb-B4 activation was studied. However, human breast tissue or
human breast cancer samples were not examined. In further contrast
to Jones, the current invention may use a simple one-step antigen-retrieval
method for determining levels of activated Stat5.
The extent to which Stat5 promotes cell proliferation or inhibits
growth by inducing cell differentiation in various tissues, including
mammary gland, is unresolved. The possibility that Stat5 activation
status is of prognostic value for breast cancer was not obvious
prior to the inventors' discovery, because a priori, it had been
argued that Stat5 activation may promote mammary tumor formation
instead of being associated with reduced risk of invasion and metastasis.
It was specifically suggested that a general anti-apoptotic effect
of Stat5 might contribute to mammary tumor progression in rodents
(Humphreys and Hennighausen 2000). This notion was supported by
the observation that in mice lacking the Stat5a gene (Stat5a-/-
mice) but overexpressing the oncogenic TGF-alpha transgene, the
rate of mammary tumor formation was reduced relative to that observed
in Stat5a+/+ mice (Humphreys and Hennighausen 1999)). This suggested
that the Stat5a transcription factor promotes mammary tumor formation.
Likewise, a positive role for Stat5 in mammary carcinogenesis indirectly
has been indicated by the reduced mammary tumor formation in mice
lacking the gene for prolactin, a major activator of Stat5 in mammary
epithelial cells (Vomachka, Pratt et al. 2000), as well as the observation
that circulating prolactin levels correlated with increased risk
of breast cancer in post-menopausal women (Hankinson, Willett et
al. 1999). Furthermore, the notion of a tumorigenic role of Stat5
in the mammary gland (Humphreys and Hennighausen 1999; Humphreys
and Hennighausen 2000) would be consistent with the prevailing view
of a tumor-promoting role of Stat5 in hematopoietic cancer (lymphomas,
leukemias) (Wellbrock, Geissinger et al. 1998; Bromberg and Darnell
2000).
Alternatively, it could be argued that Stat5 activation may suppress
breast tumor formation by acting as a growth-inhibitory differentiation
factor for mammary epithelial cells. Likewise, Stat5 regulates normal
differentiation of ovaries and prostate (Teglund, McKay et al. 1998;
Moriggl, Topham et al. 1999; Nevalainen, Ahonen et al. 2000). However,
there is currently no direct evidence available demonstrating a
role of Stat5 as a tumor suppressor in either breast or other tissues.
Therefore, the present invention and description of activated Stat5
as a marker of good prognosis in node-negative human breast cancer
was unexpected based on the published literature and prevailing
views within the scientific field. As such, the role of Stat5 in
human breast cancer development and progression had not been established,
and its use as a marker of biologic behavior of human breast tumors
had not been reported.
SUMMARY OF THE INVENTION
It has been discovered by the inventors that activated Stat5 within
human primary breast tumors correlates with reduced risk of death
from breast cancer and reduced risk of metastatic disease. This
correlation was particularly strong for node-negative breast cancer.
Such a correlation was not known or even suspected prior to the
inventors' discovery. Therefore, nuclear, activated Stat5 is a new
and novel prognostic marker of breast cancer.
The presence of nuclear, activated Stat5 in human breast cancer
indicates a higher degree of differentiation of the tumor and is
also associated with an increased survival rate within this patient
population. Activated Stat5 levels in the primary tumor of node
negative breast cancer patients is a strong positive prognostic
factor independent of other known prognostic markers, as evidenced
by multivariate Cox regression analysis.
Levels of activated, nuclear Stat5 may be analyzed with antibodies
or other binding probes. Thus, the analysis of activated Stat5 adds
a new level of information to current breast cancer markers and
is a reliable prognostic molecular/biochemical marker of cancer
in samples from untreated breast cancer patients. Additionally,
monitoring levels of activated Stat5 should be predictive of the
outcome of Stat5-targeted therapeutic strategies.
The invention involves the use of activated Stat5 as a tumor marker
that predicts the risk/prognosis and biological behavior of breast
cancer. The inventors describe a straight-forward method to determine
Stat5 activation status in histological tissue sections of tumors
by immunohistochemistry. In this regard, analysis of nuclear, activated
Stat5 levels, by univariate Cox regression analysis can also be
used as a predictive measure of a positive outcome of antiestrogen
treatment. Furthermore, it may also be predictive of the success
of other treatments where alterations in Stat5 activation levels
are involved, as well as predictive of success of breast conserving
surgery and radical mastectomy.
The invention is first directed to a diagnostic or monitoring method
comprising: a) obtaining a sample of tissue from an individual in
need of diagnosis or monitoring for cancer; b) detecting levels
of activated Stat5 antigen in said sample; c) scoring said sample
for activated Stat5 levels; and d) comparing said scoring to that
obtained from a control tissue sample to determine the prognosis
associated with said cancer. Cancers that may be diagnosed or monitored
include but are not limited to breast cancer, ovarian cancer, endometrial
cancer, thyroid cancer, prostate cancer, colorectal cancer, hematopoietic
cancer, and skin cancer.
The invention is further directed to a diagnostic or monitoring
method comprising: a) obtaining a sample of breast tissue from an
individual in need of diagnosis or monitoring for breast cancer;
b) detecting levels of activated Stat5 antigen in said sample; c)
scoring said sample for activated Stat5 levels; and d) comparing
said scoring to that obtained from a control breast sample to determine
the prognosis associated with said breast cancer. Preferably, the
cancer is a node negative breast cancer.
The invention is also directed to a diagnostic or monitoring method
comprising: a) obtaining a sample from an individual in need of
diagnosis or monitoring for breast cancer; b) contacting said sample
with an antibody or binding probe that detects activated Stat5;
c) detecting or measuring the level of activated Stat5; and d) comparing
the level of activated Stat5 to that obtained from a control breast
sample.
In all aspects of the invention a preferable embodiment involves
contacting the sample of interest with an antibody to tyrosine-phosphorylated
Stat5. Preferably the detecting is done on histological or tissue
sections or cytological preparations by immunohistochemistry or
immunocytochemistry. Additionally, detecting of activated Stat5
in the methods of the invention may be done by immunoblotting or
by Fluorescence-Activated Cell Sorting (FACS).
The invention is also directed to a method for screening compounds
comprising: a) obtaining compounds to be screened for use in breast
cancer therapy; b) contacting a cell or tissue sample with said
compound; and c) determining the effect of said compound on the
level of Stat5 activation in said cell or tissue sample relative
to a control sample. Preferably the cell or tissue sample is cells
or tissue from a breast cancer. Additionally, in the method of the
invention the effect of said compound may be determined by the binding
of an antibody to activated Stat5 to said sample relative to control
cells or tissue. Also preferably in the method said activated Stat5
is tyrosine-phosphorylated Stat5 found in the cell nucleus.
The invention is further directed to a method for screening compounds
comprising: a) obtaining compounds to be screened for altering Stat5
activation levels, b) contacting a cell or tissue of interest with
said compounds, c) determining the effect of said compound on the
level of activated Stat5 in said cell or tissue sample relative
to a control sample.
