Endocrine-Related Cancer (2001) 8 47–61

Histological and biological evolution of

human premalignant breast disease
D C Allred 1,2, S K Mohsin1,2 and S A W Fuqua1,3
1
The Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA
2
Department of Pathology, Baylor College of Medicine, Houston, Texas 77030, USA
3
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
(Requests for offprints should be addressed to D C Allred, Breast Center, Baylor College of Medicine, One
Baylor Plaza, MS600, Houston, Texas 77030, USA; Email: dcallred@breastcenter.tmc.edu)

Abstract
Most human invasive breast cancers (IBCs) appear to develop over long periods of time from certain
pre-existing benign lesions. Of the many types of benign lesions in the human breast, only a few appear
to have significant premalignant potential. The best characterized of these include atypical hyperplasias
and in situ carcinomas and both categories are probably well on along the evolutionary pathway to IBC.
Very little is known about earlier premalignant alterations. All types of premalignant breast lesions are
relatively common but only a small proportion appear to progress to IBC. They are currently defined
by their histological features and their prognosis is imprecisely estimated from indirect epidemiological
evidence. Although lesions within specific categories look alike, they must possess underlying biological
differences causing some to remain stable and others to progress. Recent studies suggest that they
evolve by highly diverse genetic mechanisms and research into these altered pathways may identify
specific early defects that can be targeted to prevent premalignant lesions from developing or becoming
cancerous. It is far more rational to think that breast cancer can be prevented than cured once it has
developed fully. This review discusses histological models of human premalignant breast disease that
provide the framework for scientific investigations into the biological alterations behind them and
examples of specific biological alterations that appear to be particularly important.
Endocrine-Related Cancer (2001) 8 47–61

Introduction Stewart 1945, Wellings & Jensen 1973, Wellings et al. 1975)
and that they were much less common in non-cancerous
Invasive breast cancer (IBC) is one of the most common and
breasts than in breasts with synchronous IBC (Foote &
lethal malignant neoplasms affecting women in Western
Stewart 1945, Wellings & Jensen 1973, Wellings et al. 1975,
cultures. The majority of IBCs are thought to develop over
Alpers & Wellings 1985). Other studies showed that women
long periods of time from certain pre-existing benign lesions.
with a history of atypical hyperplasias and in situ carcinomas
There are many types of benign lesions in the human breast
had approximately 5- and 10-fold increased relative risks,
and only a few appear to have significant premalignant
respectively, of eventually developing IBC (Page et al. 1982,
potential. The best characterized premalignant lesions
1985, Dupont & Page 1985, Palli et al. 1991, London et al.
recognized today are referred to as atypical ductal 1992, Dupont et al. 1993). The elevated risks associated with
hyperplasia (ADH), atypical lobular hyperplasia (ALH), ADH, ALH, and LCIS are bilateral, suggesting that they may
ductal carcinoma in situ (DCIS), and lobular carcinoma in only be markers rather than precursors of IBC (Page &
situ (LCIS). All these lesions possess some malignant Dupont 1993). However, these lesions are frequently
properties such as a relative loss of growth control, but they multifocal and bilateral (Foote & Stewart 1945, Wellings &
lack the ability to invade and metastasize and, in this sense, Jensen 1973, Wellings et al. 1975) which, in light of their
are premalignant. histological continuity with IBC and increased incidence in
Several types of evidence point to this handful of lesions cancerous breasts, suggests that they may be both risk factors
as being important precursors of human IBC (Table 1). For as well as precursors. DCIS is usually a unifocal disease and
example, pathologists recognized many years ago that they the associated risk for developing IBC is primarily ipsilateral,
were on a histological continuum between normal epithelium consistent with the notion that DCIS is a relatively advanced
in terminal duct lobular units (TDLUs) and IBC (Foote & and committed precursor. The most compelling evidence that

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Allred et al.: Premalignant breast disease

