Preinvasive Breast Cancer

AREV403-PM05-09 ARI 10 December 2009 17:0

Preinvasive Breast CancerDennis C. SgroiDepartment of Pathology, Molecular Pathology Research Unit, Harvard Medical School,Boston, Massachusetts 02129

Massachusetts General Hospital Center for Cancer Research, Boston, Massachusetts 02129;email: [email protected]

Annu. Rev. Pathol. Mech. Dis. 2010. 5:193–221

First published online as a Review in Advance onOctober 13, 2009

The Annual Review of Pathology: Mechanisms ofDisease is online at pathmechdis.annualreviews.org

This article’s doi:10.1146/annurev.pathol.4.110807.092306

Copyright c© 2010 by Annual Reviews.All rights reserved

1553-4006/10/0228-0193$20.00

Key Words

flat epithelial atypia, atypical ductal hyperplasia, ductal carcinoma insitu, genetics, gene expression

AbstractPreinvasive breast cancer accounts for approximately one-third of allnewly diagnosed breast cancer cases in the United States and consti-tutes a spectrum of neoplastic lesions with varying degrees of differen-tiation and clinical behavior. High-throughput genetic, epigenetic, andgene-expression analyses have enhanced our understanding of the re-lationship of these early neoplastic lesions to normal breast tissue, andthey strongly suggest that preinvasive breast cancer develops and evolvesalong two distinct molecular genetic and biological pathways that corre-late with tumor grade. Although unique epigenetic and gene-expressionchanges are not observed in the tumor epithelial compartment duringthe transition from preinvasive to invasive disease, distinct molecularalterations are observed in the tumor-stromal and myoepithelial cells.This suggests that the stromal and myoepithelial microenvironmentof preinvasive breast cancer actively participates in the transition frompreinvasive to invasive disease. An improved understanding of the tran-sition from preinvasive to invasive breast cancer will pave the way fornovel preventative and therapeutic strategies.

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Preinvasive breastcancer: a clonalproliferation ofepithelial cellsconfined within thebreast ducts withoutpenetration throughthe basementmembrane and intothe stroma

Atypical lobularhyperplasia (ALH):a clonal proliferationof breast acinar-likeepithelial cellspossessing some butnot all the features ofLCIS

Lobular carcinoma insitu (LCIS): a clonalproliferation ofacinar-like epithelialcells that appearmalignant and thataccumulate within anddistend the acini of aterminal ductal lobularunit

ER: estrogen receptor

INTRODUCTION

Breast cancer is a major health problem that af-fects the lives of millions of women worldwideeach year. In 2008 in the United States alone,approximately 180,000 women were diagnosedwith invasive breast carcinoma, and there were67,000 new cases of preinvasive breast can-cer (1). Over the past several decades, the in-cidence of breast cancer has risen, while thedeath rate from breast cancer has steadily de-creased. These seemingly contradictory obser-vations may be explained in part by increasedmammographic screening and improved diag-nostic recognition of the earliest curable prein-vasive stages of breast cancer (2).

Approximately 80% of all diagnosed prein-vasive and invasive breast cancers in the UnitedStates are of the ductal subtype (3). This spe-cific subtype is by far the most comprehensivelystudied group of breast tumors at the clinical,histopathological, immunohistochemical, andmolecular levels. Thus, with the exception ofa brief histomorphological and cytogenetic re-view of atypical lobular hyperplasia (ALH) andlobular carcinoma in situ (LCIS), the two prein-vasive lesions of the lobular subtype, this reviewfocuses predominantly on the preinvasive le-sions of the ductal subtype with special empha-sis on the molecular pathology of these ductallesions as they relate to breast cancer evolutionand progression. In addition, this review cov-ers recent advances in our understanding of thetumor microenvironment of preinvasive breastcancer and concludes with a brief discussionof the emerging area of molecular prognosticbiomarkers of preinvasive breast cancer.

THE ORIGIN OF HUMANBREAST CANCER

Currently, there are two prevailing modelsof breast cancer tumorigenesis and evolution:the stochastic model and the cancer stem cellmodel of carcinogenesis (4, 5). The stochas-tic model (also known as the clonal evolutionmodel) postulates that transformation origi-nates from random mutations in any breastepithelial cell (stem cell, progenitor, or

differentiated cell) and that the neoplasticprocess further evolves through accumulationof random genetic events that drive uncon-trolled proliferation and resistance to apoptosis(Figure 1a) (6–8). Over time, these neoplas-tic cells in resultant tumors undergo additionalgenetic and epigenetic changes and coevolvewith their microenvironment, leading to cel-lular heterogeneity within a tumor (9, 10).

According to the cancer stem cell hypoth-esis, all tissues are derived from organ-specificstem cells that are defined by their capacity toundergo self-renewal as well as to differenti-ate into the cell types that constitute each or-gan (4). In normal adult breast tissue, stem cellsare relatively quiescent, long-lived cells that aredefined by their ability to self-renew and togenerate progenitors that differentiate into thedifferent functional [estrogen receptor (ER)-positive and ER-negative luminal epithelial andmyoepithelial] cells of the breast. The cancerstem cell hypothesis has separate but relatedcomponents that include the following.

1. The cellular origin of breast cancer isthe same as that of tissue stem cells orprogenitor cells and akin to what hasbeen observed in human hematologicalmalignancies (11, 12), genetic trans-forming events occurring at differentdevelopment points in the breast stemcell continuum account for breast cancerphenotypic heterogeneity in the formof the major subtypes of breast cancer(Figure 1b) (13).

2. Breast cancers are driven by cellular com-ponents that display stem cell–like prop-erties of self-renewal (4).

Compelling and reproducible data supportboth the stochastic model and the cancer stemcell model, and it is likely that the origin of theearliest preinvasive stages of breast cancer re-sults from elements that can be encompassedby both theories. Although the origin of hu-man breast cancer is an area of intense research,this review focuses on molecular alterations thatoccur subsequent to the breast cancer–initiatingevents.

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Adult breast stem cells

Progenitor cells

Differentiated cells

Basal-like tumorsER-, PR-, HER2-

Luminal tumorsER+, PR+, HER2-

HER2 tumorsER-, PR-, HER2+

Adult breast stem cells

Progenitor cells

Basal-like tumorsER-, PR-, HER2-

Luminal tumorsER+, PR+, HER2-

HER2 tumorsER-, PR-, HER2+

Multiple genetic andepigenetic changes

Self renewal Basal-like tumors Luminal tumors HER2 tumors

Breast cancer subtypes

Breast cancer subtypes

a

b

Figure 1Hypothetical models of the origin of human breast cancer. Based on the stochastic model of breast carcinogenesis (a), any epithelial celltype (e.g., stem cell, progenitor, or differentiated cell) may be the target of the initiating event. Each breast cancer subtype is initiated ina different cell type. In the cancer stem cell model (b), the cell of origin can be the same stem cell or progenitor cell for the differentsubtypes. The tumor phenotype is then determined by a combination of genetic and epigenetic events. Abbreviations: ER+(-), presence(absence) of immunohistochemical expression of estrogen receptor (ER) expression; HER+(-), presence (absence) ofimmunohistological expression or HER gene amplification; PR+(-), presence (absence) of immunohistochemical expression ofprogesterone receptor (PR) expression.

PREINVASIVE STAGES OFBREAST CANCER: A BRIEFHISTOPATHOLOGICAL REVIEW

This section begins with a brief histomorpho-logical review of normal breast tissue and of thepreinvasive stages of breast cancer. This sum-mary provides for a more complete conceptualframework from which to build a contemporarymolecular-based model of progression.

The breast is composed of a progressivebranching system of ducts that originate at the

Terminal ductallobular unit (TDLU):the functional unit ofthe breast, composedof a terminal duct anda lobule consisting of agroup of acini

nipple and end in one of many terminal ductallobular units (TDLUs), which are the small-est functional units of the breast (Figure 2). ATDLU is composed of a single terminal duct(TD) and multiple end ductules (or acini) in-vested in a specialized stromal compartment.The ductal system of the breast and TDLU islined by two cell layers: (a) the inner luminalepithelial layer that consists of low columnarand cuboidal cells of the TD and acini, respec-tively, and (b) the outer myoepithelial layer thatabuts the basement membrane. By definition,

www.annualreviews.org • Preinvasive Breast Cancer 195

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TD

TDLU

Acinus

BMME

LE

LEMELE

b

c d

e

ME

Acinus

ME

LE

a

S

Figure 2Microanatomy of the terminal duct lobular unit (TDLU). (a) Low-power photomicrograph of a hematoxylin and eosin (H&E)-stainedtissue section of human breast with several TDLUs, each consisting of a cluster of acini (ductules) and a terminal duct (TD). High-power images of H&E-stained (b,c) and calponin-immunostained (d,e) tissue sections demonstrate the two–cell layer anatomy of theacini (b,e) and the TD (c,d ) of the TDLU. Both the acini and the TD display a continuous outer layer of myoepithelial cells (ME) thatare immunoreactive for calponin (d,e; brown elongate cells). The ME surround the cuboidal or low columnar luminal epithelial cells (LE)of the acinus and the TD, respectively. The basement membrane (BM) surrounds the ME. The TDLU is surrounded by the breaststromal (S) compartment.

the preinvasive epithelial lesions of the breastare characterized by a neoplastic epithelial cellproliferation that is confined within the ductal-lobular network of the breast without invasionthough the basement membrane into the sur-rounding breast stromal compartment (14).

