Journal of PathologyJ Pathol 2005; 205: 248–254Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1691

Review Article

Molecular evolution of breast cancer

Peter T Simpson,1† Jorge S Reis-Filho,1,2,3† Theodora Gale1 and Sunil R Lakhani4*1The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, London, UK2 IPATIMUP — Institute of Molecular Pathology and Immunology, University of Porto, Portugal3School of Health Sciences, University of Minho, Braga, Portugal4Molecular and Cellular Pathology, School of Medicine, University of Queensland, Royal Brisbane and Women’s Hospital and Queensland Institute ofMedical Research, Brisbane, Australia

*Correspondence to:Professor Sunil R Lakhani, Head,Molecular and Cellular Pathology,School of Medicine, University ofQueensland, Mayne MedicalSchool, Herston Road, Herston,Brisbane, QLD 4006, Australia.E-mail: s.lakhani@uq.edu.au

†These authors contributedequally to the writing of thisreview.

Received: 18 October 2004Accepted: 19 October 2004

AbstractMolecular analysis of invasive breast cancer and its precursors has furthered our under-standing of breast cancer progression. In the past few years, new multi-step pathways ofbreast cancer progression have been delineated through genotypic–phenotypic correlations.Nuclear grade, more than any other pathological feature, is strongly associated with thenumber and pattern of molecular genetic abnormalities in breast cancer cells. Thus, thereare two distinct major pathways to the evolution of low- and high-grade invasive carcino-mas: whilst the former consistently show oestrogen receptor (ER) and progesterone receptor(PgR) positivity and 16q loss, the latter are usually ER/PgR-negative and show Her-2 over-expression/amplification and complex karyotypes. The boundaries between the evolutionarypathways of well-differentiated/low-grade ductal and lobular carcinomas have been blurred,with changes in E-cadherin expression being one of the few distinguishing features betweenthe two. In addition, lesions long thought to be precursors of breast carcinomas, such ashyperplasia of usual type, are currently considered mere risk indicators, whilst columnarcell lesions are now implicated as non-obligate precursors of atypical ductal hyperplasia(ADH) and well-differentiated ductal carcinoma in situ (DCIS). However, only through thecombination of comprehensive morphological analysis and cutting-edge molecular tools canthis knowledge be translated into clinical practice and patient management.Copyright 2005 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: breast cancer; molecular pathology; evolution; pathways; CGH; LOH;microarrays

Introduction

Breast cancer is a heterogeneous disease encompass-ing a wide variety of pathological entities and a rangeof clinical behaviour. These are underpinned at themolecular level by a complex array of genetic alter-ations that affect cellular processes [1–3]. In seekingto characterize these aberrations, the study of cancergenetics has had a major impact in our understandingof the development and progression of breast neo-plasms.

Historically, breast cancer progression was seen as amulti-step process, akin to the Volgenstein’s model forcolon carcinogenesis [4], encompassing progressivechanges from normal, to hyperplasia with and withoutatypia, carcinoma in situ, invasive carcinoma, andmetastasis (Figure 1) [5].

Whilst most of the concepts regarding the mor-phologically defined breast cancer precursor lesionsremain valid, immunohistochemistry and moleculargenetics have changed the way that the breast can-cer multi-step model is seen [2,3,6–13]. No longerdo we perceive it as a single pathway, but as acomplex series of stochastic genetic events leading

to distinct and divergent pathways towards invasivebreast cancer. The distinction between ‘lobular’ and‘ductal’ pathways has now been blurred. In addition,some of the lesions along the pathway have been repo-sitioned and the role of others has been questioned[2,3,5–7,11,14–17].

Although some aspects of the molecular genetics ofinvasive breast carcinomas have been known for a longtime, the inability to study its putative precursors pre-cluded any direct correlation. Since the advent of reli-able methods of tissue microdissection [18] (ie lasercapture microdissection), DNA amplification [19–21],and genome and transcriptome analysis [ie cytogenet-ics, comparative genomic hybridization (CGH), lossof heterozygosity (LOH), gene expression analysis,microarray CGH], it has become possible to studythe molecular aspects of benign proliferative and pre-invasive breast lesions and their relationship to inva-sive breast carcinoma [10,15–17,22].