Table 1 General types of evidence supporting the idea that invasive breast cancers arise from certain pre-existing benign
lesions over long periods of time
Evidence ADH ALH DCIS LCIS
On a histological continuum between TDLUs and IBC Yes Yes Yes Yes
Less common in non-cancerous breasts than in breasts Yes Yes Yes Yes
with synchronous IBC (5% vs <50%) ? (5% vs <80%) (5% vs <50%)
Risk factors for developing IBC Yes Yes Yes Yes
(5-fold) (5-fold) (10-fold) (10-fold)
Shared genetic alterations with synchronous IBC Yes Yes Yes Yes

all these lesions may be precursors comes from recent studies ALH appears to arise directly within normal appearing TDLUs
showing that they share identical genetic abnormalities with as small mildly atypical epithelial cells which begin to fill and
synchronous ipsilateral IBC (O’Connell et al. 1998). Over partially distend the ducts and acini, and there is some
the past twenty years or so, all of this evidence has speculation that this may occur preferentially in relatively well
culminated in a histological model of human breast cancer differentiated type II TDLUs (Russo & Russo 1997). If the
evolution which proposes that stem cells in normal TDLUs cells accumulate until the spaces are distended to a large
give rise to atypical hyperplasias (ADH and ALH), which extent, the lesions are referred to as LCIS. Thus, the
progress to in situ carcinomas (DCIS and LCIS), which evolutionary pathways of lobular lesions (i.e. ALH and LCIS)
eventually develop into invasive and metastatic disease seem to be different from those of ductal lesions (i.e. ADH and
(Fig. 1). DCIS) and lobular lesions are less common. In a sense, the
There are many morphological differences between terms ‘ductal’ and ‘lobular’ are misleading because they imply
TDLUs and atypical hyperplasias and there are no an origin and localization to either ducts or lobules when, in
unequivocal intermediate lesions between them. In the early fact, all types of premalignant breast lesions can occupy both
1970s, Wellings and co-workers proposed that a common locations and ultimately appear to arise from stem cells in
alteration of TDLUs which they called ‘atypical lobules type TDLUs (Rudland 1993) or in ULs which themselves arise from
A’ (ALA) may be involved in the transition from TDLUs to TDLUs. Wellings and colleagues also appreciated the distinct
ADH and beyond (Wellings et al. 1975). ALAs, which are histological evolution of lobular lesions which they referred to
referred to as unfolded lobules (ULs), among other names, as ‘atypical lobules type B’ (ALB) (Wellings & Jensen 1973,
in today’s terminology, resemble TDLUs in overall Wellings et al. 1975). ALH and LCIS are bilateral risk factors
architecture but are much larger due to the proliferation and for developing IBC and the IBCs that eventually develop are
accumulation (i.e. hyperplasia) of the epithelial cells lining as likely to be infiltrating lobular carcinomas (ILCs) as
their acini. The structure of normal TDLUs themselves varies non-lobular subtypes (Page et al. 1986). However, when they
considerably as a function of hormonal status (e.g. are found in a breast with synchronous IBC, the latter is usually
menstruation, pregnancy, etc.) and they are grouped into four an ILC or an invasive lesion with prominent lobular features.
histological categories (types I through IV) on a continuum Taken together, ALH and LCIS appear to be markers of
of differentiation towards lactation (Russo et al. 1987, 1992). widespread genetic damage to breast epithelium (i.e. risk
Type I TDLUs, the least differentiated, have relatively high factors) as well as precursor lesions.
proliferation rates and are somewhat more common in This linear histological model of breast cancer evolution
cancerous breasts, suggesting that they may preferentially undoubtedly oversimplifies a very complex process. For
give rise to early growth alterations with premalignant example, it is quite possible that some IBCs arise directly
potential such as ULs (Dickson & Russo 2000). Once from morphologically normal appearing cells. In addition,
developed, ULs have the potential to evolve along several many premalignant lesions do not progress to IBC during the
diverse pathways including to microcystic disease, to a average lifespan of a woman, so progression is
common type of hyperplastic lesion referred to as usual non-obligatory. Some lesions may even revert to less
ductal hyperplasia (UDH), as well as to ADH. Furthermore, advanced phenotypes. The histological appearances of
these pathways appear to be relatively mutually exclusive. premalignant lesions within specific categories are very
Although UDH has been shown to be a weak risk factor for similar (by definition), so there must be underlying biological
developing IBC (approximately twofold) (Page & Dupont abnormalities causing some to remain stable and others to
1993), it does not fit well on the histological continuum to progress. Despite its shortcomings, however, this model has
IBC and thus may be a side branch on the evolutionary tree been very useful as a framework for scientific studies into
through shared ancestry with ULs rather than an important the biological causes of tumor progression, which may
precursor of IBC. eventually lead to strategies for breast cancer prevention.
In contrast to ADH, which seems to develop from ULs, There have been hundreds of studies during the past decade