The preinvasive stages of breast cancer, sim-ilar to the invasive stages of breast cancer, fallinto two general histologic categories: the lob-ular and ductal subtypes. The historic viewthat the lobular subtype arises from lobulesand that the ductal subtype arises from ducts

is misleading; the seminal studies by Wellingsand colleagues (15, 16) have demonstrated thatmost breast cancers (both ductal and lobulartypes) arise in the same microanatomical site,the TDLU. The distinction of the lobular sub-type from the ductal subtype is based upon dif-ferences in cell morphology. More specifically,the lobular subtype consists of small, nonpo-larized cells that resemble the low-cuboidal lu-minal cells of normal breast acini, whereas theductal subtype consists of moderate to large,frequently polarized cells that resemble the low

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columnar cells of the normal breast ducts. Thepreinvasive stages of lobular breast cancer in-clude ALH and LCIS, whereas the equivalentlesions of the ductal subtype include flat ep-ithelial atypia (FEA), atypical ductal hyperplasia(ADH), and ductal carcinoma in situ (DCIS).

ALH and LCIS, two types of preinva-sive lesion grouped under the term lobularneoplasia, are considered as nonobligate pre-cursors of invasive lobular carcinoma (ILC)(17–19). The histomorphological differentia-tion of ALH from LCIS is based on the extent ofproliferation and the distension of the TDLU(Figure 3). In ALH, a TDLU is partly colo-nized by a monomorphic population of small,round, nonpolarized, loosely cohesive cells witha high nuclear-cytoplasmic ratio. In general,the proliferation of ALH is not widespread anddoes not usually obliterate the acinar lumina.So-called classic LCIS consists of a popula-tion of cells with identical cytomorphologicalcharacteristics of ALH, but in which the col-onized acini of the TDLU are filled and dis-tended with the neoplastic cells (20). A pleo-morphic variant of LCIS has been describedand occurs less frequently than the classic type(21). Histomorphologically, pleomorphic LCISconsists of medium- to large-sized pleomor-phic cells with or without associated necrosis(Figure 3) (21). The loss of expression of mem-brane E-cadherin is a hallmark feature of ALHand LCIS (19, 22). However, this feature is notpathognomonic of these lesions, as ductal carci-nomas can also demonstrate loss of E-cadherin(23).

FEA, ADH, and DCIS are considered thenonobligate precursors of invasive ductal carci-noma (IDC). FEA—also referred to in the lit-erature as atypical cystic lobule, columnar cellchange or hyperplasia with atypia, columnar al-teration with prominent apical snouts and se-cretions with atypia, and hyperplastic enlargedlobular units (24, 25)—is characterized by aminimal proliferation and replacement of na-tive luminal cells of the TDLU by one to sev-eral layers of monomorphic cuboidal or colum-nar epithelial cells with low-grade cytologicalatypia (Figure 3). Notably, the cells of FEA do

Flat epithelial atypia(FEA): earliest stageof preinvasive ductalcancer; consists of aminimal clonalproliferation ofepithelial cells

Atypical ductalhyperplasia (ADH):intermediate stage ofpreinvasive ductalcancer; consists of aclonal proliferation ofepithelial cells thatpossess some but notall of the features ofDCIS

Ductal carcinoma insitu (DCIS): the laststage of preinvasiveductal cancer; consistsof a clonalproliferation ofepithelial cells thatappear malignant andthat accumulate withinthe lumens of thebreast ducts

IDC: invasive ductalcarcinoma

not pile up to fill the TD and acini [as they doin usual ductal hyperplasia (UDH)], but rathergrow as a single or minimally pseudostratifiedlayer that enlarges and distends the TDLU (24).FEA differs from the next stage of progression,ADH, in that the latter is characterized by bothlow-grade cytological atypia and architecturalatypia in the form of micropapillae, trabecu-lar bars, Roman arches, cribriform spaces, andsolid growth. Historically, the pathological dis-tinction between ADH and DCIS is consideredby some to be based upon the degree of archi-tectural atypia and the size and the extent ofepithelial proliferation (26, 27). Through useof conventional histomorphological parametersthat include cytomorphological and architec-tural features, DCIS is further subclassified intolow-, intermediate-, and high-grade categories.Lastly, the histomorphological feature that dis-tinguishes IDC from DCIS is the disappearanceof the outer organized myoepithelial layer alongwith growth of the neoplastic cells beyond thebasement membrane and into the surroundingstroma (14).

HISTOPATHOLOGICAL-BASEDMODELS OF HUMAN DUCTALBREAST CANCER PROGRESSION

Histopathological and epidemiological obser-vations over the past century have resulted intwo well-recognized linear models of breastcancer progression (Figure 4). The classi-cal model, derived from careful histomorpho-logical observations first described more than100 years ago (28) and further refined byWellings and colleagues (15, 16), postulates thatan initiating event within an epithelial cell ofthe TDLU gives rise to FEA. A proliferativegrowth advantage within FEA is postulated tospawn ADH, upon which additional molecu-lar alterations give rise to the last preinvasivestage of breast cancer progression, DCIS. Fi-nally, progressive molecular events give rise tothe malignant (or potentially lethal) stages ofIDC and metastatic carcinoma (29).

It was not until the influential studies byPage and colleagues (30, 31) that the alternative


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FEA ADH DCIS (low-grade)

ALH Classic LCIS

IDC

Pleomorphic LCIS ILC

DCIS (intermediate-grade) DCIS (high-grade)

Figure 3Histomorphological classification of preinvasive and invasive breast cancer. The preinvasive stages of the lobular type consist of atypicallobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS). Differentiation of ALH from LCIS is based upon the extent ofproliferation and distension of the acini within the terminal duct lobular unit (TDLU). Pleomorphic LCIS differs from so-called classicLCIS in that the pleomorphic type consists of a proliferation of cells that are highly variable in size and shape and loosely cohesive, withor without necrosis, whereas the classic type consists of a proliferation of uniform, small, loosely cohesive, nonpolarized epithelial cellsThe epithelial proliferation associated with preinvasive LCIS is confined to the ductal system, whereas invasive lobular carcinoma(ILC) infiltrates through the basement membrane into the surrounding stroma. The preinvasive stages of the ductal type consist of flatepithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS). FEA is characterized by a minimalproliferation of native luminal cells of the TDLU by one to several layers of monomorphic cuboidal or columnar epithelial cells withlow-grade cytological atypia. ADH differs from FEA in that in ADH the cells grow in a pseudostratified manner with secondaryarchitectural atypia in the form of micropapillae, Roman arches (arrow), and trabecular bars. The differentiation of ADH from DCIS isbased on the degree of architectural atypia and on the size and the extent of epithelial proliferation. Low-grade DCIS consists of small,cohesive, polarized, uniform cells of low proliferative capacity, whereas high-grade DCIS consists of large pleomorphic cells of highproliferative capacity with necrosis (asterisk). Intermediate-grade DCIS consists of small- to medium-sized polarized cells withmoderate nuclear pleomorphism and low proliferative capacity with or without necrosis (asterisk). Preinvasive carcinoma of the ductaltype (e.g., DCIS) consists of proliferation of medium- to large-sized polarized cells confined to the ductal system, whereas invasiveductal carcinoma (IDC) is characterized by the growth and invasion of neoplastic cells beyond the basement membrane and into thesurrounding stroma.

model became popular. The alternative linearmodel of breast cancer progression is identicalto that of the classical model with the exceptionof the introduction of UDH, rather than FEA,

as the direct precursor to ADH (Figure 4).Historically, the role of UDH as the directprecursor lesions to ADH was supported byepidemiologically based studies demonstrating

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Normal FEA ADH DCIS IDC Metastasis

Preinvasive stages Malignant stages

Classic Wellings model of progression a

Alternative model of progression b

Normal FEA ADH DCIS IDC Metastasis UDH

Preinvasive stages Malignant stages

Figure 4Traditional linear models of breast cancer progression. (a) Classic and (b) alternative linear multistep modelsof human ductal breast cancer progression. The two models are identical, with the exception of the placementof usual ductal hyperplasia (UDH) as the precursor to atypical ductal hyperplasia (ADH). The classic modelwas initially based on histomorphological observations, whereas the alternative model was initially proposedbased upon epidemiological observations. Recently, multiple lines of evidence (histomorphological,immunohistochemical, and molecular genetic) support the classic model and contest the alternative model ofprogression. Molecular alterations occurring in normal breast epithelium result in flat epithelial atypia(FEA). FEA leads to additional changes that give rise to ADH and ductal carcinoma in situ (DCIS), uponwhich subsequent genetic and epigenetic alterations in turn give rise to invasive ductal carcinoma (IDC).

that women with a benign breast biopsy show-ing UDH have a mildly elevated risk of breastcancer (∼1.5 to 2.0 times that of the referencepopulation) and that women with ADH have asubstantial increased risk for developing breastcancer (∼3.5 to 5.3 times that of the referencepopulation) (31). Although this epidemiologicalevidence supported UDH as the precursor toADH, recent immunohistological and molec-ular pathological evidence, as described below,strongly suggests that UDH is not a precursorto ADH and that the alternative linear modelof progression is probably invalid.