The invention is also directed to a method for screening compounds
comprising: a) obtaining compounds to be screened for use in cancer
therapy; b) contacting a cell or tissue sample with said compound;
and c) determining the effect of said compound on the level of Stat5
activation in said cell or tissue sample. Preferably the cell or
tissue sample is from a human cancer. The effect of said compound
may be determined by the binding of an antibody to activated Stat5
to said sample relative to control cells or tissue. Preferably said
activated Stat5 is tyrosine-phosphorylated Stat5 found in the cell
nucleus.
The invention is also directed to a method for screening compounds
comprising: a) obtaining compounds to be screened for their ability
to positively or negatively affect Stat5 activation; b) contacting
a relevant cell or tissue sample with said compound; and c) determining
the effect of said compound on the level of Stat5 activation in
said cell or tissue sample. Preferably the effect of said compound
may be determined by the binding of an antibody to activated Stat5
to said sample relative to control cells or tissue. Also preferably
said activated Stat5 is tyrosine-phosphorylated Stat5 found in the
cell nucleus.
The invention is also directed to a method for determining the
effect of antiestrogen treatment comprising: a) obtaining a cell
or tissue sample from an individual in need of antiestrogen treatment,
b) measuring the levels of activated Stat5 in said cell or tissue
sample; and c) comparing said levels to that of a control breast
cancer sample to predict the responsiveness to antiestrogen treatment.
The invention is also directed to a method for determining the
efficacy of breast conserving surgery (lumpectomy) for treatment
of node-negative breast cancer comprising: a) obtaining a cell or
tissue sample from an individual in need of breast conserving surgery,
b) measuring the levels of activated Stat5 in said cell or tissue
sample; and c) comparing said levels to that of a control breast
cancer sample to predict the responsiveness of said breast cancer
to breast conserving surgery.
The invention is further directed to a kit for determining the
level of activated Stat5 in a mammalian biological sample, wherein
said activated Stat5 is an indicator of the prognosis of breast
cancer, said kit comprising: a) an antibody or binding probe to
activated Stat5, b) a reagent useful for detecting the extent of
interaction between said antibody or binding probe and activated
Stat5; c) a reagent or solution useful for antigen retrieval; and
c) positive and/or negative control samples. The kit of invention
may include a monoclonal or polyclonal antibody as the antibody.
This antibody may be directly linked to an indicator reagent, wherein
said indicator reagent is selected from the group consisting of
fluorescent, colorimetric, immunoperoxidase and isotopic reagents.
Alternatively, the kit may further include a second indicator antibody
linked to an indicator reagent, wherein said indicator reagent is
selected from the group consisting of fluorescent, calorimetric,
immunoperoxidase and isotopic reagents.
The invention is further directed to a method for diagnosing a
pathological condition or a susceptibility to a pathological condition
comprising: a) obtaining a sample from an individual in need of
diagnosis for a pathological condition related to activity of Stat5,
b) determining the amount or presence of activated Stat5 in said
sample; and c) diagnosing said pathological condition or a susceptibility
to said pathological condition based on the presence or amount of
activated Stat5 relative to a control sample.
Any of the methods of the claimed invention may use either univariate
or multivariate Cox regression analysis or Kaplan-Meyer survival
analysis with log-rank statistics for analyzing the obtained results
or may analyze a sample in a tissue section, isolated cell, or isolated
nuclei (smears, cytological sample or flow cytometry.) The methods
of the invention may further comprise analyzing the levels of activated
Stat5 in conjunction with additional breast cancer markers.
The invention is further directed to a diagnostic or monitoring
method comprising: a) obtaining a sample of breast tissue from an
individual in need of diagnosis or monitoring for breast cancer;
b) treating said sample in a microwave oven or by other forms of
heat based antigen retrieval methods; c) detecting levels of activated
Stat5 antigen in said sample; d) scoring said samples for activated
Stat5 levels; and e) comparing said scoring to that obtained from
a control breast sample to determine the prognosis associated with
said breast cancer. In addition to heat based antigen retrieval
methods, other methods known in the art for antigen retrieval may
also be used.
The invention is further directed to a diagnostic or monitoring
method comprising: a) obtaining a sample of breast tissue from an
individual in need of diagnosis or monitoring for breast cancer,
b) treating said sample with an antigen-retrieval buffer, c) detecting
levels of activated Stat5 antigen in said sample; d) scoring said
samples for activated Stat5 levels; and e) comparing said scoring
to that obtained from a control breast sample to determine the prognosis
associated with said breast cancer. Preferably said antigen retrieval
solution is an aqueous buffer about pH 7 to about pH 10, such as
for example Phosphate Buffered Saline at pH 7.4. Most preferably,
the antigen retrieval solution is about 1 mM Tris having about a
pH 10.
The invention is further directed to a method for predicting disease-free
survival and overall survival in patients with node-negative breast
cancer comprising: a) obtaining a sample of breast cancer tissue
from an individual with node-negative breast cancer; b) detecting
levels of activated Stat5 antigen in breast cancer cells or breast
cancer tissue of said sample; c) scoring said samples for activated
Stat5 levels; and d) comparing said scoring to that obtained from
a control breast sample to determine likelihood of disease-free
survival and overall survival associated with said breast cancer.
Any of the methods of the invention may score the analysis by using
a scale of 0 to 4, where 0 is negative (no detectable activated
Stat5 in cell nuclei), and 4 is high intensity staining in the majority
of cell nuclei and wherein a score of 1 to 4 (i.e. a positive score)
indicates a better prognosis for disease free and overall survival
in patients with node-negative breast cancer.
The invention is also directed to a method for predicting disease-free
survival and overall survival in patients who have not received
adjuvant hormone or chemotherapy comprising: a) obtaining a sample
of breast tissue from an individual with breast cancer who has not
received adjuvant hormone or chemotherapy, b) detecting levels of
activated Stat5 antigen in breast cells or breast tissue of said
sample, c) scoring said sample for activated Stat5 levels; and d)
comparing said scoring to that obtained from a control breast sample
to determine the likelihood of disease-free survival and overall
survival associated with said breast cancer.
The invention is further directed to a method for treating breast
cancer comprising: a) obtaining a sample of breast tissue from a
patient in need of treatment of breast cancer, b) determining the
level of activated Stat5 in said breast tissue sample, c) treating
said patient with a therapeutic regime known to improve the prognosis
for breast cancer, d) repeating steps "a" and "b",
e) adjusting the therapeutic regime based on the determination of
the activated Stat5 levels and f) repeating steps a-e as frequently
as deemed appropriate.
The invention is further directed to a method for screening for
metastatic potential of breast tumors comprising: a) obtaining a
sample of breast tissue from an individual in need of screening
for metastatic potential of a breast tumor, b) reacting an antibody
to activated Stat5 with tumor tissue from said patient, c) detecting
the extent of binding of said antibody to said tissue and d) correlating
the extent of binding of said antibody with its metastatic potential
Preferably, the tumor is a node-negative breast cancer.
