A method of treating breast cancer that is at least partially ER.sup.+
is disclosed. The method comprises administering at a tumor site
in a mammalian subject a pharmaceutically acceptable form of Fe(II)
or Fe(III) in a suitable carrier. A four-part program aimed at eradicating
breast cancer includes (a) local treatment and prevention of spread
from a contained breast site, preferably using local administration
of a ferric iron composition, (b) treatment of disseminated (metastatic)
breast cancer, (c) reduction in the risk of developing breast cancer,
preferably by enhancing dimeric/polymeric IgA and polymeric IgM
inhibition of estrogen responsive cell growth, and (d) protection
against cancer causing agents.
What is claimed is:
1. A method of treating breast cancer that is at least partially
ER.sup.+ comprising administering at a tumor site in a mammalian
subject a pharmaceutically acceptable form of at least one iron
compound or complex in a suitable carrier.
2. The method of claim 1 wherein said iron compound or complex
comprises Fe(III) (ferric iron).
3. The method of claim 1 wherein said iron compound or complex
comprises Fe(II) (ferrous iron).
4. The method of claim 1 wherein said administering of said iron
compound or complex is followed by performing surgery to remove
5. The method of claim 1 comprising applying said iron compound
or complex to a mastectomy or lumpectomy site.
6. The method of claim 1 comprising applying said iron compound
or complex to surgical margins of a mastectomy or lumpectomy site.
7. The method of claim 1 comprising administering said iron compound
or complex to a disseminated/metastatic breast cancer site.
8. The method of claim 1 wherein said iron compound or complex
comprises a soluble or insoluble composition.
9. The method of claim 8 wherein said iron compound or complex
comprises a soluble form of Fe(III) chosen from the group consisting
of ferric ammonium citrate, ferric ammonium sulfate, ferric chloride,
ferric nitrate, ferric sulfate and ferric nitrate hydroxide, and
combinations, complexes or polymeric forms thereof.
10. The method of claim 8 wherein said iron compound or complex
comprises an insoluble form of Fe(III) chosen from the group consisting
of ferric deferoxamine activated insoluble matrix.
11. The method of claim 10 wherein said activated insoluble matrix
12. The method of claim 11 wherein said activated insoluble matrix
is chosen from the group consisting of dextran, starch, insoluble
protein and biodegradable synthetic polymers.
13. The method of claim 10 wherein said insoluble matrix comprises
non-biodegradable implantable beads.
14. The method of claim 1 wherein said iron compound or complex
15. The method of claim 1 wherein said iron compound or complex
comprises radiolabeled .sup.59Fe(III) or .sup.55Fe(III).
16. The method of claim 1 wherein said iron compound or complex
comprises .sup.59Fe(III) or .sup.55Fe(III) bound to transferrin.
17. The method of claim 1 wherein said iron compound or complex
comprises a radioisotope of a metal other than iron that is bound
to transferrin or apotransferrin, said radioisotope-bound transferrin
or apotransferrin molecule also being bound to at least one Fe(III)
18. The method of claim 1 wherein exposure of said tumor site to
said iron compound or complex is no more than a few days.
19. The method of claim 1 wherein said tumor site is other than
a primary localized breast tumor.
20. A method of treating breast cancer comprising ER.sup.- cancer
cells, the method comprising administering to an individual in need
thereof a pharmaceutically acceptable composition comprising means
for deterring or preventing iron uptake by said ER.sup.- cancer
21. The method of claim 20 wherein said means comprises at least
one chelator chosen from the group consisting of .alpha.-ketohydroxypyridine
22. The method of claim 20 wherein said means comprises at least
one monoclonal antibody against a transferrin receptor.
23. The method of claim 20 comprising administering said composition
at a tumor site.
24. The method of claim 17 comprising administering said composition
in the blood plasma.
25. The method of claim 24 wherein said composition comprises means
for blocking diferric transferrin from binding to a cellular transferrin
26. The method of claim 20 comprising depleting iron in the diet
of said individual.
27. The method of claim 20 wherein said composition comprises a
28. A method of treating breast cancer containing ER.sup.+ cells,
the method comprising administering to an individual in need thereof
a pharmaceutically acceptable composition comprising means for increasing
the amount of at least one immunoglobulin inhibitor chosen from
the group consisting of IgA, IgM and the Fc domain of IgA or IgM
sufficiently to kill at least a portion of said ER.sup.+ cells in
29. The method of claim 28 wherein said means comprises oral challenge
with an immunogen capable of causing an increase in the number and/or
function of IgA or IgM secreting B immunocytes in breast tissue.
30. A method to aid in deterring the occurrence, growth or progression
of breast cancer in a population of susceptible individuals, the
method comprising carrying out at least one of the following regimens:
(a) in individuals having breast cancer or at risk of developing
breast cancer, contacting the breast ductal tissue of said individuals
with a cell growth inhibiting amount of an immunoglobulin inhibitor
or an immunoglobulin inhibitor mimicking compound; (b) in individuals
with ER.sup.+ breast cancer cells, treating a localized tumor or
mastectomy or lumpectomy site with an effective amount of a pharmaceutically
acceptable form of Fe(II), Fe(III), radioactive Fe(II) or Fe(III),
or Fe(III)-bound transferrin or apotransferrin molecules that are
also bound to another radioactive metal, sufficient to kill at least
a portion of the cancer cells in said tumor or at said mastectomy
of lumpectomy site; (c) in individuals with disseminated ER.sup.+
breast cancer cells, administering said Fe(II) or Fe(III) to a metastatic
cancer site; (d) in individuals with localized or disseminated ER.sup.+
breast cancer cells, administering locally or systemically a pharmaceutically
acceptable composition comprising means for increasing the amount
of at least one immunoglobulin inhibitor chosen from the group consisting
of IgA, IgM and the Fc domain of IgA or IgM sufficiently to kill
at least a portion of said ER.sup.+ cells in said individual; (e)
in individuals with ER.sup.- breast cancer cells, administering
locally or systemically a pharmaceutically acceptable composition
comprising means for deterring or preventing iron uptake by said
ER.sup.- cancer cells; (f) in individuals with localized or disseminated
ER.sup.- breast cancer cells, treating said ER.sup.- cancer cells
with an effective amount of a pharmaceutically acceptable composition
containing radioactive Fe(II) or Fe(III), or Fe(III)-bound transferrin
or apotransferrin molecules that are also bound to another radioactive
metal, sufficient to kill at least a portion of said ER.sup.-cancer
cells; and (g) in individuals with ER.sup.- breast cancer cells,
infecting at least a portion of said ER.sup.- cancer cells with
a virus vector containing a DNA sequence coding for a poly IgA/IgM
binding receptor, and administering locally or systemically an immunoglobulin
inhibitor of cancer cell growth or a mimic thereof capable of binding
to said receptor.
31. The method of claim 30 comprising carrying out at least one
of the following regimens: (a) in individuals having breast cancer
or at risk of developing breast cancer, enhancing the number of
B immunocytes producing IgA or IgM in breast tissue; and (b) immunizing
individuals at risk of developing breast cancer against microorganisms
known to or suspected of causing breast cancer.
CROSS-REFERENCE TO RELATED APPLICATIONS
 This application claims the benefit under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Patent Application No. 60/338,037 filed Nov.
13, 2001, and is a continuation-in-part of U.S. patent application
Ser. Nos. 09/852,958 and 09/852,547, both filed May 10, 2001, the
disclosures of each of which are hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention generally relates to methods and compositions
for the eradication of cancers of the mucosal endothelial tissues.
More particularly, the present invention relates to the use of such
compositions and methods for breast cancer risk reduction, prevention
 2. Description of Related Art
 For women with breast cancer, the term "eradication"
has different meanings depending upon the state of their disease.
Additionally, for women still disease free, eradication means preventing
the development of breast cancer. Today, there are no known preventions
for breast cancer. Although many risk factors have been identified
for breast cancer (76-78), reduction of overall risk from the current
United States level of one-in-eight has not been achieved short
of the use of the anti-estrogen tamoxifen as a preventative (79).
However tamoxifen is only for use by very high-risk women (79).
It is not for use with the general population, both because of adverse
side effects and because of disruption of reproductive capacity.
Accordingly, tamoxifen is under evaluation for high-risk women,
but is not considered appropriate for use as a standard preventative
due to its serious side-effects and causation of a number of endocrine
problems. Treatment with tamoxifen for more than five years may
in fact induce breast cancer. It is now recommended that this therapy
be limited to only five years. This does not cover the span of a
women's lifetime. Based on knowledge today, it may be possible to
reduce risk by alterations of life style and diet, but these are
at best fractional gains and offer no assurances. Even women who
lead very health-risk conscious lives still develop breast cancer.
A successful mass prevention is a primary goal in the fight against
 For women diagnosed with breast cancer, the current most
effective eradication methods begin with various surgical procedures
(i.e. mastectomy or breast conservation surgery or more commonly
lumpectomy). Without doubt, removing a primary tumor is an effective
first step of eradication provided tumor cells have not escaped
to other body sites. Given that we understand that escape is a possibility,
or that multiple unrecognized tumor foci are present in one or both
breasts, many breast cancer patients opt for radiation therapy,
adjuvant chemotherapy and/or tamoxifen treatment even when they
are diagnosed as node negative. The term "node negative"
indicates that cancer cells have not moved to the axillary nodes
from the breast. Commonly however, it is thought useful to "shrink"
primary tumors before surgical removal. Today this is done by systemic
chemotherapy or radiation therapy. Patients with node negative estrogen
receptor positive (ER.sup.+) breast cancer may also be treated with
anti-estrogen (e.g. tamoxifen) as is often done for postmenopausal
women. Depending upon age and physical condition, anti-estrogen
therapy is now a common alternative for many postmenopausal women.
