||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
MALIGNANT LYMPHOMA OF THE BREAST
Institute of Oncology, Ljubljana,
Malignant lymphomas of the breast are
a rare disease. They may occur as primary or secondary tumors. Morphologically,
it is not possible to determine their primary or secondary nature1. The
criteria for defining a lymphomatous lesion in the breast as primary were
first proposed by Wiseman and Liao in 19722: 1) availability of adequate
histologic material; 2) documentation of breast involvement as a primary
site; 3) presence of breast tissue in or adjacent to lymphoma infiltrate;
4) no concurrent nodal disease except for the involvement of ipsilateral
axillary lymph nodes; and 5) no previous history of lymphoma involving
other organs or tissues. Strictly adhering to such criteria, some primary
breast lymphomas may be lost since no allowances are made for those primary
breast lymphoma cases that may present in higher clinical stages. Obviating
this, some authors consider as primary breast lymphomas all those cases
in which breast is the first or major site of presentation even though
subsequent staging procedures reveal involvement of other sites, such as
The lesion most commonly presents as
a unilateral breast mass in postmenopausal women (median age 55 to 60 years),
although it may occur at any age. In about 10% of cases, it is bilateral.
In men, breast lymphoma is exceedingly rare. A subset of patients, characteristically
from tropical Africa, are young women, during or immediately after pregnancy,
who present with massive bilateral breast swelling. The latter disease
is endemic in this part of the world. Histologic examination in these patients
reveals Burkitt’s or Burkitt-like lymphoma4. Non-African cases of this
type of lymphoma are also on record. The incidence of primary breast lymphoma
ranges from 0.04% to 0.5% of breast malignancies in most published series
1,3,5 .The incidence of secondary lymphoma in the breast is difficult to
ascertain, since many of those lesions are but one manifestation of disseminated
disease and are never biopsied.
Primary and secondary breast lymphomas
usually present grossly as a well defined uni- or multinodular mass of
soft or firmer white-gray tissue, sometimes with necrotic and hemorrhagic
foci.The tumor varies in size and may attain up to 20 cm in diameter.
The vast majority of breast lymphomas
are diffuse large cell B lymphomas as defined by recent WHO classification.
The latter lymphomas were given different names in older classification
schemes, such as reticulum cell sarcoma, histiocytic lymphoma, large cell
cleaved or noncleaved lymphomas, centroblastic or immunoblastic lymphoma,
etc. In addition to large cell B lymphomas, a variety of other types of
lymphoma may also manifest as primary or secondary tumors in the breast.
The relation of pre-existing mammary tissue and infiltrating lymphoma varies.
In some cases, the bulk of the tumor is located in subcutaneous fatty tissue
and breast parenchyma is at its periphery; in other cases, the ducts and
lobules of breast tissue are embedded in the infiltrate, while in rare
cases pre-existent tissue is overgrown by lymphoma and barely visible.
In such cases, remnants of ducts and lobules may only be revealed by using
keratin immunostaining. Stroma may be scant or more abundant, sometimes
sclerotic and hyalinized. Although most of the tumors are grossly circumscribed,
a microscopically different degree of infiltration of the surrounding tissue
is always evident.
Diffuse large cell B lymphoma1,3,5-8:
This type of lymphoma is characterized by large lymphoma cells with oval
or indented nuclei, with one to three nucleoli and with a narrow rim of
basophilic cytoplasm; such cells generally resemble centroblasts. Different
number of immunoblasts are frequently admixed, sometimes such cells are
predominant. Mitoses are usually numerous. In some cases, cells appear
more pleomorphic with wider variation in cell forms and sizes; smaller
reactive lymphocytes of B or T types are also present in the infiltrate.
Occasionally, reactive histiocytes are numerous, imparting a “starry sky”
appearance to the tumor. Adjacent mammary tissue may exhibit lobular atrophy
or lymphocytic lobulitis which may be very prominent (lymphocytic mastopathy).
Lymphoma cells are immunoreactive for CD20, CD79a, CD45RB, and negative
for CD3 and CD45RO. Cases with immunoblastic features may show light chain
restriction. Exceptionally, lymphoma cells may express CD30 antigen.
Follicular lymphoma 1,3,5,7,8: It features
neoplastic follicles composed of centrocytes and centroblasts in different
proportions and may be graded into 2 or 3 grades, depending on the number
of centroblasts inside the neoplastic follicles. Immunohistochemically,
the lymphoma cells show positivity for B cell antigens, and for CD10 and
bcl- 2, and are negative for CD5 and CD23. Follicular dendritic cells in
tight clusters, positive for CD21, delineate neoplastic follicles. Selective
infiltration of ductal-lobu- lar units by lymphomas of other types, such
as diffuse large cell B lymphoma may mimic neoplastic follicles and could
be confounded for true follicular lymphoma.
Burkitt’s lymphoma4,8: The infiltrate
in this lymphoma is composed of medium-sized cells with round nuclei, multiple
central nucleoli, coarse chromatin and rather thick nuclear membrane. The
cytoplasm is moderate in amount, basophilic with fine vacuoles containing
lipids. Mitoses are very numerous. Cells grow in a cohesive pattern, they
square off with each other. Numerous tingiblebody macrophages are evenly
scattered among lymphoma cells producing characteristic but in no way pathognomonic
“starry sky” appearance of the lymphoma. The breast parenchyma is usually
hyperplastic and secretory. Ki-67 fraction of viable cells is 100%. Immunohistochemically,
pan-B markers are positive, surface immunoglobulins, usually of IgM type,
are also positive, while CD5, bcl-2, and TdT are negative. EBV is frequently
demonstrated in endemic but not in sporadic cases. IgH and IgL genes are
rearranged. Burkitt-like lymphoma is similar in morphological appearance
but immunoblast and centroblast-like cells are also admixed.
Extranodal marginal-zone B-cell lymphoma
of MALT type1,3,5 : An undetermined number of breast lymphoma cases belong
to the category of MALT lymphoma. The breast is considered to be part of
a common mucosal immune system9 and may, during an autoimmune process,
acquire lymphoid tissue in lymphoma. Most recent series of breast lymphoma
have some cases of MALT lymphoma included. Typically, lymphoma of this
type is composed of small lymphocytes, monocytoid (marginal zone type)
cells and plasma cells. The latter may dominate the whole microscopic aspect
of the lesion. Larger blast type cells may also be present. The infiltrate
may be vaguely nodular, reactive follicles may be seen, some of them colonized
by monocytoid cells. The lymphoepithelial lesion, i.e. infiltration of
the ductal/lobular epithelium by monocytoid (centrocyte-like) cells was
originally overestimated as a diagnostic criterion for MALT lymphoma of
the breast; its presence is not a prerequisite for diagnosis. Furthermore,
it has become increasingly evident that breast epithelium is infiltrated
by lymphoma cells of a variety of lymphomas and even more commonly by T
reactive cells admixed to lymphomatous infiltrate. Immunohistochemically,
MALT lymphoma cells express pan-B markers such as CD20 and CD79a, it is
usually bcl-2 positive but negative for CD10, CD5, and CD23. The translocation
t(11;18)(q21;q21) has been identified in many MALT lymphomas; analysis
did not include breast cases10. The same holds true for recently described
trisomy 3 identified in a number of MALT lymphomas 11. Breast may also
be involved by secondary MALT lymphoma originating at another MALT site.
Some other types of lymphoma may also rarely present in the breast, as
primary or secondary lesions, including lymphoblastic lymphoma of either
B or T type, extremely rarely peripheral T cell lymphoma, and secondary
small lymphocytic lymphoma/CLL or mantle cell lymphoma.
Large cell malignant lymphomas may,
in certain instances, be misdiagnosed as poorly differentiated duct or
lobular carcinoma. Immunohistochemical reactions for keratin resolve any
possible dilemma in such cases. Myeloid cell tumors may also be confounded
for malignant lymphomas; if basic immunoreactions are inconclusive, myeloperoxidase
staining to exclude the former possibility should be used. Inflammatory
conditions may mimic MALT lymphomas; in some cases immunohistochemical
reactions, flow cytometry and molecular genetic analysis should be employed
to determine clonality of the lesion. Reactive follicular hyperplasia can
be differentiated from follicular lymphoma by using bcl-2 immunoreactions;
reactive follicles are bcl-2 negative. The issue of so-called pseudolymphoma
remains unresolved; many authors believe that they really represent MALT
Prognosis and Treatment
Primary breast lymphoma behave in a
similar way as lymphomas of corresponding types and stages in other localizations.
Localized low-grade lesions, such as MALT type lymphomas, are treated locally
by surgery and/or radiation; high grade tumors require systemic chemotherapy
with or without irradiation.
1. MATTIA AR, FERRY JA, HARRIS NL.
Breast lymphoma. A Bcell spectrum including low grade B-cell lymphoma of
mucosa associated lymphoid tissue. Am J Surg Pathol 1993;17:574-87.
2. WISEMAN C, LIAO KT . Primary lymphoma
of the breast. Cancer 1972;29:1705-12.
3. HUGH JC, JACKSON FI, HANSON J, POPPEMA
S . Primary breast lymphoma. An immunohistologic study of 20 new cases.
4. SHEPHERD JJ, WRIGHT DH . Burkitt’s
lymphoma presenting as bilateral swelling of the breast in women of child-bearing
age. Br J Surg 1967;54:776-80.
5. LAMOVEC J, JANCAR J. Primary malignant
lymphoma of the breast. Lymphoma of the mucosa-associated lymphoid tissue.
6. ABBONDANZO SL, SEIDMAN JD, LEFKOWITZ
M, TAVASSOLI FA, KRISHNAN J. Primary diffuse large B-cell lymphoma of the
breast. A clinicopathologic study of 31 cases. Pathol Res Pract 1996;192:37-43.
7. BOBROW LG, RICHARDS MA, HAPPERFIELD
LC, ISAACSON PG, LAMMIE GA, MILLIS RR. Breast lymphomas: a clinicopathologic
review. Hum Pathol 1993;24:274-8.
8. LIN Y, GOVINDAN R, HESS JL. Malignant
hematopoietic breast tumors. Am J Clin Pathol 1997;107:177-86.
9. BIENENSTOCK J, BOFUS AD. Review:
mucosal immunology. Immunology 1980;41:249-70.
10. OTT G, KATZENBERGER T, GREINER
A, KALLA J, ROSENWALD A, HEINRICH U, OTT MM, MULLER-HERMELINK HK . The
t(11;18)(q21;q21) chromosome translocation is a frequent and specific aberration
in low-grade but not high-grade malignant non-Hodgkin’s lymphomas of the
mucosa-associated lymphoid tissue (MALT) type. Cancer Res 1997;57:3944-8.