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 Endocrine-Related Cancer (2001) 8 47–61

Figure 1 Histological model of breast cancer evolution and representative photomicrographs of important premalignant lesions.
Terminal duct lobular units (TDLUs) are thought to be the major stem cell compartment giving rise to all types of premalignant
breast lesions. Unfolded lobules (ULs) are TDLUs that are greatly expanded due to hyperplasia of their lining epithelium and
may represent the earliest morphologically recognizable premalignant change. Atypical ductal hyperplasias (ADHs) are small
clonal outgrowths of low-grade epithelium (circle) that often arise in ULs. Ductal carcinoma in situ (DCIS) are large outgrowths
of epithelium that greatly distend ductal and lobular spaces and vary on a histological continuum from low to high grade lesions.
Atypical lobular hyperplasias (ALHs) represent relatively normal sized TDLUs that are partially filled by low grade neoplastic
epithelium. Lobular carcinoma in situ (LCIS) represent TDLUs that are greatly distended by cells that are cytologically identical
to those of ALH. ALH and LCIS are essentially the same disease on a quantitative continuum.

evaluating dozens of biological pathways in premalignant lesions (Table 2). Proliferation in TDLUs averages only
breast disease. This review discusses a few that appear to be about 2% overall (Meyer 1977, Ferguson & Anderson
particularly important and that have been studied in a 1981, Joshi et al. 1986, Longacre & Bartow 1986, Russo
relatively comprehensive manner. et al. 1987, Going et al. 1988, Potten et al. 1988,
Kamel et al. 1989, Schmitt 1995, Visscher et al. 1996,
Mohsin et al. 2000a). In premenopausal women, the rate
Growth characteristics of premalignant
fluctuates with the menstrual cycle and is twofold higher in
breast disease the luteal than in the follicular phase (Potten et al. 1988).
Even though microscopic in size, all types of premalignant The association between hormonal status and proliferation
breast lesions are ‘tumors’ which expand TDLUs and emphasizes the importance of estrogen and progesterone as
proximal ducts to many times their normal size (Fig. 2). mitogens for normal breast epithelium (Pike et al. 1993).
Many studies, using a variety of techniques, have measured Proliferation has not been evaluated in ULs with the
the magnitude of proliferation in TDLUs and premalignant exception of one preliminary study reporting an average rate

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Allred et al.: Premalignant breast disease

Figure 2 All types of premalignant breast lesions are ‘tumors’ which expand terminal duct lobular units (TDLUs) and proximal
ducts to many times their normal size. This example shows a normal TDLU on the left compared with one being distended by
ductal carcinoma in situ (DCIS) on the right.

Table 2 Growth (proliferation and apoptosis) in premalignant this diversity (Berardo et al. 1996a). Proliferation is
breast lesions proportional to differentiation along this histological
TDLU UL ADH DCIS ALH LCIS continuum with rates averaging as low as 1% in the lowest
Average % 2% 5% 5% 15% low 2% grade to more than 70% in the highest grade lesions (Bobrow
proliferation et al. 1994, Berardo et al. 1996a). Proliferation has not been
Average % apoptosis 0.6% low 0.3% 5% low low formally studied in ALH but is probably similar to LCIS
where the reported average is about 2% (Fisher et al. 1996,
Rudas et al. 1997, Libby et al. 1998, Querzoli et al. 1998).
of about 5%, which is still two- to threefold higher than in The overall growth of premalignant breast lesions can be
normal TDLUs (Mohsin et al. 2000a). Studies of ADH also viewed simplistically as a balance between cell proliferation
observed rates averaging about 5% (De Potter et al. 1987, and cell death. On average, the cells in all types of
Hoshi et al. 1995, Mohsin et al. 2000a). Proliferation has premalignant lesions proliferate faster than normal cells in
been studied more extensively in DCIS than in any other type TDLUs (Fig. 3), contributing to their positive growth
of premalignant breast lesion (Meyer 1986, Locker et al. imbalance. Much less is known about cell death in this
1990, Bobrow et al. 1994, Poller et al. 1994, Zafrani et al. setting (Table 2). One preliminary study reported
1994, Albonico et al. 1996, Berardo et al. 1996a, Mohsin et significantly lower rates of apoptosis in ADH compared with
al. 2000a). Rates average about 5% in histologically TDLUs in the same breasts (0.3% vs 0.6% respectively),
low-grade ‘non-comedo’ DCIS compared with 20% in suggesting that the growth of ADH may be the result of both
high-grade ‘comedo’ lesions. The widespread practice of increased proliferation and decreased cell death compared
dichotomizing DCIS into non-comedo and comedo subtypes with normal cells (Prosser et al. 1997). However, a few
is misleading in the sense that, similar to IBC, DCIS shows studies have reported rates of apoptosis in DCIS that are up
tremendous histological diversity along a continuum ranging to 10-fold higher than typically seen in normal cells (Bodis
from very well to very poorly differentiated, and grading et al. 1996, Harn et al. 1997, Prosser et al. 1997), yet DCIS
systems have been developed which more accurately convey have a large positive growth imbalance, suggesting that the