 For breast cancer patients who are axillary node positive
(either ER.sup.+ or ER.sup.-), additional treatment is essential
including those noted above. There is no doubt that these modalities
have a major positive impact on long term survival, but it is likewise
clear to basic cancer investigators and cancer clinicians alike
that the failure rate is significant even despite frequent quoting
of favorable statistical data. Currently, node positive women without
other evidence of dissemination can be treated by standard chemotherapy
and/or radiation therapy. However, node positive patients with ER.sup.-
tumors are at risk no matter the therapy utilized. For these women,
the use of anti-hormone therapy is not as effective as with patients
with ER.sup.+ tumors.
 It is with the diagnosis of metastases to liver, bone, brain,
lung, etc., that another more serious level of the eradication issues
arises, and standard chemotherapy is most often not effective. For
reasons both known and unknown, these cases have a very poor prognosis.
Current chemotherapy can sometimes retard metastatic cancer growth,
but as cancers spread they become progressively more therapy resistant.
The level of public concern about this issue is clear from anticipation
of the benefits of such new drugs as Herceptin.RTM., which is a
monoclonal antibody against the HER2 receptor. Introduction of Herceptin.RTM.
was accompanied by widespread reports in the news media that heightened
expectations. Unfortunately, even this new family of biopharmaceuticals
is not an effective means of eradication. At best, Herceptin.RTM.
provides only a small increase in survival time and then only in
combination with chemotherapy and only with a minority of treated
patients (70). While other types of drugs are under investigation,
the magnitude of the disseminated cancer problem remains undiminished.
 Iron deprivation has been discussed as a means of eliminating
cancer cells (39,40), but the focus has been on two technologies
that, used alone, have not worked. First, those investigators have
considered iron deprivation via treatment with chelators that bind
the metal and thereby render it metabolically inactive. Chelation
alone has not removed enough iron from the body to be effective
as an anticancer program (41). This is to be expected, for it is
known that iron is retained in organs and tissues with a biological
half-life of about 2000 days. Most likely, chelation alone will
not be an effective therapy.
 Currently, the primary effort in breast cancer research
aimed at eradication of the disease is intensely focused by powerful
technology that permits identification of a large number of genes,
and by the human genome project that promises to solve the cancer
problem. To date, these technologies have provided valuable information
but have failed to move to the next level of application, cancer
 A brief historical overview of breast cancer research over
the past four decades points to numerous periods of advancement,
each with its own promise of defeating cancer. For example, during
the 1960s investigators were encouraged by newly identified enzyme
and metabolic changes in cancer cells. It then seemed clear that
these changes were the cause of cancer and that its end was near.
During the 1970s, the beginnings of molecular biology (then called
microbial genetics) yielded new found information that was thought
certain to lead to the end of many human diseases, including cancer.
Investigations in the field of endocrine cancer research, during
the 1980s, focused on how hormones caused cell growth and developed
animal models to study hormone dependent cancer. At that time, serum-free
defined animal cell culture was being developed (1) and new substances
called growth factors were being explored (2). Also during that
period, another major advancement was the discovery of the estrogen
receptor (ER) and the hypothesis that it alone controlled estrogen
dependent cell growth (3-5). Some investigators did not accept all
of the ER hypothesis (6-8), however, and thought that estrogen-inducible
growth factors (estromedins) were necessary (6,9,10). It appeared
clear at that time that growth factor research would untangle the
cancer enigma. Today cancer scientists know this is not the case.
 Along with the growth factor research came the "oncogene"
explosion of the 1990s, which promised an end to cancer. Today,
cancer investigators are inundated by scores of gene changes in
cancer. The list grows weekly. A GENBANK search of "breast
cancer hot spots" yielded more than 100 "hits" on
several chromosomes. This cornucopia of genetic information obscures
two facts: First, very few breast cancers can be traced to germ
line DNA changes (11). Most are not inherited. Notable exceptions
are BRCA1 and BRCA2, which represent at most 1-10% of breast cancers
in the United States (31-33). Given that the incidence of breast
cancer now approaches 1 in 8, the majority of breast cancers have
other origins. Second, sophisticated new molecular technology has
identified changes in expression of at least 100 mRNAs in breast
cancer cells (11). There is promise of hundreds of gene/expression
changes (11-13). It is very unlikely they are all causative or even
critical to breast cancer. The tempting scenario is to investigate
each mRNA or gene to define its role. Of course, this represents
years of work for researchers, and still leaves open the question:
"Will this lead to breast cancer eradication"?
 It is known that eighty percent or more of breast cancers
are invasive ductal carcinomas that arise from ductal cells (85,86)
or precursors of ductal cells (85,87). Based on the current state
of knowledge, there is no genetic lesion to explain the 70% of breast
cancers now termed "sporadic". Certainly the BRCA1 and
BRCA2 genes are responsible for at most a small percentage of breast
cancers in this country (88-91). Lesions in the p53 gene were initially
thought to be important in as many as 15 to 50% of breast cancers
(92-94). However, it was far from clear which mutations are causally
related to breast cancer onset or which actually constitute secondary
changes leading to loss of function of this tumor suppressor gene
in the different types of breast cancer (i.e. ER.sup.+ or ER.sup.-).
A more recent study has rightly pointed out the confusion regarding
p53 mutations and breast cancer patients (96). Studies of p53 mutations
have yielded a wide range of results depending upon the methods
employed (97). One useful fact is that the results average about
30 to 40% for loss of heterozygosity at the p53 gene (97). This
means the remaining gene may be a "hot spot" (i.e., a
chromosomal loci or gene that is frequently altered in breast cancer
specimens). However, at this time, there is not sufficient evidence
to support the use of p53 as a guide to selection of therapy modalities
for breast cancer (98).
 In fact, today it is very difficult to explain the great
many mutations and other types of genetic expression alterations
that are known in breast cancer cells (11). Based on the findings
with breast and other types of mucosal cancers, such changes include
mutations, translocations, amplifications of oncogenes, loss of
heterozygosity (LOH), and allelic imbalances (12,13,100-102). How
do all of these happen? Are environmental carcinogens in such high
abundance that they explain these data? Despite the concentrated
focus given to environmental carcinogens as causes of breast cancer
(20,95,99), that hypothesis has failed to move forward to the level
of accepted scientific fact. Ways to reduce the risk of developing
breast cancer, ways of preventing its occurrence, and ways to treat
existing cases of localized and metastatic breast cancer are urgently
needed. Even with the very best of treatments currently available,
a longer-term plan is still needed in which prevention is the first
line of eradication. A successful prevention will be, preferably,
safe and have low or negligible side effects. It should be capable
of reducing risk for the majority of women, independent of their
economic circumstances. It should cause little or no disruption
of life-style or reproductive capacity.
SUMMARY OF PREFERRED EMBODIMENTS
 While continued gene searching may not lead to the goal
of eradication of breast cancer in the near or even mid-range future,
the present invention offers eradication technology that can be
applied today--no matter how many gene changes are ultimately associated
with cancer development. Eradication is approached from a unique
perspective based on discoveries described in more detail below
and in co-pending U.S. patent application Ser. Nos. 09/852,547 entitled
"Compositions and Methods for the Diagnosis, Treatment and
Prevention of Steroid Hormone Responsive Cancers" and Ser.
No. 09/852,958 entitled "Compositions and Methods for Demonstrating
Secretory Immune System Regulation of Steroid Hormone Responsive
Cancer Cell Growth", and in corresponding International Patent
Applications PCT/US01/15171 (WO 01/86307 and PCT/US01/15183 (WO
01/85210), also identified in the list of References, below, as
items 29 and 30, and hereby incorporated herein by reference).
 The present invention specifically focuses on the eradication
of breast cancer, and overcomes many of the problems and barriers
in breast cancer research today. In certain embodiments of the invention,
(ferric) iron-based treatment of local breast cancer tumors and
lumpectomy sites is provided. New treatment strategies for three
conditions are disclosed, which include (i) treatment of mastectomy
sites to eliminate residual cancer cells, (ii) treatment of primary
tumors before surgery, (iii) treatment of the surgical margins of
mastectomy sites to eliminate undetected residual cancer cells.
 In certain embodiments of the invention, treatment of disseminated/metastatic
breast cancer is addressed from a cell nutrition perspective. Both
ER.sup.+ and ER.sup.- metastatic breast cancers are highly growth
dependent upon diferric transferrin as a source of metabolic iron
required for cell growth (29,30,71,72) and more specifically DNA
synthesis (73,74), as described in the above cited USPTO pending
patent applications, using newly developed serum-free medium cell
culture methods. Because of this strict requirement for diferric
transferrin, manipulation of iron metabolism is employed to kill
disseminated cancer cells.