11. WOTHERSPOON AC, FINN TM, ISAACSON PG. Trisomy 3 in low-grade B-cell
lymphomas of mucosa-associated lymphoid tissue. Blood 1995;85:2000-4.
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
RECENT ADVANCES IN MOLECULAR GENETICS
OF BREAST CANCER
K. Pavelić, K. Gall-Trošelj
Ruđer Bošković Institute, Division
of Molecular Medicine, Zagreb, Croatia
Breast cancer is among the most common
tumors affecting women. It is characterized by a number of genetic aberrations.
Five to 10% of all cases are estimated to be inherited. The hereditary
breast and ovarian cancer syndrome includes genetic alterations of various
susceptibility genes, particularly BRCA 1 and BRCA 2. Breast tumors in
patients with a germ-line mutations in the BRCA 1 and BRCA 2 gene have
an increase in additional genetic defects compared with sporadic breast
tumors. Accumulation of somatic genetic changes during tumor progression
may follow a specific and more aggressive pathway of chromosome damage
in these individuals. Recent advances in genomics and bioinformatics, particularly
in DNA-sequencing approaches and DNA-chip technology are revolutionizing
target identification of small molecules. Here we review some new findings
in the function of BRCA 1 gene function. A major BRCA 1 downstream target
gene is the DNA damage-responsive gene GADD 45. Induction of BRCA 1 triggers
apoptosis through activation of c-Jun N-terminal kinase/stress-activated
protein kinase ( JNK/SAPK). BRCA 1 interacts with the SWI/SNF complex which
controls DNA structure. SWI/ SNF is a chromatin remodeling complex important
in gene expression. New knowledge about the genetic portrait of breast
tumor is coming from differential gene expression profiling using microarrays.
Human genome studies as well as development of “DNA chips” provide a window
for observing patterns of gene activity in cells, which will revolutionize
cancer classification. Knowledge of the molecular characteristics of breast
tumor has already made it possible to identify those breast cancer patients
who could benefit from therapies that target these features. Progress in
basic research in signaling provides the opportunity to attack signal-transduction
targets involved in proliferation, survival, invasion, angiogenesis, metastasis
and resistance. Exciting knowledge in breast cancer biology is rapidly
accumulating in parallel with recent developments in rational selection
and validation of relevant targets that provide unique opportunities for
development of “intelligent” therapeutics.
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
IDENTIFICATION OF NEW MOLECULAR
TARGETS FOR THE TREATMENT OF BREAST CANCER
E. A. G. Blomme1, F. Del Piero2,
K. L. Kolaja1
1Pharmacia Corporation, Molecular
and Experimental Toxicology and Pathology, Skokie, IL, USA, and 2University
of Pennsylvania, School of Veterinary Medicine, Department of Pathobiology
and Department of Clinical Studies, New Bolton Center, PA, USA
SUMMARY - The completion of the human
genome sequence provides unique opportunities to identify new molecular
targets for a variety of diseased conditions, especially for neoplastic
diseases. Breast cancer is an ideal disease for the implementation of the
recently developed, sophisticated genomic technologies, which permit the
study of expression of many genes or proteins simultaneously, an approach
known as molecular profiling. This approach is considered a major step
forward in the development of new drugs that are more effective and less
toxic than the current generation of antitumor agents. In this paper, we
briefly review the current and future genomics technologies, such as DNA
microarrays and proteomics techniques, and their use in the identification
of new molecular targets for the treatment of breast cancer. We also discuss
the challenge associated with the development of bioinformatics tools to
analyze the massive number of data points generated by these technologies.
Proof of principle is now emerging, demonstrating that selective agents
against abnormal or mutated gene products can indeed be useful in the treatment
of cancer. However, despite heavy investment in genomics research by the
pharmaceutical industry, the full impact of genomics on drug discovery
has yet to be fully demonstrated.
Key words: Genomics; Microarrays;
Proteomics; Molecular target
The completion of the human genome
sequence provides a quantum lead towards identifying new molecular targets
for a variety of diseased conditions, especially for neoplastic diseases1,2.
The repositories of genes and their regulatory sequences represent the
starting point of a new challenge, understanding how the 30,000-40,000
genes present in the human genome and their protein products interact and
function. In addition of providing the unique opportunity to better understand
basic biology and to identify the molecular basis of diseases, the annotation
of the human genome offers the promise of an increased rate of drug discovery
and development. Breast cancer is a major health problem worldwide and
consequently, a large amount of research effort has been focused on the
molecular understanding of this disease3. The medical treatment of cancer
still has many unmet needs4. The main curative therapies (surgery and radiation)
are usually successful only at an early stage, and existing chemotherapeutic
treatments are largely palliative. The majority of the current antitumor
agents have been unveiled during screening in cytotoxicity assays, although
some have also been designed to act on defined molecular targets. However,
none of the established cancer drugs were developed in the light of a clear
understanding of the molecular differences between neoplastic and normal
cells. Breast cancer is an ideal disease for the implementation of the
recently developed, sophisticated genomic technologies, which permit to
study the expression of many genes or proteins simultaneously, an approach
known as molecular profiling. This approach is considered a major step
forward in the development of new drugs that are more effective and less
toxic than the current generation of antitumor agents. In this paper, we
will review the application of genomic technologies for the rational identification
of new therapeutic targets for breast cancer. DNA Microarray and Molecular
Transcription Profiling Molecular transcription profiling is the large-scale
analysis of gene expression using DNA array technologies5,6. DNA microarrays
have only been recently introduced to the scientific community7. Microarrays
consist of rows and rows of microscopic spots, each of which contains an
identical single-stranded polymeric molecule of deoxyribonucleotide (typically
oligonucleotides or cDNAs, the probe) attached to a solid support, such
as a glass slide or a miniature silicon chip. These arrays can accommodate
up to tens of thousands of spots and can be used for high-throughput studies
of genomic structure and studies of active gene expression. Figure 1 provides
an illustration of these DNA arrays. These arrays use the principle of
specific DNA base pairing, i.e. A-T and GC, to allow the large-scale analysis
of mRNA abundance as an indicator of gene expression, to detect polymorphisms
within a population, or to detect new genes, as unknown DNA sequences can
be analyzed8. Over the past few years, a number of different commercially
available array products have been introduced. Although most of these products
remain relatively expensive, their cost is regularly decreasing, and these
products should soon become affordable for most laboratories. Therefore,
it is critical that most cancer scientists become familiar with this technology.
Recently, customized or in-house microarrays have grown in popularity to
help investigators focus on their particular area of interest without being
distracted by the huge volume of data generated by some commercial microarrays9.
It should be mentioned that, although the microarray technology is the
most widely used technique for gene expression analysis, others are available,
including serial analysis of gene expression (SAGE) , differential hybridization,
differential display or GeneCalling®10. The application of arrays to genomic
studies includes the search for single nucleotide polymorphisms (SNPs)
and a powerful application of these studies is in the field of pharmacogenomics6.
Because each individual has a slightly different genetic makeup, each will
have a unique set of SNPs. SNPs are the most frequent form of genetic variation
(~3 million/person or approximately 1 SNP/kb), are highly stable, and are
relatively easy to identify. Programs to detect and map SNPs in the human
genome are well underway with the ultimate aim of establishing a SNP map
of the genome11. When SNP analysis is used in conjunction with analytical
techniques, such as genetic-linkage mapping or association analysis, a
genetic propensity for predisposition to disease, unique metabolism, or
adverse events can be identified.Although these SNPs may not be the actual
cause of disease, their utility lies in their potential to help predict
how an individual may respond to a particular drug. Pharmacogenomics may,
therefore, help identify at-risk patients prior to treatment and prevent
adverse drug reactions. Furthermore, SNPs may be useful to predict whether
a certain drug would be effective in a patient with a specific disease.
Although most marketed drugs are efficacious in a vast majority of the
patient population, pharmacogenomics offers an opportunity to resurrect
drugs that have been discarded because of low efficacy or adverse effects
in the entire patient population. In this regard, pharmacogenomics can
undoubtedly contribute to a better design of clinical trials in the future.
Molecular transcription profiling analyses have already profoundly enhanced
our understanding of many diseases, including breast cancer5,12,13. Cell
function can be best understood by determining the transcription level
of all genes in the genome (the transcriptome). The next step for transcriptional
analysis will be the rapid identification and evaluation of potential therapeutic
targets. In cancer, the accumulation and combinatorial effects of abnormalities,
driving the initiation and progression of cancer, result from mutations
and/or changes in expression level of cancercausing genes14. Therefore,
therapeutic agents that would target the key molecular abnormalities that
lead to malignant progression, have the potential of being more selective
than the current non-specific cytotoxic agents, and therefore, more efficacious
and less toxic. Proof of principle is now emerging that these selective
agents can indeed be useful for the treatment of cancer14. For instance,
the HER- 2/neu oncogene is overexpressed in approximately 30 percent of
breast cancers, and these tumors are more aggressive and somewhat more
resistant to chemotherapy than those not overexpressing the oncogenes15.
These observations have led to the development of a monoclonal antibody
(Herceptin® or trastuzumab) against the extracellular domain of this receptor
tyrosine kinase4. Several clinical trials have demonstrated an improved
response rate, a prolongation of the time to disease progression, and an
increased overall survival compared to the standard of care, demonstrating
the power of this genes-to-drugs paradigm for drug discovery.Herceptin®
was approved by the U.S. Federal Drug Administration (FDA) in 1998 for
the treatment of HER-2-positive breast cancer. Other receptor tyrosine
kinases are frequently overexpressed in cancer. Therefore, several epidermal
growth factor (EGF) receptor tyrosine kinase inhibitors, such as ZD-1839
(Iressa®), are currently being developed for the treatment of various cancer
types, and results of preclinical studies and preliminary clinical trials
indicate that the EGF receptor is indeed a valid target for anticancer
therapy16,17. Because microarray technologies examine the expression profile
of thousands of genes simultaneously at the mRNA level, it is now possible
to study the sum total differences in gene expression between normal and
diseased cells. This clearly will lead to the identification of new subtypes
of tumors and will not only help the pathologist provide a more refined
biological-based diagnosis, but will also enable scientists to identify
previously unrecognized therapeutic targets in a rapid and efficient manner9,13,18.
A major limitation of transcript profiling
is that transcriptional activity does not necessarily reflect the activity
of the protein product of a particular gene. This is mostly due to variation
in cellular location and to complex and versatile protein regulation mechanisms,
such as context-dependent post-translational phosphorylation, sulphation
and glycosylation5. In addition, assigning a role for a protein based on
a gene sequence information is not always feasible, because gene sequence
reveals little information about protein function and disease relevance19.
Therefore, a recent focus has been shifted towards proteomics, a protein-based
approach to provide functional and expression information for proteins
on a genome-wide scale (the proteome).
Currently, the proteomics tools consist
mostly of electrophoresis or chromatography coupled with mass spectrometry19.