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Figure 3 Examples of typical proliferation rates in premalignant breast lesions as assessed by immunohistochemistry using the
Ki67 antibody (small dark nuclei represent dividing cells). Terminal ductal lobular units (TDLUs) in premenopausal (pre) women
usually contain more proliferating cells than TDLUs in postmenopausal (post) women due to the mitogenic effects of estrogen.
Unfolded lobules (ULs), atypical ductal hyperplasias (ADHs), and low-grade ‘non-comedo’ ductal carcinoma in situ (ncDCIS)
contain, on average, two to three times more proliferating cells than normal TDLUs. Typically, a large proportion of cells are
proliferating in high-grade ‘comedo’ DCIS (cDCIS). Proliferation is usually quite low in atypical lobular hyperplasia (ALH) and lobular
carcinoma in situ (LCIS).

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Allred et al.: Premalignant breast disease

relationship between cell proliferation and death may not such as that between progesterone and PgR. The average
always be accurately portrayed by the static methods used to proportion of ER-positive cells in TDLUs in postmenopausal
measure these dynamic processes. Like proliferation, women is somewhat higher (about 50%) and stable in the
apoptosis seems to vary with histological differentiation in absence of hormone replacement therapy (Mohsin et al.
DCIS, being much lower in non-comedo than comedo lesions 2000a). Very little is known about ER expression in ULs,
(averaging about 1% vs 5% respectively) (Prosser et al. although one preliminary study reported that virtually all
1997). Disturbances of the equilibrium between cell express the receptor in over 90% of cells (Mohsin et al. 2000a).
proliferation and cell death probably result from alterations A few studies have evaluated ER in ADH and collectively
of several normal growth-regulating mechanisms including agree that nearly all lesions express very high levels in nearly
those involving sex hormones, oncogenes, tumor suppressor all cells (Barnes & Masood 1990, Schmitt 1995, Mohsin et al.
genes, and many other as yet unknown genetic and epigenetic 2000a). Many studies have evaluated ER in DCIS and, on
abnormalities, some examples of which are discussed below. average, about 75% of all cases express the receptor (Giri et
al. 1989, Helin et al. 1989, Masood 1990, Pallis et al. 1992,
Chaudhuri et al. 1993, Poller et al. 1993b, Zafrani et al. 1994,
Hormones and receptors in premalignant
Leal et al. 1995, Albonico et al. 1996, Barnes & Berardo et al.
breast disease 1996a, Bose et al. 1996, Karayiannakis et al. 1996, Mohsin et
Estrogen, mediated through the estrogen receptor (ER), plays al. 2000a). Expression varies with histological differentiation,
a central role in regulating the growth and differentiation of being highest in non-comedo lesions, where up to 100% show
normal breast epithelium (Henderson et al. 1988, Pike et al. expression in over 90% of cells, and lowest in comedo lesions,
1993). It stimulates cell proliferation and regulates the where only about 30% show expression in a minority of cells.
expression of other genes including the progesterone receptor ER is not expressed in about 25% of DCIS and these are
(PgR). PgR then mediates the mitogenic effect of predominantly high-grade comedo lesions. Over 90% of
progesterone, further stimulating proliferation (Henderson et LCIS express high levels of ER in nearly all cells (Giri et al.
al. 1988, Pike et al. 1993). Many additional factors 1989, Pertschuk et al. 1990, Pallis et al. 1992, Fisher et al.
collectively referred to as ‘coactivators’ and ‘corepressors’ 1996, Rudas et al. 1997, Libby et al. 1998, Querzoli
have been discovered recently which appear to modulate the et al. 1998), which is probably similar in ALH although formal