 In certain embodiments of the invention, risk reduction
via oral "immunization" is provided, i.e., oral administration
of immunogens that result in increased content of secretory immunoglobulins
(IgA and IgM) in breast tissue. This approach to risk reduction
is based on the very well established fact that DNA synthesis (i.e.
cell replication) is required to achieve the full effects of mutagens
(80-85). The secretory immunoglobulins IgA and IgM are inhibitors
of breast cell DNA synthesis (29,30) and therefore reduce the probability
of mutations that lead to breast cancer later in life.
 Certain embodiments of the present invention provide methods
for eradicating breast cancer by conventional oral or standard immunization
against bacteria or other microorganisms existing in the breast
duct system that release or cause formation of mutagenic agents
that lead to causative genetic changes in the exposed ductal cells.
This approach addresses the problem of what single source might
give rise to a process that can cause so many mutations and different
genetic changes that accumulate over the known prolonged period
required to develop breast cancer. Identification of the causative
bacteria/microorganisms makes possible the exploitation of the body's
secretory immune system to develop secretory immunity or to use
standard immunization to transmit immunity to the ductal fluids.
Protection from the underlying causative agents will provide the
best means of ultimate eradication of breast cancer. A microbial
origin of breast cancer does not appear to have been previously
described or suggested in the scientific literature.
 In preferred embodiments of the present invention, all four
parts of the breast cancer eradication program are applied to appropriate
groups of affected or at-risk individuals, including (1) local treatment
and prevention of spread from a contained breast site; (2) treatment
of disseminated (metastatic) breast cancer; (3) reduction in the
risk of developing breast cancer; and (4) protection against cancer
causing agents. In some embodiments, one or more parts of the program
are employed for treatment, reduction of risk, or prevention of
breast cancer in a single individual or a one or more groups of
individuals. Full implementation of the preferred four-part integrated
program is expected to eradicate breast cancer within the next decade.
These and other embodiments, features and advantages of the present
invention will become apparent with reference to the following description.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
 The breakthrough in breast cancer research disclosed in
U.S. patent application Ser. Nos. 09/852,958 and 09/852,547, and
which also applies to cancers of other mucosal epithelial tissues
in the body, is further implemented and extended herein. The disclosures
of those applications are hereby incorporated herein by reference.
Purification of new "serum factor(s)" that regulate estrogen
responsive breast cancer cell growth in culture is described in
those preceding applications. The purification yielded dimeric/polymeric
immunoglobulin A (IgA) and pentameric immunoglobulin M (IgM) as
the active regulators. These immunoglobulins ("immunoglobulin
inhibitors") arrested estrogen target tumor cell growth completely
at low nanomolar concentrations, and their inhibitory effects were
entirely reversible by picomolar concentrations of estrogens. That
disclosure revealed a previously unknown function for the secretory
immune system. In the above-identified patent applications, a major
role for TGF.beta. in breast growth regulation is also identified:
it is a cytokine that controls IgA/IgM immunocytes. Breast cancer
growth is best defined as negative paracrine control by secretory
immunoglobulins (immunoglobulin inhibitors) and positive direct
control by estrogens. In conjunction with this work, the longstanding
problem of the regulation of estrogen dependent cell growth in culture
under serum-free defined medium conditions was solved. These results
have great physiological relevance. IgA and IgM are secreted by
B immunocytes located in the lamina propria of estrogen target tissues
including breast. They are more than 90% of the immunoglobulins
secreted into breast milk. The positioning of the immunocytes in
the tissue adjacent to the epithelial cells and the secretion of
the immunoglobulins is hormone regulated.
 In the course of developing suitable serum-free defined
culture media for studying estrogen effects on breast cancer cell
growth, it was discovered that both soluble iron (FeIII) and diferric
transferrin had special roles with regard to estrogen receptor positive
(ER.sup.+) and ER.sup.- breast cancer cell growth. Even more surprising,
the results point to a new estrogen receptor ("ER.gamma.")
with a higher affinity for steroid hormone than that of the known
receptors ER.alpha. and ER.beta.. In addition, this work led to
the identification of a mediating receptor for IgA/IgM that shares
the properties of the classical immunoglobulin transcytosis Poly-Ig
receptor and is an Fc receptor superfamily member. This receptor
maps to a gene location linked to allelic imbalances in 75% of breast
cancer specimens. These discoveries lend themselves to major advancements
in breast cancer eradication, which are further developed and described
herein in a four-part program that has been devised to achieve the
goal of eradication of breast cancer. This four-part breast cancer
eradication program discloses specific new solutions to the four
most pressing aspects of breast cancer eradication:
 (1) Local treatment and prevention of spread from a contained
 (2) Treatment of disseminated (metastatic) breast cancer
 (3) Reduction in the risk of developing breast cancer
 (4) Protection against cancer causing agents
 For the nearly 180,000 women in the United States who will
be diagnosed with breast cancer this year, the most important issue
is eradication. For some women, eradication means eliminating existing
localized disease. Accordingly, in Part I, below, a new non-toxic
method for treatment of localized cancers is based on direct application
of a soluble form of iron that kills early forms of cancer. Another
approach is direct "immuno-therapy" with immunoglobulin
inhibitors of cancer cell growth. For other women, eradication means
destroying cancer that has moved from the breast to other locations
in the body, i.e., disseminated or metastatic breast cancer. Today,
even with the best treatments metastatic disease has a poor prognosis.
In Part II, below, a number of new therapeutic approaches are presented
herein, which exploit cellular nutritional requirements for growth
of breast cancer cells. For disseminated breast cancer, a different
aspect of iron metabolism is exploited to kill the tumor cells.
In addition, the use of a new, previously unrecognized breast cancer
gene identified in U.S. patent application Ser. Nos. 09/852,958
and 09/852,547/PCT Published Application Nos. WO 01/86307 and WO
01/85210 as a poly-Ig (Fc) receptor or a poly-Ig-like (Fc) receptor
that mediates IgA/IgM inhibition of cancer cell growth is employed
in the present eradication program. Until now, the only breast cancer
genes known were BRCA1 and BRCA2. The problem has been, however,
that BRCA1 and BRCA2 are important for only 1 of 400 women in this
country. This leaves 70% or more of breast cancers with no known
genetic origin. The new gene shows "allelic imbalances"
in 75% of breast cancers. It is a likely candidate for the 70% of
breast cancers not explained today. The new breast cancer gene,
coding for the poly-Ig (Fc) receptor or poly-Ig-like (Fc) receptor
is a potentially valuable candidate for a new gene therapy for disseminated
breast cancer because it can restore immune inhibition and anti-estrogen
inhibition to a cell and can even lead to cancer cell death.
 The surest means of breast cancer eradication is prevention,
which is the focus of Part III, below. The Inventor's studies in
cell culture have proven that the immunoglobulins IgA and IgM from
the secretory immune system can serve to kill early breast cancer
cells by terminally arresting their growth. It is now proposed that
oral challenge will be successfully employed to reduce the risk
of cancer causing mutations in breast cells, as described in more
detail in Part IV, below. Immunity is transferred from the gut to
the breast via the secretory immune system. This strategy is consistent
with the fact that breast cancer incidence is lowest in areas of
the world where oral immune challenges are common and highest in
the Western countries where similar challenges are restricted. The
"hygiene hypothesis" that we are "too clean"
will be applied to reduce the current Western world risk of breast
cancer (1 in 6-8) to the 1 in 40-100 rate of the non-Western world.
 In another view, prevention can mean full eradication worldwide.
As proposed in U.S. patent application Ser. No. 09/852,547/PCT Published
Application No. WO 01/86307, cancer causing agents are located in
the ducts of the breast gland and infectious agents are responsible
for the development of breast cancer. It has been known for many
years that the majority (i.e. 75%) of breast cancers arise from
the cells lining the ducts of the gland. In light of the present
disclosure, this is an extraordinary clue to the cause of breast
cancer. Microbes and possibly viruses inhabit the milk ducts. Furthermore,
there are strong recent clues that cancers may be of microbial origin
by virtue of their metabolic products or secreted proteins. The
causative organisms will be sought from breast tissue specimens
as well as human milk. Conventional-type oral immunizations will
be employed to kill the culprit organisms in the ducts. This approach
is consistent with other time-tested methodologies, such as Sabin's
oral vaccination against the poliomyelitis virus, which is based
on the same secretory immune responses that, in the present case,
are directed against breast cancer causing bacteria. Alternatively,
standard inoculation immunizations with modified microorganisms
or fragments of the suspect microbes will be employed to elicit
production of natural immunoglobulin inhibitors of cancer cell growth.
No matter the general or technical approaches that are ultimately
successful, immunizations have been used worldwide to eradicate
human diseases. A similar attack is expected to bring an end to
breast cancer for a majority of women.
Part I: Local Treatments that Prevent Further Spread from a Contained
Iron Effects on Local Breast Tumors
 Methods and compositions for treating localized breast cancer
involve iron effects on local breast tumors, and employ nutritional
information developed from previous studies of the differential
roles of iron metabolism in steroid hormone receptor positive (ER.sup.+)
and steroid hormone receptor negative (ER.sup.-) breast cancer cells.
These studies were done with well-known cell lines (29,30,110) grown
under serum-free defined conditions (29,30,110) that permitted precise
control of free iron (FeIII) and diferric transferrin concentrations
(21-30). In U.S. patent application Ser. Nos. 09/852,958 and 09/852,547/PCT
Published Application Nos. WO 01/86307 and WO 01/85210 the need
to reduce free or soluble Fe(III) concentrations to very low levels
to achieve sex steroid hormone or thyroid hormone dependent epithelial
cell growth in culture is disclosed, and such disclosure is hereby
incorporated herein by reference.