Several new technologies have recently been introduced for high-throughput
protein characterization and discovery, such as protein arrays and proteome-scale
screens for generic enzyme activities (e.g., protease and phosphatase)19.
Applying these technologies to various diseases, and to breast cancer in
particular, can work in concert with genomic technologies to identify new
potential therapeutic targets. The challenge facing proteomics is, however,
enormous, since it is estimated that approximately 75 percent of proteins
in multicellular organisms have, as of yet, no known cellular function.
Furthermore, human genes are fairly complex, incorporating variable numbers
of protein domains into sophisticated functional products, with further
protein diversity provided by alternative splicing17. Finally, protein-based
technologies are extremely low throughput and more challenging to develop
compared to transcription profiling techniques. Laser Capture Microdissection
Genomic and proteomic analysis of cells in their native environment can
provide the most accurate picture of the alterations that occur in vivo
during the disease state. In vitro, cells are not subject to the endocrine
and paracrine signals that regulate their overall behavior. However, studying
tissues is not an easy task, mostly because tissues are complex three-dimensional
structures, composed of large numbers of perpetually interacting cell populations.
In the case of breast cancer, neoplastic cells may only account for a small
proportion of the tissue analyzed, and the overall genomic and proteomic
analyses may be confounded by the presence of large numbers of non-neoplastic
cells, such as fibroblasts, endothelial cells or macrophages.
To overcome these confounding factors,
powerful analytical algorithms have been developed to gauge the relative
abundance of an unknown cell subpopulation within tissue samples. Such
algorithms use known genes associated with particular subpopulations of
cells as reference values to estimate the proportion of cancer cells, stromal
cells and inflammatory cells5. An alternative to the use of these algorithms
is the implementation of microdissection techniques. Laser capture microdissection
(LCM) is a technology for rapid and easy procurement of a microscopic and
pure cell subpopulation from its complex tissue milieu12. The advantage
is that LCM permits the investigator to focus directly on the disease subpopulation
or compare several subpopulations of tissue cells from the same patient’s
sample. In addition, recent data suggest that LCM would allow quantitative
gene expression analysis in formalin-fixed, paraffin-embedded tissues,
allowing to take advantage of large numbers of archived pathological tissue
specimens20. The disadvantages of LCM are that it is resource intensive
and provides only limited amounts of cellular material to study, although
the recent development of reliable amplification protocols has partially
solved this problem12. LCM has been successfully applied in the study of
breast cancer pathogenesis and the identification of potential therapeutic
targets, and it is likely that this technology will become a necessary
component of the drug discovery process, as well as the cancer biology
A critical step after the identification
of a putative therapeutic target is to validate the target’s relevance
to the disease process21. An important part of this validation step can
be achieved through the development of appropriate preclinical animal disease
models. In particular, scientists now have the ability to genetically manipulate
the mouse through transgenesis and gene targeting to test hypotheses regarding
gene function and their role in disease. These knockout or transgenic mouse
models (an important part of functional genomics) provide a powerful tool
to the gene-to-drug paradigm for drug discovery22. The literature contains
an enormous number of examples where a genetically engineered mouse model
has helped better define the relevance of a specific gene product in a
disease model. For instance, p53 knockout mice rapidly develop neoplasms
in various tissues, as seen in patients with the Li-Fraumeni syndrome who
have germline mutations of p5323-25. Other molecular tools are also available
for target validation in animal models, such as antisense oligonucleotides,
ribozymes and neutralizing antibodies4,26.
Another method used to validate a target
for breast cancer is analysis of its expression in a large population of
breast tumors by immunohistochemistry and/or in situ hybridization10. While
such a validation step can still be achieved analyzing one slide at a time,
this can be very tedious and laborious. Hence, the recent development of
tissue microarrays to increase the throughput of the process27. Tissue
microarrays consist of hundreds to thousands cylindrical tissue biopsies,
ranging from 0.6 up to 4 mm in diameter, each from a different patient,
all distributed on a single glass slide. The tissue microarray technology
has been tested and validated in several cancer types, including breast
cancer28. The data confirmed many of the clinicopathological correlation
of gene amplifications or immunostaining reactions reported with conventional
techniques on the basis of whole tumor analysis5. Full automation of tissue
array creation and screening is being developed to expeditiously validate
the large numbers of newly identified potential therapeutic targets for
While the cancer biologist will definitely
benefit from these new technologies, it is clear that one of the major
current challenges is to develop informatics techniques to facilitate the
processing and analysis of this large amount of data. Interpretation of
data and the development of models that facilitate the understanding of
specific biological phenomena have become priorities. A common characteristic
of contemporary drug discovery projects is their increasing complexity
compared to the past, where discovery efforts were largely dominated by
chemistry and pharmacology. Genomics techniques have led to the creation
of a new research discipline, called bioinformatics.
Initially, the focus of bioinformatics
was on the analysis, processing, and archiving of genomic sequence data.
Because of the rapid progress of the large-scale genome sequencing projects
culminating in a “first draft” of the human genome, bioinformatics is now
moving from the genome to the transcriptome and proteome level, with the
focus shifting from the evaluation and annotation of genomic sequence data
to the analysis of actual gene products29. The use of DNA microarrays generates
a massive number of individual data points, which must then be analyzed
by using data mining tools sufficiently sophisticated to categorize all
these data group them in a meaningful manner8. These analytical tools are
aimed toward the hunting of potential drug targets, the deciphering of
possible cellular pathways, and the generation of hypotheses regarding
the potential roles of certain genes. Protein-focused bioinformatics efforts
aim to better understand the cellular expression, post-translational modifications,
family relationships, structures and functions of proteins, as well as
to evaluate their potential as suitable drug targets29.
Several private and public databases
exist for genome mapping, nucleic acid and protein sequences, and protein
structures. In particular, Internet resources provide invaluable information
and make available databases with relevance to drug discovery and genomic
technologies21. For instance, the DNA sequence information available in
these public databases can be used to identify transcripts differentially
expressed in normal breast epithelial cells and breast tumor cells30. Similar
approaches can easily be adapted and applied to other tumor types with
sufficient transcript sequences available in the public databases.
In drug discovery, initial expectations
of new technologies are often too high. Despite large-magnitude efforts
and resource commitments to new technologies (such as high-throughput screening
and combinatorial chemistry) in the last decade, our ability to produce
high-quality drug candidates has not become significantly enhanced. Obviously,
the long period of time it takes for novel drugs to reach the market makes
it very difficult to assess the immediate impact of a technology. In the
1990s, the primary goal of genomics research in the pharmaceutical industry
was to increase the number of identified molecular targets and to gain
proprietary rights to use those targets31. This goal has partly been successful:
many more targets have been identified, intellectual property abounds,
and proof of principle that these targets are valid has been demonstrated.
However, an increased productivity of the pharmaceutical industry has yet
to be demonstrated. Will genomics have its expected impact on drug discovery?
First, it is important to remember that these technologies add a substantial
new level of complexity to the drug discovery process, which will require
the formation of large multidisciplinary teams to properly integrate these
tools in the existing drug discovery and development process. In addition,
in the drug discovery and development process, a long road with many hurdles
separate the identification of a relevant gene target and the regulatory
approval of an innovative drug: is the new gene a drugable target? Can
adequate pharmacokinetic properties be achieved? Does the drug modulate
the target in patients? Are there side effects and if so, are they manageable?
Is there significant therapeutic benefit? What biomarker can be used to
quickly predict efficacy? Are combinatorial genomebased therapies necessary
for efficacy? What is the appropriate patient population? While drug development
has not become much easier or faster, the promise of a major impact of
genomics on drug discovery is still largely intact: it is likely that the
genomics technologies will provide scientists with a large new collection
of molecular targets for the treatment of various diseases, including breast
cancer. However, success will require the proper and effective use of complementary
technologies, expertise and innovative design of clinical trials, and proper
integration of these various aspects is likely to become the definitive
competitive advantage to pharmaceutical companies.
1. International Human Genome Sequencing
Consortium. Initial sequencing and analysis of the human genome. Nature
2. VENTER JC, ADAMS MD, MYERS EW, LI
PW, MURAL RJ, SUTTON GG, et al. The sequence of the human genome. Science
3. HORTOBAGYI GN. Treatment of breast
cancer. N Engl J Med 1998;339:974-84.
4. GIBBS J. Mechanism-based target
identification and drug discovery in cancer research. Science 2002;287:1969-73.
5. LIOTTA L, PETRICOIN E. Molecular
profiling of human cancer. Nat Rev Genet 2000;1:48-56.
6. GRAVES DL. Powerful tools for genetic
analysis come of age. Trends Biotechnol 1999;17:127-34.
7. SCHENA M, SHALON D, DAVIS RW, BROWN
PO. Quantitative monitoring of gene expression patterns with a complementary
DNA microarray. Science 1995;270:368-9.
8. MAUGHAN NJ, LEWIS FA, SMITH V. An
introduction to arrays. J Pathol 2001;195:3-6.
9. ALIZADEH AA, ROSS DT, PEROU CM,
Van de RIJN M. Towards a novel classification of human malignancies based
on gene expression patterns. J Pathol 2001;195:41-52.
10. PEALE FVJr, GERRITSEN ME. Gene
profiling techniques and their application in angiogenesis and vascular
development. J Pathol 2001;195:7-19. 11. BENTLEY DR. The human genome project:
an overview. Med Res Rev 2000;20:189-96.
12. SGROI DC, TENG S, ROBINSON G, LeVANGIE
R, HUDSON JR, ELKAHLOUN AG. In vivo gene expression profile analysis of
human breast cancer progression. Cancer Res 1999;59: 5656-61.
13. LAKHANI SR, ASHWORTH A. Microarray
and histopathological analysis of tumours: the future and the past? Nat
14. CLARKE PA, te POELE R, WOOSTER
R, WORKMAN P. Gene expression microarray analysis in cancer biology, pharmacology,
and drug development: progress and potential. Biochem Pharmacol 2001;62:1311-36.
15. SLAMON DJ, CLARK GM, WONG SG, LEVIN
WJ, ULLRICH A, McGUIRE WL. Human breast cancer: correlation of relapse
and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177-82.
16. ADJEI AA. Epidermal growth factor
receptor tyrosine kinase inhibitors in cancer therapy. Drugs Fut 2001;26:1087-92.
17. WORKMAN P, CLARKE PA. Innovative
cancer drug targets: genomics, transcriptomics and clinomics. Expert Opin
18. ALIZADEH AA, EISEN MB, DAVIS RE,
MA C, LOSSOS IS, ROSENWALD A, et al. Distinct types of diffuse large B-cell
lymphoma identified by gene expression profiling. Nature 2000;403 (6769):503-11.