Invasive and in situ breast carcinomas

In the past 15 years, molecular pathology of invasivebreast cancer has received great attention. Attempts

Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular evolution of breast cancer 249

Figure 1. Multi-step model of breast cancer progression based solely on morphological features and epidemiological studies, nowconsidered oversimplistic with conceptual flaws

to redefine breast cancer taxonomy [23–25], refinethe prognostic indicators, and predict recurrences andmetastases have been carried out [24–32].

Classification of breast cancer into prognosticallymeaningful groups has traditionally been done by his-tological (sub)type; however, it has become increas-ingly clear that grade (degree of differentiation) isa better predictor of outcome than type [2,12,13].Therefore, it is not surprising that molecular methodshave demonstrated that grade, more than any otherclinico-pathological parameter, strongly reflects theextent, complexity, and type of genomic aberrations[2,9,12,13].

The genetic aspects of low- and high-grade breastcancer not only differ in quantitative terms, but are alsosegregated by the type of aberrations: grade I and tubu-lar breast carcinomas show a low number of genomicalterations with highly recurrent losses of 16q, whereasgrade III breast carcinomas show complex genotypesfrequently harbouring loss of 11q, 14q, 8p, 13q; gainof 17q, 8q, 5p; and high-level gains (amplifications)on 17q12, 17q22–24, 6q22, 8q22, 11q13, and 20q13[2,12,13]. Since the molecular profiles of grade I andgrade III tumours are so different, they suggest thatfor the most part, progression from low- to high-gradebreast carcinoma is rare [2,12,13]. For instance, phys-ical loss of 16q, the most frequent genomic changeobserved in grade I tumours, is exceedingly infre-quent in high-grade breast carcinomas [2,12,13,33].Although loss of 16q is observed in a subset of gradeIII carcinomas, it has recently been shown to occurby a distinct mechanism (LOH in combination withmitotic recombination) [34].

CGH and conventional cytogenetic studies haveshown that there is some degree of variation in thepattern of genetic alterations between different histo-logical types of invasive breast cancer [2,10,35–46].Although differences between histological subtypes doexist, this association is not as strong as it happens tobe with grade [2,9,12]. Comparative analyses betweeninvasive ductal (IDCs) and lobular breast carcinomas(ILCs) have demonstrated that overall a lower numberof genetic changes are found in ILCs relative to IDCs[10,35,36,44–46]. Although some specific chromoso-mal abnormalities are found at a significantly differentfrequency in each histological type [10,35,36,44–46],this may only highlight the fact that most ILCs areof lower nuclear grade. Interestingly, several recurrent

unbalanced changes, including physical loss of 16q,were common to both types, indicating that ILCs andlow-grade IDCs may arise via common pathways oftumourigenesis [35,36,44–46].

This finding has blurred the boundary betweenductal and lobular lesions and has raised the questionas to whether the designations ‘ductal’ and ‘lobular’are appropriate. In fact, the majority of neoplasticbreast diseases arise from the terminal duct-lobularunit (TDLU) and so this terminology is not intendedto reflect the micro-anatomical site of origin, but adifference in cell morphology [2,47]. It should alsobe stressed that although loss of 16q is observed inboth grade I IDC and ILC, the culprit genes mightdiffer in these two lesions [47–51]. The likeliestcandidate gene involved in loss of 16q in atypicallobular hyperplasia (ALH)/lobular carcinoma in situ(LCIS) (see below) and ILC is CDH1 (E-cadherin),which maps to 16q22.1 [2,47,50,52,53]. It is acceptedthat most of ALH/LCIS and ILC harbour loss of 16q,followed by gene mutation, promoter methylation orfurther loss of CDH1. Loss of E-cadherin, a criticalcell adhesion molecule [2,47,52,53], is reflected at themorphological level by the characteristic discohesivenature of individual cells and overall growth patternof lobular carcinomas. However, CDH1 is almostcertainly not the target gene in grade I IDCs, as lossof E-cadherin expression and CDH1 gene mutationsare exceedingly rare in these tumours [48]. The huntfor the gene actually involved in grade I ductalcancers is ongoing and proving exceedingly difficult[48,50,51].