The invention is further directed to a method for screening for
metastatic potential of solid tumors comprising: a) obtaining a
sample of tumor tissue from an individual in need of screening for
metastatic potential of a solid tumor; b) reacting an antibody to
activated Stat5 with tumor tissue from said patient; c) detecting
the extent of binding of said antibody to said tissue and d) correlating
the extent of binding of said antibody with its metastatic potential.
Preferably, the tumor is a node-negative cancer arising from the
ovary, large bowel (colorectal cancer), uterus (endometrial cancer),
thyroid gland, prostate, or skin.
Any of the methods of the invention involving analysis of the levels
of activated Stat5 may be used in conjunction with additional breast
cancer markers readily known to those of skill in the art.
The invention is further directed to a monoclonal antibody, wherein
said antibody a) is generated against the phosphopeptide KAVDG(phospho
Y)VKPOIK (SEQ ID NO: 1); b) specifically recognizes tyrosine phosphorylated
isoforms of Stat5, but not unphosphorylated isoforms; c) does not
recognize Stat5 mutants in which the Tyr694 residue has been substituted
with phenylalanine; and d) recognizes phosphorylated Stat5 following
an antigen retrieval treatment that does not use a protease.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1--Cutpoint analysis of scores quantifying levels of activated
Stat5. The effect of various levels of activated Stat5 on overall
survival of breast cancer patients is shown. Patients with primary
tumors with no detectable level of activated Stat5 (Score 0) has
significantly lower rates of overall survival than patients with
detectable levels of activated Stat5 in their tumors (Scores 1-4).
However, no significant difference was noted between scores 1-4
in terms of overall survival rate. Scores 1-4 were therefore recoded
into a single categorical parameter ("Positive Stat5 activation
status"), whereas score 0 was given the designation "negative
Stat5 activation status".
FIGS. 2A-2B--Detection of activated Stat5 by AX1 antibody. Immunocytochemistry
(FIG. 2A) and immunoblotting (FIG. 2B) of activated Stat5 in T47D
human breast cancer cells before and after prolactin stimulation.
FIG. 3--Antibody AXI specifically detects tyrosine phophorylated
Stat5 by immunoblotting. Immunoblotting of activated Stat5 in COS-7
kidney cells transfected with either wild type Sta5 or a tyrosine
phosphorylation-defective mutant, Stat5-Y694F.
FIG. 4--Antibody AX1 specifically detects tyrosine phophorylated
Stat5 by immunocytochemistry. Immunocytochemical detection of activated
Stat5 in COS-7 kidney cells transfected with either wild type Stat5
or a tyrosine phosphorylation-defective mutant, Stat5-Y694F.
FIG. 5--Immunohistochemical detection of levels of activated Stat5
in normal and malignant human breast tissues using antibody AX1.
Immunohistochemical detection of activated Stat5 in formalin-fixed,
paraffin-embedded normal and malignant human breast tissues.
FIG. 6--Actuarial curves. Kaplan-Meyer actuarial curves for overall
survival in breast cancer patients with Stat5 positive versus Stat5
negative tumors
FIG. 7--Survival Function. Kaplan-Meyer actuarial curves for recurrence
of metastatic disease (relapse) in node negative breast cancer patients
who had undergone lumpectomy (breast conserving surgery) with Stat5
positive versus Stat5 negative tumors. Note that positive Stat5
activation status is associated with no relapse, indicating that
lumpectomy is a safe procedure for this group of patients.
FIG. 8--Survival function for Stat5 activation status. Cox regression
curves for overall survival in breast cancer patients with either
positive or negative Stat5 activation status of the primary tumor.
Note that positive Stat5 activation status predicts improved response
to antiestrogen therapy on this overall material of node-positive
and node-negative breast cancer material.
FIG. 9--Immunohistochemical analysis of activated Stat5 with Ax1
anti-phosphoTyr-Stat5 antibody and a general antibody to Stat5.
This figure documents that at least in post-lactational mouse mammary
glands (involution), when Stat5 is known to be turned off, AX1 does
not detect activated Stat5 in cell nuclei (left panel), whereas
a general Stat5 antibody detect significant levels (right panel).
Thus, a general Stat5 antibody will not accurately detect levels
of active Stat5 even if Stat5 is located in the cell nucleus.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
In order to provide a clearer understanding of the specification
and claims the following definitions are provided.
The terms a and an should be understood to refer to at least "one"
item, but are not limited to reference to only "one",
unless such is specifically indicated. Thus, for example, reference
to "a" cell refers to one or more cells.
Activated Stat5--Activated Stat5 is Stat5 that is detected in the
cell nucleus in the tyrosine phosphorylated form. Tyrosine phosphorylated
Stat5 has been reported to exist in a dimeric structural configuration
that is capable of binding to specific DNA sequences. Activated
Stat5 may include Stat5a and Stat5b isoforms, gene products, and/or
posttranslationally modified variants thereof, such as for example
proteolytically truncated forms (Wang, Stravopodis et al. 1996;
Azam, Lee et al. 1997; Kirken, Malabarba et al. 1997; Meyer, Jucker
et al. 1998; Grimley, Dong et al. 1999; Lee, Piazza et al. 1999;
Piazza, Valens et al. 2000).
Stat5 is activated by two distinct, sequential events. The initial
activation switch of Stat5 involves phosphorylation of a tyrosine
residue within a conserved C-terminal segment that causes dimerization
of Stat5 molecules (Gouilleux, Wakao et al. 1994). A second coordinated
activation event involves translocation of dimerized Stat5 from
the cytoplasm into the cell nucleus, which permits Stat5 to bind
to gene regulatory promoter elements and regulate transcription
of specific genes (Gouilleux, Wakao et al. 1994; Kazansky, Kabotyanski
et al. 1999). Because Stat5 not only requires phosphorylation of
a specific tyrosine residue, but also needs to translocate into
the cell nucleus in order to function as an active DNA-binding transcription
factor, amounts of tyrosine phosphorylated Stat5 located within
the cell nucleus will more accurately reflect the levels of activated
Stat5 than overall cellular levels of tyrosine phosphorylated Stat5.
For instance, aberrant tyrosine phosphorylation of Stat5a by hyperactive
Src tyrosine kinase has been shown not to be accompanied by nuclear
translocation (Kazansky, Kabotyanski et al. 1999). This observation
illustrates that quantitation of tyrosine phosphorylation status
alone without assessing nuclear localization is not sufficient for
accurate determination of levels of activated Stat5. Correspondingly,
Stat transcription factors may become dephosphorylated within the
cell nucleus and loose the ability to bind to DNA (Haspel and Darnell
1999), making assays that detect nuclear Stat5 protein levels alone
also not sufficient for determining levels of activated Stat5. The
definition of activated Stat5 therefore refers to both nuclear localization
and tyrosine phosphorylation.
