How Breast Cancer is Diagnosed
Introduction:
In spite of major advances in cell and molecular biology, cancer diagnosis still
depends on recognizing large-scale morphologic alterations of cells and tissues by light
microscopy. It is expected that the diagnostic appearances of cancer cells and their
precursors reflect basic carcinogenic mechanisms. The purpose of this mini-review is to
make these diagnostic criteria accessible to the cell/molecular biologists studying breast
cancer. To learn breast cancer diagnosis within roughly one hour, we will ignore lobular
and stromal lesions and limit ourselves to the following:
- Normal duct histology and benign ductal lesions
- Early probable precursors to ductal carcinoma (proliferative fibrocystic changes and
papillomas)
- Advanced precursor to ductal carcinoma: atypical ductal hyperplasia
- Non-invasive ductal carcinoma
- Invasive ductal carcinoma
- Cytologic diagnosis of breast cancer
This covers about 75% of breast cancer and the best known precursors. The reader may
consult one of several comprehensive texts to read about the appearance of other breast
lesions(1,2,3). 40 images are provided. Words written in italics are defined in a
glossary. At the end of this session the reader should be able to recognize a typical
invasive breast cancer, some related benign ductal proliferations, and know general
features of glandular proliferations outside the breast. The last 7 images will be
a self-assessment test. Supplementary text and answers to the self-assessment are included
in the reference section.
The accumulation of diagnostic concepts in breast cancer during the past 150 years has
been laborious, depending primarily on correlations of histologic patterns with clinical
outcomes. In order to infer how various histologic patterns develop, pathologists have
relied on finding lesions with intermediate appearances. Surprisingly there are very few
models in pathology to explain why cancer cells grow in these various patterns. Without a
framework, breast cancer diagnosis is reduced to pattern recognition, with little interest
to the researchers who want to know about carcinogenic mechanisms.
To allow non-trained observers to appreciate diagnostic patterns within a short period
of time, it has proved useful to first construct a framework for envisioning why the
patterns look the way they do. In this framework, cancer is viewed as an evolutionary
process in which increasingly fit clones of ductal cells emerge within the complex
environment of the breast(4,5,6). The framework permits evolutionary principles to be
applied to understanding the histologic patterns, similar to the way evolutionary
principles are successfully applied in community-level ecology research. In essence, the
patterns that distinguish the various stages of cancer progression reflect changes in
"fitness". Note that in evolution, increasing fitness does not necessarily mean
increasing the speed of reproduction (growth rate), nor to increasing longevity (decreased
apoptosis). In evolution, increasing fitness is simply the ability to grow better or
expand relative to other populations. In development of cancer, the greatest competition
is often with the other cousin clones. The assumptions about the nature of the change in
fitness of the ductal cells, or the key consequences of the apparent change in fitness
that allow the patterns to be recognized will be marked in bold type.(7)
Normal histology and benign ductal proliferations
Figure 1 shows the H & E appearance of normal postpubertal, non-lactating
female breast tissue.
Figures 1 and 2
The lobule is composed of numerous small acini that empty into a terminal
ductule. The terminal duct empties into progressively larger ducts which in turn connect
to the nipple. In lactation the acini produce milk and the duct system conveys the milk to
the nipple. The ducts themselves are not greatly altered in appearance during lactation.
Figure 2 shows a higher magnification of a duct. The environmental components of
diagnostic significance include the ductal cells which form a single row on the inner-most
part of the duct, myoepithelial cells external to the ductal cells, the basal lamina,
extracellular matrix, periductal fibroblasts, and periductal blood vessels. In this image,
the extracellular matrix contains abundant collagen, as manifest by the dense eosinophilia
(asterisks). Myoepithelial cells are not always as easy to distinguish from ductal cells.
A detailed description of the immunohistochemical features of each of these components,
their proposed origin, and biochemical features are described elsewhere(8). Myoepithelial
cells are mitotically inactive and appear to arise from terminal differentiation of ductal
cells(9,10). BrdU labeling studies show that mitoses can occur anywhere along the duct(10)
although there appears to be a zone of relatively higher proliferation in the terminal
ductule (short arrow in Figure 1)(11).