 For in vivo treatment of localized breast cancer, preferred
forms of iron used and the preferred compositions are described
as follows: The most cancer cell toxic forms of soluble Fe(III)
presently identified are complexes of ferric ammonium citrate (about
16% iron by weight) and ferric ammonium sulfate. These solid salt
mixtures are dissolved in water at high concentrations (1.0-250
mg/ml), and after filter sterilization, and preferably used immediately.
These mixtures are light sensitive. Mixtures stored at 4 to 27.degree.
C. under normal room light conditions (for example, 3 to 5 days
or up to 30 days) show an increase in cytotoxicity of 2 to 10-fold.
Higher intensity lights are also effective, using wavelengths ranging
from low ultraviolet to visible. Addition of sodium chloride at
0.001 to 0.5 M facilitates the light sensitivity. Addition of up
to 0.01 M sodium phosphate, pH 7.0, has a similar toxicity enhancing
effect provided it does not cause precipitation. More acidic pH
is also effective. More basic pH precipitates the iron complexes.
Elevated temperatures to 60.degree. C. increased toxicity. Longer-term
storage of up to 30 days increased toxicity in the presence of light
or in light shielded containers. It is preferred to use the iron
composition immediately after preparation for maximum consistency,
particularly when handled under surgical conditions, and to provide
a more uniform base for testing efficacy. However, in some situations
of use the long shelf-life of the iron compositions is advantageous,
and the amount administered can be adjusted for the increased potency
of the stored composition. Ferric chloride, ferric nitrate and ferric
sulfate were effective, but less so than the iron-ammonium-citrate
complexes. This is likely due to greater solubility of the iron-ammonium-citrate
complexes. Still other suitable compounds or complexes of ferric
iron may exist that also have the desired cancer cell toxicity properties,
and it is expected that one or more of those forms of Fe (III) could
be substituted for the above-identified forms. In addition to the
ferric iron compositions, there are also ferrous iron compositions
(e.g., ferrous ammonium citrate) that also kill cancer cells when
applied immediately after preparation to a localized breast cancer
site, or a mastectomy or lumpectomy site. Preferably the ferrous
salt is dissolved fresh and used immediately, as air (oxygen) converts
Fe(II) to Fe(III) very quickly. Without wishing to be limited to
a particular theory, it is believed that the Fe(II) (ferrous) compounds
shower the cancer cells with oxidative products that cause cell
death as the Fe(II) is converted to the Fe(III) form. The Fe(III)
conversion product then operates to provide further cell killing
effects, as described above.
 In an in vitro model cell culture system as described in
U.S. patent application Ser. Nos. 09/852,958 and 09/852,547/PCT
Published Application Nos. WO 01/86307 and WO 01/85210, exposure
of ER.sup.+ breast cancer cells to sufficient iron causes cell death
in .ltoreq.48 hours. ER.sup.- breast cancer cells are insensitive
to Fe(III) killing under similar in vitro test conditions. These
in vitro model systems are believed to be predictive of the in vivo
effect of iron treatment on ER.sup.+ breast cancer cells.
 Another alternative form of iron administration for killing
localized cancer cells is prepared by adding .sup.59Fe(III) to one
of the unlabeled (non-isotope) ferric or ferrous salts described
above to increase effectiveness. The radioactive and non-radioactive
iron can be prepared as the same salt or the admixture of two or
more salts of Fe(III). The effectiveness of the composition is increased
because any tumor cells not reached by the soluble iron will be
exposed to DNA fragmenting .gamma. radiation via .sup.59Fe that
penetrates at greater distances.
 One other polymeric form of Fe(III) has high inhibitory/toxic
activity. The combination of ferric nitrate and bicarbonate is prepared
chemically as described (75). After permitting precipitation of
ferric hydroxide, and its removal, the polymeric form of ferric-nitrate-hydroxid-
e remains in clear solution indefinitely at room temperature. It
is used in this clear solution form.
 A pharmaceutical (FDA approved) iron dextran product (INFeD.RTM.,
Watson Pharmaceuticals, Inc., Los Angeles, Calif.) is available
today (137) that has the properties of one suitable preparation
for non-radioactive iron treatment of localized breast cancer. It
is supplied as a single injection 100 mg dose of iron in 2.0 ml.
A series of warnings are supplied concerning the use of INFeD and
precautions to be taken with this preparation. There is an indication
that animals treated with high doses may develop cancers after repeated
injections, however the incidence of such complications in humans
is either not clear or very low. However, it is clear that in the
older literature sarcomata were found at the injection sites of
intramuscular iron (158-160). Evaluation of the literature reveals
that sarcomas at the site of iron dextran injection are (i) species
specific, with rodents most likely to develop tumors (156), especially
after repeated injections (157), (ii) dose and threshold dependent,
(iii) residual characteristics at the site, and (iv) latent period
relative to the life span of the test species. As reviewed in 1977,
introduction of intramuscular iron therapy more than 22 years before
then resulted in only nine malignancy reports in man. Of these,
only one appears causally related to the iron injections (156).
Care must be given to various aspects such as methods of delivery,
dilution of drug, and overall iron status of the patients (161).
Attention must be given to anaphylactic reactions (161). Current
use of intramuscular iron preparations with proper techniques in
humans does not appear to bear a significant risk (161). The iron
in the preparation associates with serum ferritin or hemosiderin,
and to a lesser extent with apotransferrin (i.e. transferrin without
bound iron). Care is taken to monitor serum ferritin levels to determine
potential iron overload. With intramuscular injection, the majority
of the iron dextran is absorbed in 72 hours. The remaining iron
dextran is absorbed over the next 3 to 4 weeks. This rate of absorption
is compatible with use as a treatment for local breast cancer. For
additional cancer cell killing capability, .sup.59Fe(III) can be
incorporated into the iron dextran to increase effectiveness, as
discussed above with respect to it use in iron salt solutions.
 In still another way in which iron can be used to effect
tumor cell death, diferric transferrin is prepared radio-labeled
with .sup.59Fe(III) or .sup.55Fe(III) (132,133) and is used to irradiate
breast cancer cells to cause cell death. Each apotransferrin molecule
accepts two Fe(III). These bind at neutral pH with high affinity
(i.e. K.sub.a=10.sup.20) to similar "N" and "C"
lobes of the transferrin molecule (136). When breast cancer cells
are grown in serum-free cell culture medium containing 0.01 to 20
.mu.g/ml of .sup.59Fe-transferrin, the radioactive iron from the
transferrin becomes incorporated into many cellular components.
Subsequent radiation induced DNA damage leads to cell death within
24 to 168 hours. This effect was seen with both ER.sup.+ and ER.sup.-
breast cancer cells grown in serum-free defined culture medium.
Other radioisotopes such as .sup.131I-transferrin and .sup.125I-transferrin
(132,133) as well as several other radioisotopes of metals that
bind to apotransferrin can be expected to serve an equivalent function.
The use of .sup.59Fe is preferred because of its 44.6-day half-life
that is suitable for radiation therapy effectiveness. Yttrium-90
is also a consideration because of its high-energy .beta. emission
and 64.1 hour half-life. Of the two iodine isotopes, each has advantages.
The .sup.125I has a longer 60-day half-life compared to .sup.131I
with an 8-day half-life. The .sup.131I has higher energy. The advantages
of the different isotopes in killing of ER.sup.+ and ER.sup.- breast
cancer cells will be evaluated first in cell culture and then using
in vivo rat mammary tumor induction models as described herein.
 Another alternative method and composition employs an insoluble
form of radio-labeled iron, which is prepared by methods previously
described (27). When it is necessary or desirable to limit the amount
of radiation to a restricted site, such as with mastectomy or lumpectomy,
or to a specific set of axillary nodes, the .sup.59Fe can be delivered
via a complex of deferoxamine-Sepharose. Deferoxamine is covalently
attached to Sepharose (27), or to any other "activated"
insoluble matrix (human compatible) by the methods described or
derivative methods. Deferoxamine is a low molecular weight bacterial
product that is currently in use as DESFERAL.RTM. (Novartis Pharmaceuticals
Corp., East Hanover, N.J.) to treat human iron overload patients
(138). Deferoxamine binds Fe(III) with a very high affinity (i.e.
10.sup.23). The complex of deferoxamine-Fe(III) does not dissociate
under body conditions. Thus, placement of insoluble deferoxamine
bound .sup.59Fe in any site will effectively expose a local area
to high-energy .gamma. radiation with escape of only minimal labeled
 It may be preferable to select a biodegradable matrix (e.g.
dextran, starch or insoluble protein or biodegradable polymer) in
some cases or a stable/non-degradable matrix (e.g. cellulose or
synthetic biomatrix) in others. Another application includes attachment
of the deferoxamine to non-degradable "biobeads" for implantation
directly into the local tissue for specific periods of time. The
visible non-immunogenic beads can be removed at times deemed desirable
or when the desired effect has been achieved, or they can be removed
at the time of mastectomy or lumpectomy.