Alternatively, antibodies or binding reagents that specifically
detect Stat5 in its active, dimerized (structural) configuration
may also be used to detect Stat5 that has become phosphorylated
and translocated to the cell nucleus. For description of such conformation-specific
antibody-derivatives that may not bind directly to a phosphorylation
site but still detect the active form of other effector molecules,
including cellular oncogenes Ras and receptors for epidermal growth
factor and platelet-deriverd growth factor, see (Panneerselvam,
Reitz et al. 1995; Bishayee, Beguinot et al. 1999; Horn, Wittinghofer
et al. 1999). Detection of activated, nuclear Stat5 is used to predict
the biological behavior of breast tumors and may also be useful
for diagnosing or monitoring other pathological conditions involving
changes in the activation state and expression levels of Stat5,
including other forms of cancer.
Antibody--An "antibody" (interchangeably used in plural
form) is an immunoglobulin molecule capable of specific binding
to a target, such as a polypeptide, through at least one antigen
recognition site. As used herein, the term encompasses not only
intact antibodies, but also fragments thereof, mutants thereof,
fusion proteins, humanized antibodies, and any other modified configuration
of the immunoglobulin molecule that comprises an antigen recognition
site of the required specificity. An antibody against activated
Stat5 is used in the methods of the invention.
Antigen--The term "antigen" refers to the target molecule
that is specifically bound by an antibody through its antigen recognition
site, such as for example, the activated Stat5 antigen. The antigen
may, but need not be chemically related to the immunogen that stimulated
production of the antibody. The antigen may be polyvalent, or it
may be a monovalent hapten. Examples of different kinds of antigens
that can be recognized by antibodies include polypeptides, polynucleotides,
other antibody molecules, oligosaccharides, complex lipids, drugs,
and chemicals. An "immunogen" is an antigen capable of
stimulating production of an antibody when injected into a suitable
host, usually a mammal.
Compounds may be rendered immunogenic by many techniques known
in the art, including crosslinking or conjugating with a carrier
to increase valency, mixing with a mitogen to increase the immune
response, and combining with an adjuvant to enhance presentation.
Antigen Retrieval Reagent--An "antigen retrieval reagent"
facilitates and/or allows binding of immunostaining reagents with
epitopes masked by formalin-fixation, by natural binding moieties,
or by structural constraints such as protein folding, or any combination
of these factors. Antigen retrieval reagents can be used alone or
in combination with other physical or physicochemical procedures
such as heating or microwave treatment (Boon and Kok 1994; Fresno,
Molina et al. 1997; Shi, Cote et al. 1997; Brown 1998; McNicol and
Richmond 1998; Mighell, Hume et al. 1998; Krenacs, Krenacs et al.
1999).
Such a reagent expands the range of antibodies useful in immunohistochemistry
as well as reduces the incidence of false negative staining in over-fixed
tissues. Methods of antigen retrieval are known in the art such
as described, for example, in U.S. Pat. Nos. 5,244,787 and 5,578,452
and in (Boon and Kok 1994; Fresno, Molina et al. 1997; Shi, Cote
et al. 1997; Brown 1998; McNicol and Richmond 1998; Mighell, Hume
et al. 1998; Krenacs, Krenacs et al. 1999).
Any embodiement of the invention may use an antigen-retrieval buffer
of about 1 mM Tris at a pH of about 10.
Binding Probe--Binding probes are not antibody-based (immunoglobulin
based) but still bind with high specificity and affinity to an antigen
or antigenic site. For instance, following routine molecular engineering
methods such as those set forth in (Ausubel 1988; Sambrook, Maniatis
et al. 1989), those skilled in the art may develop a binding probe
containing the Stat5 SH2 (src-homology-2) domain, which is known
to bind with high affinity and specificity to the tyrosine-phosphorylated
Stat5 molecule (Liu and Roth 1995; Igarashi, Shigeta et al. 1998;
Ariyoshi, Nosaka et al. 2000). Such a binding probe could contain
one (monovalent) or several (mulitivalent) Stat5 SH2 domains. This
binding probe could be engineered or chemically modified to contain
detection label, which could consist of isotope, fluorescence, enzyme
or one or more antigenic sites or "tags" to be recognized
by secondary antibodies, which in turn may have similar detection
labels attached. Thus a non-antibody based binding probe could be
generated that is able to specifically detect activated Stat5 that
is tyrosine phosphorylated and present in the cell nucleus.
Rational genetic engineering, random mutagenesis, or targeted molecular
evolution in vitro may lead to Stat5-SH2 domains with improved binding
characteristics (Ariyoshi, Nosaka et al. 2000). Alternatively, peptide-based
binding probes may be generated from scratch by selection of random
chemical or genetic libraries for interaction with Stat5 in its
activated, dimeric conformation, for example by binding to tyrosine
phosphorylated Stat5. General approaches to selection of these types
of binding probes have been described by numerous authors (Kelly,
Liang et al. 1996; Dente, Vetriani et al. 1997; Gram, Schmitz et
al. 1997; Doi and Yanagawa 1998; Pellegrini, Liang et al. 1998;
Doi and Yanagawa 1999; Gram 1999; Cochrane, Webster et al. 2000;
Illgen, Enderle et al. 2000; Messmer, Benham et al. 2000; Zhang,
Zhu et al. 2000).
Cancer Cell--The terms "cancerous cell" or "cancer
cell", used either in the singular or plural form, refer to
cells that have undergone a malignant transformation that makes
them pathological to the host organism. Malignant transformation
is a single- or multi-step process, which involves in part an alteration
in the genetic makeup of the cell and/or the gene expression profile.
Malignant transformation may occur either spontaneously, or via
an event or combination of events such as drug or chemical treatment,
radiation, fusion with other cells, viral infection, or activation
or inactivation of particular genes. Malignant transformation may
occur in vivo or in vitro, and can if necessary be experimentally
induced. Malignant cells may be found within the well-defined tumor
mass or may have metastasized to other physical locations.
A feature of cancer cells is the tendency to grow in a manner that
is uncontrollable by the host, but the pathology associated with
a particular cancer cell may take any form. Primary cancer cells
(that is, cells obtained from near the site of malignant transformation)
can be readily distinguished from non-cancerous cells by well-established
pathology techniques, particularly histological examination. The
definition of a cancer cell, as used herein, includes not only a
primary cancer cell, but any cell derived from a cancer cell ancestor.
This includes metastasized cancer cells, and in vitro cultures and
cell lines derived from cancer cells.
Cell line--A "cell line" or "cell culture"
denotes higher eukaryotic cells grown or maintained in vitro. It
is understood that the descendants of a cell may not be completely
identical (either morphologically, genotypically, or phenotypically)
to the parent cell. Cells described as "uncultured" are
obtained directly from a living organism, and have been maintained
for a limited amount of time away from the organism: not long enough
or under conditions for the cells to undergo substantial replication.
Clinical Sample--It is understood that a "clinical sample"
encompasses a variety of sample types obtained from a subject and
useful in the procedure of the invention, such as for example, a
diagnostic or monitoring test of activated Stat5 levels. The definition
encompasses solid tissue samples obtained by surgical removal, a
pathology specimen, an archived sample, or a biopsy specimen, tissue
cultures or cells derived therefrom and the progeny thereof, and
sections or smears prepared from any of these sources. Non-limiting
examples are samples obtained from breast tissue, lymph nodes, and
breast tumors. The definition also encompasses blood, bone marrow,
spinal fluid, and other liquid samples of biologic origin, and may
refer to either the cells or cell fragments suspended therein, or
to the liquid medium and its solutes.