The ducts can proliferate in response to unknown signals. Figure 3 shows such a ductal
proliferation in a pattern called blunt duct adenosis.
Figures 3 and 4
This pattern is not generally regarded as a direct precursor to ductal cancer, and the
benign nature of a proliferation such as this can be reproducibly recognized if one
envisions that there is no increase in ductal cells compared to the other cellular
components of their environment. All the cellular constituents of the duct
microenvironment have increased in number, so there is no change in fitness of the ductal
cells themselves. Figure 4 shows that the ductal cells form a single layer in close
proximity to the myoepithelial cells and basal lamina compartments which in turn have a
normal relationship to the stroma.
Early potential precursors to breast cancer: Proliferative fibrocystic changes, usual
ductal hyperplasia, epitheliosis, and intraductal papilloma
There is evidence indicates that these can be precursors to breast cancer, and that the
progression is via an evolutionary process. Patients shown in a biopsy to have marked
proliferative fibrocystic changes have a slight (2 fold) increased relative risk of
subsequent development of invasive ductal carcinoma(2,12). One study using FISH found
single or multiple clonal cytogenetic abnormalities in short term cultures in 11 of 15
proliferative ductal lesions(6). Further, these proliferative lesions contain some of the
same karyotypic abnormalities as reported in other cases of invasive breast carcinomas(6).
The patterns of loss of heterozygosity have been shown to be shared between these
proliferative lesions and concomitant intraductal or invasive carcinomas(5). Finally,
about 25% of proliferative lesions show grossly aneuploid DNA content(14) suggesting the
presence of genetic instability. There is controversy concerning the question of whether
proliferative fibrocystic changes are a precursor to cancer, or merely a marker of
increased risk (see reference 2 for discussion).
The morphologic features of this group is dazzlingly complex, suggesting many potential
mechanisms for increasing "fitness". The complexity also suggests that numerous
trophic interactions are able to influence the ductal cells(15). The patterns have been
given many different names. Two classes of such early ductal proliferations are often
separated. The first group is referred to variously as "proliferative fibrocystic
changes", "usual ductal hyperplasia", or "epitheliosis". The
hallmark of this group is that the ductal cells increase in number by proliferating as
a multilayered epithelium, thus the ductal cells have achieved greater numbers relative to
other cells of their environment. Figures 5 through 9 show two examples of usual
ductal hyperplasia with multilayering of the ductal cells.
Figures 5 through 9
This group of ductal proliferations are still benign, and the following three features
predict their benign nature: 1-Myoepithelial cells are present. Myoepithelial cells are
not always easy to tell from ductal cells and it is generally sufficient to identify
varied cell types within the duct to diagnose it as benign (albeit proliferative).
Although other interpretations are possible, the morphologic evidence is compatible with
the following: the ductal cells in these benign lesions still seem to undergo the
asymmetric cell divisions that leads to the presence of the terminally differentiated
myoepithelial cells. The small pyramidal shaped cells in Figure 9, distinctly
different from the ductal cells, are probably myoepithelial. 2-The ductal cells that grow
away from the basal lamina zone have a less active cytologic appearance compared to
the cells growing in the apparently more choice area next to the basal lamina environment,
as if the ductal cells maintain a partial trophic dependence on the basal lamina zone
in this group of benign lesions. The term "active cytologic
appearance" needs to be described. A basic theorem in cytology is that the appearance
of the nucleus reflects the functional state of the cell(16). A cell that is metabolically
active has abundant euchromatin (pale areas in the nucleus that presumably reflect
transcriptional activity/competence), while a transcriptionally inactive terminally
differentiated cell often has dark condensed heterochromatin. In the center of the ducts
shown in Figure 6, the cells have inactive heterochromatic nuclei, while at the edge of
the duct the cells look more active. The cytoplasm also changes appearance when the cells
are away from the basal lamina environment. In most cases, the cytoplasm becomes less
abundant and more eosinophilic, (presumably indicating increased numbers of intermediate
filaments), while in other cases the cytoplasm can become pale and more abundant. The
important benign feature is that the cytoplasm changes predictably according to the
distance of the cells from the edge of the duct. 3-The ductal cells appear to align their
long axes to point in the same direction(17) as if they assembled cell junctions in a
coordinated manner with their neighbors. This third benign feature is shown in Figures
7-9. The typical pattern-recognition terms that are taught to pathologists in training
include "swirling" of the cells in the same direction (box), or
"streaming" of ductal cells with formation of "attenuated (or
"weak") bridges" around the incompletely filled "slitlike
spaces".