MOLECULAR CLASSIFICATIONOF INVASIVE DUCTALBREAST CANCER

Major progress in the molecular classificationof invasive breast cancer has been achievedthrough the combined use of immunohis-tochemistry, gene-expression, and genomic-based technologies. The molecular classifica-tion of invasive breast cancer serves as an

Tumor grade:an importanthistopathologicalmeasurement ofcellular differentiationthat correlates with theclinical behavior of thetumor

important reference point for the following dis-cussion of preinvasive breast cancer.

Genomic Analysis of InvasiveDuctal Carcinoma

Tumor heterogeneity is a well-recognizedand clinically relevant, but poorly under-stood, property of invasive breast cancers.Traditionally, classification of IDCs into clin-ically meaningful groups has been performedthrough histological grading systems (seesidebar, Histopathological Grading) (32, 33).Several groups studying the relationship of ge-netic alterations with histological tumor gradehave made important contributions to our un-derstanding of breast cancer classification andbreast cancer evolution (34–36). These groupsdemonstrated that specific tumor grades exhibitdistinct genomic differences in which fewerchromosomal aberrations occur in low-gradetumors as compared with high-grade tumors.These general quantitative genomic differencesare also associated with distinct qualitative


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HISTOPATHOLOGICAL GRADING

Invasive and in situ breast cancers display enormous histologicaldiversity; they range from well-differentiated, slow-growing tu-mors to poorly differentiated, rapidly growing tumors. Given thisdiversity, investigators have proposed multiple histological grad-ing schemes to subclassify invasive and in situ carcinomas intoclinically meaningful prognostic categories. Histological gradingdescribes the microscopic growth pattern of breast cancer as wellas the cytological features of differentiation. Pathologists employdifferent grading systems to evaluate invasive and in situ carcino-mas. For IDCs, the most commonly accepted histomorpholog-ical grading system—the modified Bloom-Richardson system—evaluates three distinct tumor factors, namely tubule formation(glandular differentiation), nuclear pleomorphism (a reflectionof DNA content), and mitotic rate (cellular proliferation). Thisgrading system also classifies breast tumors into three distinct cat-egories: low-, intermediate-, and high-grade malignancies. For insitu ductal carcinomas, a number of DCIS grading schemes havebeen proposed that use various measurements of architecturalpatterns (cellular polarization), nuclear morphology, mitosis, andnecrosis. In general, for both invasive and in situ cancers, a low-grade (grade I) tumor consists of malignant epithelial cells thatlack significant nuclear pleomorphism and mitotic activity andthat form glandular (acinar-like) structures. At the opposite ex-treme, a high-grade (grade III) tumor consists of malignant cellspossessing marked nuclear pleomorphism and high mitotic activ-ity that completely fail to form glandular elements and that oftenexhibit necrosis. Clinically, it is well established that high- andlow-grade tumors are associated with the highest and lowest ratesof recurrence and the shortest and longest recurrence times, re-spectively. Intermediate-grade breast cancers display phenotypicand clinical behavioral characteristics that lie in between low-and high-grade tumors, and it is this group of tumors that posesthe greatest interobserver grading challenge for pathologists andthe greatest treatment challenge for oncologists. However, as de-scribed in the main text, gene-expression and genomic data sug-gest that this group of intermediate-grade tumors consists of ei-ther low grade–like or high grade–like tumors and that DCIS andIDCs should be classified into two, rather than three, biologicallyand clinically meaningful categories.

differences. Grade I tumors (grade I tumors arewell differentiated, whereas grade III tumorsare poorly differentiated) display frequentrecurrent chromosomal loss of 16q and gainsof 1q, 16p, and 8q, whereas high-grade tumors

display frequent high-level amplifications of17q12 and 11q13; losses of 8p, 11q, 13q, 1p,and 18q; and gains of 1q, 8q, 17q, 20q, and16p (Figure 5) (35, 36). Intermediate-gradetumors share genomic alterations common toeither low-grade or high-grade carcinomas,suggesting that this population of tumors con-sists of a mixture of each type (37). The mostsignificant finding as it relates to breast cancerclassification and breast cancer evolution isthe frequent versus infrequent loss of 16q inlow-grade and high-grade IDCs, respectively(35, 36). This distinct pattern of chromosomalloss has led some investigators to speculatethat the majority of grade I carcinomas donot evolve to grade III tumors through ded-ifferentiation, as such an evolutionary schemewould necessitate the recovery of lost geneticmaterial (29, 35). Taken together, all of theseobservations support a hypothesis of earlydivergence between the two main subtypes ofIDCs (Figure 5) and support a modified modelof breast cancer progression.

Gene-Expression Analysis of InvasiveDuctal CarcinomaOver the past seven years, multiple indepen-dent research groups have conducted genome-wide expression profiling studies in an effortto identify novel diagnostic, prognostic, andpredictive classification schemes as a means toguide clinical decision making (38–49). In aninfluential study, Perou et al. (50), using mi-croarray gene-expression profiling, revealed aremarkably consistent molecular classificationscheme in which breast cancers can be classifiedinto four distinct intrinsic categories that in-clude (a) two subtypes of ER-negative tumors,the basal-like and ERBB2 subtypes, and (b) twoER-positive tumors, the luminal A and lumi-nal B subtypes. The basal-like tumors typicallylack expression of ER, progesterone receptor(PR), and human epidermal growth factor re-ceptor 2 (HER2) and express cytokeratins 5/6(basal cytokeratins) and epidermal growth fac-tor receptor (EGFR) (50, 51). The ERRB2 tu-mors are characterized by expression of HER2and by lack of expression of ER and PR

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Normal

Low-grade IDC –16q, +1q, ER+, PR+

Low grade–like gene-expressionmolecular pathway

Intermediate-grade IDC

Low-grade IDC


Luminal Asubtype

Luminal Bsubtype

HER2-

HER2+


High-grade IDC


High-grade IDC

HER2subtype

Basalsubtype

HER2+

HER2-

+17q12, –13q, +11q13, ER-, PR-

High grade–like gene-expressionmolecular pathway

a

b

Figure 5Molecular classification of invasive ductal breast cancer. Genetic and gene-expression data classify invasiveductal carcinomas into two distinct molecular biological and clinicopathological pathways. (a) The lowgrade–like gene-expression molecular pathway is characterized by chromosome 16q loss, predominantestrogen receptor (ER) and progesterone receptor (PR) expression (ER+, PR+) and a low-gradegene-expression profile populated with genes associated with ER positivity (ER+). (b) The high grade–likegene-expression molecular pathway is characterized by loss of chromosome 13q, gain of 11q13 and/oramplification of 17q12, infrequent expression of ER and PR, and a high grade–like gene-expressionsignature populated with genes associated with cell cycle, centrosomal function, and DNA repair. Thelow-grade tumors express a unique set of genes that are rarely expressed in high-grade tumors, and viceversa. The luminal A and luminal B subtypes constitute the majority of invasive breast cancers in the lowgrade–like gene-expression molecular pathway and generally display indolent clinical behavior, whereas thehuman epidermal growth factor receptor 2 (HER2) and basal subtypes constitute the majority of cancers inthe high grade–like pathway and generally display aggressive clinical behavior. Light blue rectangles denotemorphological ductal subtypes. Abbreviations: HER+(-), presence (absence) of immunohistologicalexpression or HER gene amplification; IDC, invasive ductal carcinoma; −16q, loss of chromosome 16q;+1q, gain of chromosome 1q; −13q, loss of chromosome 13q; +11q13, gain of chromosomal region 11q13;+17q12, amplification of chromosomal region 17q12.

(50, 51). Luminal A tumors are characterized byER and PR expression with the lack of HER2overexpression, whereas luminal B tumors typ-ically express ER and overexpress HER2 withor without PR expression (50, 51). Subsequentfollow-up studies demonstrated that beyondgene-expression differences these four specificmolecular subtypes are associated with distinctclinical outcomes (39, 40).

PR: progesteronereceptor

Since these initial observations, multi-ple seemingly novel breast cancer gene-expression signatures have been identified(41, 44, 45, 52, 53). Such signatures con-sist of fairly unique gene sets with very littleto no overlap, yet they all have a similarability to predict breast cancer outcome. Im-portant issues are whether these differentgene-expression signatures share a unifying


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HER2: humanepidermal growthfactor receptor 2

Gene-expressionsignature/profile: theexpression of a set ofgenes that is associatedwith a distinct clinicaland/or biologicalphenotype

biological principle that translates into a com-mon clinical overlap in their prognostic infor-mation and whether combining several of thesesignatures would provide more accurate risk as-sessment. To address this issue, Fan et al. (54)compared the prognostic performance of fourdifferent breast cancer gene-expression signa-tures and demonstrated that all four signa-tures were highly concordant in classifying pa-tients into low- and high-risk groups. A com-bination of the four signatures did not sig-nificantly improve upon the prognostic pre-dictive accuracy of each signature alone, sug-gesting that the prognostic information cap-tured by these signatures is largely overlap-ping and that it probably reflects a commonbiological principle (54). All four signatureswere significantly correlated with tumor grade,suggesting that the unifying biological prin-ciple captured by these signatures may berooted in this well-established pathological pa-rameter (54). Subsequent comparative gene-expression studies indicate that these seeminglydisparate breast cancer gene-expression signa-tures share common molecular pathways cen-tered on cell-cycle regulation and cell prolif-eration (55–58). Thus, the common biolog-ical principle of cell proliferation, which isthe most important component of histopatho-logical grading (59–62), is the main drivingforce behind the prognostic power of thesebiomarkers.