Over the last few years, a pleomorphic vari-ant of lobular carcinoma (PLC) has been described[47,53–57]. Briefly, in pleomorphic LCIS and ILC,neoplastic cells show the typical discohesivenessof lobular neoplasms; however, they are of highgrade and show features of apocrine differentia-tion. Although molecular data on the PLC are scant[47,53–57], these tumours have overlapping geneticchanges with both classic ILC and grade III inva-sive ductal breast carcinomas, harbouring recurrentloss of 16q and lack of E-cadherin expression, butalso showing overexpression of Her-2 [47,53–57]. Inaddition, anecdotal evidence suggests that PLC mayhave a more aggressive biological behaviour than ILC[53,55–57].

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Studies using expression arrays have also providednew insights into breast cancer taxonomy. Breast car-cinomas can be classified into four categories accord-ing to their transcriptome: oestrogen receptor (ER)-positive, HER-2-positive, basal, and normal breast-likecarcinomas [23]. It is interesting that even this sepa-ration is fundamentally into ER-positive and -negativegroups, which for the most part fall into low- and high-grade carcinomas, respectively. In following publica-tions, it was shown that these groups have importantprognostic implications; namely, patients with HER-2-positive or basal carcinomas (high-grade) do worsethan patients with ER-positive or normal breast-liketumours (low-grade) [24,25]. However, recent dataalso suggest that each of these groups may be hetero-geneous at the genetic and clinical levels [26,58,59].In addition, it is still unclear whether there is anycorrelation between the expression profile, moleculargenetic features, and histopathological parameters ofthese tumours.

The frequent association and morphological simi-larities between invasive and some forms of prolifer-ative breast diseases have led pathologists to assumethat certain entities would be biologically related (egLCIS and ILC, DCIS and IDC) [2,5]. The complexityof these relationships has been thoroughly exploredusing the advancement in molecular pathology. Forexample, immunohistochemistry, LOH, CGH, andgene expression analysis have recapitulated the geno-typic/phenotypic patterns observed in invasive ductaland lobular carcinomas in ADH/DCIS and ALH/LCIS[2,5,9,11,16,44,60–62]. The distinct molecular geneticfeatures found in different grades of invasive carci-nomas are also mirrored in pre-invasive lesions ofcomparable morphology [2,5,11].

In hindsight, it seems clear that there are two majorarms in the multi-pathway model of multi-step breastcancer progression (Figure 2): one comprising well-differentiated DCIS that progress to grade I IDC, andthe other encompassing poorly differentiated DCISthat progress to grade III IDC. In the ‘low-grade arm’,

Figure 2. Multi-step model of breast cancer progression redefined using the current morphological, immunohistochemical, andmolecular features. Note the two distinct low- and high-grade pathways of progression. Connectors drawn with continuous linesrepresent links between morphological entities for which there is supporting morphological and/or molecular data. Connectorsdrawn with dotted lines represent hypothetical correlations/evolutionary links yet to be proven. Note: boxes show morphologicalentities. +: presence of immunohistochemical expression or gain of genetic material; −: lack of immunohistochemical expression orloss of genetic material; +/−: heterogeneous immunohistochemical expression; −/+: infrequent immunohistochemical positivity;ADH: atypical ductal hyperplasia; ALH: atypical lobular hyperplasia; CCL: columnar cell lesion; CDH1: human E-cadherin gene;CKs: cytokeratins; DCIS: ductal carcinoma in situ; ER: oestrogen receptor; HUT: hyperplasia of usual type; IDC: invasive ductalcarcinoma; ILC: invasive lobular carcinoma; Interm.-Dif.: intermediately differentiated; LCIS: lobular carcinoma in situ; LOH: loss ofheterozygosity; PgR: progesterone receptor; Poor-Dif.: poorly differentiated; Well-Dif.: well differentiated