19. EDWARDS AM, ARROWSMITH CH, des
PALLIERES B. Proteomics: new tools for a new era. Modern Drug Discov 2000;3:
20. SPECHT K, RICHTER T, MUELLER U,
WALCH A, WERNER M, HOEFLER H. Quantitative gene expression analysis in
microdissected archival formalin-fixed and paraffin-embedded tumor tissue.
Am J Pathol 2001;158:419-29.
21. SAWYER TK. Deciphering therapeutic
targets. Biotechniques 2001;30:1086-90.
2. WEST DB, IAKOUGOVA O, OLSSON C,
ROSS D, OHMEN J, CHATTERJEE A. Mouse genetics/genomics: an effective approach
for drug discovery and validation. Med Res Rev 2000;20: 216-30.
23. DONEHOWER LA, HARVEY M, SLAGLE
BL, McARTHUR MJ, MONTGOMERY CAJR, BUTEL JS et al. Mice deficient for p53
are developmentally normal but susceptible to spontaneous tumors. Nature
24. HARVEY M, McARTHUR MJ, MONTGOMERY
CAJr, BUTEL JS, BRADLEY A, DONEHOWER LA. Spontaneous and carcinogen-induced
tumorigenesis in p53-deficient mice. Nat Genet 1993;5:225-9.
25. VARLEY JM, EVANS DG, BIRCH JM.
Li-Fraumeni syndrome: a molecular and clinical review. Br J Cancer 1997;76:1-14.
26. TAYLOR MF. Target validation and
functional analyses using antisense oligonucleotides. Expert Opin Ther
27. KONONEN J, BUBENDORF L, KALLIONIEMI
A, BARLUND M, SCHRAML P, LEIGHTON S, et al. Tissue microarrays for high-throughput
molecular profiling of tumor specimens. Nat Med 1998;4:844-7.
28. HEISKANEN M, KONONEN J, BARLUND
M, TORHORST J, SAUTER G, KALLIONIEMI A, et al. CGH, cDNA and tissue microarray
analyses implicate FGFR2 amplification in a small subset of breast tumors.
Anal Cell Pathol 2001;22:229-34.
29. BAJORATH J. Rational drug discovery
revisited: interfacing experimental programs with bio- and chemo-informatics.
Drug Discovery Today 2001;6:989-95.
30. LEERKES MR, CABALLERO OL, MACKAY
A, TORLONI H, O’HARE MJ, SIMPSON AJG, et al. In silico comparison of the
transcriptome derived from purified normal breast cells and breast tumor
cell lines reveals candidate upregulated genes in breast tumor cells. Genomics
31. WARD SJ. Impact of genomics in
drug discovery. Biotechniques 2001;31:626-34.
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
PROGNOSTIC VALUE OF HER-2/NEU IN
BREAST CARCINOMA PATIENTS
University Department of Pathology,
Zagreb University Hospital Center, Zagreb, Croatia
The development and spread of malignant
tumors is a multi-step process, involving a variety of alterations in the
mechanisms controlling cell proliferation, differentiation and genetic
alterations. Understanding of the biological process involved in tumorigenesis
has practical application in the clinical areas of diagnosis, prognosis
and treatment1,2. The goal of clinicians managing patients with malignancy
is to create therapy to give maximum benefit to each individual patient.
The decisions are usually based in part on predictors of the likely biological
behavior of a given tumor. The major tumor characteristics in breast cancer
known to be of value in prognosis include tumor size, tumor histologic
type and grade, axillary lymph node status, steroid hormone receptor status,
ploidy, and cell kinetics3,4. Establishing prognosis based on these parameters
is successful to a large extent, but they still fail to accurately predict
the clinical course of all patients. Therefore, the search for better means
of integrating prognostic data and for new prognostic markers in breast
cancer patients still remains a major goal.
The human epidermal growth factor receptor-2
(HER-2) protooncogene encodes 185 transmembrane glycoprotein, often simply
calledHER-2/neu or c-erbB- 2 protein receptor. In vitro and animal studies
have indicated that HER-2/neu gene amplification and protein overexpression
play a role in oncogenic transformation, tumorigenesis and metastasis.
Furthermore, the growth of tumors and human breast carcinoma cell lines
overexpressing HER-2/neu receptor is inhibited by anti- HER-2/neu monoclonal
antibody, opening a new avenue for targeted cancer therapy. In 1987, Slamon
et al.6 first reported a significant relationship between amplification
of the HER-2/neo oncogene and adverse clinical outcome in patients with
breast cancer. Although subsequent studies have largely confirmed this
in patients with node positive disease, whether or not HER-2/neo gene amplification
or overexpression is an independent prognostic factor in patients with
node-negative breast cancer remains a matter of controversy5-10. Although
still somewhat controversial, the majority of clinical studies suggest
that HER-2/neu is amplified and overexpressed in approximately 20%-30%
of breast carcinomas, and that among the new biological indicators of tumor
aggressiveness it is potentially useful in predicting the outcome of patients
with breast carcinoma and can be used effectively to improve the identification
of high-risk patients6,7,11,12.It is generally accepted that HER-2 overexpression
is associated with shorter overall survival, low level of ER, and higher
tumor grade.However, considerable variation in the incidence of amplification/overexpression
and prognostic significance of HER-2/neu has been reported. Some investigators
found amplification in only 10% of patients and no correlation to clinical
outcome13, whereas others found overexpression in up to 50% of patients
and a strong association with outcome4-14. Indeed, in our study the incidence
of HER-2/neu overexpression was 42.7% with some marginal association with
outcome in univariate analysis (p=0.059) and no significant influence on
survival in multivariate analysis15. Conflicting results of numerous studies,
including our study, highlight some of the persisting controversies surrounding
the use of HER-2/neu as a prognostic marker. Also, these results emphasize
the importance of considering HER2/neu status in the light of information
provided by other prognostic variables. For this reason, we tried to assess
the prognostic significance of HER-2/neu overexpression in association
with other known prognostic factors, and showed association with tumor
size (p=0.041) and grade (p=0.037), DNA ploidy (p=0.046) and cathepsin
D expression in stromal macrophages (p=0.024). These findings pointed to
HER2/neu overexpression as an indicator of prognosis in grade II breast
carcinoma, suggesting that determination of both tumor size and DNA ploidy
in combination with HER- 2/neu overexpression appear to enhance the ability
to recognize the patients at different risk15. More recently, there has
been considerable interest in the potential role of HER-2/neu gene amplification
and overexpression as a predictor of response to various therapeutic modalities
in patients with breast cancer. In particular the results of recent clinical
trials have indicated that treatment with monoclonal antibody to HER-2/neu
protein (Herceptin®) may be useful in prolonging the survival of patients
with metastatic disease. In our small group of 17 advanced breast carcinoma
patients who were enrolled in the study from the beginning of 1999 until
July 2000 at the Zagreb University Hospital Center, all were treated with
Herceptin® and Taxol® in combination. All patients had tumors positive
for HER2/neu by HercepTest. Partial response to therapy was observed in
47%, stable disease in 29% and progression of disease in 24% of patients.
However, complete response was not
observed in the investigated group of patients. In this way, the overall
therapy benefit (stable disease and partial response) was found in 76%
of patients. Complications of therapy include neutropenia, thrombocytopenia,
diarrhea and onycholysis, but there were no signs of congestive heart failure16.
Furthermore, some studies have indicated that tumors with HER-2/neu overexpression
may show resistance to certain forms of cytotoxic therapy and sensitivity
to others. Finally, some recent experimental and clinical studies have
suggested that HER-2/neu overexpression is associated with resistance to
tamoxifen even when tumors were ER positive, and therefore the success
of Herceptin® therapy depends upon the selection of the most appropriate
patients for treatment. Candidates for Herceptin® therapy can be identified
by the evaluation of tumor cells for the presence of altered HER-2/neu.
As a result of this information, there is a growing clinical demand for
HER-2/neu analysis of current and archived breast cancer specimens. There
are a variety of methods available to determine the HER-2/neu status of
breast cancers. These include assays to evaluate: 1) gene amplification
as Southern blot, slot blot, dot blot, polymerase chain reaction (PCR),
in situ hybridization and fluorescence in situ hybridization (FISH), 2)
assays to determine mRNA overexpression such as Northern blot analysis,
slot blot, and in situ hybridization, and 3) methods to assess protein
overexpression (Western blot analysis, immunoassay, and immunohistochemistry
(IHC). Many of these methods are beyond the scope of most pathology laboratories
for technical reasons. Furthermore, most of these assays require prospective
collection of fresh tissue and are not applicable to archival material.
The IHC method performs well and gives a clear picture of the heterogeneity
ofprotein expression in tumor cells; it distinguishes tumor cells from
normal cells and is easy to perform on routine paraffin-embedded material.
HercepTest is a semi-quantitative IHC assay to determine HER-2/ neu protein
overexpression in breast cancer tissues routinely processed for histologic
evaluation. For the determination of HER-2/neu protein overexpression,
only the membrane staining intensity and pattern should be evaluated using
scale 0-3+. Even if the standardized HER-2/ neu protocol is followed, the
subjective nature of histologic scoring can lead to spurious results. The
need to distinguish between “faint” and “weak” staining is subject to bias
even when positive control tissues are included in each stain run.Based
on the problems associated with IHC (i.e. lack of standardization and subjective
bias), some experts argue that FISH is a better alternative. FISH measures
HER-2/neu gene amplification but tends to be more expensive, complicated,
and time-consuming than IHC, and HER-2/neu must be scored on the invasive
component of breast cancer (because a high % of breast in situ lesions
have altered the HER-2/neu status).
Although each of these methods has
its advantages and disadvantages, direct comparisons of these two assays
have been few and are limited by small numbers of cases. Utility of IHC
versus FISH for the selection of breast cancer therapy requires thorough
analysis of the results of clinical trials now underway that address this
issue. Any correlative study comparing IHC with FISH without statistically
significant outcome data of patients treated with Herceptin® is of limited
value for resolving the IHC- FISH controversy. Wang et al.17correlated
IHC results with FISH over the past 18 months. These results shoved excellent
correlation in over 98 percent of HercepTestnegative cases (0, 1+) lacking
HER-2/neu gene amplification, and in tumors with high expression (3+) demonstrating
amplification. A poor correlation was found between cases considered to
be weakly positive (2+) with the HercepTest and amplification with FISH.
However, only 11 percent of all breast cancer cases demonstrated 2+ IHC
staining. Based on these results, the authors suggest that clinically and
economically, significant value is testing for HER-2/neu by HercepTest
(IHC) with reflex to FISH only in cases of weakly positive results (2+).
The cost of reflex testing of all 0 or 1+ cases is significant, since this
group comprises up to 88 percent of all breast cancer cases. Controversy
still exists as to whether the cases that show 0 or 1+ staining but demonstrate
amplification (two percent of all cases examined) will respond to Herceptin.