Although the histopathological gradingsystem recognizes three distinct clinico-pathological subgroups, the above-describedgenomic studies suggest that a two-categorygrading system may be more biologicallyand clinically relevant. Using a so-calledbottom-up approach, Sotiriou and colleagues(38) and Ma and colleagues (63) identifiedboth complex [97-gene genomic grade index(GGI)] and simple [five-gene molecular gradesindex (MGI)] tumor-grade gene-expressionsignatures, which are differentially expressedbetween low- and high-grade tumors. Con-sistent with the view that molecular gradingis superior to subjective histomorphologicalgrading, these signatures successfully classified

intermediate-grade tumors into two groupswith similar clinical outcomes: low grade–likeand high grade–like tumors (Figure 5) (38).Given the aforementioned data, which suggestthat most gene-expression signatures reflecttumor grade and proliferation, Sotiriou andcolleagues (58) recently performed an unbiasedcomparison of 70- and 76-gene-expressionsignatures to the GGI in the TRANSBIGclinical trial series and demonstrated that GGI,a pure tumor grade–based gene-expressionbiomarker, exhibited prognostic performancehighly similar to those of both the 70- and the76-gene-expression signatures. Another studyby Sotiriou’s group (64) demonstrated that GGIand the 21-gene signature had similar prognos-tic performance, and our group (63) recentlydemonstrated that the simple five-gene MGIbiomarker possesses prognostic equivalence tothe more complex 97-gene GGI assay. There-fore, despite investigators’ differing approachesto the development of various gene-expressionsignatures, most if not all gene-expressionsignatures are an objective surrogate measureof tumor grade and proliferation and are highlysimilar in terms of prognostic performance. Insummary, the molecular interrogation of inva-sive breast cancer strongly suggests that tumorgrade, more than any other clinicopathologicalparameter, strongly reflects the extent and typeof underlying genetic and gene-expressionalterations, and that these alterations representtwo distinct evolutionary, biological, andclinicopathological pathways (Figure 5).

MOLECULAR CLASSIFICATIONOF THE PREINVASIVE STAGESOF DUCTAL BREAST CANCERPROGRESSION

Until recently, a significant impediment to abetter understanding of breast cancer progres-sion was our inability to readily and accuratelyinterrogate and assess the molecular events as-sociated with the early preinvasive stages asthey relate to invasive carcinoma. However,over the past decade the successful combina-tion of highly specific tissue-microdissection

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technologies with advanced high-throughputgenomic, gene-expression, and proteomic tech-nologies has reshaped our view of the prein-vasive stages of breast cancer progression. Asdescribed below, significant genomic and gene-expression parallels exist between the prein-vasive and invasive stages of breast cancer.The molecular characterization of DCIS is de-scribed first, as this well-characterized lesionserves as the logical reference point for ADHand FEA.

GENOMIC ANALYSIS OF THEPREINVASIVE STAGES OFDUCTAL BREAST CANCER

Similar to what has been observed for IDC,DCIS represents a spectrum of neoplastic dis-eases. Some of these behave in an indolent man-ner, whereas others behave in a more aggres-sive manner (see sidebar, Natural History ofPreinvasive Breast Cancer). Multiple morpho-logically based schemes have been proposed asa means to better classify DCIS biologically andclinically (65–68). Several comparative studieshave revealed that DCIS is a genetically ad-vanced lesion and that different morphologi-cal subtypes of DCIS mirror distinct genomicalterations characterized by loss of 16q in low-grade DCIS and by amplification of 17q12 inhigh-grade DCIS (69, 70). More specifically,Buerger et al. (69) performed a comparative ge-nomic hybridization (CGH)-based analysis ofDCIS and invasive carcinoma and revealed thatlosses of 16q were seen almost exclusively inlow- and intermediate-grade DCIS, whereas ahigher frequency of 1q gain and 11q loss wasobserved in intermediate-grade DCIS. High-grade DCIS, however, demonstrated complexgenomic alterations characterized by loss of 8p,11q, 13q, and 14q; by gains of 1q, 5p, 8q, and17q; and by high-level amplifications of 17q12and 11q13 (69). Analysis of CGH data gener-ated from synchronous and metachronous IDCand DCIS lesions revealed a near-identical pat-tern of genetic change, supporting a direct pre-cursor relationship between DCIS and IDC(36, 37, 69). In a recent study utilizing CGH

NATURAL HISTORY OF PREINVASIVEBREAST CANCER

Preinvasive breast cancer accounts for approximately one-thirdof all newly diagnosed breast cancer cases in the United States.Although women do not die of preinvasive disease, it is wellknown that a subpopulation of these women will subsequentlydevelop invasive cancer. Women diagnosed with ADH and ALHdemonstrate a 3.5- to 5.0-fold-increased risk of developing inva-sive breast cancer, compared with the general population. Thedevelopment of invasive breast cancer in women diagnosed withLCIS is estimated at 7 to 9 times the relative risk of women inthe general population, whereas women diagnosed with DCIShave an estimated 4 to 12 times relative risk of developing inva-sive breast cancer. In general, the degree of relative risk directlycorrelates with biologically aggressive features that are capturedby tumor grade; for instance, low-grade in situ carcinomas tendto have a better prognosis than do their high-grade counterparts.

in conjunction with serial analysis of gene ex-pression (SAGE), Yao et al. (71) demonstratedan overall trend toward an increase in the num-ber and amplitude of gains and losses duringbreast cancer progression, which supports theconcept that the early neoplastic stage of DCISis a direct precursor to IDC. Taken together,these data provide evidence that DCIS is a di-rect precursor to IDC and, furthermore, thatdistinct genetic pathways captured by the clin-icopathological parameter of tumor grade existwithin the morphologically diverse spectrum ofDCIS.

Histomorphologically, ADH shares somebut not all of the architectural and cytologicalfeatures of low-grade DCIS, and the diagnosticcriteria separating the two lesions are predomi-nantly quantitative, rather than qualitative, innature (26, 27). These pathological features,along with clinical and epidemiological data,support the role of ADH as the precursor tolow-grade DCIS. Additional evidence in sup-port of this hypothesis is provided by the resultsof multiple loss of heterozygosity (LOH)-basedand CGH-based studies. Several small stud-ies revealed losses of chromosomal regions 16qand 17p in ADH (72–74) and showed that the


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frequency of chromosomal losses in these re-gions is similar to that observed in DCIS andIDC. A comprehensive study by O’Connelland colleagues (75), who analyzed LOH in 399preinvasive breast lesions, revealed LOH in atleast 1 of 15 loci studied in 42% and 44% ofADH lesions from noncancerous and cancerousbreasts, respectively. Furthermore, this groupidentified chromosome 16q as an LOH hot spotin ADH and showed that this hot spot was morefrequently shared with low-grade (noncomedo)DCIS than with high-grade DCIS (75). Giventhe considerable difficulties in histomorpholog-ically discriminating between ADH and low-grade DCIS, it is not surprising that these twopreinvasive lesions share a common chromo-somal abnormality. The morphological overlapthat is reflected at the molecular level in thesetwo lesions has led some researchers to questionthe validity of classifying ADH and low-gradeDCIS as distinct and separate pathological en-tities (76). Currently, ADH is the accepted pre-cursor lesion for low-grade DCIS. However,the precursor lesion for the majority of high-grade DCIS cases (those with 17q12 amplifica-tion; see Figure 6) remains elusive.

As mentioned above, some epidemiologicaland clinical data support an alternative modelof breast cancer progression in which UDH ispostulated to be the precursor lesion of ADH.However, multiple lines of evidence suggestotherwise and point to FEA, rather than UDH,as the precursor to ADH and low-grade DCIS.First, morphologically speaking, the cells thatconstitute FEA are characterized by variabledegrees of low-grade cytological atypia thatforms a continuum from normal lobules to duc-tal carcinoma in situ, whereas UDH consistsof a population of immature mammary epithe-lial cells that can undergo divergent differen-tiation as either glandular epithelial cells ormyoepithelial cells (77). Second, immunohisto-chemical profiles of FEA are nearly identical tothose of ADH and low-grade DCIS, with FEAdemonstrating diffuse positivity for ER, PR,and cytokeratin 19 (CK19) (70, 78); variablebut increased staining for cyclin D1 (78); andnearly uniform negativity for HER2 (70, 79)

and CK5/6 (70). Immunohistochemical profilesof UDH, however, show a mixed populationof cells with a variable proportion of CK5/6-positive myoepithelial and basal cells andER+, PR+, and CK8/18/19-positive glandu-lar (luminal) epithelial cells (80, 81). Third, atthe molecular level FEA’s genetic profile over-laps with those of synchronous low-grade DCISand low-grade invasive carcinoma (82). Morespecifically, Moinfar and colleagues (82) noteda particularly high rate of LOH at chromosome16q, a locus commonly altered in low-gradeDCIS. A more recent comprehensive CGH-based study by Simpson et al. (70) extendedthese observations by demonstrating that FEAexhibits recurrent chromosomal copy numbergains and losses (gains on 15p, 16p, and 19;losses on 16q, 17p, and X) and that these recur-rent genetic alterations have significant over-lap with those observed in both ADH andlow-grade DCIS. However, although LOH isobserved in UDH, the pattern of LOH is no-tably different from that associated with ADHand DCIS (75, 83–88). More specifically, onlyrare and fairly randomly distributed chromoso-mal changes, or no changes at all, occur in UDH(a pattern similar to that observed in phenotyp-ically normal breast tissue and nonproliferativefibrocystic change), whereas recurrent, non-randomly distributed chromosomal changes (inparticular 16q loss) occur more frequently inADH and DCIS (69, 75, 85, 86, 88–90). Takentogether, these observations support the role ofFEA as the precursor to ADH. They also ques-tion the role of UDH as a precursor to ADHand, thus, the validity of the alternative modelof breast cancer progression.