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tumours are of low nuclear grade, usually ER and PgR-positive, negative for Her-2 and basal markers, andharbour low genetic instability and recurrent 16q loss,whereas in the ‘high-grade arm’, the lesions show ahigher degree of nuclear atypia, are more frequentlyhormone receptor-negative, frequently positive foreither Her-2 or basal markers, and are geneticallyadvanced lesions, showing a combination of recurrentgenomic changes including loss of 8p, 11q, 13q,14q; gain of 1q, 5p, 8q, 17q; and amplifications on6q22, 8q22, 11q13, 17q12, 17q22–24, and 20q13[2,5,9,12,13]. Based on their pathological and geneticfeatures, classic LCIS and ILC are remarkably similarto those tumours in the ‘low-grade arm’ [2,44,47].However, in contrast to well-differentiated DCIS/gradeI IDC, the vast majority of these tumours lack E-cadherin expression owing to genetic and/or epigeneticchanges in the CDH1 gene [48,50–52]. On the otherhand, the overlapping morphological features of PLCwith both classic lobular and grade III carcinomas,and the combination of E-cadherin (16q) loss withoccasional Her-2 positivity [53–57] add another levelof complexity to these molecular pathways to breastcancer tumourigenesis.

Hyperplasias and columnar cell change

Apart from ADH and ALH, which bear remarkablemorphological and molecular resemblance to well-differentiated DCIS and LCIS, respectively, the othernon-obligate/premalignant lesions have proven moredifficult to characterize and establish their actual posi-tion along the pathways [2,16,60–67]. Interestingly,ADH and low-grade DCIS show identical immunopro-files and low numbers of chromosomal abnormalities,comprising recurrent loss of 16q (see ref 2 and refscited therein). The similarities between ALH and LCISare not restricted to the morphological level, but alsothe immunohistochemical and genetic features [47].In fact, differentiating between them is arbitrary andsubjective, being based on subtle quantitative morpho-logical features. Hence, it is well accepted that bothADH and ALH are non-obligate precursors to thedevelopment of well-differentiated DCIS and LCIS,respectively. Alternatively, one could view them justas small DCIS or LCIS.

For a long time, hyperplasia of usual type (HUT)was seen as the precursor of ADH and DCIS(see Figure 1). However, its role in the multi-stepmodel of breast carcinogenesis has been questioned(for a review see ref 2 and refs cited therein)[2,5,15,16,64–67]. The morphological features andimmunoprofile of HUTs are in stark contrast withthose of the accepted precursors, since they are com-posed of a mixed population of cell types with avariable proportion of ER, PgR-positive luminal cellsand myoepithelial/basal marker-positive cells. At themolecular level, no or only rare and fairly randomchromosomal changes are observed [2,15,65]. Despite

the controversies, there is evidence to suggest that asmall proportion of HUTs may be clonal, neoplasticproliferations (adenomas) that may putatively progressto ADH or DCIS, whereas the majority of them fail toshow any evidence of a neoplastic/monoclonal natureusing existing technology [2,3,16,66].

A more likely precursor to ADH and well-diffe-rentiated DCIS are columnar cell lesions(CCLs) [8,68–70], which are characterized by thepresence of tightly packed columnar-shaped epithelialcells, with ovoid-to-elongated nuclei and prominentapical cytoplasmic snouts and intraluminal secretions.In fact, they comprise a spectrum of lesions with vary-ing degrees of architectural and mild nuclear atypia.At the lower end of the spectrum are lesions com-posed of variably dilated acini lined by a single layerof the characteristic cells. At the higher end of thespectrum, lesions resemble ADH, with stratification,bridging, and punched out spaces. Throughout thespectrum, CCLs show an immunoprofile similar to thatof ADH/well-differentiated DCIS [8]. However, thedegree of proliferation, architectural and cytologicalatypia are mirrored at the genetic level, with a stepwiseincrease in the number and complexity of chromoso-mal copy number changes as defined by CGH [70].Moreover, the hallmark genetic feature of ‘low-grade’lesions, loss of 16q, is the most frequently detectedrecurrent change and in addition, there is some degreeof overlap in the molecular genetic profile of CCLand associated more advanced lesions [69,70]. Inter-estingly, it is not infrequent to observe ALH/LCIS inthe context of multifocal CCLs. Hence CCLs may bethe link between normal breast and ADH, as well asbetween ‘ductal’ and ‘lobular’ neoplasia.