Control Sample--A control sample is a source of cells or tissue
for comparison purposes. A control sample may include, inter alia,
cancer-free breast or mammary tissue or an archived pathology sample
containing activated Stat5 at various levels for use as positive
control, and breast tumor tissue or other tissue showing no Stat5
activation as negative control samples.
Diagnostic Method--A "diagnostic method" may include,
but is not limited to determining the metastatic potential of a
tumor or determining a patient's prognosis following discovery of
a breast tumor. Such diagnostic methods may also be used for determining
the effectiveness of a therapeutic regime used to treat cancer or
other disease involving the presence of activated Stat5. An example
of such a therapeutic treatment is antiestrogen treatment for breast
cancer. The terms "diagnostic method" or "monitoring
method" are often used interchangeably.
Differential Result--A "differential" result is generally
obtained from an assay in which a comparison is made between the
findings of two different assay samples, such as a cancerous cell
line and a control cell line or a cancerous tissue and a control
tissue. Thus, for example, "differential levels" of a
marker protein, such as Stat5 are observed when the level of Stat5
is higher in one tissue sample than another.
Disease-Free Survival--"Disease-free survival" should
be understood to mean living free of the disease being monitored.
For example, if activated Stat5 is used to diagnose or monitor breast
cancer, disease-free survival would mean free from detectable breast
cancer.
Metastatic Potential--Metastasis refers to the condition of spread
of cancer from the organ of origin to additional sites in the patients.
Therefore, "metastatic potential" as it relates to for
example, breast cancer may be considered to be the risk of progression
of primary node-negative cancer from localized disease to disseminated,
metastatic disease.
Monitoring Method--A "monitoring method" may include,
but is not limited to, following a patient's progress or response
to a therapeutic regime after discovery of a breast tumor. Such
monitoring methods may also be used for determining the effectiveness
of a therapeutic regime used to treat cancer or other diseases involving
the presence of activated Stat5. An example of such a therapeutic
treatment is antiestrogen treatment for breast cancer. Antibodies
to activated Stat5 are used in monitoring methods of this invention.
The terms "diagnostic method" or "monitoring method"
are often used interchangeably.
Node Negative Breast Cancer--"Node negative breast cancer"
is breast cancer that is localized to the breast without detectable
metastasis to nearby lymph nodes, thereby indicating a low risk
for recurrence of the cancer after surgery of the primary tumor.
Pathology--The "pathology" caused by cancer cells within
a host is anything that compromises the well-being or normal physiology
of the host. This may involve, but is not limited to abnormal or
uncontrollable growth of the cancer cell, metastasis, release of
cytokines or other secretory products at an inappropriate level,
manifestation of a function inappropriate for its physiological
milieu, interference with the normal function of neighboring cells,
aggravation or suppression of an inflammatory or immunological response,
or the harboring of undesirable chemical agents or invasive organisms.
Pharmaceutical Candidate--A "pharmaceutical candidate"
or "drug candidate" is a compound believed to have therapeutic
potential, that is to be tested for efficacy against a specific
condition, such as for example a condition having altered activated
Stat5 levels (such as breast cancer). The "screening"
of a pharmaceutical candidate refers to conducting an assay that
is capable of evaluating the efficacy and/or specificity of the
candidate. In this context, "efficacy" refers to the ability
of the candidate to affect Stat5 activation levels and/or affect
the cell or organism it is administered to in a beneficial way:
for example, the limitation of the pathology of cancerous cells.
Prognosis--"Prognosis" as used in this application means
the likelihood of recovery from a disease or the prediction of the
probable development or outcome of a disease. For example, if a
sample from a patient with breast cancer is positive for nuclear
staining with an antibody to activated Stat5, then the "prognosis"
for that patient is better than if the sample was negative for activated
Stat5 staining. Samples may be scored for activated Stat5 levels
on a scale from 0-4 for levels of antibody staining, where 0 is
negative and 1-4 represents positive staining at four semiquantitative
steps of increasing intensity. Scores 1-4 can be recoded as positive
because each positive score was associated with significantly reduced
risk for relapse and fatal disease when compared to score 0 (negative),
but increasing intensity among the positive scores did not provide
additional risk reduction. Cox semiparametric proportional hazard
analysis can be used to estimate the prognostic value of activated
Stat5. Cutpoint analysis has shown that scores 1-4 differ significantly
from 0 in terms of predicting overall survival among node-negative
breast cancer patients, but did not differ significantly from each
other (See FIG. 1). Additional refinement of the quantification
procedure may reveal a better quantitative relationship with the
prognosis. The term positive or negative "Stat5 activation
status" of tumors used in this description refers to scores
0 or scores 1-4, respectively.
The prognosis of a patient with breast cancer may be based, inter
alia, at least in part on the metastatic potential of the breast
cancer and a relationship to activated Stat5 levels. This description
is not meant to limit the basis for the determination of a patient's
prognosis, because those of skill in the art would be aware of other
related bases for determination of the prognosis.
Relative Amount--The term "relative amount" is used where
a comparison is made between a test measurement and a control measurement.
Thus, the relative amount of a reagent forming a complex in a reaction
is the amount reacting with a test specimen, compared with the amount
reacting with a control specimen. The control specimen may be run
separately in the same assay, or it may be part of the same sample
(for example, normal tissue surrounding a malignant area in a tissue
section).
Scoring--A sample may be "scored" during the diagnosis
or monitoring of breast cancer. In its simplest form, scoring may
be categorical negative or positive as judged by visual examination
of samples by immunohistochemistry. More quantitative scoring involves
judging the two parameters intensity of staining and the proportion
of stained ("positive") cells that are sampled. Based
on these two parameters numbers may be assigned that reflect increasing
levels of positive staining. Allred et al (Allred, Harvey et al.
1998) have described one way of achieving this, which involved scoring
both parameters on a scale from 0 (negative) to 4, and summarizing
the scores of the individual parameters to an overall score. This
results in a scale with possible scores of 0, 2, 3, 4, 5, 6, 7 or
8. (Note that a score of 1 is not possible on Allred's scale). A
somewhat simpler scoring method integrates the intensity of nuclear
staining and the proportion of cells that display stained nuclei
into a combined scale from 0 to 4. In practice, the scores 7 and
8 of Allred's scale correspond to 4 on the simplified scale. In
the same way, scores 5 and 6 correspond to 3, scores 3 and 4 to
score 2, score 2 corresponds to 1, and, 0 corresponds to 0 on both
scales. Either scoring method may be applied to scoring intensity
and proportion of staining of activated Stat5 in the cell nuclei.
The terms positive or negative "Stat5 activation status"
of tumors used in the present description refers to levels of activated
Stat5 that correspond to scores 0 or 1-4 on the simplified scale,
respectively.