GENE-EXPRESSION ANALYSISOF THE PREINVASIVE STAGESOF DUCTAL BREAST CANCER

Over the past several years, there has beenconsiderable research interest in understand-ing the gene-expression changes that occurduring the early preinvasive stages of breastcancer (91–97). It is particularly noteworthythat these studies focused primarily on the

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+17q12, –13q, +11q13, ER-, PR-

High grade–like gene-expressionmolecular pathwayb

–16q, +1q, ER+, PR+

Low grade–like gene-expressionmolecular pathway

a

Normal

Intermediate-grade DCIS

High-grade DCIS

Low-grade DCIS

Classic LCIS

High-grade IDC

Pleomorphic LCIS

ADHFEA


Low-grade IDC

ALH Classic ILC

Pleomorphic ILCPleomorphic ALH

Intermediate-grade DCIS Intermediate-grade IDC

High-grade DCIS High-grade IDC

Intermediate-grade DCIS Intermediate-grade IDC

High-grade DCIS High-grade IDC

HER2+

HER2–

HER2+ or HER2–

HER2+ or HER2–

Figure 6Contemporary multistep, two-dimensional model of human breast cancer progression derived from morphological,immunohistochemical, genetic, and gene-expression data. Distinct molecular events occur in normal breast epithelium, giving rise totwo distinct divergent molecular pathways within which linear pathological stage progression (horizontal black arrows) and intrastageheterogeneity (i.e., tumor-grade evolution; vertical red dashed arrows) occur. (a) The low grade–like gene-expression molecular pathwayis characterized by chromosome 16q loss, predominant estrogen and progesterone receptor expression (ER+, PR+), and a low-gradegene-expression profile populated with genes associated with ER positivity (ER+). This low-grade pathway is observed in preinvasivelesions of both the ductal subtype (light blue rectangles) and the lobular subtype ( green rectangles). (b) The high grade–likegene-expression molecular pathway is characterized by loss of chromosome 13q; gain of 11q13 and/or amplification of 17q12;infrequent expression of ER and PR; and a high grade–like gene-expression signature populated with genes associated with cell cycle,centrosomal function, and DNA repair. Although pleomorphic atypical ductal hyperplasia (ALH), pleomorphic lobular carcinoma insitu (LCIS), and pleomorphic invasive lobular carcinoma (ILC) phenotypically resemble high-grade tumors, immunohistochemical (ERpositivity) and genetic (16q loss and 1q gain) data support an evolutionary association with the low grade–like gene-expressionmolecular pathway. Recent immunohisto-chemical, morphological, and genetic data support the concept of intrastage tumor-gradeprogression (red dashed arrows), which probably accounts for the observation of intratumoral heterogeneity. Light blue and greenrectangles denote ductal and lobular morphological subtypes, respectively. Abbreviations: ALH, atypical lobular hyperplasia; DCIS,ductal carcinoma in situ; FEA, flat epithelial atypia; HER+(-), presence (absence) of immunohistological expression or HER geneamplification; IDC, invasive ductal carcinoma; −16q, loss of chromosome 16q; +1q, gain of chromosome 1q; −13q, loss ofchromosome 13q; +11q13, gain of chromosomal region 11q13; +17q12, amplification of chromosomal region 17q12.

gene-expression changes within the neoplasticepithelial cells that constitute ADH and DCIS;comprehensive gene-expression profiling ofFEA has not been reported to date. One of the

earliest and most comprehensive studies is thatdone by Ma and colleagues (94), in which bothpatient-matched phenotypically normal breastepithelium (N) from the TDLU and epithelium


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constituting ADH, DCIS, and IDC weremicrodissected and hybridized to a comple-mentary DNA (cDNA) microarray containing12,000 genes. Not unexpectedly, comparativegene-expression profile analysis of patient-matched N versus ADH, N versus DCIS, andN versus IDC revealed that the most pro-nounced transcriptional changes occur at theN-to-ADH transition and that such transcrip-tional alterations are maintained throughoutthe later stages (DCIS and IDC) of progression(94). Unexpectedly, however, on a global levelno consistent major transcriptional changes be-tween the preinvasive and invasive stages wereidentified. This finding has been reported intwo additional studies (93, 95). Taken together,these data support the idea that the differentstages of breast cancer progression are evolu-tionary products of the same clonal origin andsuggest that gene-expression patterns expressedin the preinvasive stages (ADH and DCIS) ofdisease may, in fact, reflect the progressive po-tential of the pathological lesion. The conceptthat gene expression of early-stage breast cancermay predict future clinical behavior is sup-ported in the literature by the repeated obser-vations that gene-expression patterns in early-stage invasive breast cancer predict the risk ofdistant metastases (39–41, 43, 44, 98–102).

Despite their use of different gene-expression microarray platforms (cDNAarrays, oligonucleotide arrays, and SAGE),several studies have demonstrated that thetransition from the preinvasive stage of DCISto invasive carcinoma is associated with quan-titative, rather than qualitative, differences ingene expression (94–96). More specifically,these studies have identified subsets of genesthat are consistently overexpressed in IDCrelative to patient-matched DCIS. Similarly,analyses of breast cancer development in sev-eral transgenic mouse models also demonstratethat the transition from a preinvasive to aninvasive stage of progression is associated withquantitative, rather than qualitative, differencesin gene expression (103, 104). This quantitativerelationship is most prominent in high-grade

(poorly differentiated/grade III) samples,revealing an intriguing link between tumorgrade and tumor-stage progression (94). All ofthese observations suggest that breast cancerprogression may be more complex than envi-sioned by the current linear theory of activationand inactivation of oncogenes and tumor-suppressor genes, respectively, and that it maybe dependent upon such contingencies as quan-titative levels and timing of gene expression.

Although the study by Ma and colleagues(94) did not identify gene-expression differ-ences that are specific to the distinct prein-vasive and invasive stages of breast cancer,unique gene-expression alterations have beenassociated with different tumors grades (38, 94,105, 106). Notably, similar to what has beenobserved with invasive breast cancer, distinctgene-expression signatures are present in low-and high-grade DCIS lesions (94). Low-gradeDCIS and ADH lesions share a common gene-expression signature populated with genes as-sociated with the ER phenotype, whereas high-grade DCIS lesions possess a gene-expressionsignature populated with genes associated withincreased mitotic-activity and cell-cycle pro-cesses (94). Thus, taken together these gene-expression data further support the conceptthat low-grade and high-grade preinvasive neo-plasms arise from two distinct evolutionarypathways (Figure 6) and that intermediate-grade DCIS represents a mixture of low- andhigh-grade neoplasms (38, 94, 106).

MOLECULAR ANALYSIS OFINVASIVE AND PREINVASIVELOBULAR BREAST CANCER

As compared with those of ductal breast can-cer, molecular analyses of the invasive andpreinvasive stages of lobular breast cancer aresomewhat limited. Conventional cytogenetic-and array CGH–based studies demonstratesome differences in the patterns of genetic al-teration in ILC as compared with those ofIDC (36, 107–110). As the majority of ILCsconsist of low-nuclear-grade malignant cells

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(i.e., classic ILCs), it is not surprising thatILCs share a recurrent loss of 16q with low-grade IDCs (107–110). This finding supportsthe hypothesis that ILCs and low-grade IDCsmay share a common pathway of tumorigene-sis (29). In a minority of ILCs, the tumor con-sists of high-nuclear-grade malignant cells (i.e.,pleomorphic ILC). Interestingly, these tumorsshare genetic changes with both classic ILCsand high-grade IDCs in the form of (a) 16qloss and 1q gain and (b) amplification of 17q12,respectively (111, 112). However, a recent com-parative analysis of array CGH data demon-strated that the overall molecular features ofpleomorphic ILC are more closely related tothose observed in ILC than those seen in IDC,suggesting that pleomorphic ILCs share a com-mon evolutionary association with classic ILCalong the low-grade, ER-positive pathway ofneoplastic development and that they do notrepresent high-grade IDCs (112).