In our hands also HercepTest provides
excellent reproducibility and standardization, and close to 90% of all
breast cancers can be adequately and reliably determined by IHC, with molecular
evaluation reserved for borderline cases. Our own findings in 45 patients
tested by IHC and FISH for HER-2/neu detection showed excellent correlation
between IHC HER-2/neu analysis and the molecular technique for HER-2/neu
amplification (FISH), except for the weakly positive (2+) IHC results as
determined by the FDA-approved HercepTest. By IHC 12/45 (26.6%) were HER-2/neu
positive. Six out of seven IHC high positive specimens (3+) showed gene
amplification by FISH (85.7%), and 3/5 IHC medium positive specimens (2+)
showed no gene amplification (60%). None of the cases negative by IHC showed
expression of HER-2/neu by FISH. Concordances between FISH and IHC results
were seen in 42 out of 45 cases (93.5%). We conclude that all 2+ IHC cases
can in turn be subject to FISH analysis to confirm the presence of an altered
HER-2/neu gene. This combination of assays would help ensure that patients
who are most likely to benefit from Herceptin are identified. FISH testing
is used to determine HER-2/neu status in equivocal circumstances, but the
use of HercepTest as a screening assay allows for improved cost control
and turnaround time without detriment to the patient.
It is known that the FISH procedure
required more technician time and more interpretation time per case for
the pathologist than IHC. Reagent costs were subsequently higher for FISH
than for IHC. There is a high level of correlation between FISH and IHC
in the evaluation of HER-2/neu status of breast cancers using formalin-
fixed, paraffin-embedded specimens, although the choice of which assay
to use should be left for individual laboratories to make based on technical
and economic considerations. The results published to date may make it
difficult to justify the routine use of FISH for detection of HER2 status
in breast cancers18-20. It is generally accepted, at this level of knowledge
that the best approach is to combine both IHC and FISH assays, and to use
the IHC assay as a triage step, followed by FISH to analyze the IHC medium
and high positive cases18-20.
1. FEARON ER, VOGELSTEIN B. A genetic
model for colorectal tumorigenesis. Cell 1990;61:759-67.
2. THOMPSON AM, STEEL CM, CHETTY U,
CARTER DC. Evidence for the multistep theory of carcinogenesis in human
breast cancer. Breast 1992;1:29-35.
3. ELLEDGE RM, MCGUIRE WK, OSBORNE
CK. Prognostic factors in breast cancer. Semin Oncol 1992;19:244-53.
4. MILLER WR, ELLIS IO, SAINSBURG JRC,
DIXON JM. Prognostic factors - ABC of breast diseases. BMJ 1994;309:1573-
5. CLARK GM, WONG SG. Human breast
cancer: correlation of relapse and survival with amplification of the HER2/neu
oncogene. Science 1987;235:177-82.
6. SLAMON D, CLARK GM, WONH SH, LEVIN
WJ, ULLRICH A, MCGUIRE WL. Human breast cancer: Correlation of relapse
and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177-82.
7. BORG A, TANDON AK, SIGURDSSON H,
et al. HER2/neu amplification predicts poor survival in node-positive breast
cancer. Cancer Res 1990;50:4332-7.
8. LOVEKIN C, ELLIS IO, LOCKER A, et
al. c-erbB-2 Oncoprotein expression in primary and advanced breast cancer.
Br J Cancer 1991;63:439-43.
9. QUENEL N, WAFFLART J, DONICHON F,
et al. The prognostic value of c-erbB-2 in primary breast carcinomas: a
study on 942 cases. Breast Cancer Res Treat 1995;35:283-91.
10. ROSEN PP, LESSER ML, ARROYO CD,
et al. Immunohistochemical detection of HER2/neu in patients with axillary
lymph node negative breast caracinoma: a study of epidemiologic risk factors,
histologic features, and prognosis. Cancer 1995;75:1320-6.
11. REVILLION F, BONNETERRE J, PEYRAT
JP. ERBB2 oncoprotein in human breast cancer and its clinical significance.
Eur J Cancer 1998;34:791-808.
12. PRESS MF, BERNSTEIN L, THOMAS PA,
et al. HER2/neu gene amplification characterized by fluorescence in situ
hybridization: poor prognosis in node-negative breast carcinoma. J Clin
13. ZHOU DJ, AHUJA H, CLINA MJ. Proto-oncogene
abnormalities in human breast cancer: c-erbB-2 amplification does not correlate
with recurrence of disease. Oncogene 1989;4:105-8.
14. NOGUCHI M, KAWASAKI N, NAGAYOSHI
O, et al. C-erbB- 2 oncoprotein expression versus internal mammary lymph
node metastases as additional prognostic factors in patients with axillary
lymph node-positive breast cancer. Cancer 1992;69:2953-60. Acta clin Croat
2002; 41:135-186 Conference papers 148 Acta clin Croat, Vol. 41, No. 2,
15. JAKI.-RAZUMOVI. J, PETROVE»KI M,
UAAREVI. B, GAMULIN S. Mutual predictive value of c-erbB-2 overexpression
and various prognostic factors in ductal invasive breast carcinoma. Tumori
16. MRSI. M, GRAGI. M, BUDI©I. Z, PODOLSKI
P, BOGDANI. V, LABAR B, JAKI.-RAZUMOVI. J, RESTEKSAMARAIJA N, GO©EV M.
Trastuzumab in the treatment of advanced breast cancer: single-center experience.
Ann Oncol 2001;12 (Suppl):95-6.
17. WANG S, SABOORIAN MT, FRENKEL E,
HYNAN L, GOKASLAN ST, ASHFAQ R. Laboratory assessment of the status of
HER2/neu protein and oncogene in breast cancer specimens: comparison of
immunohistochemistry assay with fluorescence in situ hybridization assays.
J Clin Pathol 2000;53:374-81.
18. JACOBS TW, GOWN AM, YAZIJI H, BARNES
MJ, ASHNITT SJ. Comparison of fluorescence in situ hybridization and immunohistochemistry
for the evaluation of HER2/neu in breast cancer. J Clin Oncol 1999;17:1974-82.
19. DOWSETT M, COOKE T, ELLIS IO, GULLICK
WJ, GUSTERSON B, MALLON E, WALKER R. Assessment of HER2 status in breast
cancer: why, when and how? Eur J Cancer 2000; 36:170-6.
20. HENDRICKS JB. Histopathology at
the trailing edge (editorial). J Histotechnol 2000;23:297.
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
FACTORS INFLUENCING PROGNOSIS AND
SURVIVAL IN EARLY (TN0M0) BREAST CARCINOMA
Institute of Oncology, Ljubljana,
Carcinoma of the breast is the most
common malignant tumor in women in the western world. Its incidence has
been rising steadily over the past decades. Despite considerable progress
in early diagnosis and treatment, mortality remains relatively high and
approximately every third or second woman with breast cancer will ultimately
die of the disease.
TNM stage is generally accepted as
the most important determinant of outcome in breast cancer. In addition
to hormonal status of the patient (pre- or postmenopausal) and the presence
of steroid receptors in the tumor, TNM stage has long been the only factor
used by clinicians to make therapeutic decisions.
During the past two decades, there
has been a gradual change in the treatment of breast cancer with a tendency
toward less radical surgery and more adjuvant systemic therapy. In the
early era of chemotherapy, this type of treatment was mainly used in patients
with advanced disease; however, meta-analyses of large clinical trials
have clearly shown that systemic chemotherapy and hormonal therapy also
reduce the risk of cancer recurrence and mortality in patients with early
breast cancer. Accordingly, the question of who should be treated by systemic
therapy has eventually changed to the question of who should not be so
Due both to more widespread public
education and to early diagnosis by mammography screening programs, the
percentage ofpatients with node negative breast cancer (N0) is increasing.
The majority of these patients (about 70%) do not experience disease recurrence
after surgery and/or radiotherapy alone (local therapy); therefore, it
seems inappropriate to suggest systemic adjuvant therapy for all node negative
breast cancer patients. Since the relative risk reduction is constant across
different tumor stages and risk groups, it is obvious that the absolute
benefit from adjuvant therapy may be quite small in some specific low-risk
group of patients. It is reasonable to attempt to avoid excessive treatment
morbidity and cost by using selective prognostic markers to identify prognostically
relevant subsets of patients. However, an “ideal” prognostic indicator
or a widely accepted combination of markers able to identify patients at
low versus high risk has yet to be clearly defined. Numerous studies have
reported that several clinicopathologic features have prognostic importance
in nodenegative breast carcinoma patients. These features include tumor
size, histologic grade, and histologic type of tumor, vascular invasion
and some new biological markers. Only a few studies have simultaneously
evaluated the relative prognostic weight of various newer biological markers
compared with all conventional clinicopathological markers by performing
a multivariate analysis. Also, the majority of these studies included heterogeneous
group of patients regarding tumor size.
Recently, an International Consensus
Panel proposed a 3-tiered risk classification for patients with negative
axillary lymph nodes (Table 1) and defined the low-risk group as those
with tumor size of 1 cm or less, positive ER or PR status and histologic
grade 1; they suggested that this is the only group in which adjuvant systemic
therapy could be omitted. Since this proposal is not based on multivariate
analyses of large data sets, its true prognostic value remains uncertain.
Although the patients with stage I
(T1N0M0) breast carcinoma (i.e. tumor measuring 2 cm or less without regional
lymph node and distant metastasis) have an excellent short term prognosis,
approximately 20% will eventually develop distant metastases and die of
the disease. However, the remaining majority would be cured by surgery
alone and gain no benefit from adjuvant systemic therapy. Unfortunately,
there is no general agreement how to best identify the latter group. Less
than 15 published studies evaluated the prognostic value of different factors
in T1N0M0 breast carcinoma and only few of them included the newer biologic
indicators, such as c-erbB-2, p53, bcl2, Ki67 (MIB-1), flowcytometric DNA
ploidy or S-phase fraction determination. Although the results of these
studies are somewhat controversial, histologic or nuclear grade, proliferative
activity (as assessed by either mitosis counting, SPF determination or
Ki67 expression), vascular invasion and tumor size most often emerged as
significant prognostic factors. We recently investigated a group of 270
patients with T1N0M0 breast carcinoma who were treated at the Institute
of Oncology Ljubljana and were followed for a median of 12.5 years. All
original slides were reviewed and examined for histologic type, mitotic
index (MI), Nottingham histologic grade (NHG) and its components (extent
of tubule formation, pleomorphism, mitotic counts) and presence of vascular
invasion. Representative tumor slides were stained immunohistochemically
for p53, bcl-2, c-erbB-2, MIB-1(Ki67), CEA, ER and PR using LSAB method
and Dako TechMate 500 automatic immunostainer. The prognostic value of
investigated features was evaluated using univariate and multivariate survival
analysis. Survival of our patients (84.4% cancer-specific survival, CSS,
and 77.4% metastasis-free survival, MFS, at 10 years) was similar to that
reported in other series of patients with T1N0M0 tumors. In keeping with
other reports, late recurrences were not uncommon. The prognostic value
of tumor size was not confirmed in our study: although survival was somewhat
worse in patients with tumors larger than 1 cm (T1c) than in those with
tumors measuring 1 cm or less (T1ab), the difference was not significant.