The precursor of poorly differentiated DCIS hasbeen elusive. Based on morphological, immunohisto-chemical, and molecular findings, CCL, ADH, andwell-differentiated DCIS would be unlikely candi-dates. Although apocrine change has long been con-sidered a metaplastic process in breast tissues, usuallyassociated with ageing, this concept has come intoquestion with the application of molecular findings[2,17,71–73]. At least a subset of lesions with apoc-rine morphology show molecular changes, includingLOH/allelic imbalance at 1p (MYCL1), 11q (INT2),13q, 16q and 17q [71,72], and recurrent chromosomalchanges as defined by CGH, including loss of 1p, 2p,10q, 16q, 17q and 22q, and gain of 1p, 2q and 13q[17]. These findings are more frequently observed inapocrine adenosis and apocrine hyperplasia comparedwith apocrine cysts. For the large part, these obser-vations have been ignored by the diagnostic patholo-gist. It is remarkable that a proliferative lesion withthe architecture of a micropapillary DCIS is consid-ered benign because it happens to have abundant pinkcytoplasm! The lesion regarded as papillary apocrinehyperplasia would be seriously considered DCIS bymany if it was not apocrine in nature. Whether thisprejudice is justified should be questioned. It may turnout to be wrong but we would suggest that there is

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compelling molecular data that at least some of theselesions may be the precursors of high-grade DCIS andinvasive cancer. There is a real need for further workin this area.

Benign proliferative breast lesions andnormal breast

Little is known about the presence of moleculargenetic changes in benign proliferative breast lesions,such as adenosis, papillomas, and tubular adeno-mas [2,65,73]. Although gross chromosomal aberra-tions are not observed in normal breast, most non-proliferative breast lesions, and papillomas, there aresome data to suggest that non-recurrent LOH at someloci may be found [2,65].

Although there are changes detected by LOH insome benign proliferative breast lesions, these showlittle overlap to the most prevalent genetic changesdescribed in DCIS and invasive carcinomas and arethus insufficient to make them non-obligate precursorsof DCIS or invasive breast cancer [2,65,73]. In addi-tion, whilst LOH in neoplastic lesions encompasses allinformative markers mapping to a particular chromo-some arm, LOH in adjacent normal-appearing epithe-lium involves only single, random markers across thegenome [74,75]. Although the genetic abnormalitiesfound in normal cells show little or no overlap withthose of neoplastic lesions, these changes may indicatedefective mechanisms for the maintenance of genomicintegrity, which may contribute to the carcinogenicprocess and denote increased risk for developing breastcancer.

Conclusion and future prospects

Molecular analysis has been extensively utilized in thecharacterization of breast lesions and has been instru-mental in unravelling a direct relationship betweengenotype and phenotype, identifying new subtypes,illustrating molecular pathways in the progression toinvasive carcinoma, defining relationships betweendifferent histological variants, and providing invalu-able prognostic parameters.

Now, the mission of pathologists and scientistsalike is to translate our current understanding ofthe molecular evolution of breast cancer and thecomplexity of microarray data into practical methodsthat are suitable for diagnostic pathology and patients’management.

Acknowledgements

J Reis-Filho is the recipient of the Gordon Signy InternationalFellowship Award and is partially supported by PhD GrantSFRH/BD/5386/2001 from the Fundacao para a Ciencia ea Tecnologia, Portugal, and Programa Operacional Ciencia,Tecnologia e Inovacao POCTI/CBO/45157/2002.

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