Relatively few studies have focused on thegenomic analysis of the preinvasive stages oflobular breast cancer (113–116). Lu and col-leagues (114) performed a CGH-based analysisof ALH and LCIS; they demonstrated a similarpattern of chromosomal imbalance, with fre-quent chromosomal loss of material from 16p,16q, 17p, and 22q. Notably, no statistically sig-nificant differences between ALH and LCISwere identified, providing evidence that thesetwo lesions are not only related but are also ata similar stage of progression (114). More re-cently, Mastracci et al. (115) further refined themolecular signature of ALH and LCIS by iden-tifying novel loss of 7p11 and 22q11 and gainof 2p11 in ALH and novel loss of 19q13 andgain of 20q13 in LCIS. Most importantly, thesestudies, as well as that by Morandi and cowork-ers (116), demonstrated the common loss of 16qin ALH and LCIS with ILC, supporting an evo-lutionary link among the three types of lesions.Gene-expression profiling of classic ILC andLCIS reveals a gene-expression signature thatoverlaps significantly with that observed in low-grade IDCs (117). Thus, these gene-expressiondata, in conjunction with the CGH data,

support a common evolutionary −16q, low-grade gene-expression pathway (Figure 6) thatencompasses (a) ILC and its precursor lesions(ALH and LCIS) and (b) low-grade IDC and itsprecursor lesions (FEA, ADH, and low-gradeDCIS).

A CONTEMPORARYEPITHELIAL-CENTRICMODEL OF BREASTCANCER PROGRESSION

The traditional linear model of carcinogene-sis put forth by Wellings and colleagues (15,16) is one of progression from normal breastepithelium to FEA to DCIS to IDC. How-ever, both mathematical (118, 119) and molec-ular biological analyses question the validityof this morphological-based model. As out-lined above, comparative genomic and gene-expression analyses of the different stages ofbreast cancer suggest that breast carcinogen-esis evolves along one of two distinct, branchedpathways of progression defined by tumorgrade and loss of chromosome 16q (the two-pathway model; see Figure 5). Thus, thesemolecular studies account for phenotypic het-erogeneity among different tumors. However,such studies do not address the phenomenonof phenotypic heterogeneity, which is definedby variation in the degree of tumor cell differ-entiation (i.e., tumor grade) and is frequentlyencountered in both the in situ and the in-vasive stages of ductal breast cancer progres-sion. Recently, breast cancer intrastage het-erogeneity was specifically addressed by Allredand colleagues (25), who performed immuno-histochemical, morphological (nuclear grade),and gene-expression comparative analysis of200 cases of pure DCIS. The authors showedthat multiple histologic grades, biomarker phe-notypes, and intrinsic subtypes often coexistwithin the same DCIS lesion (25). Impor-tantly, these data support further refinement ofour model of breast cancer progression to in-clude intrastage heterogeneity in which a sub-set (∼9%) of high-grade DCIS can evolve from


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MMP: matrixmetalloproteinase

their low-grade counterparts (25). Although theobservations by Allred and colleagues appearincongruent with a simple two-pathway modelof progression, recent data by Natrajan et al.(120) may provide a unifying corollary to thetwo-pathway model. Specifically, Natrajan andcolleagues demonstrated increased frequencyof 16q loss in high-grade tumors of the luminaltype as compared with the other intrinsic types,suggesting that the evolutionary progression ofa low-grade tumor to a high-grade tumor maypreferentially occur in breast cancers of theluminal (low-grade, ER-positive) phenotype(120). Thus, in light of the recent data by Allredet al. and Natrajan et al., our contemporary two-pathway model of progression must be furtherrefined to account for intrastage heterogeneitythrough the process of intrastage tumor-gradeevolution within the −16q low-grade gene-expression molecular pathway (Figure 6).

MOLECULAR ANALYSIS OF THENONEPITHELIAL CELLS OF THETUMOR MICROENVIRONMENT

Most breast cancer researchers have tradition-ally employed a reductionist approach to ex-ploring tumors by focusing on the cancer cellsand the genes within them. Although this tu-mor epithelial–centric approach is conceptuallysatisfying, it may be too simple. The neoplasticepithelial cells of breast cancer coexist with sev-eral types of nonneoplastic cells that togethercreate the tumor microenvironment. Myoep-ithelial and inflammatory cells constitute theintraluminal tumor microenvironment of thepreinvasive stages of breast cancer, whereas fi-broblasts, myofibroblasts, inflammatory cells,and endothelial cells constitute the stromalmicroenvironment of invasive breast cancer.Although these nonneoplastic cells have gen-erally been considered silent or reactive by-standers within breast tumors, several lines ofevidence suggest that active bidirectional sig-naling between malignant breast epithelial cellsand nonneoplastic cells of the tumor microm-ilieu plays a critical role in breast tumorigene-sis and progression. First, both in vitro– and in

vivo–based assays of breast tumorigenesis havedemonstrated that experimental manipulationof the stromal microenvironment has profoundeffects on tumor cell growth, invasion, andmetastasis (121–124). Second, normal myoep-ithelial cells have been demonstrated to exert anautocrine- and paracrine-mediated pleiotropicsuppressive effect on breast cancer progression(125, 126). Third, multiple epithelial-centric,comprehensive gene-expression profiling stud-ies have failed to identify breast cancer stage–specific alterations. These findings stronglysuggest that tumor-stromal microenvironmen-tal alterations, rather than tumor-intrinsicalterations, may orchestrate tumor-stage pro-gression (94, 95). As a result of these observa-tions, there has been an intense resurgence ofinterest in the tumor microenvironment of bothinvasive and preinvasive breast cancer.

To better understand the potential roleof the tumor microenvironment in humanbreast tumorigenesis, Allinen et al. (127)performed a comprehensive genomic and gene-expression analysis of ex vivo–isolated neoplas-tic epithelium as well of as the nonneoplasticcells that constitute the tumor microenviron-ment of preinvasive and invasive breast cancer.Allinen and colleagues demonstrated consis-tent and dramatic gene-expression changes inthe nonneoplastic myofibroblastic cells of thetumor-stromal microenvironment and in thenonneoplastic myoepithelial cells of the intra-luminal tumor microenvironment of DCIS, ascompared with myofibroblastic and myoepithe-lial cells of normal breast tissue. Closer analy-sis of these changes demonstrated that DCIS-associated myoepithelial cells, when comparedwith normal myoepithelial cells, show upreg-ulation of genes encoding proteases such ascathepsins F, K, and L and matrix metallo-proteinase 2 (MMP2), as well as chemokinessuch as CXCL12/SDF-1 and CXCL14, both ofwhich have been implicated as regulators of cellgrowth, migration, and invasion (128–131). Re-cent findings by Orimo et al. (132) stronglysuggest that human breast cancer–associatedstromal cell secretion of CXCL12/SDF-1 pro-motes neoplastic epithelial growth in invasive

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carcinomas by direct paracrine stimulation.Consistent with these findings, Allinen et al.(127) observed an increased frequency of mi-totic activity in malignant epithelial cells adja-cent to the myoepithelial cells as compared withother regions of DCIS. This finding suggeststhat heterotypic paracrine signaling involvingchemokines occurs in the preinvasive stage ofbreast cancer.

To determine whether the dramatic gene-expression changes in the neoplastic epithelialcells, as well as in the myoepithelial and my-ofibroblastic cells, could be due to underlyinggenetic alterations, Allinen et al. (127) furtherperformed a comprehensive array CGH–basedanalysis of such ex vivo–procured cells in nor-mal breast tissue as well as in DCIS and inva-sive breast cancer samples. As expected, theydetected no genetic alterations in the epithe-lial or myoepithelial cells isolated from normalbreast tissue located adjacent to these tumors.Furthermore, in agreement with previous stud-ies (69, 133), the authors observed significantgenetic alterations in the neoplastic epitheliumof both preinvasive and invasive breast cancers.However, in contrast to previous observations(134–136), no genomic changes were detectedin tumor-associated myofibroblastic or myoep-ithelial cells. To further demonstrate the lackof genomic alterations in the myoepithelial andmyofibroblastic cells, the authors performed acomprehensive genome-wide single nucleotidepolymorphism (SNP) array analysis, as well asdirect sequencing of SNPs, and found no con-clusive evidence for genetic alterations in thesestromal cells. A lack of clonal genetic changein breast cancer associated stromal cells hasbeen further confirmed by Qiu and colleagues(137). These data as they specifically relate totumor-associated stromal cells are discrepantwith previous findings. However, as pointedout by Polyak and colleagues, this discrepancyis probably due to technical limitations asso-ciated with the previous published approaches(9, 127). Taken together, these data demon-strate that although nonneoplastic tumor-associated myoepithelial cells of DCIS and my-ofibroblastic cells of invasive carcinoma are

phenotypically different (i.e., gene expres-sion) from their normal counterparts, genomicchanges detected by comprehensive array CGHand SNP analyses are limited to the neoplasticepithelial cells (127).

Significant gene-expression changes in non-neoplastic stromal and myoepithelial cells,without obvious genetic alterations, suggestthat epigenetic modifications may be respon-sible for alterations in these cells. Utilizingmethylation-specific digital karyotyping, Huet al. (138) explored the possibility that epige-netic alterations underlie the relatively stablegene-expression phenotype observed in non-neoplastic stromal cells of preinvasive and in-vasive breast cancer samples. First, as expected,distinct epigenetic alterations between normalbreast epithelium and tumor epithelium wereobserved. Second, epigenetic changes betweennormal stroma and tumor stroma were alsoseen. Third, and most notably, similar to stro-mal epigenetic differences observed betweennormal breast and invasive breast cancer tis-sues, distinct recurrent epigenetic alterationswere observed in DCIS-associated myoepithe-lial cells as compared with their normal coun-terparts. The latter finding strongly suggeststhat epigenetic changes in nonneoplastic my-oepithelial cells play an important role in theestablishment and maintenance of the abnor-mal tumor microenvironment in the preinva-sive stage of breast cancer (138).