Figures 10 and 11
A second group of benign ductal proliferations are called intraductal papillomas.
In papillomas, the ductal cells seem to proliferate along with an increase in the
stromal fibroblastic compartment, as if the stromal fibroblast proliferations were
stimulated along with ductal cells. Both the ductal cells and myoepithelial cells
comprising papillomas are monoclonal(18, 19). Stratification (increased numbers of cells
lining the basal lamina) is not always prominent in papillomas.
Figures 10 and 11 show a small papilloma with the characteristic admixture of stromal
fibroblasts. The benign nature of this is still manifest by one or more of the three
features described above: 1-a mixture of myoepithelial cells (or varied cell types) within
the proliferation, 2-an alteration of the cytologic features of the ductal cells depending
on how close they are to the basal lamina compartment. 3-a tendency to develop a similar
orientation of their long axis. (Boxes in Figure 11)
Figures 12 and 13
Figures 12-13 show a larger intraductal papilloma. See if you can find the features
indicative of a benign ductal proliferation without looking at the figure legend.
Advanced probable precursor to invasive ductal carcinoma: Atypical ductal hyperplasia
Atypical ductal hyperplasia (ADH) appears more evolved or closer to intraductal
carcinoma than the former groups of proliferative fibrocystic changes, and its occurrence
predicts a 4 fold increased relative risk for subsequent invasive ductal
adenocarcinoma(2,12). The morphologic features of ADH can overlap with the proliferative
ductal lesions at one end of a spectrum, and with intraductal carcinoma at the other
end(20). The increase in fitness of the ductal epithelial cells in atypical ductal
hyperplasia compared to the proliferative ductal lesions previously described can be
recognized given the assumptions: The ductal cells do not require the trophic support
provided by the basal lamina environment, and asymmetric cell divisions that give rise to
myoepithelial cells become very rare. In ADH, the ductal cells seem to have similar cytologic
features whether or not they are near the basal lamina or myoepithelial environments, and
the orientation of one ductal cell seems to be nearly independent of the orientation of
neighboring cells. The low magnification impression is that the duct becomes expanded by a
nearly homogeneous (clonal appearing) population of cells.
Figure 14
Figure 14 shows atypical ductal hyperplasia arising in the midst of a proliferative
ductal lesion. The asterisk is in an area with usual hyperplasia (with more condensed
nuclei in the center of the duct) and the arrow heads show two foci with an apparent clone
of ADH in which the ductal population appears more uniform and the cell growth is about as
robust in the center of the proliferation as it is next to the basal lamina compartment.
Figure 15
Figure 15 shows a different patient's focus of ADH. The distinction from intraductal
carcinoma is somewhat arbitrary but would include the following: 1-the cells have slightly
different cytologic features in one area of the duct (boxed area Figure 15) than the rest
of the population, and they are focally oriented in the same direction (just above the
box). Thus the cells still appear to show some very mild trophic dependence or
responsiveness to their environment. 2-Arbitrarily, if the proliferation does not extend
over 2 mm, ADH rather than intraductal carcinoma is diagnosed(3). It is as if the
proliferation must show an ability to expand in order to warrant a diagnosis of
intraductal carcinoma.
Intraductal carcinoma
This proliferation is also known as ductal carcinoma in situ or DCIS. This
lesion presents no immediate threat to the patient since the cells are not
"invasive" and cannot metastasize. The diagnostic features of the various forms
of DCIS can be accurately appreciated given the following assumption about the ductal
cells' fitness: The ductal cells have no dependence on the basal lamina environment for
trophic support. Thus they grow equally well at any polarity anywhere within the duct. "Grow
equally well at any polarity" means the cytologic features of the cells are the same
anywhere in the duct and cells have a random orientation of their long axes relative to
each other (except as occasionally noted in(17)). Figures 16 and 17 show one subtype of
DCIS (called "micropapillary" DCIS).