Treatment--"Treatment" of an individual or a cell is
any type of intervention in an attempt to alter the non-treated
course of the individual or cell. For example, treatment of an individual
may be undertaken to decrease or limit the pathology caused by a
cancer harbored in the individual. Treatment includes but is not
limited to a) administration of a composition, such as a pharmaceutical
composition, b) administration of a surgical procedure (such as
lumpectomy or modified radical mastectomy), or c) administration
of radiation therapy, and may be performed either prophylactically,
subsequent to the initiation of a pathologic event or contact with
an etiologic agent.
Tyrosine-phosphorylated Stat5--"Tyrosine-phosphorylated Stat5"
refers to Stat5a phosphorylated on amino acid residue Tyr694 or
Stat5b phosphorylated on the homologous amino acid residue Tyr699.
This tyrosine phosphorylation causes the Stat5 molecules to dimerize,
and is critical for the ability of Stat5 to bind to DNA. Tyrosine
phosphorylated Stat5 is therefore equated with activated Stat5,
although only tyrosine phosphorylated Stat5 that is found in the
nucleus may strictly reflect properly activated Stat5. Tyrosine
phosphorylated Stat5 that remains located in the cytoplasm is not
functionally activated in the sense that it remains unable to interact
with DNA in the cell nucleus and regulate gene transcription.
Diagnostic Antibodies
The present invention relates to the use of antibodies against
activated Stat5, antibody fragments against activated Stat5 and
Stat5 binding probes. Examples of binding probes that may be used
to detect activated Stat5 that are not antibody or immunoglobulin
based include proteins derived from the phosphotyrosyl-binding SH2
(src-homology-2) domain of Stat5 (Wakao, Gouilleux et al. 1994;
Ariyoshi, Nosaka et al. 2000), or binding proteins that have been
selected for their ability to bind to activated Stat5 by using screening
methods for large chemical or molecular libraries similar to those
described in the literature (Kelly, Liang et al. 1996; Dente, Vetriani
et al. 1997; Gram, Schmitz et al. 1997; Igarashi, Shigeta et al.
1998; Cochrane, Webster et al. 2000). The principles for development
of such binding reagents have been described in detail for other
binding probes, and provide means for those skilled in the art to
use similar approach to develop probes that bind to activated Stat5.
Further elaboration is now provided for terms related to diagnostic
antibodies and assays.
"Fragment" is defined as at least a portion of the variable
regions of the immunoglobulin molecule which binds to its target,
i.e. the antigen binding region. Some of the constant region of
the immunoglobulin may be included.
"Antigen-binding region" means that part of the antibody,
the fusion protein, or the immunoconjugate of the invention which
recognizes the target or portions thereof
"Directly" means the use of antibodies coupled to a label.
The specimen is incubated with the labeled antibody, unbound antibody
is removed by washing, and the specimen may be examined.
"Indirectly" means incubating the specimen with an unconjugated
antibody, washing and incubating with a marker-conjugated antibody.
The marker may be a fluorochrom, enzyme, isotope, metal, etc. The
second or "sandwich" antibody thus reveals the presence
of the first.
The term "Stat5 antibody" as used herein includes whole,
intact polyclonal and monoclonal antibody materials, and chimeric
antibody molecules. The Stat5 antibody described above may include
any fragments thereof containing the active antigen-binding region
of the antibody such as Fab, F(ab')2 and Fv fragments, using techniques
well established in the art (see, e.g., (Rousseaux, Rousseaux-Prevost
et al. 1986)). The Stat5 antibody used in the invention also includes
fusion proteins.
In addition, the present invention encompasses use of antibodies
that are capable of binding to the same antigenic determinant as
the activated Stat5 antibodies and competing with the antibodies
for binding at that site. These include antibodies having the same
antigenic specificity as the Stat5 antibodies but differing in species
origin, isotype, binding affinity or biological functions (e.g.,
cytotoxicity). For example, class, isotype and other variants of
the antibodies of the invention having the antigen-binding region
of the Stat5 antibody can be constructed using recombinant class-switching
and fusion techniques known in the art (see, e.g., (Thammana and
Scharff 1983; Neuberger, Williams et al. 1984; Spira, Paizi et al.
1996).
One skilled in the art will appreciate that the invention also
encompasses the use of immunoglobulin fragments that retain recognition
of the antigen. Such immunoglobulin fragments may include, for example,
the Fab', F(ab')2, F(v) or Fab fragments, or other antigen recognizing
immunoglobulin fragments. Such immunoglobulin fragments can be prepared,
for example, by proteolytic enzyme digestion, using enzymes such
as pepsin or papain, reductive alkylation, or recombinant techniques.
The materials and methods for preparing such immunoglobulin fragments
are well-known to those skilled in the art. See generally, (Matthew
and Reichardt 1982; Parham, Androlewicz et al. 1982; Lamoyi and
Nisonoff 1983; Parham 1983).
An immunoglobulin can be a "chimeric antibody" as that
term is recognized in the art. Also, the immunoglobulin may be a
"bifunctional" or "hybrid" antibody, that is,
an antibody which may have one arm having a specificity for one
antigenic site, such as a tumor associated antigen while the other
arm recognizes a different target, for example, a hapten which is,
or to which is bound, an agent lethal to the antigen-bearing tumor
cell. Alternatively, the bifunctional antibody may be one in which
each arm has specificity for a different epitope of a tumor associated
antigen of the cell to be therapeutically or biologically modified.
In any case, the hybrid antibodies have a dual specificity, preferably
with one or more binding sites specific for the hapten of choice
or one or more binding sites specific for a target antigen, for
example, an antigen associated with a tumor, an infectious organism,
or other disease state.
Biological bifunctional antibodies are described, for example,
in European Patent Publication, EPA 0 105 360, to which those skilled
in the art are referred. Such hybrid or bifunctional antibodies
may be derived, as noted, either biologically, by cell fusion techniques,
or chemically, especially with cross-linking agents or disulfide
bridge-forming reagents, and may be comprised of whose antibodies
and/or fragments thereof Methods for obtaining such hybrid antibodies
are disclosed, for example, in PCT application WO83/03679 and published
European Application EPA 0 217 577. Particularly preferred bifunctional
antibodies are those biologically prepared from a "polydome"
or "quadroma" or which are synthetically prepared with
cross-linking agents such as bis-(maleimideo)-methyl ether ("BMME"),
or with other cross-linking agents familiar to those skilled in
the art.
In addition the immunoglobin may be a single chain antibody ("SCA")
These may consist of single chain Fv fragments ("scFv")
in which the variable light ("V(L)") and variable heavy
("V(H)") domains are linked by a peptide bridge or by
disulfide bonds. Also, the immunoglobulin may consist of single
V(H) domains (dAbs) which possess antigen-binding activity. See,
e.g., (Ward, Gussow et al. 1989; Glockshuber, Malia et al. 1990;
Winter and Milstein 1991).
As used herein, the term "chimeric antibody" refers to
a monoclonal antibody comprising a variable region, i.e. binding
region, from one source or species and at least a portion of a constant
region derived from a different source or species, usually prepared
by recombinant DNA techniques. Such murine/human chimeric antibodies
are the product of expressed immunoglobulin genes comprising DNA
segments encoding murine immunoglobulin variable regions and DNA
segments encoding human immunoglobulin constant regions. Other forms
of chimeric antibodies encompassed by the invention are those in
which the class or subclass has been modified or changed from that
of the original antibody. Such "chimeric" antibodies are
also referred to as "class-switched antibodies". Methods
for producing chimeric antibodies involve conventional recombinant
DNA and gene transfection techniques now well known in the art.