More recently, Ma et al. (93) used laser cap-ture microdissection and oligonucleotide mi-croarrays to conduct an in vivo–based com-parative global gene-expression analysis in theepithelial and stromal compartments duringbreast cancer progression from normal topreinvasive to IDC. Again, consistent withprevious observations, major epithelial gene-expression changes were shown to occur at thenormal-to-DCIS transition, whereas no ma-jor epithelial gene-expression differences wereidentified at the transition from DCIS to IDC.In the stromal compartment, as in the epithelialcompartment, thousands of gene-expressionchanges occurred at the transition from nor-mal to DCIS. The top differentially expressed


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genes between DCIS-associated stroma andnormal breast stroma include several signal-ing pathways previously implicated in humanbreast cancer. Two antagonists of WNT re-ceptor signaling WIF1 and SFRP1, were con-sistently downregulated in the DCIS stroma,while GREM1 and INHBA, two transform-ing growth factor beta (TGF-β) family mem-bers, were strongly induced in DCIS-associatedstroma. These observations confirm previousfindings (139, 140) and strongly suggest thatdecreased expression of WIF1 and SFRP1 andincreased expression of GREM1 and INHBAin the tumor-stromal microenvironment mayplay an important role in the early stagesof breast cancer progression. However, un-like the epithelial compartment that demon-strates no or rare gene-expression changes atthe transition from DCIS to invasive carci-noma, the stromal compartment demonstratessignificant gene-expression change at this tran-sition point. More specifically, whereas onlythree epithelial genes were differentially reg-ulated at the transition from DCIS to inva-sive cancer, 76 stromal genes were upregulatedand 229 stromal genes were downregulated atthe identical transition point (93). To obtainan overview of the biological processes associ-ated with these differentially expressed stromalgenes at the preinvasive-to-invasive transitionpoint, the authors (93) performed gene setenrichment analysis. The stromal genes thatwere differentially expressed at this transi-tion demonstrated the highest correlation withgene sets that featured the components of theextracellular matrix and the MMPs respon-sible for matrix remodeling. At an individ-ual gene level, MMP2, MMP11, and MMP14showed significant increased expression in IDCas compared with DCIS. These findings sup-port the notion that stroma-produced MMPsmay be key players driving the DCIS-to-IDCtransition.

Taken together, these gene-expression andepigenetic data support the view that the tumormicroenvironment is an important coconspira-tor rather than a passive bystander during breastcarcinogenesis. These results also suggest that

the tumor microenvironment participates intumorigenesis even before tumor cells invadeinto the stroma and that it may play a criticalrole in the transition from preinvasive to inva-sive growth.

THE TRANSITION FROMPREINVASIVE TO INVASIVEBREAST CANCER

The watershed event in breast cancer progres-sion is the transgression of tumor cells of thepreinvasive stage of DCIS through the base-ment membrane into the surrounding stromalcompartment. This transition is poorly under-stood, and exploring the molecular events thatdrive this transition continues to be of greatinterest. Phenotypic, genetic, and epigeneticchanges have been detected in the neoplas-tic epithelium of the preinvasive DCIS stageof breast cancer progression. However, stage-specific molecular alterations within the neo-plastic epithelial cells have not been identifiedin human breast cancer samples (93–95, 127,138). Multiple experimental lines of evidencehave highlighted the potential importance ofmolecular alterations in the nonneoplastic cellsof the tumor microenvironment, rather than inneoplastic epithelial cells, during the transitionfrom invasive to metastatic breast cancer (132,141–144).

Recently, Hu et al. (145) provided exper-imental evidence to support a similar phe-nomenon in the DCIS-to-IDC transition.Using a cell line model for DCIS (146) knownas MCFDCIS, the authors demonstrated thatthe spontaneous transition from DCIS to IDCis not associated with additional molecular al-terations within the neoplastic epithelial cells(145). Instead, Hu et al. showed that theDCIS-to-IDC transition is promoted by fi-broblasts and suppressed by myoepithelial cellsthat constitute the nonneoplastic stromal andintraluminal microenvironment of DCIS (145).The authors provided evidence for extensivecross talk among the TGF-β, Hedgehog, celladhesion, and p63 signaling pathways in theMCFDCIS cells, which results in the loss of

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myoepithelial differentiation and acceleratedprogression to invasive disease (145).

On the basis of these recent molecular ge-netic data, our view of breast cancer progres-sion as an epithelial-centric driven process hasevolved, and two models of the DCIS-to-IDCtransition have been proposed (147). First, theso-called escape model emphasizes the roleof neoplastic DCIS epithelial cells and pro-poses that genetic changes in combination withclonal selection give rise to a subpopulation ofneoplastic cells with the ability to disruptthe myoepithelial layer, degrade the basementmembrane of the duct, and invade into the sur-rounding stromal tissue. The so-called releasemodel, however, hypothesizes that alterationsin the DCIS microenvironment such as pheno-typic alterations of myoepithelial, myofibrob-lastic, and fibroblastic cells and infiltration ofinflammatory cells lead to the degradation ofthe basement membrane with subsequent in-vasion of the neoplastic epithelial cells. Cur-rent evidence supports a combination of the twomodels, in which changes in both the neoplas-tic epithelial cells and the nonneoplastic my-oepithelial and stromal cells result in a tumormicroenvironmental signaling network that ul-timately facilitates the transition from preinva-sive to invasive breast cancer (Figure 7).

MOLECULAR PROGNOSTICSFOR PREINVASIVEBREAST CANCER

Given that a minority (∼15%–30%) of womendiagnosed with pure DCIS develop a subse-quent breast tumor event within the first decadeafter treatment with lumpectomy, and that amajority (∼70%) of women with pure DCISare treated with lumpectomy in conjunctionwith radiation and antihormonal treatment, itis likely that many women with pure DCISare being overtreated (148, 149). Thus, thereis an unmet clinical need to identify a prog-nostic biomarker to accurately predict the clin-ical behavior of DCIS. Identification of such abiomarker will assist in the selection of the mostappropriate treatment regimen. Those patients

expected to develop indolent disease could betreated with lumpectomy alone, whereas thoseexpected to develop aggressive disease could betreated more aggressively.

Immunohistochemical and gene-expressionprofiling has confirmed the presence of molec-ular subtypes in DCIS that parallel the dis-tinct molecular subtypes observed in invasivebreast cancer (25, 91–93). The use of molec-ular profiling technologies to identify distinctfeatures that predict the future behavior of in-vasive disease is well documented. However, theapplication of such approaches to the identifi-cation of molecular predictors of clinical be-havior (i.e., invasive recurrence) of preinvasivedisease has been hampered by several problems.First, because preinvasive disease is frequentlymicroscopic in size, all of the tissue is processedthrough use of standard pathological formalin-fixed paraffin-embedded (FFPE) processes andutilized for clinical diagnostic purposes. Sec-ond, standard FFPE processes pose a significanttechnical challenge for high-throughput array-CGH and gene-expression microarray profil-ing. Third, and most importantly, large clinicalcohorts and clinical trials of preinvasive diseasewith well-annotated clinical samples and long(10–20 years) clinical follow-up are lacking.

Recently, Gauthier et al. (149) used aknowledge-based approach to study the rela-tionship of stress-induced senescence with pro-gression of the basal subtype of DCIS to inva-sive breast cancer. The authors demonstratedthat DCIS with high p16 and/or high COX2 inthe absence of cell proliferation reflects a nor-mal protective stress-activation response andthat this phenotype is associated with disease-free progression (149). However, DCIS withhigh p16 and/or high COX2 in the presence ofhigh cellular proliferation was shown to reflectan abrogated (or abnormal) response to cellu-lar stress, and this phenotype is associated withprogression of DCIS to invasive breast cancer(149). This biomarker may represent a definingbiological signature in the progression path-way of basal-like breast cancers, and it couldprovide a useful tool in the clinical manage-ment of patients with basal-like DCIS. These


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Myoepithelial cellssecrete cathepsins,MMPs, CXCL12, and

CXCL14

Stromal myofibroblasts and fibroblasts

Upregulated in stroma: MMPs, CXCL12, GREM1, TGF-βDownregulated in stroma: WIF1, SFRP1

Preinvasive tumor cellssecrete cytokines/chemokines

Figure 7Tumor-stromal interactions during the preinvasive-to-invasive breast cancer transition. Multiple lines ofevidence demonstrate marked genetic, epigenetic, and gene-expression alterations in the neoplasticepithelium of preinvasive breast cancer as compared with normal breast epithelium. At the transition frompreinvasive to invasive breast cancer, the neoplastic epithelium is not associated with additional qualitativemolecular alterations. Instead, the nonneoplastic tumor microenvironment cells, in particular myoepithelialcells and stromal fibroblasts, myofibroblasts, and leukocytes, display significant molecular alterations that areassociated with and may contribute to the transition from in situ to invasive breast carcinoma. In preinvasivebreast cancer (such as that illustrated in this hematoxylin and eosin–stained section), overexpression ofcytokines and chemokines (as compared with normal breast epithelium) by neoplastic epithelium can inducean autocrine pathway that directly stimulates tumor growth. These tumor epithelial cell–derived cytokinescan also induce several paracrine pathways. In the first paracrine pathway, tumor epithelial cell–derivedcytokines and chemokines induce decreased stromal expression of growth pathway antagonists (i.e., WIF1and SFRP1) that, in turn, favors tumor epithelial cell growth. Furthermore, the tumor epithelium can inducestromal cell expression of proteases [matrix metalloproteinases (MMPs) and cathepsins] that are involved inextracellular matrix remodeling, angiogenesis, and basement membrane (black arrowheads) degradation. Inthe second paracrine pathway, epithelial cytokines induce myoepithelial cells to secrete both proteases(MMPs) that degrade the basement membrane, thereby facilitating the transition from in situ to invasivecarcinoma, and cytokines (CXCL12 and CXCL14) that promote tumor epithelial cell growth, migration,and invasion. Abbreviation: TGF-β, transforming growth factor beta.

preliminary results generate optimism thatprognostic biomarkers of the different sub-types of DCIS exist, and identification of suchbiomarkers will further improve the clinicalmanagement of patients diagnosed with prein-vasive breast cancer.