 Localized Breast Cancer Eradication. In earlier studies
of the role of nutrients, hormones and growth factors in hormone
responsive pituitary tumor cell growth in serum-free chemically
defined culture, it was observed that 1 .mu.M soluble iron in the
form of Fe(III) inhibited growth (21-28). Exposure to 10 .mu.M Fe(III)
killed these cells. The results were thought initially to be applicable
only to rat pituitary cells. However, they have proven useful with
ER.sup.+ breast cancer cells (29,30). To develop in vivo confirmation
that iron can be used to locally treat breast tumors, a series of
experimental animal models will be investigated.
 The use of iron for the treatment of cancer is a clear departure
from the widely held belief or paradigm that Fe(III) cannot be (or
should not be) administered locally in vivo. It is commonly cited
(87) that Fe(III) released from cellular ferritin induces (.cndot.OH)
free radical formation and that this reactive species modifies proteins,
lipids and nucleic acids (120). Thus, investigators generally view
iron as cancer initiator or promotor (87,121-125). That paradigm
is not pertinent to the present therapeutic forms of Fe(III), however,
because in the present case the metal will act only short term.
The Fe(III) applied to the tumors is extracellular and has little
or nothing to do with the complex models developed for explaining
the putative role of intracellular ferritin H chain in oxidative
damage to cells (120,121). Notably, as is indicated (121), much
of the ferritin oxidative model is presumptive and unsubstantiated.
Furthermore, ER.sup.+ breast cancer cells appear to be exquisitely
sensitive to a putative burst of extracellular oxidative products.
These cells die very quickly when non-protein bound Fe(III) is added
to culture medium. It should be noted that free Fe(III) does not
support epithelial growth. Diferric transferrin is required. For
cancer treatment, the period of exposure in vivo will be limited
by the fact that within a few days the Fe(III) will be converted
to the inactive but metabolically useful forms of monoferric and
diferric transferring and ferritin. Free/soluble Fe(III) is expected
to bind to apoferritin and apotransferrin under physiologic conditions.
Plasma contains about 2 mg/ml of apotransferrin and 1 mg/ml diferric
transferrin (i.e. transferrin is 66% unsaturated with iron in plasma).
Since Fe(III) cell killing happens in less than a few days, the
risk of other adverse effects of the iron are minimized. Certainly
long-term mutagenic effects are minimized. The time of exposure
and dose schedule of free Fe(III) will be kept to the minimum needed
to achieve therapeutic results. This is the same principal used
with short doses of .gamma. radiation and short-term applications
of chemotherapy used today to treat breast cancer. In fact, the
very same argument can be made against the radiation protocols used
today to treat localized breast cancer, which run contrary to the
paradigm that excess radiation can induce tumors. Likewise, several
of the current chemotherapy chemicals are actually mutagenic. Therefore,
they are used in regimens that kill tumor cells but stop short of
causing a substantial increase in other cancers.
 Further support for the value of this approach comes from
the Physician's Desk Reference information (137) discussed above,
in which humans are treated with intramuscular injections of iron
dextran (INFeD) to correct iron deficiencies that are not treatable
by oral therapy. While there have been individual reports of the
appearance of sarcoma tumors at the injection site in humans (137),
such reports could not be confirmed by the manufacturer at the present
time (personal communication with Watson Pharmaceuticals, Inc.).
The frequency of such tumors as a proportion of the total injections
per year or patients treated per year is not available, but is presumed
to be very low.
 The use of systemic iron or orally administered iron causes
an increase in the body content of this metal and in plasma ferritin
and diferric transferrin levels. One report states that increased
dietary iron facilitates carcinogen induction of rat mammary tumors
and estrogen induction of Syrian hamster kidney tumors (139). Another
report (140) states that excess iron again appeared to facilitate
carcinogen induced rat mammary tumors, but there was more care given
to control the effects of various iron status states on body weight
gain and hematocrit. The effects of excess iron were only apparent
later in that study. In another, study, support for a critical role
of iron was not found with the rat mammary tumor models (141). In
the present case, it is concluded that increased saturation of apotransferrin
by dietary iron results in greater growth rates in carcinogen induced
rat mammary tumor cells. This is consistent with a previous showing
with a carcinogen induced rat mammary tumor cell model in culture
that diferric transferrin is absolutely required for growth (142,143).
Apparently, the systemic elevation of plasma iron is conducive to
growth of breast cancer cells. Any therapy with Fe(III) for treatment
of breast cancer is therefore, preferably local and is subject to
natural elimination within a period of a month. Preferably the doses
are managed such that they do not substantially elevate plasma ferritin
or the iron saturation percent of transferrin.
 Today, women with localized breast cancer have two initial
surgical options: mastectomy or breast conserving surgery as known
as "lumpectomy". With increasing frequency, pretreatment
is done to shrink primary or nodal tumors before surgery. According
to the present plan, an animal model will be used to test whether
iron in the form of soluble ferric ammonium complexes can destroy
existing tumors, or can eliminate undetected cells within mastectomy/lumpectomy
sites. Initially, this program will include testing the direct effect
of Fe(III) on estrogen growth responsive tumors developed from rat
mammary MTW9/PL2 cells in W/Fu female rats (34,35). Studies will
test treatment by application directly into tumors or into their
immediate blood supply. Additionally, after tumors have developed
they will be resected and the surgical site treated with soluble
Fe(III) to determine effect on recurrence. It is known that without
any treatment, there is a 40 to 60% recurrence rate in four months
 With a different model based on CD-rats, environmental carcinogens
will be used to induce rat mammary primary tumors as described (36)
before initiating localized Fe(III) treatment. The primary carcinogen
induced rat model selected has many characteristics of human breast
cancer (37) and therefore is considered relevant.
 Another model also has special relevance. It is now clearly
established and almost universally accepted that estrogens promote
target tissue cell growth (109). There is still a question about
the exact DNA and functional sequence of the receptor that mediates
this response (29,30). However, these steroids may have a second
function. Investigators have long proposed that estrogens (or their
metabolites) are genotoxic and cause mutations (107,108). Estrogens
are considered central to human female breast cancer development
even beyond their growth promoting function. There is a rat mammary
tumor model that mimics this dual effect. Estrogen (17.beta.-estradiol)
treatment of female ACI rats induces mammary tumors in 100% of the
population within 197 days (49). The tumors are estrogen growth
responsive. This model will also be used to induce tumors and determine
the effects of iron therapy on the primary neoplasms. Positive results
with this model will have special applicability to human cancers.
Local treatment with Fe(III) provides an entirely new first line
of eradication of breast cancer.
 In parallel studies, the animal tumor models described above
will be injected or otherwise treated with the .sup.59Fe-deferoxamine-Sepharos-
e complex and the effects on tumor mass monitored. This same procedure
will be assessed for its effect on recurrence of resected tumors.
This is believed to be a completely new approach to local breast
cancer eradication. The effects of soluble Fe(III) versus those
of immobilized .sup.59Fe will be compared for tumor regression,
survival of the hosts and effects of both treatments on the physiological
health of the animals. Confirmation obtained in these in vivo rodent
studies will indicate the applicability of, and will supply partial
evidence for FDA approval for, human trials. Today, the only other
direct local breast cancer treatment without systemic effects is
radiation, which causes healing problems post-surgery and other
chest wall and organ complications.
Part II: Treatment of Disseminated (Metastatic) Breast Cancer
Methods of Treating Disseminated Breast Cancer
 The problem of eliminating disseminated or metastatic breast
cancer is profoundly different than eradication of primary localized
breast disease. These forms of breast cancer are most often chemotherapy
resistant and nearly always fatal. Today there is no satisfactory
chemotherapy or any other therapy for these cancers. In a marked
departure from conventional theories and methodologies, effective
treatments for both ER.sup.+ and ER.sup.- disseminated breast cancers
have been devised.
 Iron Metabolism and Disseminated Breast Cancer. It has been
found that diferric transferrin is unconditionally required for
both ER.sup.+ and ER.sup.- human breast cancer cell growth. It is
clear that ER.sup.+ cells are under both hormone and growth factor
control. However, ER.sup.- cancer cells no longer require growth
factors or hormones for proliferation (29,30). Only nutrients are
required. It is a common observation that hormone autonomous breast
cancer cells have also escaped the requirement for exogenous growth
factors. However, without an adequate supply of iron delivered by
transferrin, both ER.sup.+ and ER.sup.- breast cancer cells fail
to survive. Iron is required for DNA synthesis and other key metabolic
processes (38). Of the known types of chelators, the present studies
indicate that only those that remove iron from diferric transferrin
and serum ferritin will be useful for iron deprivation in tumors.
An example of a class of chelators that are able to successfully
withdraw iron from serum ferritin and diferric transferrin are the
.alpha.-ketohydroxypyridine chelators (144). Other classes are also
known, and some of these may also be suitable for use as described
herein for combined treatment modalities. Deferoxamine does not
remove iron that is already bound to transferrin.
 A second or alternative approach is to use monoclonal antibodies
against the transferrin receptor to prevent iron uptake. This has
yet to be developed into an effective treatment (42-44). Monoclonal
antibody therapies alone are often ineffective because (i) there
is a large supply of the competing natural ligand available that
competes with the antibody for receptor binding, (ii) the natural
ligands often have higher affinity for the receptor than the blocking
monoclonal antibody, (iii) antibodies often do not escape the blood
readily, and (iv) humanized antibodies are required for repeated/prolonged
 Another approach that can be employed as part of the breast
cancer eradication program is to block transferrin directly in the
plasma so that it will not be a source of iron for cancer cells.