See, e.g., (Morrison, Johnson et al. 1984).
In addition, the invention encompasses within its scope use of
immunoglobulins (as defined above) or immunoglobulin fragments to
which are fused active proteins, for example, an enzyme of the type
disclosed in (Neuberger, Williams et al. 1984), PCT application,
WO86/01533. The disclosure of such products is incorporated herein
by reference.
Furthermore, as noted above, the immunoglobulin (antibody), or
fragment thereof, used in the present invention may be polyclonal
or monoclonal in nature. Monoclonal antibodies are the preferred
immunoglobulins, however. The preparation of such polyclonal or
monoclonal antibodies now is well known to those skilled in the
art who, of course, are fully capable of producing useful immunoglobulins
which can be used in the invention. See, e.g., (Kohler and Milstein
1975). In addition, monoclonal antibodies which are produced by
such hybridomas and which are useful, with the appropriate antigen
retrieval procedures, in the practice of the present invention are
publicly available from sources such as Advantex BioReagents LLP,
11950 White Oak Landing, Conroe, Tex. 77385, or Zymed, Inc, 458
Cartlon Court, South San Francisco, Calif. 94080.
Particularly preferred antibodies for use in the present invention
are monoclonal antibodies which recognize tyrosine phosphorylated,
activated Stat5.
Diagnostic Techniques
Diagnostic techniques involve the detection and quantitation of
antigens of patients thought to be suffering from carcinoma. Such
antigens can be detected using techniques known in the art such
as immunohistochemistry and immunocytochemistry wherein an antibody
reactive with the antigen is used to detect the presence of the
antigen in a tissue sample. These assays, using anti-active Stat5
antibodies can therefore be used for the detection in tissue of
the antigen with which the anti-active Stat5 antibodies react and
thus predict the metastatic potential of the tumor. Thus, it is
apparent from the foregoing that the Stat5 antibodies can be used
in most assays involving antigen-antibody reactions. These assays
include, but are not limited to, standard radioimmunoassays (RIA)
techniques, both liquid and solid phase, as well as enzyme-linked
immunosorbent assays (ELISA) assays, ELISPOT, immunofluorescence
techniques, and other immunocytochemical assays (see, e.g., (Sikora
and Smedley 1984)). Preferably, the assay is one which can be used
in situ such as in a biopsy sample to be diagnosed or pathological
archived material to directly detect levels of activated Stat5 within
the tumor cell nuclei.
The invention also encompasses diagnostic kits for carrying out
the assays described above. In one embodiment, the diagnostic kit
comprises at least anti-active Stat5 monoclonal antibody, fragments
thereof, fusion proteins or chimeric antibody of Stat5, or a non-antibody
based binding probe specific for activated Stat5, and a conjugate
comprising a specific binding partner for the Stat5 antibody or
binding probe and a label capable of producing a detectable signal.
The reagents can also include ancillary agents such as buffering
agents, antigen retrieval solutions and reagents, and protein stabilizing
agents (e.g., polysaccharides). The diagnostic kit can further comprise,
where necessary, other components of the signal-producing system
including agents for reducing background interference, control reagents
or an apparatus or container for conducting the test.
In another embodiment, the diagnostic kit comprises at least a
conjugate of the Stat5 antibodies and a label capable of producing
a detectable signal. Ancillary agents as mentioned above can also
be present.
Flow Cytometry (FACS Analysis)
Flow cytometry (FCM), an automated, laser optics-based technique,
is used to detect and quantify the levels of antigens or chemically
reactive substances in isolated cells or cell nuclei in suspension.
The uptake or binding of fluorescent molecules that diagnose changes
in nuclear DNA content as measured by staining of DNA with propidium
iodide; changes in cell size and granularity as measured by forward
light scatter and 90 degree side light scatter; down-regulation
of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins
or other antigens as measured by reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the
binding of fluorescein-conjugated Annexin V protein to the cell
surface. Methods in flow cytometry are discussed in (Ormerod 2000).
Flow cytometric quantitation in breast cancer cells obtained from
biopsies or fine needle aspirates provides relevant information,
and allows the characterization and quantitation of breast cancer
associated parameters, like over or under-expression or activation
as compared to normal counterparts, that is suitable for the diagnosis
of malignancy or for residual disease evaluation. It may improve
scoring systems for prognostic markers of breast tumors. It allows
to find original prognostic parameters and improves the comparison
of different series due to a better definition of positivity (more
quantitative).
Flow cytometry is now widely used for immunophenotyping purposes.
It allows, in addition to the determination of the percentage of
positive cells, to determine the intensity of fluorescent staining,
that can be converted into antigen density provided that reagents
are used under saturating concentrations and correct standards of
fluorescence are tested in parallel. The concept of antigen density
evaluation appears to improve the efficiency of immune techniques
in the monitoring of hemopoietic malignancies (Lavabre-Bertrand,
George et al. 1994).
Therapeutic Regimes for Treating Breast Cancer
Nearly all patients with breast cancer will have some type of surgery.
"Lumpectomy" removes only the breast lump and the surrounding
area, or margin, of normal tissue. In most cases, lumpectomy is
combined with 6 to 7 weeks of radiation therapy following surgery.
This combination of lumpectomy and radiation is often referred to
as "breast conserving" therapy. Alternatively, in a "modified
radical mastectomy", surgeons remove the entire breast and
some of the axillary (underarm) lymph nodes. Modified radical mastectomy
is the most common surgery for patients with breast cancer in whom
doctors remove the whole breast. Systemic therapies for breast cancer
includes adjuvant antiestrogen treatment and chemotherapy.
Breast conserving surgery--"Lumpectomy" removes only
the breast lump and the surrounding area, or margin, of normal tissue.
If cancer cells are present at the margin (the edge of the excisional
biopsy or lumpectomy specimen), a re-excision can usually be done
to remove the remaining cancer. In most cases, lumpectomy is combined
with 6 to 7 weeks of supplementary radiation therapy following surgery.
Mastectomy--In a "simple (total) mastectomy" procedure
surgeons remove the entire breast but do not remove any lymph nodes
from under the arm, or muscle tissue from beneath the breast. In
a "modified radical mastectomy", surgeons remove the entire
breast and some of the axillary (underarm) lymph nodes. Modified
radical mastectomy is the most common surgery for patients with
breast cancer in whom doctors remove the whole breast. "Radical
mastectomy" removes not only the entire breast, but axillary
lymph nodes, and the chest wall muscles under the breast as well.
The modified radical mastectomy has proved as effective as radical
mastectomy, which is nowadays rarely performed due to disfiguration
and frequent side-effects.