Figures 16 and 17
The ductal cells have a uniform appearance throughout many duct profiles, reflecting
the expansion of a clone of cells. The defining feature of malignancy is that the cells in
the center of the duct well away from their natural environment of the basal lamina, have
the same cytologic features as the cells near the basal lamina. A few myoepithelial
cells may rarely be seen in DCIS, probably left from the previous populations that
inhabited the duct before being colonized by DCIS. Figure 18 and 19 show a different
example of micropapillary DCIS.
Figures 18 and 19
Again, the defining feature is the ability of the cells in the center of the duct to
grow as well as the cells near the native basal lamina zone. The reader is cautioned that
occasionally a benign condition called apocrine metaplasia (defined by an appearance of
the cytoplasm resembling apocrine glands) can show a stable multilayering of the
cells without evidence of an altered nuclear appearance (discussed in 1,2,3).
Free to orient randomly and grow equally well anywhere in the duct, highly regular
geometric patterns are often formed in the ducts that are incompletely filled with the
ductal cells. Figures 20 and 21 show the appearance of cribriform DCIS, with nearly
spherical holes randomly placed within an incompletely filled duct.
Figures 20 and 21
Spherical holes in the incompletely filled ducts indicates that the surface area that
is not in contact with a neighboring cells is minimized. In cribriform DCIS, it is as if
cell junctions still cause cells to stick to each other, but the junctions were devoid of
any apparent biologic tethering. The cells stick to each other to minimize the surface
area, but the orientation of any cell contacts seems random. Figures 22 and 23 show
"roman arches", smoothly arching trabeculae with a uniform thickness, composed
of otherwise randomly oriented ductal cells.
Figures 22 and 23
Compare the cytologic and architectural uniformity of the DCIS in Figure 23 with the
cells in Figure 6.
Comedo carcinoma (Figure 24 and 25) is a variant of DCIS defined by the presence of
central necrosis within the duct lumen.
Figures 24 and 25
Typically the line that demarcates the necrosis is always the same distance--usually
about 1 mm from the basal lamina--and it is generally assumed that the necrosis is due to
a critical lack of a diffusible nutrient such as oxygen. The cells in comedo carcinoma
probably need more oxygen that the cells of non-comedo DCIS because they tend to have high
mitotic rates. A striking feature about comedo carcinoma is the ability of the cells to
appear to maintain identical cytologic features whether the cells are located next to the
basal lamina or next to the sharp line of necrosis. In Figure 25, numerous mitoses are
present including some next to the zone of necrosis (arrow heads). Combinations of
cribriform and comedo patterns are possible (Figure 26).
Figure 26
The basic feature of DCIS--that the cells seem to be able to grow with random polarity
and with equal ability whether or not they are adjacent to a basal lamina--is also
essentially diagnostic of adenocarcinoma in situ in most other glandular
tissues, including the lung, the pancreas, the gall bladder and biliary tree, and the
endocervix. (One difference: There are no apparent cells equivalent to myoepithelial cells
in these sites.) Due to arbitrary differences in nomenclature used by pathologists
specializing in different organ systems, however, the name given to the same type of
lesion is sometimes different in other organs. In the gastrointestinal tract, (including
the colon, small intestines, stomach, and glandular metaplasia of the esophagus--Barrett's
esophagus), the proliferation that shows the features described for DCIS is currently
usually called high grade dysplasia. For the ovary, the lesion that corresponds
most closely to the definition of DCIS is called adenocarcinoma of borderline malignancy.
The concept of in situ prostatic adenocarcinoma is controversial. The thyroid gland forms
two types of adenocarcinomas, neither of which seem to progress through phases analogous
to DCIS.
It is likely that other histologic patterns besides the ones recognized by pathologists
are precursors to breast cancer. This is discussed in supplementary text (21).