Current results support the view that diferric transferrin can bind
to cellular transferrin receptors via either the "N" or
"C" lobes (118) or that binding is primarily via the "C"
lobe (119). Because the lobes have similar amino acid sequences,
it is likely that the same receptor recognition sequence is present
in both. Most notably however, the amino acid sequence of diferric
transferrin that codes for receptor binding is not known. This sequence
will be identified using techniques that are well known in the art
(e.g. by phage display technology) and specific monoclonal antibodies
will be raised to block transferrin binding to cellular receptors,
employing standard techniques. This will prevent the use of serum-borne
diferric transferrin by cancer cells and thus starve cancer cells
for iron. This approach does not suffer from several of the problems
of receptor binding monoclonal antibodies just cited above. The
anti-receptor recognition monoclonal antibody is not required to
leave the general circulation to be effective.
 Iron Chelation Depletion and Combined Modalities. To deplete
iron from the diet, deferoxamine can be given intravenously or other
chelators used orally. This will further lower the blood plasma
content of diferric transferrin and increase the effectiveness of
the receptor sequence monoclonal antibody just described above.
This treatment should be combined with a low iron diet. In addition,
many other oral drugs are available to reduce the effective body
load of iron. These can be used in combination therapies to deprive
the cancer cells of necessary iron sources.
 Genetically and Chemically Modified Transferrin. In addition,
genetically and chemically modified transferrin can be used to introduce
lethal doses of specific toxins and cytolytic enzymes that kill
cancer cells. For example, RNAse can be genetically engineered or
chemically attached to transferrin, using techniques that are well
known in the art. Delivery of this cytotoxic enzyme via the transferrin
receptor can be expected to cause cell death. Most importantly for
this disclosure, the most dangerous (i.e. rapidly growing and spreading)
ER.sup.- breast cancers over-express the transferrin receptor (129).
Genetically modified transferrin will be developed that is cytotoxic
and/or unable to act as iron donor to cells. This strategy focusing
directly on transferrin has the advantage of acting systemically
without regard to the issue of tissue penetration by receptor blocking
monoclonal antibodies or the necessity of developing "humanized"
monoclonal antibodies. In some instances it will be preferred to
combine modalities that interfere with iron metabolism in order
to achieve the most satisfactory and effective results. The above-described
rat mammary tumor models will be employed to confirm the suitability
of this treatment modality for human trials.
 Immunotherapy of ER.sup.+ Breast Cancer. As discussed above,
studies in cell culture with ER.sup.+ breast cancer cells have shown
that contact with IgA and IgM causes cell death within three weeks
(29,30). These results will be employed in immunotherapy for breast
cancer based on the smaller, more tissue penetrating, Fc domains
of polymeric IgA and IgM. The use of Fc fragments is planned because
of data indicating their importance in causing cell growth inhibition.
The rat mammary tumor models described above will be used to continue
testing in vivo.
 The natural forms of IgA and IgM, as well as the Fc fragments,
can be administered intravenously. Several immunoglobulins including
IgA and IgM are already FDA approved for human use. Those preparations,
as well as other secretory immunoglobulin preparations, are expected
to be useful for inhibiting cancer cell growth when administered
in various pharmacologic amounts. A suitable pharmaceutical composition
must provide IgA and/or IgM in a form that is capable of producing
the above-described inhibitory effect on estrogen dependent breast
cancer cell growth, and the immunoglobulin component must be able
to bind to the poly-Ig (Fc) receptor or poly-Ig-like (Fc) receptor
that mediates such inhibition. The preferred active compositions
(i.e., cell growth inhibitory) contain dimeric/polymeric IgA and
pentameric IgM and an activity-stabilizing medium (e.g., a steroid
hormone stripped non-heat inactivated serum or purified compositions
containing calcium), as described in U.S. patent application Ser.
Nos. 09/852,958 and 09/852,547 (PCT Published Application Nos. WO
01/86307 and WO 01/85210), incorporated herein by reference. To
treat localized breast cancer, oral immunization challenge will
also be used to increase the number and function of IgA and IgM
secreting B immunocytes in the breast tissue and thereby provide
more inhibitory/killing immunoglobulins. This treatment has the
advantage of not requiring a clinical setting for administration
and being applicable to all women regardless of age or physical
condition. This immunotherapy can be combined with tamoxifen anti-estrogen
or combined with other immune modulating drugs that increase the
function of the secretory immune system in the breast. Preventative
and risk reduction methods and compositions are described in co-pending
U.S. patent application Ser. No.______ (Atty. Dkt. No. 1944-01301)
entitled "Anti-estrogen and Immune Modulator Combinations for
Treating Breast Cancer," the disclosure of which is incorporated
herein by reference. This treatment mode may have special application
to breast cancer in situ, a form of the disease that has not left
the ducts and often recurs in the same breast after lumpectomy or
in the other breast.
 Gene Therapy and Anti-estrogen Therapy Combined for ER.sup.+
Breast Cancer. A new function for the well-known anti-estrogen tamoxifen
has been discovered (29,30). Tamoxifen mimics the estrogen reversible
inhibitory properties of immunoglobulins IgA and IgM to inhibit
ER.sup.+ breast cancer cell growth. Tamoxifen acts in the complete
absence of estrogens. This indicates a function unrelated to its
classical interaction with the ER.sup.- (79). In related studies
(29,30) it has been recognized that the cell surface receptor mediating
the growth inhibitory effects of IgA and IgM is a member of the
immunoglobulin superfamily of receptors and shares the properties
of the poly-Ig receptor and Fc family receptors. The receptor properties
have been described and discussed (29,30). Data indicate that tamoxifen
only inhibits the growth of cells containing this immunoglobulin
receptor. Furthermore, the immunoglobulin mediating receptor appears
to be under the control of either sex steroid hormones or thyroid
hormones. Hence, loss of the sex steroid hormone receptor (or the
appropriate thyroid hormone receptor from among several thyroid
hormone receptors) causes an equal loss of the superfamily receptor.
This information can be combined to develop a new, more effective
gene therapy for breast cancer. ER.sup.- cells lacking the superfamily
receptor can be restored to immunoglobulin control by infection
with an immunoglobulin binding receptor DNA bearing virus vector,
using conventional DNA transfection techniques. As a result, tamoxifen
will then become effective even with the transfected ER.sup.- breast
cancer cells, and can be used to kill these cancers as is done today
with ER.sup.+ cancers. This is an entirely new combination of gene
therapy and anti-estrogen therapy.
 Gene Therapy and Immunotherapy Combined for ER.sup.- Breast
Cancer. Another approach to eradication of ER.sup.- disseminated
breast cancer has been devised. Gene therapy can be targeted to
human breast cancers via the over-expressed transferrin receptor.
Gene therapy using the poly-Ig-like Fc receptor will be used to
reestablish immune negative control of autonomous breast cancers
that would otherwise grow uncontrolled. In related studies (29,
30) it has been established that exposure of breast cancer cells
bearing this receptor to IgA or IgM leads to cell death by arresting
cancer cell growth. By developing gene therapy and coupling it with
either oral "immunization" to boost systemic immunoglobulins
or direct intravenous use of suitable human immunoglobulins, new
treatments for disseminated breast cancer can be established. The
rat mammary tumor models described above will also be used to establish
the first applications in vivo. Use of athymic nude mice and human
breast cancer xenografts will be used to establish human relevance.
 Disseminated or Metastatic Breast Cancer Eradication using
Radioactive .sup.59Fe. The radioisotope .sup.59Fe is a high-energy.gamma.
emitter that disrupts breast cancer cells in culture by fragmenting
DNA. This proposal plans the use of transferrin as a .sup.59Fe delivery
system to both disseminated ER.sup.+ and ER.sup.- breast cancers.
Because these cancers have such a high requirement for iron bound
to transferrin, the delivery system may only need low concentrations
of this high-energy isotope. Rat mammary tumor models are available
to investigate this therapy, as described above. It is expected
to be effective because growing cells concentrate more iron than
static normal cells. This modality may be especially effective with
blood replacement therapy, described as follows.
 Blood Replacement Therapy and Cancer Eradication. A number
of companies are now well advanced in the development of "artificial
blood". The FDA expects that within a year or two a blood substitute
will become available. Many treatments, such as gene therapy and
the above-described iron therapies may be enhanced. Others such
as standard adjuvant chemotherapy are expected to be more selective
and effective in substitute blood where the levels of interfering
substances can be regulated. This approach may also increase the
effectiveness of tamoxifen and the newer "pure" antiestrogens.
Since each treatment round is expected to last only a few weeks
to a month, a substitute blood product may be employed in one of
the above-described therapies, as part of the present breast cancer
 This approach may have special application to the treatment
of disseminated breast and other cancers because artificial blood
can be prepared free of diferric transferrin, which is usually present
in physiological human blood at about 1.0 gram per Liter. Addition
of low concentrations of radiolabled diferric transferrin to artificial
blood will avoid the dilution caused by the natural blood diferric
transferrin and therefore increase the effective dose of radioisotope
to cancer cells without exposing the body to high levels of radiation.