Lymph node surgery--Regardless of whether a breast cancer patient
has a mastectomy, or a lumpectomy for invasive cancer, the physicians
need to determine whether the cancer has spread. The regional lymph
nodes in the underarm drain lymph from the breast, and are typically
the first sites of spread. Furthermore, lymph node involvement increases
the likelihood that cancer cells have spread through the blood-stream
to other parts of the body.
While lymph node surgery itself does not improve the chance for
a cure, this is the only way to accurately determine if the cancer
has spread to the lymph nodes. This usually means removing some
or all of the lymph nodes in the armpit. Typically 10 to 20 lymph
nodes in the armpit are examined by an operation called "axillary
lymph node dissection". Although axillary lymph node dissection
is a safe procedure with low rates of serious side effects, efforts
are ongoing to develop new ways of detecting the spread of cancer
to lymph nodes that are less invasive and do not involve a full
lymph node dissection. Such alternative methods include the "sentinel
lymph node biopsy" (Orr, Hoehn et al. 1999; Sugg, Ferguson
et al. 2000), and new detection methods for breast cancer cells
in bone marrow and blood (Berois, Varangot et al. 2000; Braun, Pantel
et al. 2000; Fetsch, Cowan et al. 2000; Ikeda, Miyoshi et al. 2000;
Kraeft, Sutherland et al. 2000; Zhong, Kaul et al. 2000). It is
possible that these newer methods in the future may replace lymph
node dissection as a means of determining micrometastatic spread
of cancer.
Sentinel lymph node biopsy--In the sentinel lymph node biopsy procedure
the surgeon finds and removes the `sentinel node`--the first lymph
node into which a tumor drains, and therefore the one most likely
to contain cancer cells. In a sentinel lymph node biopsy the surgeon
injects a radioactive substance and/or a blue dye into the area
around the tumor. Lymphatic vessels carry these materials into the
sentinel node. The doctor can either see the blue dye or detect
the radioactivity with a geiger counter, and then cuts out the node
for examination. If the sentinel node contains cancer, the surgeon
will have to perform an axillary dissection-removal of more lymph
nodes in the axilla (armpit). If the sentinel node is cancer-free,
the patient and her physicians may consider avoiding more lymph
node surgery and the potential side effects. Although the sentinel
node procedure is relatively new and its long-terin effectiveness
is uncertain (Orr, Hoehn et al. 1999; Sugg, Ferguson et al. 2000),
it is possible that it will turn out to be equally as effective
in determining lymph node spread as the full lymph node dissection.
Detection of disseminated cancer cells in blood and bone marrow--Recent
methods for detecting metastatic breast cancer cells in blood (Berois,
Varangot et al. 2000; Fetsch, Cowan et al. 2000; Kraeft, Sutherland
et al. 2000) or in bone marrow (Braun, Pantel et al. 2000; Ikeda,
Miyoshi et al. 2000; Zhong, Kaul et al. 2000) are typically based
on the presence of breast cell-specific cytokeratin markers by immunodetection
or by genetic testing. These new methods may also lead to an alternative
approach to lymph node dissection for determining whether a breast
cancer has spread beyond the local tumor area.
Radiation therapy--Radiation is used to destroy cancer cells left
behind in the breast, chest wall, or lymph nodes after surgery.
Radiation treatments usually take place 5 days a week over a period
of 6 to 8 weeks. Side effects most likely to occur include swelling
and heaviness in the breast, sunburn-like skin changes in the treated
area, and fatigue. Changes to the breast tissue and skin usually
go away in 6 to 12 months. In some women, the breast becomes smaller
and firmer after radiation therapy. Radiation therapy of axillary
(armpit area) lymph nodes can also cause lymphedema. Although generally
safe, it is evident that radiation therapy comes at a considerable
expense and with potentially serious side-effects. Radiation therapy
also involves a major risk for abnormal fetal development, and cannot
be used to treat pregnant women with breast cancer.
Chemotherapy--Systemic treatment with anti-cancer drugs given intravenously
(injected into a vein) or by mouth. Either way, the drugs travel
in the bloodstream and move throughout the entire body. Doctors
who prescribe these drugs (medical oncologists) generally use a
combination of medicines proven more effective than a single drug.
For women with node-negative breast cancer the most frequently used
chemotherapy options are CMF (cyclophosphamide, methotrexate, and
fluorouracil), CAF (cyclophosphamide, doxorubicin), and AC (doxorubicin
(Adriamycin) and cyclophosphamide).
Hormone therapy--Estrogen, a female sex hormone produced by the
ovaries, promotes growth of some breast cancers. Doctors use several
approaches to block the effect of estrogen or to lower estrogen
levels. The most commonly used antiestrogen drug is tamoxifen, taken
daily in pill form, usually for 5 years. Studies show that tamoxifen
can reduce the chances of breast cancer coming back after surgery
if the breast cancer cells contain receptors for estrogen or progesterone.
Tamoxifen may be used to treat metastatic breast cancer, but also
a significant number of patients with node-negative cancer receive
tamoxifen treatment.
Anti-estrogen treatment with tamoxifen is, however, associated
with potentially serious morbidity and side-effects For instance,
studies have shown an increase of early-stage endometrial cancer
(which occurs in the lining of the uterus) among post-menopausal
women taking tamoxifen (Cardosi and Fiorica 2000). Another potential
side-effect of tamoxifen is deep vein thrombosis, a condition in
which blood clots form in the deep blood vessels of the legs and
groin. The blood clots sometimes break off and spread to the lungs
as a life-threatening complication. The risk of stroke is also somewhat
increased. Other side effects are hot flashes, mood swings, and
cataracts (Rennie 1993; Gail, Costantino et al. 1999).
EXAMPLE 1
Analysis of Levels of Activated Stat5 in Breast Cancer to Predict
Prognosis
It has been established in the present invention that activation
of Stat5, a transcription factor which is constitutively activated
in normal breast epithelial cells, is gradually lost in lesser differentiated
human breast tumor cells. Based on analysis of human normal and
malignant breast tissue samples, a positive correlation was observed
between Stat5 activation and degree of cell differentiation. Cell
differentiation, as measured by low histological grade, of a tumor
is a known general prognostic factor for cancer. Tumors of higher
grade, i.e. lower degree of tumor cell differentiation, is associated
with poor prognosis. The prognostic value of Stat5 activation for
breast cancer outcome therefore was examined. A simple procedure
for antigen retrieval of tyrosine-phosphorylated Stat5 in formalin-fixed
cells and tissues was established. The procedure validated the specificity
of detection of antibodies directed to this phosphorylated epitope.
The new technique was then applied to a material from 553 primary
tumors obtained from breast cancer patients with known disease history
and well-characterized tumors.
The results showed that Stat5 was activated in approximately 50%
of primary breast cancers, and that activated Stat5 was correlated
with reduced rate of recurrence and increased overall survival rate.
This correlation was especially strong in patients with node-negative
disease. Activated Stat5 may therefore be the first tumor marker
that will significantly help to identify a subgroup of low-risk
breast cancer patients with excellent prognosis.
Materials and Methods
Cell culture and Transfections used for Validation of Anti-pTyr-Stat5
Antibody AXI
T47D human breast cancer cells--T47D cells (American Type Culture
Collection, 10801 U |