CONCLUSIONSOver the past decade, translational and ba-sic science researchers have witnessed manynew approaches to the study of the invasiveand preinvasive stages of breast cancer andbreast cancer progression. Significant advances

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in our knowledge of the molecular biology ofinvasive breast cancer have allowed us to bet-ter understand the heterogeneity of this dis-ease as it relates to its biological and clini-cal behavior. Such advances have resulted inthe development of prognostic and predictivemolecular assays that are currently used in themanagement of patients with invasive breastcancer. Through combined use of advancedmicrodissection and ex vivo isolation tech-niques with high-throughput genomic, gene-expression, and epigenetic technologies, re-search efforts have begun to piece togetherthe complex molecular genetic and molecular

biological interrelationship between preinva-sive stages of breast cancer and invasive breastcancer. Preinvasive breast cancer appears toarise from two distinct molecular genetic evo-lutionary pathways. However, despite evidencefor the existence of two evolutionary pathways,preinvasive breast cancer constitutes a spectrumof lesions with heterogeneous biology and clin-ical behavior. Thus, the next challenge facingbreast cancer researchers is successfully inte-grating our knowledge of the molecular bi-ology of preinvasive breast cancer to identifypredictive biomarkers of disease-progressionrisk.

SUMMARY POINTS

1. The ductal (FEA, ADH, and DCIS) and lobular (ALH and LCIS) preinvasive stages ofthe breast cancer are nonobligate precursors to invasive disease. These lesions constitutea spectrum of morphological entities with variable clinical behavior.

2. Genetic and gene-expression studies reveal two distinct molecular pathways of breastcancer progression that, in general, correlate with the histopathological parameter oftumor grade, resulting in a contemporary model of breast cancer progression that alsoaccounts for intrastage heterogeneity.

3. Distinct genetic, epigenetic, and gene-expression alterations occur in breast epitheliumat the transition from normal to preinvasive breast cancer. However, additional molecularchanges appear to be infrequent at the transition from preinvasive to invasive disease. Thelatter observation suggests that preinvasive epithelium possesses the molecular machineryfor aggressive behavior and that progression may depend upon other factors, such asquantitative levels and the timing of molecular events.

4. The tumor-stromal and myoepithelial microenvironments demonstrate unique epige-netic and gene-expression alterations at the normal-to-preinvasive transition as well asat the preinvasive-to-invasive transition.

5. Recent gene-expression and epigenetic data strongly suggest that the stromal and my-oepithelial microenvironment of preinvasive breast cancer actively participates in thetransition from preinvasive to invasive disease. Current data point to a complex stromal-epithelial signaling interplay between matrix remodeling (e.g., secretion of MMPs andcathepsins by stromal and myoepithelial cells) and signal transduction pathways linkedto growth and migration (e.g., secretion of CXCL12, CXCL14, SFRP1, and WIF1).

6. Translational research over the past two decades has resulted in the successful applicationand use of molecular diagnostics in the clinical management of invasive breast cancer.Although application of molecular diagnostics to preinvasive breast cancer lags behind itsapplication to the invasive counterpart, the recent discovery of a p16- and COX2-basedprognostic signature in basal-like DCIS has generated optimism that clinically usefulmolecular biomarkers of preinvasive breast disease will be discovered in the near future.


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FUTURE DIRECTIONS

1. Identification of the precise cell or cells of origin responsible for the preinvasive stagesof breast cancer would provide a critical starting point for a better understanding of themolecular events underlying the development of human breast cancer.

2. Because the transition from preinvasive cancer to invasive cancer is the critical water-shed event in breast cancer progression, a more complete understanding of the basicmechanisms underlying breast cancer invasion is needed.

3. Unraveling the complex heterotypic signaling in the microenvironment of preinvasivebreast cancer will require the development of improved in vivo and in vitro models ofbreast cancer progression. More specifically, it will require the development of mod-els that represent the various subtypes (ER+/HER−, ER+/HER+, ER−/HER+, andER−/PR−/HER−) of preinvasive disease, as each subtype is associated with distinctmolecular biological pathways and clinical behavior.

4. Preinvasive breast cancer constitutes a spectrum of lesions with heterogeneous biologyand clinical behavior. Currently, it is not possible to specify with certainty which prein-vasive lesion will progress to invasive disease and which will not. Thus, there is a criticalneed to develop a molecular classification system or to identify biomarkers for preinvasivebreast cancer that will accurately predict risk of local recurrence and, more importantly,risk of subsequent invasive disease.

5. Identification of prognostic (prediction of local recurrence and invasion) and treatment-predictive biomarkers for preinvasive breast cancer will require the establishment of alarge international cohort of patients with long-term clinical follow-up data linked topathology tissue repositories.

6. Development and implementation of novel molecular-based imaging reagents and meth-ods for the detection of microscopic preinvasive breast cancer will be of tantamountimportance, as they will allow for potentially curative therapeutic intervention.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

I extend special thanks to many collaborators for thoughtful discussions over the years; to RyanMcMullin, Elena F. Brachtel, and Miguel Rivera for critical review of the manuscript; and to F.Koerner for providing the LCIS photomicrograph. I apologize to all investigators whose researchcould not be appropriately cited owing to space limitations. I am supported in part by NationalInstitutes of Health grant RO1-1CA112021-01, U.S. Department of Defense grant W81XWH-04-1-0606, Susan G. Komen Breast Cancer Foundation grant BCTR0402932, and the AvonFoundation.

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AR403-FM ARI 9 December 2009 12:46

Annual Review ofPathology:Mechanisms ofDisease

Volume 5, 2010Contents

Molecular Pathogenesis of Necrotizing FasciitisRandall J. Olsen and James M. Musser � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

The Pathobiology of Glioma TumorsCandece L. Gladson, Richard A. Prayson, and Wei Michael Liu � � � � � � � � � � � � � � � � � � � � � � � � � � �33

Mutational Heterogeneity in Human Cancers: Origin andConsequencesJesse J. Salk, Edward J. Fox, and Lawrence A. Loeb � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �51

Fibrogenic Reactions in Lung DiseaseJun Araya and Stephen L. Nishimura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �77

The Senescence-Associated Secretory Phenotype: The Dark Sideof Tumor SuppressionJean-Philippe Coppe, Pierre-Yves Desprez, Ana Krtolica, and Judith Campisi � � � � � � � � � � �99

Epithelial Barriers in Homeostasis and DiseaseAmanda M. Marchiando, W. Vallen Graham, and Jerrold R. Turner � � � � � � � � � � � � � � � � � � 119

Nonalcoholic Fatty Liver Disease: Pathology and PathogenesisDina G. Tiniakos, Miriam B. Vos, and Elizabeth M. Brunt � � � � � � � � � � � � � � � � � � � � � � � � � � � � 145

Pathogenesis of PreeclampsiaBrett C. Young, Richard J. Levine, and S. Ananth Karumanchi � � � � � � � � � � � � � � � � � � � � � � � � 173

Preinvasive Breast CancerDennis C. Sgroi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 193

The Pathogenesis of Acute Pulmonary Viral and Bacterial Infections:Investigations in Animal ModelsMary F. Lipscomb, Julie Hutt, Julie Lovchik, Terry Wu, and C. Rick Lyons � � � � � � � � � � � 223

Mammalian Sirtuins: Biological Insights and Disease RelevanceMarcia C. Haigis and David A. Sinclair � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Mitochondrial Energetics and TherapeuticsDouglas C. Wallace, Weiwei Fan, and Vincent Procaccio � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 297

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p63 in Epithelial Survival, Germ Cell Surveillance, and NeoplasiaChristopher P. Crum and Frank D. McKeon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 349

Indexes

Cumulative Index of Contributing Authors, Volumes 1–5 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 373

Cumulative Index of Chapter Titles, Volumes 1–5 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 376

Errata

An online log of corrections to Annual Review of Pathology, Mechanisms of Disease articlesmay be found at http://pathol.annualreviews.org

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Preinvasive Breast Cancer

Documents

Transcript of Preinvasive Breast Cancer