functions of these hormones and receptors, including their studies are lacking.
mitogenic activity (Horwitz et al. 1996). Prolonged estrogen exposure is an important risk factor
Several studies have assessed ER expression in normal for developing IBC, perhaps by allowing random genetic
breast epithelium and premalignant lesions (Table 3). Most alterations to accumulate in normal cells stimulated to
were immunohistochemical studies focusing presumably on proliferate (Henderson et al. 1988), which may also be true
ER-alpha, although the potential cross-reactivity for ER-beta for cells in premalignant lesions. The very high levels of ER
of all the different antibodies used in these studies is not observed in nearly all premalignant lesions (Fig. 4) may
entirely clear. Mindful of this qualification, studies of normal contribute to their increased proliferation relative to normal
TDLUs reported that nearly all (over 90%) express ER, but in a cells by allowing them to respond more effectively to any
minority of cells (averaging about 30%) for all ages combined level of estrogen, even the low concentrations observed in
(Allegra et al. 1979, Peterson et al. 1986, Ricketts et al. 1991, postmenopausal women (Mohsin et al. 2000a). In addition to
Schmitt 1995, Mohsin et al. 2000a). In premenopausal women increased levels of expression, however, there may be other
the average proportion of ER-positive cells in TDLUs is alterations of ER resulting in increased growth. For example,
somewhat lower (about 20%) and varies with the menstrual proliferation in TDLUs occurs predominantly in ER-negative
cycle, being twice as high during the follicular phase as during epithelium (Clarke et al. 1997, Russo et al. 1999), whereas
the luteal phase (Ricketts et al. 1991). Proliferation in TDLUs the majority of dividing cells in premalignant lesions are ER
peaks during the luteal phase (Potten et al. 1988), suggesting positive (Shocker et al. 1999), so the normal
that the normal mitogenic effect of estrogen may be partially compartmentalization of hormonally regulated growth
delayed, or indirect and mediated by downstream interactions appears to be disrupted early on. As another example, one
recent study measured proliferation in TDLUs and
premalignant lesions from the same breasts in a large number
Table 3 Estrogen receptor expression in premalignant breast
lesions of patients stratified by menopausal status (Mohsin et al.
2000a). Proliferation rates in TDLUs were nearly threefold
TDLU UL ADH DCIS ALH LCIS
lower in postmenopausal compared with premenopausal
% Containing 90% 95% 95% 75% high 95% women, consistent with the expected mitogenic effect of
ER-positive cells estrogen in normal cells. In contrast, the difference in
Average % 30% 90% 90% 45% high 90% proliferation in premalignant lesions stratified by menopausal
ER-positive cells
status was less than half that of normal cells, again

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 Endocrine-Related Cancer (2001) 8 47–61

Figure 4 Examples of typical estrogen receptor (ER) expression in premalignant breast lesions as assessed by
immunohistochemistry (small dark nuclei are ER-positive cells). Terminal duct lobular units (TDLUs) in premenopausal (pre)
women usually contain relatively few ER-positive cells. In contrast, the majority of cells in TDLUs of postmenopausal (post)
women express ER. Most premalignant breast lesions show very high levels of ER in nearly all cells, including unfolded lobules
(ULs), atypical ductal hyperplasias (ADHs), low grade ‘non-comedo’ ductal carcinoma in situ (ncDCIS), atypical lobular
hyperplasias (ALHs), and lobular carcinoma in situ (LCIS). The only significant exception is high grade ‘comedo’ DCIS (cDCIS)
which often show low or no ER expression.