Part III: Reduction of Risk of Developing Breast Cancer
 Overview. The risk of developing breast cancer for women
in the United States has been rising steadily for the past several
decades. It will soon approach one in eight. It is fortunate that
new treatments and more effective screening methods have kept mortality
rates from rising dramatically. Nonetheless, more than one hundred
women are lost per day to breast cancer in the United States. Researchers
and health care providers understand that the first line of defense
against this disease is prevention. If the long term outlook for
all women is to be improved, and especially if our daughters to
be free of this threat, the focus must now be on finding a prevention.
 As described above, a recent breakthrough in understanding
how breast cancer grows reveals that, in its initial stages, breast
cancer cells are inhibited and even killed by the secretory immune
system (29,30). That means a part of our immune system can stop
early cancer cells from growing. During adult life, breasts produce
milk or milk-like fluids. These fluids contain high concentrations
of three immunoglobulins, IgA, IgM and IgG1. These are passed from
mother to child during breast-feeding, and protect the child from
bacterial infections. As a result of this discovery, and considering
the fact that long duration breast-feeding is known to reduce the
risk of breast cancer, it is proposed that these immunoglobulins
are likely to protect the mother against breast cancer via this
newly discovered inhibitory mechanism. This presents an unexpected
opportunity to rethink the problem of prevention and to apply new,
 The present plans for a new type of oral immunization for
breast cancer were advanced by another remarkable fact about the
secretory immune system. The immunocytes of this system permit mothers
to protect their suckling offspring from infectious pathogens. Because
both mother and young child are exposed to the same infectious agents
at the same time, but only the mother can develop immunity (or already
has it), how does the young offspring fight off disease? The answer
is that infectious agents orally entering the mother's body cause
an antibody response in areas of the intestinal mucosa called Peyer's
patches. These lymphoid structures cause the production of B immunocytes
that ultimately populate breast tissue. Once there, the B cells
secrete milk-borne immunoglobulins with specificity against the
offending infectious agents.
 It is proposed that oral "immunization" is expected
to be most effective during a first susceptibility age range "window"
(e.g., during puberty, or 9 and 19 years of age), and/or during
a second "window," after menopause, when secretory immune
system function decreases sharply. If the secretory immune system
of the breast is stimulated at times when women are known to be
most susceptible to breast cancer, it might be prevented or at least
the risk of occurrence considerably reduced.
 Finally, recent developments in mucosal cancers other than
breast cancer suggest another application of this discovery. As
discussed above, it is now understood that at least one mucosal
cancer is of bacterial origin. The bacterium Helicobacter pylori
is a Class I carcinogen thought to cause gastric cancer. This fact,
coupled with the discovery of new secretory immune functions in
the breast, supports the proposal that breast cancer might arise
from an infectious agent. Plainly, there is no known cause for 70%
or possibly more of breast cancers, although environmental carcinogens
are most often named as culprits. Nonetheless, bacterial participation
or a bacterial origin remains entirely possible. If such was proven,
the development of pathogen specific breast immunity via oral challenge
would offer a unique approach to immunization.
 In the history of cancer research, oral immunization has
been investigated mostly from the point of view of treatment. However,
there has been no serious application to either the treatment or
prevention of breast cancer, which is surprising since oral immunization
is so readily adaptable to mass populations of women of all ages
and all circumstances. Applying oral immunization as a new means
of preventing breast cancer is a preferred part of the breast cancer
total eradication program.
Oral Immunization to Reduce the Risk of Breast Cancer
 During adult life, breasts produce milk or milk-like fluids
that contain high levels of three immunoglobulins, IgA, IgM and
IgG1. In cell culture these immunoglobulins not only block early
breast cancer cell growth, but if elevated for a period of time,
will kill breast cancer cells. This discovery presents the unexpected
opportunity for a new oral immunization for breast cancer, taking
advantage of a basic function of the secretory immune system: the
natural production of IgA and IgM.
 It is proposed that oral immunization be administered when
it might be most effective, i.e., during the same susceptibility
age ranges that were identified in data collected from survivors
of the atomic bomb blasts during World War II, which showed an unexpected
pattern of breast cancer development. Those exposed to the radiation
between 9 and 19 years of age developed breast cancer at much higher
rates than survivors who were 30 years or older (45). This meant
there was a "window" early in life when breast tissue
was highly susceptible to DNA modifying mutations. This same pattern
became clear again when women survivors of Hodgkin's disease were
studied. Treatment of Hodgkin's requires agents that cause DNA damage.
Women treated between 11 and 19 develop breast cancer at higher
rates than women whose treatment began at later ages (46). Again,
young women appeared to have a "window" of susceptibility.
This same "window" is well known in experimental animals
exposed to carcinogens (47,48).
 Young women might not be alone in experiencing increased
susceptibility, however, as secretory immune system function decreases
sharply after menopause. This coincides with the time when breast
cancer rates achieve near their highest levels (145), and may represent
a second "window" not previously recognized. If the secretory
immune system of the breast is stimulated at times when women are
known to be most susceptible to breast cancer, it might be prevented
or at least the risk of occurrence considerably reduced.
 Rat mammary tumor models are being used to define the conditions
for increasing breast tissue IgA/IgM secreting immunocytes and determining
if this protects against the DNA synthesis dependent damage effects
of estrogens as carcinogens (49) as well as the effects of environmental
carcinogens (36). Conditions and appropriate oral bacterial challenges
(i.e. non-pathogenic and pathogenic E. coli) to increase breast
cell B immunocytes and therefore increased secretion of IgA/IgM
are being defined. It is known that women from the non-Western world
have high levels of antibodies to fecal E. coli in their milk. An
example study compares women from the United Kingdom and Sri Lanka
(146,148). These women also have the lowest risks of any worldwide.
In the present disclosure, it is now proposed that these microorganisms,
live or attenuated, or fragments or molecules from them, will be
potential agents for inducing breast cancer immunity (153). Oral
immunization is not the only administration route to be considered.
Nasal, rectal and vaginal administration must also be considered
(150,154). Antigen challenge may be required on a routine schedule
since oral immunity may not be permanent. Multiple challenges are
most likely necessary to maintain full immunity. This means placing
the challenge in a delivery form suitable to the site of challenge.
For orally administered treatments a tablet, a food product, a food
drink, or the like may be most useful and readily accepted by women.
When increased secretory immune function has been established, the
effect of the immunoglobulin cell-growth inhibitors on attenuation
of pre-malignant changes in breast ductal cells will be evaluated
as described (56-58).
 Another route for presenting the bacterial antigens to women
of all ages on a routine schedule is in genetically engineered food.
Potatoes, tomatoes and bananas can be genetically engineered to
express foreign antigens such as those from a virus or bacteria
(151,152). Oral immunity expressed by mucosal tissues can then be
achieved on a routine basis by consumption of the generically developed
food product (151-155). This route for administration has worldwide
applications. This technology has not been previously applied to
immunization of breast, prostate, colon, kidney, ovary or endometrial
PART IV: ERADICATION BY CONVENTIONAL IMMUNIZATION AGAINST THE INFECTIOUS
AGENTS THAT CAUSE BREAST CANCER
Infectious Origin of Breast Cancer and Mass Immunization
 A bacterial origin of cancer is a growing theme, however
this has not always been the case. The work of Marshall & Warren
in 1982-1984 was a milestone, in which their identification of a
genus Campylobacter organism in gastric ulcers from biopsy specimens
changed our thinking completely. Notably, H. pylori was unheard
of 20 years ago. The notion that gastric ulcers were caused by a
bacterium was unprecedented. Initially, the skeptical scientific
community rejected this idea. Today it is a recognized etiology
of a major human disease (15).
 Finding a clue to the origin of breast cancer comes from
the fact that 75% of breast cancer is invasive ductal carcinoma,
it is now proposed that the agent causing ductal carcinoma exists
in the ducts. As with all mucosal tissues, breast ducts are open
to the exterior; it is likely that infectious agents enter the ducts.
The question is can these organisms cause breast cancer?
 Today it is recognized that bacteria are long-term participants
in cancer development (14,18,19,103) including gastric (15,16),
colon (50,51), cervical (17), and very likely prostate (104) cancer.
The concept that bacteria are involved in colon cancer is more applicable
to breast cancer than the H. pylori model. Investigators studying
the colon model offer some important insights. The bacteria involved
in colon cancer are not obvious at histologic examination and do
not cause ulcers or severe inflammation as does H. pylori. Instead,
Bacteroides in colon produce fecapentaenes that are potent mitogens
(i.e. they cause cells to grow and divide) (51). By way of comparison,
breast atypical hyperplasia (i.e. increased growth rates above normal)
is known to be a pre-cancerous condition that also does not show
severe ulceration or major inflammation. It is expected that breast
bacteria also cause DNA synthesis and cell growth necessary to make
cancer-causing mutations permanent.
 Human milk contains many microorganisms (52-55). They are
believed to be skin and nipple contamination of expressed breast
milk. Then again, in some samples, the organisms were not usual
skin flora (52). In one larger study, several pathogenic organisms
were found along with non-pathogens (53). No virus particles were
identified in the milk. The fact that bacteria are present on the
nipple and surrounding skin certainly leaves open the possibility
that they might migrate into the ducts. To date, no microbes have
been investigated from the perspective of causative agents of breast
 Data supporting the likely presence of at least low levels
of bacteria in ducts comes from two different sources. First, human
milk lipids have been modified by chemical reactions to generate
genotoxic agents (56-58). The presence of these agents is measured
directly by DNA damage of breast cells obtained from milk. Because
genotoxic components are in freshly expressed milk, the products
are thought to be endogenous to the breast. In another study, samples
of freshly expressed human milk contained N-nitroso-dimethylamine,
nitrate and nitrate reducing microorganisms (59). This study concluded
that the compounds arose endogenously. These reactive nitrogen compounds
are likely cancer causing agents in other tissues. It highly probable
that reactive lipids and nitrogen compounds are formed endogenously
in human milk ducts.