Invasive ductal carcinoma
In invasion, the ductal cells appear able to induce a new trophic relationship with
the stromal fibroblasts and blood vessels outside the duct. This process is called
desmoplasia. Figure 27 shows invasive ductal carcinoma on the left with a focus of DCIS
(comedo type) on the right.
Figure 27
The stromal tissue around the DCIS is eosinophilic (pink), while the stroma
around the invasive tumor has a pale purple color. The purple color is from the presence
of mucopolysaccharides, a product of activated fibroblasts. Figure 28 shows the junction
between the DCIS and the infiltrating carcinoma.
Figure 28
The DCIS has a smooth outer contour (like a normal duct) since the stromal cells in
this vicinity have produced this architecture long ago when the gland was benign. The
stromal fibroblasts around the DCIS are inactive-appearing as manifest by the relatively
condensed chromatin and inapparent nucleoli (arrows). In contrast, the fibroblasts next to
the invasive tumor have been recently activated as indicated by the presence of a
euchromatic nucleus, nucleoli, cytoplasmic basophilia (reflecting large amounts of
polyribosomes/rough endoplasmic reticulum), and increased secreted mucopolysaccharides in
the extracellular space. Compare the appearance of the stromal compartment in Figure 2.
Invasive tumor cells often have a shaggy outer contour and individual malignant cells can
sometimes be seen in the midst of the active fibroblasts (arrow).
Infiltrating ductal carcinoma is often highly desmoplastic. It is the proliferation of
the fibroblasts and the resulting production of collagen that makes some of the tumors
feel hard. A consequence of the desmoplasia is that a sample of infiltrating ductal
carcinoma typically contains many stromal cells compared to the number of ductal cells.
Desmoplasia appears very close and may be identical to the reaction of fibroblasts in
normal wound healing. Thus, the appearance of the fibroblasts in Figures 27 and 28 and
their production of mucopolysaccharides closely matches the appearance of fibroblasts
about one week following a wound.While collagenases and proteases appear to play a role in
invasion (22), the nature of their role may not merely be to "open the barn
doors" to let the cells out of the duct. There are many natural examples of in
situ carcinomas in which the basal lamina is physically disrupted, but invasion does
not seem to develop. For example, there is no histologic evidence that invasive carcinomas
develop next to any of the multiple transected ducts filled with DCIS in the innumerable
patients for whom mastectomy follows a surgical biopsy. In such cases, one may find the
DCIS cells next to the interface of the transected duct, but they appear to show no
ability to grow out into the stroma. This is even more surprising since the tissue in
which they find themselves has activated stromal cells very similar to the activated cells
that a truly invasive carcinoma is able to induce. It is as if the increasing fitness
displayed by invasive tumor cells were due to an acquired resistance to a growth
inhibitory activity outside of the ductal microenvironment, an idea expressed by Petersen
et. al. (8).
The histologic criteria for invasion are similar for many other epithelial
malignancies. In some cancers, (including some ductal carcinomas and most lobular
carcinomas) the desmoplasia is not as well-developed. The features diagnostic of invasion
in such cases are hard to teach in just a few pages, but would include the finding of
individual detached cancer cells, occasional increases in the amount of cytoplasm in the
tumor cells, and the finding of cells growing without evidence of coordination with the
stroma.
How cytologic features can be used to diagnose breast cancer
Cytopathology is the subspecialty of pathology whereby the characteristics of only tiny
fragments, or sometimes even individual cells (obtained by fine needle aspiration, or from
a nipple discharge) can be used to diagnose malignancy. In fine needle aspirates of the
breast, the ductal epithelium and myoepithelial cells (if present) tend to peel away as a
group from the basal lamina or stroma. These tiny tissue fragments can be diagnosed using
the same framework for diagnosing proliferative ductal lesions and DCIS in tissue
sections. Thus, the relation of ductal cells to each other or to any myoepithelial cells
can provide diagnostic information. For example, tiny tissue fragments from a benign duct
show a mixture of myoepithelial cells with ductal cells in essentially a 2 dimensional
sheet (Figure 29, Papanicolaou stained).