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Allred et al.: Premalignant breast disease

demonstrating that the hormonal regulation of proliferation in premalignant breast disease, but the majority of studies have
these lesions is fundamentally abnormal. Another particularly been small and have not been validated (see reviews: Berardo
interesting recent study (Fuqua et al. 2000) found a somatic et al. 1996b, Allred et al. 1997, Libby et al. 1999, Allred &
mutation in the ER gene in 30% of hyperplastic breast lesions Mohsin 2000). Exceptions include the erbB2 oncogene and
(UDH) which, when transfected into breast cancer cell lines, p53 tumor suppressor gene, which have both been evaluated
showed much higher transcriptional activity and proliferation in a large number of studies.
than wild-type ER at very low concentrations of estrogen erbB2 is amplified and/or overexpressed in 20–30% of
such as seen in postmenopausal women (Fig. 5). The mutated IBCs (Ravdin & Chamness 1995). These abnormalities are
ER also showed increased binding to the co-activator TIF-2, associated with increased proliferation, poor clinical
which may partially explain its increased functional outcome, and altered responsiveness to various types of
responsiveness to estrogen. Whatever the mechanisms, the adjuvant therapies (De Potter 1994, Ravdin & Chamness
hypersensitivity to estrogen associated with this mutation 1995, DiGiovanna 1999). erbB2 may also promote cell
may play a very important role in the early development and motility (De Potter & Quatacker 1993, De Potter 1994),
progression of premalignant breast disease. which could contribute to the ability of tumor cells
overexpressing erbB2 to invade and metastasize. Nearly all
Oncogenes and tumor suppressor genes studies of erbB2 in premalignant breast disease have used
immunohistochemistry to detect overexpression of the
in premalignant breast disease
oncoprotein, which is highly correlated with gene
In addition to proliferation and ER, a large number of other amplification (Venter et al. 1987). Overexpression has not
biological characteristics have been evaluated in human been observed in TDLUs (De Potter et al. 1989, Allred et al.

Figure 5 Somatic point mutation (Lys for Arg at position 303) of the estrogen receptor (ER) gene identified in a high proportion of
hyperplastic breast lesions that results in functional ‘hypersensitivity’ to estrogen. The mutated ER has normal binding affinity for
estrogen but, when transfected into breast cancer cell lines, results in markedly increased transcriptional activity and proliferation in
response to estrogen. In the growth curves shown, note the much higher rates of growth at very low estrogen (E2) concentrations in
the cells transfected with mutated compared with wild type (WT) ER. These phenomena, especially the increased proliferation,
could be very important in the early development of premalignant breast lesions and their progression to cancer.