We confirmed the prognostic significance of NHG: both MFS and CSS were
significantly better in patients with grade 1 than in those with grade
2 or 3 tumors. Apart from NHG, in univariate analysis, MI, vascular invasion
and c-erbB-2 expression were significant predictors of MFS and CSS. In
addition, CEA expression and MIB-1 reactivity were significantly related
to MFS, and histologic type to CSS. Age, menopausal status, type of treatment,
PR or ER status and expression of bcl-2 or p53 were not significantly associated
with survival. The relative importance of prognostic variables was tested
in Cox’s proportional hazard model. When all variables were entered in
the model, MI, histologic type, vascular invasion and CEA expression emerged
as significant independent prognostic factors for both MFS and CSS.
MI was the single most important prognostic
factor for MFS and CSS; however, our findings suggest that optimal cutoff
values for different prognostic groups may be lower than those proposed
in NHG. When testing various multivariate models to predict CSS, NHG retained
its independent prognostic value only in the model that did not include
MI and histologic type, whereas it was replaced by MIB-1 reactivity in
multivariate analysis of MFS.
By combining four independent factors
(MI, histologic type, vascular invasion and CEA expression) into a prognostic
index, the patients could be allocated into three prognostic groups. Patients
in the first group (15%) developed metastatic disease in almost one half
of cases and those in the second (60%) in one third of cases. In the third
group (25%), prognosis was excellent, with more than 90% MFS at 15 years
after surgery. In the latter group, the use of adjuvant chemotherapy may
be unnecessary. By applying the aforementioned Consensus Panel criteria,
the group of patients in whom adjuvant systemic therapy could be omitted
would be considerably smaller.
1. EIFEL P, AXELSON JA, COSTA J, et
al. National Institutes of Health Consensus Development Conference Statement:
adjuvant therapy for breast cancer, November 1-3, 2000. J Natl Cancer Inst
2. Polychemotherapy for early breast
cancer: an overview of the randomised trials. Early Breast Cancer Trialists’
Collaborative Group. Lancet 1998; 352:930-42.
3. Systemic treatment of early breast
cancer by hormonal, cytotoxic, or immune therapy. 133 randomised trials
involving 31,000 recurrences and 24,000 deaths among 75,000 women. Early
Breast Cancer Trialists’ Collaborative Group. Lancet 1992; 339:1-15.
4. Early stage breast cancer: consensus
statement. NIH Consensus Development Conference, June 18-21, 1990. Cancer
Treat Res 1992; 60:383-93.
5. GOLDHIRSCH A, WOOD WC, SENN HJ,
et al. Meeting highlights: international consensus panel on the treatment
of primary breast cancer. J Natl Cancer Inst 1995; 87:1441-5.
6. YARBRO JW, PAGE DL, FIELDING LP,
et al. American Joint Committee on Cancer Prognostic Factors Consensus
Conference. Cancer 1999; 86:2436-46.
7. FITZGIBBONS PL, PAGE DL, WEAVER
D, et al. Prognostic factors in breast cancer. College of American Pathologists
Consensus Statement 1999. Arch Pathol Lab Med 2000; 124:966-78. Acta clin
Croat 2002; 41:135-186 Conference papers 150 Acta clin Croat, Vol. 41,
No. 2, 2002
8. SAUERBREI W, HÜBNER K, SCHMOOR C,
et al. Validation of existing and development of new prognostic classification
schemes in node negative breast cancer. German Breast Cancer Study Group.
Breast Cancer Res Treat 1997; 42:149-63.
9. MOON TE, JONES SE, BONADONNA G,
et al. Development and use of a natural history data base of breast cancer
studies. Am J Clin Oncol 1987; 10:396-403.
10. QUIET CA, FERGUSON DJ, WEICHSELBAUM
RR, et al. Natural history of node-negative breast cancer: a study of 826
patients with long-term follow-up. J Clin Oncol 1995; 13:1144-51.
11. ROSEN PP, GROSHEN S, KINNE DW.
Survival and prognostic factors in node-negative breast cancer: results
of long-term follow- up studies. J Natl Cancer Inst Monogr 1992; 11:159-62.
12. ROSEN PP, GROSHEN S. Factors influencing
survival and prognosis in early breast carcinoma (T1N0M0-T1N1M0). Assessment
of 644 patients with median follow-up of 18 years. Surg Clin North Am 1990;
13. RAUSCHECKER HF, SAUERBREI W, GATZEMEIER
W, et al. Eight-year results of a prospective non-randomised study on therapy
of small breast cancer. The German Breast Cancer Study Group (GBSG). Eur
J Cancer 1998; 34:315-23.
14. JOENSUU H, PYLKKÄNEN L, TOIKKANEN
S. Late mortality from pT1N0M0 breast carcinoma. Cancer 1999; 85:2183-9.
15. LEE AK, LODA M, MACKAREM G, et al. Lymph node negative invasive breast
carcinoma 1 centimeter or less in size (T1a,b NOMO): clinicopathologic
features and outcome. Cancer 1997; 79:761-71.
16. LEITNER SP, SWERN AS, WEINBERGER
D, et al. Predictors of recurrence for patients with small (one centimeter
or less) localized breast cancer (T1a,b N0 M0). Cancer 1995; 76:2266-74.
17. STIERER M, ROSEN H, WEBER R. Nuclear
pleomorphism, a strong prognostic factor in axillary node-negative small
invasive breast cancer. Breast Cancer Res Treat 1992; 20:109-16.
18. STAEL O, DUFMATS M, HATSCHEK T,
et al. S-phase fraction is a prognostic factor in stage I breast carcinoma.
J Clin Oncol 1993; 11:1717-22.
19. STENMARK-ASKMALM M, STAEL O, OLSEN
K, et al. p53 as a prognostic factor in stage I breast cancer. South-East
Sweden Breast Cancer Group. Br J Cancer 1995; 72:715-9.
20. RAILO M, LUNDIN J, HAGLUND C, et
al. Ki-67, p53, Er-receptors, ploidy and S-phase as prognostic factors
in T1 node negative breast cancer. Acta Oncol 1997; 36:369-74.
21. FRKOVI. GRAZIO S, BRA»KO M. Long
term prognostic value of Nottingham histological grade and its components
in early (pT1N0M0) breast carcinoma. J Clin Pathol 2002; 55:88-92.
Table 1. Risk categories for
women with node-negative breast cancer
Low-risk Intermediate-risk High-risk
(has all (between the other (has at
least listed factors) 2 categories) 1 listed factor)
Tumor size ??1cm 1-2 cm > 2cm
ER or PR status positive positive negative
Tumor grade grade 1 grade 1-2 grade
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
ADVANCES IN BREAST FNA
Instituto de Patologia e Imunologia
Molecular da Universidade do Porto . IPATIMUP e Faculdade de Medicina da
Universidade do Porto, Porto, Portugal
Fine-needle aspiration cytology (FNA)
is a simple, rapid, accurate and cost-effective method that has become
a standard of care in the evaluation of breast lesions. Over recent years,
FNA has become the method to assess palpable and non-palpable breast lesions,
and contributes to management decisions at surgical and medical levels,
being the source of primary diagnosis in several cases. In addition, cytology
is also largely used to diagnose lymph node metastasis and to evaluate
pleural effusions in patients with breast cancer. As the treatment planning
is frequently made preoperatively based on cytological material, the diagnosis
should be as precise as possible, and as much prognostic information should
be gained from the cytologic specimens as possible.
FNA has largely replaced frozen sections
and, in cases candidates for primary chemotherapy it can provide hormonal
assessment as well as other useful parameters relevant for the prognosis
and prediction of therapeutic response. Furthermore, FNA material can also
be used for some special studies such as immunohistochemistry, cytometry,
in situ hybridization and molecular biology techniques.
The aim of this review is to report
the major application of the new technologies in cytologic material obtained
from breast FNA biopsies.
Identification of Myoepithelial Cells
The presence of myoepithelial cells
is one of the most important criteria to support a diagnosis of benign
lesion in FNA of the breast. However, on cytologic examination, the correct
identification of myoepithelial cells is sometimes difficult, as they might
be confused with apoptotic cells, stromal cells, and even epithelioid histiocytes.
Since some years ago, several markers have been used in cytologic material
in an attempt to identify myoepithelial cells. However, most of these markers
(such as smooth-muscle actin, calponin, H-caldesmon, cytokeratins 5/6/14)
have shown cross-reaction with other cells (myofibroblasts, luminal cells,
stromal cells, pericytes) and are expressed at the cytoplasm of myoepithelial
cells that can be lost in smears.
Recently, two novel markers have been
used to identify myoepithelial cells at the histologic and cytologic levels.
P63, a p53-homologue nuclear transcription factor, is a protein that is
necessary for the maintenance of the basal compartment of several multilayered
epithelia and is selectively expressed in basal cells of stratified epithelia,
in the basal cells of prostate and myoepithelial cells of the breast, salivary
and lacrimal cells. Recently, Barbareschi et al. and our group have shown
that p63 is a reliable myoepithelial cell marker in histologic sections.
Moreover, some preliminary studies have pointed out that p63 could be better
than other conventional myoepithelial cell markers because, as it is localized
in the nuclei of myoepithelial cells, it overcomes the cytoplasmic fragility
of myoepithelial cells in FNA. Studying more than 90 cases of breast FNA
smears with immunocytochemistry for p63 antibody, we demonstrated that
this marker highlighted the nuclei of two distinct cell populations: 1)
all of the oval-tospindle- shaped cells with dark nuclei on epithelial
cell clusters, and 2) all of the naked nuclei observed in the background
of the smears. No immunoreactivity for p63 was observed in the cells admixed
with fibrillary matrix of fibromyxoid stroma or in those isolated cells
with oval nuclei and sparse-to-moderate cytoplasm. Based on these results,
we strongly suggest that p63 is a reliable marker for myoepithelial cells
in breast FNAs and that the majority of naked nuclei, defined as oval-to-spindle-shaped
cells without any discernible cytoplasm, show a myoepithelial origin and
thus, they might be included in the major criteria to diagnose benign breast
lesions. Moreover, we observed that p63 helps identify myoepithelial cells
overlying malignant cell clusters that we found consistently in cases of
ductal carcinoma in situ. However, further studies with large series of
patients using similar methodology are required in order to define how
specific and sensitive this finding is in ruling out invasion in FNA of
The other myoepithelial cell marker
used by our group is maspin. Maspin is a member of the serpin family of
serine protease inhibitors, and it has been claimed to be a tumor and metastasis
suppressor and to have antiangiogenic properties. Maspin is consistently
expressed by myoepithelialcells and our group has shown that myoepithelial
tumors of the breast are positive for maspin. Although initial studies
demonstrated cytoplasmic expression only, we showed for the first time
that maspin is also expressed in the nuclei, a finding recently confirmed
by other groups.