 Considering that the development of breast cancer is a multi-step
process that often leads to tumor cell heterogeneity (111), it is
now proposed that the steps leading from normal duct epithelium
to hyperplasia to pre-malignant changes and finally to in situ carcinoma
and invasive ductal carcinoma (112) are caused by a relatively continuous
source of mutagenic agents that are present over a number of years.
Furthermore, because each change at each step requires DNA replication
in order to become permanent, it is likely that both estrogens and
the causative bacteria induce cell proliferation. Since bacterially
induced mammalian cell proliferation related to cancer development
is known, the proposed model for bacterial causation takes into
account the very well known (112) progression of normal breast epithelium
as it transitions to invasive ductal carcinoma (i.e. the form in
75% of breast cancers).
 In further studies, human milk, breast cancer samples and
normal breast tissue (reduction mammoplasty) will be examined for
bacterial content. They will be analyzed for bacterial content by
culture (aerobic and anaerobic) and PCR methods (62-65). Example
techniques to be applied include (i) use of specific PCR primers
for known and new bacteria, (ii) PCR amplification of conserved
16S rRNA sequences, and (iii) RDA-PCR which is also called "reverse
PCR". These can be used to identify unique infectious agents
in tissues, even in paraffin embedded specimens (61). PCR technology
pertinent to the identification of most microbes that this study
might encounter is now being applied.
 Next, colony-derived bacteria will be used in the "Ames
test" to identify mutagen production (66). Culture medium from
the bacteria isolated can be tested directly for mutagenic activity
using any of several strains of Salmonella. Candidate organisms
can be grown plus and minus human milk components to determine the
source of the mutagenic agents. The different types of existing
screening methods have been reviewed (67). Improvements in the Ames
test have been introduced to provide more quantitative evidence
that the assay is providing significant results with respect to
cancer bioassays (68,69). The results of this test will establish
which bacterial isolates produce mutagenic metabolites. The Ames
test can also be applied to demonstrate that the bacteria cause
an "oxidative burst" mediated by neurophils and macrophages.
In this case, the leukocytes are incubated with the bacteria to
generate the active mutagenic species. This approach resolves the
issue of whether the products of the bacteria are the mutagens themselves
or if the activation of leukocytes is required.
 Bacteria that meet the criteria described above will be
cultured and the medium tested with non-tumorigenic human breast
epithelial cells (Clonetics, San Diego, Calif.) or epithelial cells
derived from human milk (56,58) for transformation activity. The
human milk derived HBL-100 non-tumorigenic cells are also candidates
for this assay. The cells will be tested for colony formation in
soft agar. Tumor or transformed cells will form colonies. There
is a strong correlation between colony formation in soft agar and
tumorgenicity in animals. This approach will confirm that the Ames
test translates to transformation of human breast cancer cells.
Candidate cell lines will be analyzed for tumor formation in athymic
 In addition to the above analyses, the bacterial isolates
are expected to have an additional immunoprotective mechanism. Breast
secretions contain high concentrations of secretory IgA that kill
bacteria by the known antigen-antibody recognition function. This
is a first line of protection against breast duct infections by
many strains of bacteria. However, some bacteria can escape IgA
killing by secreting proteases that cleave the IgA into inactive
Fab and Fc fragments.
 In a final test, an animal model will be sought to determine
if infection leads to breast cancer development. Several strains
of inbred and outbred rats are highly susceptible to breast cancer
induction. Candidate bacteria will be introduced into the breast
by milk pump (60) and tumorigenesis monitored.
 The final step will be to use the attenuated organism, or
entities derived from the organism, to test for oral induced immunity
in human breast milk as has been described (148,149). Once immunity
against the mutagenic bacteria is established in human milk, studies
can move forward to determine if this method reduces the risk of
developing breast cancer.
 Surrogate end points will be used to estimate the effectiveness
of oral route administration of immunogen. Because a full clinical
trial of an oral immunization may require five or more years to
establish efficacy, DNA changes in cells isolated from milk or breast
fluids of women being administered the treatment will be studied.
By using this surrogate end point approach, a reduction in genotoxic
(i.e. mutagenic) events can be identified within months. This will
provide data to support more expensive long-term clinical studies.
 The same antigen preparation protocols can be used with
direct immunizations by standard methods such as intramuscular injection.
Many approaches to standard immunizations are known and commonly
employed in worldwide programs to eradicate infectious diseases.
The oral route of immunization utilizing the secretory immune system
is considered preferable because the antibody is delivered directly
into the intraductal space where the causative bacteria are then
 The program and procedures described above advance directly
to the core problem of eradication of breast cancer. Today there
are women battling breast cancer and others already developing the
pre-malignant changes leading to the disease. For them eradication
means availability of effective treatments, especially for chemotherapy
resistant metastatic cancer. Treatment will likely remain an important
issue for several years to come, and must therefore be given serious
continuing consideration. Very definitely, this means developing
new methods of eradicating metastatic breast cancer. One aspect
of the present eradication program departs from the usual chemotherapy
regimens to exploit the nutritional requirements for growth of breast
cancer. Cancer cells grown in culture invariably require iron in
the form of diferric transferrin for growth. With the advent of
the very powerful tool of serum-free defined cell culture (113-118),
this requirement could be established conclusively (29,30,113-117,135).
Variations in this requirement usually result from differences in
the level of storage of iron as ferritin (126) in the cells. When
ferritin stores are depleted, iron must be acquired from the outside
of the cells via the specific iron carrier transferrin (136) and
internalization via specific cell surface transferrin receptors
(127,128,134). The same logic/hypothesis employed in the breast
cancer eradication program is extendable to all epithelial cancers
(80% of the total cancers in humans) and for lymphoid origin cancers,
sarcomas (i.e. cancer of the connective tissues and muscle) and
cancers of the bone and nervous system. Indeed, it is absolutely
clear that iron is required for the growth of all cells because
of its involvement in metabolic processes and DNA synthesis (124,130,131).
It can be readily appreciated that the disclosed concepts and procedures
involving the exploitation and/or interruption of iron metabolism
required for cancer cell growth apply to many if not most cancers
 The eradication of breast cancer will likely come from a
different direction than the common themes of genes and cell signaling
pursued by so many investigators today. The work leading to the
new eradication program comes from the discovery of the role of
the secretory immune system in estrogen responsive breast cancer
cell growth, described in the two U.S. patent applications cited
above. Based on this discovery, the plan for an "oral immunization"
to protect breast cells from DNA synthesis dependent mutagenic events
has been devised. The goal is to reduce risk from the current 1
in 8 to a level of 1 in 40 or 50. This plan is consistent with the
growing, but still widely ignored, concept that the population of
the Western world is placed at higher risk for mucosal cancers because
we are "too clean." Our immune systems are not challenged
sufficiently to protect us.
 An additional immune based eradication plan finds support
in an unexpected source. It is known that bacterium Helicobacter
pylori is the most common cause of gastric ulcers. It also is the
first bacterium to be a definite cause (Class I) of a human cancer
as rated by the International Agency for Cancer Research (14,15).
A number of infectious agents have been documented to cause or contribute
to human cancers (14-19). However, this information has had little
or no impact on the search for the origin of breast cancer. More
commonly, it has been proposed that environmental chemicals cause
the possible majority of human cancers. Indeed, despite advances
in defining various breast cancer risks due to chemical exposure
(20), the perplexing element of randomness without clear indications
of chemical exposure has not been explained. An infectious origin
is random, and therefore a reasonable alternative. Breast cancer
risk is sometimes familial but most often is not genetic (i.e. inherited)
for most women. Indeed, this is a characteristic of the incidence
pattern of ulcers in H. pylori infected families (16). While not
all infected cohabitants develop gastric ulcers or gastric cancer,
members of a family tend to have higher incidences than average.
By seeking infectious agents as either the cause or as major contributors
to breast cancer development, and then using that information to
develop appropriate immunizations, it is expected that the incidence
of breast cancer will be reduced or eliminated.
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 While the preferred embodiments of the invention have been
shown and described, modifications thereof can be made by one skilled
in the art without departing from the spirit and teachings of the
invention. The embodiments described herein are exemplary only,
and are not intended to be limiting. Many variations and modifications
of the invention disclosed herein are possible and are within the
scope of the invention. For example, the foregoing discussion specifically
focuses on the eradication of breast cancer, however the same or
similar approaches can be employed to eradicate other types of cancers
of mucosal tissues, including prostate, ovary, endometrium, cervix,
vagina, colon, kidney, lung and nasopharynx. Cancers of those tissues,
together with breast cancer, account for 80% of all human cancer.
The disclosures of all patents, patent applications and publications
cited above are hereby incorporated herein by reference. The discussion
of certain references in the Description of Related Art, above,
is not an admission that they are prior art to the present invention,
especially any references that may have a publication date after
the priority date of this application.