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1992) and it has been detected only rarely in ADH low-grade non-comedo lesions, and relatively common
(Gusterson et al. 1988, De Potter et al. 1989, Lodato et al. (about 40%) in high-grade comedo lesions. Abnormalities of
1990, Allred et al. 1992). Many studies have evaluated p53 have been detected in only about 5% of LCIS (Domagala
erbB-2 in DCIS (van de Vijer et al. 1988, Bartkova et al. et al. 1993, Younes et al. 1995), which is probably similar
1990, Lodato et al. 1990, Ramachandra et al. 1990, Barnes to ALH. Mutations of p53 may contribute to the development
et al. 1991, Walker et al. 1991, Allred et al. 1992, Barnes et and progression of premalignant breast disease by several
al. 1992a, Schimmelpenning et al. 1992, Somerville et al. mechanisms, including interference with DNA repair through
1992, De Potter et al. 1993, 1995, Tsuda et al. 1993, Bobrow loss of an important G1 cell-cycle checkpoint, leading to
et al. 1994, Poller et al. 1994, Zafrani et al. 1994, Leal et al. replication of a damaged DNA template and genetic
1995, Albonico et al. 1996, Berardo et al. 1996a). The instability, and also perhaps by clonal expansion through
average incidence of amplification and/or overexpression inhibition of programmed cell death (Levine 1997).
was about 10% in non-comedo compared with 60% in Most of the biological abnormalities responsible for the
comedo lesions. However, as with many other biological development and progression of premalignant breast lesions
features in DCIS, alterations of erbB2 vary directly with are still unknown. Recent genetic studies demonstrate that
differentiation on a histological continuum (Berardo et al. their biological evolution is very complex. Many recent
1996a). Studies of erbB2 in ALH have not been published, studies have assessed allelic imbalance (AI) by loss of
although several have addressed LCIS and reported heterozygosity (LOH) analysis or comparative genomic
abnormalities in about 2% (Gusterson et al. 1988, Lodato et hybridization (CGH) (Table 4). These methods can identify
al. 1990, Ramachandra et al. 1990, Porter et al. 1991, the general chromosomal locations of non-functional tumor
Somerville et al. 1992, Midulla et al. 1995, Fisher et al. suppressor genes (through losses) or amplified oncogenes
1996). Just how alterations of erbB-2 lead to the development (through gains) that may be important in the development of
and progression of premalignant breast disease is not entirely premalignant disease.
clear, although both the increased proliferation and motility Studies of AI in premalignant lesions from non-
of cells associated with overexpression may contribute. cancerous breasts (i.e. without synchronous IBC) are an ideal
Whatever the mechanisms, the absence of overexpression in setting to identify genetic alterations that may be important
normal TDLUs and ADH, compared with the relatively high in the early development of these lesions. Those assessing
rate in DCIS, suggests that alterations of erbB2 are an atypical hyperplasias (ADH and ALH) have shown that up
important event in early malignant transformation. to 50% contain one or more AIs among more than 30 genetic
p53 also appears to play an important role in the loci distributed over 10 chromosomes that have been
evolution of premalignant breast disease. This tumor evaluated so far (Lakhani et al. 1995b, Rosenberg et al. 1996,
suppressor gene is mutated in about 30% of IBCs, which is 1997, Chauqui et al. 1997, Nayar et al. 1997, O’Connell et
associated with generally aggressive biological features and al. 1998). Not surprisingly, AIs were more common in
poor clinical outcome (Elledge & Allred 1994, Chang et al. non-invasive carcinomas (DCIS and LCIS) than in
1995). Most are missense point mutations resulting in an hyperplasias. Nearly all DCIS showed at least one AI among
inactivated but stabilized protein that accumulates to very more than 100 genetic loci on 17 chromosomes studied so
high levels in the cell nucleus (Davidoff et al. 1991a). Hence, far, consistent with the notion that they represent a relatively
measuring protein levels is a relatively easy and accurate late stage of evolution (Radford et al. 1993, 1995a,
surrogate assay for detecting mutations and most studies of O’Connell et al. 1994, 1998, Aldaz et al. 1995, Munn et al.
premalignant disease have used immunohistochemistry to 1995, Stratton et al. 1995, Fujii et al. 1996a,b, Man et al.
assess p53 status. With the exception of morphologically 1996, Chappell et al. 1997, Waldman et al. 2000). LCIS has
‘normal’ breast epithelium in Li-Fraumeni patients with also shown multiple gains and losses involving at least 8
inherited mutations (Barnes et al. 1992b), abnormalities of chromosomes (Lakhani et al. 1995a, Nayar et al. 1997, Lu
p53 have not been reported in TDLUs (Bartek et al. 1990, et al. 1998). In contrast to atypical hyperplasias, which
Davidoff et al. 1991b, Eriksson et al. 1994, Rajan et al. usually show only one or two imbalances individually, in situ
1997). p53 also appears to be normal in nearly all ADH carcinomas typically demonstrate many, especially comedo
(Bartek et al. 1990, Umekita et al. 1994, Chitemere et al. DCIS which in one study had as many as eight in a single
1996). Similar to erbB2, many studies have assessed p53 in lesion (O’Connell et al. 1998). The highest rates of AI in
DCIS (Walker et al. 1991, Poller et al. 1993a, Tsuda et al. DCIS approach 80% and involve loci on chromosomes 16q,
1993, Bobrow et al. 1994, Eriksson et al. 1994, O’Malley et 17p, and 17q, suggesting that altered genes in these regions
al. 1994, Zafrani et al. 1994, Leal et al. 1995, Schmitt 1995, may be particularly important in the development of DCIS.
Albonico et al. 1996, Berardo et al. 1996a, Bose et al. 1996, The genetic diversity of DCIS and LCIS assessed by LOH
Chitemere et al. 1996, Siziopikou et al. 1996, Rajan et al. and CGH rivals the complexity observed in IBC.
1997) and found alterations to correlate directly with Several studies have evaluated AI in premalignant
histological differentiation, being quite rare (about 5%) in lesions from non-cancerous breasts compared with