Estrogen and Progesterone(ER/PR)
Besides giving prognostic information,
hormonal receptor analysis is a useful tool to predict hormonal response
in human breast cancer. According to the College of American Pathologists
(CAP) Consensus Statement 1999, concerning the prognostic factors in breast
cancer, hormone receptor analysis should be performed routinely in all
primary breast carcinomas. Although hormone receptor analysis has been
traditionally performed on surgically removed specimens, FNA offers a suitable
alternative for this determination in a number of situations: a) inoperable
cases and metastatic or recurrent tumors in which the size and accessibility
to surgical biopsy presents a problem; b) cases in which preoperative irradiation
or presurgical therapy is the initial treatment option; and c) advanced
tumors in which serial hormone receptor studies may provide information
regarding response to therapy.
The assessment of hormone receptors
on cytologic material has been performed by immunocytochemistry with good
correlation with histologic and biochemistry determinations. Some years
ago, we described a method of immunocytochemical assessment of estrogen
receptor status on alcohol-fixed smears obtained by FNA from breast cancer
patients, using a commercially available monoclonal antibody with antigen
retrieval, and the results were compared with the assessment by ER immunocytochemical
assay using the same procedure on formalin- fixed tissue and with assessment
by ER-ICA assay on frozen sections. The results were scored semiquantitatively
using a five grade scoring system. Although we have been critical in the
use of score system in cytologic specimens, especially in relation to the
extension ofimmunoreactivity, we found a good correlation between the results
obtained on the cytologic specimens and on the histologic material. The
heterogeneity of ER expression within the tumor should be taken in consideration
whenever using FNA material for semi-quantification of ER because it might
cause discrepant results. The putative usefulness of quantification in
cytochemical hormone receptor assays remains controversial, and no consensus
about the use of semiquantitative scoring system or mere division of tumors
into positive and negative ones has been attained so far. Although it has
not been shown that quantitative values beyond a defined level are helpful
in selecting treatment options, some authors using FNA material showed
better response to treatment with tamoxifen in patients with more than
50% ER-positive tumor cells. HER2/neu Assessment
The proto-oncogene c-erbB2 is localized
on chromosome 17 and encodes a 185-kD transmembrane glycoprotein with tyrosine
kinase activity, which possesses a close sequence homology with the epidermal
growth factor receptor. This oncogene is overexpressed in about one third
of breast cancers and its overexpression is associated with high histologic
grade, reduced survival, lower responsiveness to methotrexate-based treatment
regimens and hormone receptor modulators such as tamoxifen, as well as
higher responsiveness to doxorubicin-based regimens. Recently, a humanized
antibody against HER2/neu was developed (Herceptin - Genentech, Inc, South
San Francisco, CA), and might be used as a novel neoadjuvant primary therapy
for HER2/neu positive breast cancer patients. Some authors showed that
it is possible to determine the immunocytochemical expression of HER2/neu
in previously Papanicolaou-stained aspirates of breast carcinomas. They
found a strong correlation between HER2/ neu immunocytochemistry of aspirates
and their corresponding tissue biopsies. In fact, the determination of
HER2/neu in smears and cytoblock preparations may be as sensitive as, or
even more sensitive than that of formalin- fixed, paraffin-embedded tissue.
In cytology, the pattern of expression of HER2/neu is relatively uniform
and is evidenced by membrane staining. NCL-CB11, a monoclonal antibody
against the internal domain of the HER2/ neu protein, gave better results
in cell smears with strongest reaction and least background staining. The
clinical use of Herceptin requires the evaluation of HER2/neu amplification
from every potentially eligible patient. Fluorescence in situ hybridization
(FISH) is currently regarded by the FDA as the gold standard method for
detecting HER-2/neu amplification. A big deal of discussion has emerged
recently concerning the accordance between the immunohistochemical assessment
of HER2/ neu overexpression and the real amplification of the gene assessed
by FISH. For tissue sections, the semiquantitative immunohistochemical
approach accepted by FDA is defined as positive membranous staining in
more than 10% of the neoplastic cells. Partial or incomplete, weak to moderate,
and moderate to strong membranous staining in more than 10% of the tumor
cells must be scored as 1+ (negative), 2+ (weak positive), and 3+ (strong
positive), respectively. Several reports showed a good correlation between
a 3+ immunoexpression of HER2/neu and the amplification of the gene using
formalin-fixed paraffin embedded tissue; however, in 1+ and 2+ cases, there
is no correlation between these parameters and these patients may have
benefits assessing c-erb-B2 amplification by FISH analysis. In the last
years, some authors have successfully assessed the amplification of HER2/neu
by FISH analysis in archival cytologic fine needle aspirates and showed
a good concordance with paraffin-embedded tissue.
The findings reported so far support
that FNA cytologic samples might constitute the most cost-effective and
easiest way to assess the HER2/neu amplification and overexpression, however,
further studies are required to characterize the method for semiquantitative
analysis of its immunocytochemical expression. Besides, FISH analysis requires
microscopes with special filters and complex image analysis system to interpret
the fluorescent signal. With the aim to supervene this drawback, the chromogenic
in situ hybridization (CISH), a new modification of FISH, that enables
detection of HER-2/neu gene copies with conventional peroxidase reaction
in breast cancer specimens using regular microscopes, has been tested with
outstanding results. Further studies in this front are needed to verify
the applicability of this new method in FNA cytologic samples.
P53 encodes a 53 kD nuclear phosphoprotein
with tumor suppressor activity. The wild type P53 protein, which is a transcription
regulator, is present in the nuclei of all mammalian cells where it appears
to be involved in the regulation of cell proliferation and apoptosis. Recent
clinical evidence supports a critical role of P53 status in providing prognostic
information, mainly in node-negative breast cancer patients. In fact, there
is increasing evidence that tumors lacking normal P53 function are clinically
more aggressive as they acquire a selective growth advantage becoming more
resistant to ionizing radiation and some anticancer drugs. P53 immunostaining
is nuclear and could be determined in previously Papanicolaou-stained aspirates
of breast carcinomas. When we compare P53 gene mutations versus overexpression,
the data obtained by molecular biology methods for assessment of mutations
give better prognostic information than immunohistochemistry performed
with PAb 1801 monoclonal antibody. In histologic material using a semi-quantitative
approach we found an association between the presence of mutation in SSCP
analysis and strong P53 staining and absence of mutation in cases with
scarce and weakly positive neoplastic cells, however, we do not test this
system in FNA smears. Since it is possible to detect P53 mutations and
deletion in FNA material using polymerase chain reaction single strand
conformation polymorphism (PCR-SSCP) and DNA sequencing analyses in DNA
extracted from cell suspensions or archived stained smears and that specific
mutations in the P53 gene are associated to primary resistance to chemotherapy,
the assessment of P53 mutations on FNA material could be very useful to
predict clinical behavior and responsiveness to therapy in breast cancer.
This is of particular value in a primary chemotherapy setting, as complete
tumor regression may occur and FNA-based pre-chemotherapy information may
represent the only available information unaffected by therapy. The application
of molecular biology techniques to the existing archival smears may become
a valuable tool to detect genetic changes in samples from breast cancer
aspirates, making FNA a reliable and helpful tool for the diagnosis, prognostic
assessment and therapeutic management of breast cancer patients.
Sialyl-Tn (STn) is a core region carbohydrate
antigen formed by the premature 2-6 sialylation of N-acetylgalactosamine
whose expression is associated with some human malignancies. In fact, neoplastic
transformation is almost invariably associated with marked changes in cell
membrane glycoconjugates due to abnormal expression or depression of DNA
encoding glycosyl transferases and the expression of simple mucin-type
antigens, including STn is highly restricted in normal adult tissues. In
our experience, the expression of this marker in breast cancer is associated
with the presence of axillary metastases, lack of hormonal receptor, and
high histologic grade. Moreover, some authors have demonstrated that STn
positivity appears to be a marker of resistance to adjuvant chemotherapy.
Using immunocytochemistry, we documented STn expression in mammographically
detected breast lesions diagnosed by FNA cytology. Therefore, the determination
of expression of STn in breast aspirates could be a useful marker to assess
resistance to chemotherapy as well as to identify cases with high risk
of axillary metastases.
Assessment of Telomerase Activity
Telomeres are repetitive sequences
at the ends of chromosomes that protect chromosomes from incomplete replication,
nuclease degradation, and end-to-end fusion during replication, playing
a major role during DNA replication. In most somatic cells, after each
cell division, the telomeres are eroded, leading to a progressive shortening
of their length. When one telomere reaches the critical point, the cell
stops dividing, and senesces. The maintenance of telomeres depends on the
telomerase activity. Telomerase is a ribonucleoprotein complex responsible
for de novo telomere synthesis and addition of telomeric repeats to existing
telomeres. Telomerase activity (TA) is almost restricted to embryonic cells,
germ cells, and malignant neoplastic cells; very low levels of this enzyme
have been detected in somatic tissues, mainly restricted to the basal cell
layer ofseveral epithelia and cells in the terminal ducts of the breast.
Telomerase activity can be measured in vitro by using the telomeric repeat
amplification protocol (TRAP), including cells obtained by FNA specimens.
The presence of TA was demonstrated in 80% to 90% of breast carcinoma FNA
samples; however, some benign lesions presented some level of telomerase
activity, including fibroadenomas. Further evaluation of the sensitivity
and specificity of TA for malignant cells is required before this technique
could be accepted as a new marker in routine cytology.
1. BARBARESCHI M, PECCIARINI L, CANGI
MG, MACRI E, RIZZO A, VIALE G, DOGLIONI C. P63, a p53 homologue, is a selective
nuclear marker of myoepithelial cells of the human breast. Am J Surg Pathol
2. FITZGIBBONS PL, PAGE DL, WEAVER
D, THOR AD, ALLRED DC, CLARK GM, RUBY SG, O’MALLEY F, SIMPSON JF, CONNOLLY
JL, HAYES DF, EDGE SB, LICHTER A, SCHNITT SJ. Prognostic factors in breast
cancer. College of American Pathologists Consensus Statement 1999. Arch
Pathol Lab Med 2000;124:966-78.