Figure 29
The myoepithelial cells often have heterochromatic nuclei, and appear in a different
plane from the ductal cells (arrow). Figure 30 shows a three dimensional architecture of
cells, indicating that the duct is lined by cells more than one cell layer thick.
Figure 30
The cells appear to be a uniform population with little tendency to vary in their
nuclear features in any direction (reflecting the putative ability of the cells to grow
well whether or not they are next to the basal lamina), and there is no common polarity
between adjacent cells. The findings were "suspicious for malignancy" and found
to be atypical ductal hyperplasia on biopsy. Figure 31 shows unequivocal three dimensional
growth of a monoclonal-appearing cell population in a different fine needle aspiration.
Figure 31
The same features diagnostic of DCIS in tissue sections are evident in Figure 31.
Invasive ductal carcinoma cannot be reliably distinguished from DCIS by cytology.
Cancer cells do not always have to be in tissue fragments to be diagnosed. In fact, the
discohesion of cancer cells--presumably reflecting decreased cell-cell contacts--is
itself useful for recognizing cancer in cytology smears. The "cytologic criteria of
malignancy"(16) allow cancer cells to be distinguished from normal ductal cells based
on nuclear changes. These changes are not absolute and can be sometimes seen to some
extent in normal cells. The changes are displayed well with alcohol fixation or air
drying, and are harder to see in formalin fixed tissue sections. Most cancers anywhere in
the body show nuclear changes compared to normal cells. The types of nuclear changes vary
from cancer to cancer and there are important examples of specific nuclear structural
changes diagnostic of certain types of cancer. Figures 32 and 33 show some of the nuclear
features characteristic of many cancers: sharp and variably deep indentations of the
nuclear membrane contour, lobulated nuclear contours, aneurism-like swelling of parts of
the nuclear membrane, variable thickness of the nuclear membrane associated
heterochromatin, asymmetry in the distribution of heterochromatin/euchromatin, large
variations in any one cell in the size and shape of heterochromatin aggregates, and
nucleoli that have angular contours or are inappropriately large for the apparent needs of
the cell.
Figure 32 and 33
The apparent needs of the cell are gauged by the degree of basophilia of the
cytoplasm (reflecting the number of mRNA-containing polyribosomes), the prominence of a
golgi zone, and the amount of secretory material produced. Normal cells are typically
uniform and symmetric, with smooth contoured nuclear membranes and evenly textured
chromatin, and nucleoli that appear rounded and predictable from cell to cell and are
coordinated with the apparent needs for ribosome synthesis(16).
There are problems applying the cytologic criteria of malignancy to individual cells in
breast cancer diagnosis. Under some circumstances, cells believed on the basis of their
patterns in a tissue section to be benign can closely resemble cancer cells. Often, but
not always, this circumstance can be deduced from the appearance and mixture of other
cells present. There are not enough studies of such examples to know whether these cells
are truly neoplastic(21). Another problem is that some breast cancers do not show many
nuclear changes. Thus, the architectural features described above are often more
diagnostically useful than the changes in individual cell nuclei. When present, the
nuclear changes are seen early in the apparent evolution. Comedo carcinoma nearly always
shows nuclear changes diagnostic of malignancy. The degree of nuclear abnormality has
prognostic significance(3).
Some, but not all, of the malignant nuclear features appear closely related to genetic
instability. Tumors in which cell to cell heterogeneity and aneuploidy are most marked
often have the most asymmetric chromatin distributions. It is not clear, however whether
the nuclear abnormalities are a consequence or a cause of the genetic instability. Nuclear
changes do not always occur in aneuploid cells, and in cell culture systems, oncogene
expression per se, rather than karyotypic changes, seem to be associated with nuclear
structural changes(23). Figure 34 shows an abnormal mitotic figure (highly predictive of
aneuploidy). Such asymmetric mitoses are typically found in the cells with asymmetry in
interphase. Aneuploidy cannot explain all of the abnormal nuclear features in cancer
cells, and the biochemical basis or functional significance any of these nuclear
characteristics of cancer are unknown.
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