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Table 4 General chromosomal locations of allelic imbalances (gains and losses) in premalignant breast lesions from studies
assessing loss of heterozygosity and comparative genomic hybridization
Category Losses Gains
ADH 1q, 2p, 6q, 9p, 11p, 11q, 13q, 14q, 16q, 17p, 17q, Xq Unknown
ALH 11q, 16p, 16q, 17p, 22q 6q
DCIS 1p, 1q, 2p, 2q, 3p, 3q, 4p, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 11p, 11q, 12p, 13q, 1q, 3q, 6p, 6q, 8q, 17q, 20q, Xq
14q, 15q, 16p, 16q, 17p, 17q, 18q, 21q
LCIS 11q, 13q, 16p, 16q, 17p, 17q, 22q 6q

histologically similar lesions from cancerous breasts as a Studies of LOH, CGH, and many other methodologies
strategy to identify alterations which might be important in over the past decade provide crude but compelling evidence
the progression to invasive disease (Allred & Mohsin 2000). that IBC evolves from premalignant lesions by highly diverse
Following this strategy, one recent study of a marker on genetic and epigenetic mechanisms. Hopefully, future studies
chromosome 11p (D11S988) showed rates of LOH will provide more detailed information about specific
increasing from 10% to 20% in UDH, 10% to 40% in ADH, mechanisms which can be manipulated to prevent the
and 20% to 70% in DCIS (O’Connell et al. 1998). The gene development and progression of premalignant disease.
for cyclin D1 resides near this locus, suggesting that Progress in the past has been hampered by a reliance on
alteration of its function may be important in tumor correlative studies of small archival tissue samples from
progression, although many other genes in this region are patients that are difficult to obtain – due in part to a lack
probably also involved. In the same study, comedo DCIS of appropriate cell lines and animals to support mechanistic
showed significant increases in LOH at several other loci studies. Fortunately, cell lines and animal models are
including D2S362 on 2q (10% to 40%), D13S137 on 13q beginning to emerge to support the mechanistic studies
(10% to 40%), and D17S597 on 17q (5% to 40%), suggesting necessary for more fundamental progress (Allred & Medina
that high-grade DCIS is particularly unstable genetically and 2000), such as the MCF10AT cell line that can mimic certain
that several alterations may be important in tumor aspects of ADH and DCIS (Dawson et al. 1996, Shekhar et
progression. al. 1998).
Studies of AI in premalignant breast lesions from
cancerous breasts also provide an opportunity to assess Prognostic factors in premalignant breast
shared alterations with synchronous IBC as an indication of
disease
their evolutionary relatedness. In one recent study
(O’Connell et al. 1998) assessing LOH at 15 loci on 12 Premalignant lesions are very common and they are being
chromosomes, 50% of ADH shared their LOH phenotypes diagnosed more frequently due to increasing public
with synchronous IBC, providing novel and compelling awareness and screening mammography. They are currently
genetic evidence that ADH is a direct precursor of IBC. defined by their histological features and their prognosis is
Many studies of DCIS and a few of LCIS have shown that imprecisely estimated based on indirect epidemiological
nearly all lesions share several identical AIs with evidence (Page & Dupont 1993). While lesions within
synchronous IBC, providing convincing if not surprising specific categories look alike histologically, there must be
evidence that they too are evolutionarily related (Radford et underlying biological differences causing a subset to progress
al. 1995b, Stratton et al. 1995, Zhuang et al. 1995, Fujii et to IBC. Studies identifying biological prognostic factors in
al. 1996a, Ahmadian et al. 1997, Dillon et al. 1997, premalignant disease are beginning to emerge.
O’Connell et al. 1998). Synchronous DCIS and IBC may For example, preliminary results from two recent studies
occasionally show distinct AIs, suggesting that there may suggest that increased levels of ER in normal breast
also be divergent aspects to their evolution (Fujii et al. epithelium (Khan et al. 1998) and certain premalignant
1996a). lesions (UL, ADH, and DCIS) (Mohsin et al. 2000b) may be
An interesting study by Deng and colleagues (Deng et associated with a two- to threefold increased risk of
al. 1996) noted that histologically normal TDLUs shared developing IBC, and assessing ER status may eventually be
LOH for markers on 3p, llp, and 17p with closely adjacent important in clinical management. Its most promising role
IBC, while TDLUs farther away in the same breast did not, may be in identifying patients with high-risk premalignant
suggesting that even normal appearing epithelium may have lesions who might benefit from hormonal therapy. In the
genotypic abnormalities associated with an elevated risk for recent National Surgical Adjuvant Breast Project (NSABP)
developing breast cancer. P-1 chemoprevention clinical trial (Fisher et al. 1998),

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 Endocrine-Related Cancer (2001) 8 47–61

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