3. HIYAMA E, SAEKI T, HIYAMA K, TAKASHIMA
S, SHAY JW, MATSUURA Y, YOKOYAMA T. Telomerase activity as a marker of
breast carcinoma in fine-needle aspirated samples. Cancer 2000;90:235-8.
4. KLIJANIENKO J, COUTURIER J, GALUT
M, EL-NAGGAR AK, MACIOROWSKI Z, PADOY E, MOSSERI V, VIELH P. Detection
and quantitation by fluorescence in situ hybridization (FISH) and image
analysis of HER-2/neu gene amplification in breast cancer fine-needle samples.
5. LAVARINO C, CORLETTO V, MEZZELANI
A, DELLA TORRE G, BARTOLI C, RIVA C, PIEROTTI MA, RILKE F, PILOTTI S. Detection
of TP53 mutation, loss of heterozygosity and DNA content in fine-needle
aspirates of breast carcinoma. Br J Cancer 1998; 77:125-30.
6. MOORE JG, TO V, PATEL SJ, SNEIGE
N. HER-2/NEU gene amplification in breast imprint cytology analyzed by
fluorescence in situ hybridization: direct comparison with companion tissue
sections. Diagn Cytopathol 2000;23:299-302.
7. NIZZOLI R, BOZZETTI C, NALDI N,
GUAZZI A, GABRIELLI M, MICHIARA M, CAMISA R, BARILLI A, COCCONI G. Comparison
of the results of immunocytochemical assays for biologic variables on preoperative
fine-needle aspirates and on surgical specimens of primary breast carcinomas.
8. POREMBA C, SHROYER KR, FROST M,
DIALLO R, FOGT F, SCHAFER KL, BURGER H, SHROYER AL, DOCKHORN- DWORNICZAK
B, BOECKER W. Telomerase is a highly sensitive and specific molecular marker
in fine-needle aspirates of breast lesions. J Clin Oncol 1999;17:2020-6.
9. REIS-FILHO JS, SCHMITT FC. Taking
advantage of basic research: p63 is a reliable myoepithelial and stem cell
marker. Adv Anat Pathol 2002; in press. Acta clin Croat 2002; 41:135-186
Conference papers 154 Acta clin Croat, Vol. 41, No. 2, 2002
10. REIS-FILHO JS, MILANEZI F, SILVA
P, SCHMITT FC. Maspin expression in myoepithelial tumors of thebreast.
Pathol Res Pract 2001;197:817-22.
11. SCHMITT FC, BENTO MJ, AMENDOEIRA
I. Estimation of estrogen receptor content in fine-needle aspirates from
breast cancer using the monoclonal antibody 1D5 and microwave oven processing:
correlation with paraffin embedded and frozen sections determinations.
Diagn Cytopathol 1995;13:347-51.
12. SCHMITT FC. Comments on p53 protein
expression, cell proliferation and steroid hormone receptors in ductal
and lobular in situ carcinomas of the breast. Eur J Cancer 1997; 33:1903.
13. SCHMITT FC, SOARES R, CIRNES L,
SERUCA R. P53 in breast carcinomas: association between presence of mutation
and immunohistochemical expression using a semiquantitative approach. Pathol
Res Pract 1998;194:815-19.
14. SCHMITT FC, MARINHO A, AMENDOEIRA
I. Expression of sialyl-Tn in fine-needle aspirates from mammographically
detected breast lesions: a marker of malignancy? Diagn Cytopathol 1998;
15. SOLOMIDES CC, ZIMMERMAN R, BIBBO
M. Semiquantitative assessment of c-erbB-2 (HER-2) status in cytology specimens
and tissue sections from breast carcinoma. Anal Quant Cytol Histol 1999;21:121-5.
16. TANNER M, GANCBERG D, DI LEO A,
LARSIMONT D, ROUAS G, PICCART MJ, ISOLA J. Chromogenic in situ hybridization:
a practical alternative for fluorescence in situ hybridization to detect
HER-2/neu oncogene amplification in archival breast cancer samples. Am
J Pathol 2000;157:1467-72.
|F. Del Piero
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
MAMMARY NEOPLASIA IN CAST AND DOGS
F. Del Piero
University of Pennsylvania, School
of Veterinary Medicine, Departments of Pathobiology and Clinical Studies,
New Bolton Center, Philadelphia, USA
Mammary neoplasms are very prevalent
in dogs and cats, but are rare in other domesticated species. Because of
their prevalence in these companion animals, and because they are a model
for human breast cancer, they are deeply investigated clinically, histologically,
immunohistochemically and with other molecular pathology techniques. Mammary
neoplasms are the third most frequently occurring tumor in female cats,
following hematopoietic neoplasms and skin tumors. The incidence of mammary
tumors in this species is less than half that of humans and dogs, nevertheless,
these tumors account for 17% of neoplasms in female cats. The great majority
(about 80%) of feline mammary tumors are malignant (adenocarcinomas). Breed
predisposition (Siamese) has been speculated but not proven. Mammary tumors
occur primarily in intact cats from 9 months to 23 years of age, with a
mean age of 10 to 12 years. Several reports have documented an association
between the prior use of progesterone-like molecules and the development
of benign or malignant feline mammary neoplasms. While low concentrations
of progesterone receptors have been found in the cytoplasm of some feline
mammary tumors, dihydrotestosterone receptors have not been identified.
Only 10% of the feline tumors examined were positive for estrogen receptors:
a much higher percentage of estrogen receptor positive tumors are seen
in dogs and humans. Tubular, papillary, and solid adenocarcinomas are the
most common malignant tumors and the majority present a combination of
tissue types in each tumor. Sarcomas, mucinous carcinomas, duct papillomas,
adenosquamous carcinomas, and adenomas are rarely seen. Mammary gland dysplasia,
while infrequent, needs to be differentiated from malignant neoplasms.
Lobular hyperplasia and fibroepithelial hyperplasia are types of non-inflammatory
hyperplasia identified in the mammary gland of the cat. These hyperplastic
conditions are relatively common, and thought to be associated with hormonal
stimulation of the glandular tissue, which decreases following ovariohysterectomy.
Benign tumors include simple and complex adenomas, lowand high-cellularity
fibroadenomas, benign mixed tumors and duct papillomas. Feline mammary
gland neoplasm can involve any or all of the glands and is distributed
equally between the left and right sides. Multiple gland involvement occurs
in more than half of affected cats. Metastatic lung and thorax involvement
may be extensive. Tumor size is the single most important prognostic factor
for malignant feline mammary tumors. Other significant prognostic factors
affecting recurrence in and survival of feline malignant mammary tumors
are the extent of surgery and histologic grading of the tumor. Cats with
a tumor size of greater than 3 cm in diameter will have a median survival
time of 4 to 6 months. Cats with a tumor size of 2 to 3 cm in diameter
will have a significantly increased survival time with a median of about
2 years. Cats with tumor less than 2 cm in diameter have a median survival
time of more than 3 years. Thus, early diagnosis and treatment is a very
important prognostic factor for malignant feline mammary tumors. Mammary
tumors are extremely common in intact female dogs, and account for at least
50% of all reported neoplasms. At least 70% of intact bitches will develop
a clinically detectable mammary tumor if they live to 15 years of age,
and almost all of them will have microscopic tumor foci. Ovariohysterectomy
prior to the first heat cycle greatly reduces the risk (more than 80%)
of developing mammary neoplasia later in life, with the benefits reduced
with time and no benefits of spaying after the second heat. Some report
that 50% of canine mammary tumors aremalignant and 50% of the malignant
neoplasms metastasize, others reports that metastasis is much less frequent.
The most reliable predictor of true behavioral malignancy in canine mammary
tumors is local invasion. While local invasion is readily detected by histologic
examination of the excised tumors, it can also be predicted with accuracy
by clinical examination. Tumors that seem to be fixed to the underlying
tissue, cross the midline, or are otherwise obviously infiltrative are
likely to be true malignancies. A bitch with one detected mammary tumor
very often will have numerous microscopic foci in the same or other glands.
Malignant tumors of the dog include several types of carcinomas: noninfiltrating
(in situ), complex, simple, tubulopapillary, solid, anaplastic and some
special types of carcinomas including spindle, squamous, mucinous, and
lipid rich carcinomas. Sarcomas, which are much less frequent, include
fibrosarcomas, osteosarcomas, and other sarcomas. In addition, carcinosarcoma
and carcinoma or sarcoma within a benign neoplasm have been described.
Benign tumors include simple, complex, basaloid adenomas, fibroadenomas,
benign mixed tumors, duct papillomas. Other described changes in the canine
mammary gland include ductal, lobular, and epithelial hyperplasia, adenosis,
cysts, duct ectasia, fibrosclerosis and gynecomastia.
||Acta clin Croat 2002; 41:135-186
Acta clin Croat, Vol. 41, No. 2,
DIAGNOSTIC IMAGING FOR THE IDENTIFICATION
OF CARDIAC TUMORS IN HUMANS AND DOGS
C. M. Bussadori* S. Biasi** C.
Quintavalla*** D. Pradelli*** L. Marconato****
*Clinica Veterinaria G.Sasso, Milan,
Italy **Unità Operativa di Cardiologia Clinica S.Carlo, Paderno
Dugnano, Milan, Italy ***Istituto di Clinica Medica Facolta’ di Medicina
Veterinaria, Universita di Parma ****School of Veterinary Medicine, Department
of Pathobiology, University of Pennsylvania, USA
Diagnostic imaging (particularly echocardiography
and MRI) has increased the frequency of identification of cardiac tumors.
Although still uncommon, cardiac neoplasia is more than an academic curiosity.
In human patients, benign tumors are more frequent than malignant, with
myxomas being most prevalent. A sex predisposition has been observed, with
a higher prevalence in females. Cardiac tumors are diagnosed in patients
of all ages. The most frequent cardiac tumors in dogs are hemangiosarcomas,
chemodectomas, ectopic thyroid carcinomas, and pericardial mesothelioma.
For most of these tumor types there is a demonstrated breed predisposition;
for example chemodectomas are most commonly found in brachiocephalic breeds.
Diagnostic imaging is needed to guide surgical treatment: surgical removal
of these tumors has grown following the introduction of echocardiographic
examination. Diagnostic imaging is useful even in prenatal diagnosis of
cardiac neoplasias such as rhabdomyoma and rhabdomyosarcoma. Almost all
cardiac related tumors could be identified by echocardiography, particularly
intracardiac ones. When transthoracic echocardiography is unable to fully
evaluate the edges of cardiac masses, transesophageal echocardiography
can be used to provide more complete diagnostic information. MRI synchronized
with cardiac cycle can achieve additional information. Echocardiography
and MRI are complementary diagnostic tools. In the dog, cardiac tumors
are commonly typified by echocardiography based on their peculiar morphological
features. Even in human patients, echocardiography and MRI give enough
information to identify the type of cardiac tumor.