Molecular evolution of breast cancer

Transcription

1 Journal of Pathology Published online in Wiley InterScience ( DOI: /path.1691 Review Article Molecular evolution of breast cancer Peter T Simpson, 1 Jorge S Reis-Filho, 1,2,3 Theodora Gale 1 and Sunil R Lakhani 4 * 1 The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, London, UK 2 IPATIMUP Institute of Molecular Pathology and Immunology, University of Porto, Portugal 3 School of Health Sciences, University of Minho, Braga, Portugal 4 Molecular and Cellular Pathology, School of Medicine, University of Queensland, Royal Brisbane and Women s Hospital and Queensland Institute of Medical Research, Brisbane, Australia *Correspondence to: Professor Sunil R Lakhani, Head, Molecular and Cellular Pathology, School of Medicine, University of Queensland, Mayne Medical School, Herston Road, Herston, Brisbane, QLD 4006, Australia. s.lakhani@uq.edu.au These authors contributed equally to the writing of this review. Received: 18 October 2004 Accepted: 19 October 2004 Abstract Molecular analysis of invasive breast cancer and its precursors has furthered our understanding of breast cancer progression. In the past few years, new multi-step pathways of breast cancer progression have been delineated through genotypic phenotypic correlations. Nuclear grade, more than any other pathological feature, is strongly associated with the number and pattern of molecular genetic abnormalities in breast cancer cells. Thus, there are two distinct major pathways to the evolution of low- and high-grade invasive carcinomas: 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 overexpression/amplification and complex karyotypes. The boundaries between the evolutionary pathways 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 between the two. In addition, lesions long thought to be precursors of breast carcinomas, such as hyperplasia of usual type, are currently considered mere risk indicators, whilst columnar cell lesions are now implicated as non-obligate precursors of atypical ductal hyperplasia (ADH) and well-differentiated ductal carcinoma in situ (DCIS). However, only through the combination of comprehensive morphological analysis and cutting-edge molecular tools can this knowledge be translated into clinical practice and patient management. Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: breast cancer; molecular pathology; evolution; pathways; CGH; LOH; microarrays Introduction Breast cancer is a heterogeneous disease encompassing a wide variety of pathological entities and a range of clinical behaviour. These are underpinned at the molecular level by a complex array of genetic alterations that affect cellular processes [1 3]. In seeking to characterize these aberrations, the study of cancer genetics has had a major impact in our understanding of the development and progression of breast neoplasms. Historically, breast cancer progression was seen as a multi-step process, akin to the Volgenstein s model for colon carcinogenesis [4], encompassing progressive changes from normal, to hyperplasia with and without atypia, carcinoma in situ, invasive carcinoma, and metastasis (Figure 1) [5]. Whilst most of the concepts regarding the morphologically defined breast cancer precursor lesions remain valid, immunohistochemistry and molecular genetics have changed the way that the breast cancer multi-step model is seen [2,3,6 13]. No longer do we perceive it as a single pathway, but as a complex series of stochastic genetic events leading to distinct and divergent pathways towards invasive breast cancer. The distinction between lobular and ductal pathways has now been blurred. In addition, some of the lesions along the pathway have been repositioned and the role of others has been questioned [2,3,5 7,11,14 17]. Although some aspects of the molecular genetics of invasive breast carcinomas have been known for a long time, the inability to study its putative precursors precluded any direct correlation. Since the advent of reliable methods of tissue microdissection [18] (ie laser capture microdissection), DNA amplification [19 21], and genome and transcriptome analysis [ie cytogenetics, comparative genomic hybridization (CGH), loss of heterozygosity (LOH), gene expression analysis, microarray CGH], it has become possible to study the molecular aspects of benign proliferative and preinvasive breast lesions and their relationship to invasive breast carcinoma [10,15 17,22]. Invasive and in situ breast carcinomas In the past 15 years, molecular pathology of invasive breast cancer has received great attention. Attempts Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

2 Molecular evolution of breast cancer 249 Figure 1. Multi-step model of breast cancer progression based solely on morphological features and epidemiological studies, now considered oversimplistic with conceptual flaws to redefine breast cancer taxonomy [23 25], refine the prognostic indicators, and predict recurrences and metastases have been carried out [24 32]. Classification of breast cancer into prognostically meaningful groups has traditionally been done by histological (sub)type; however, it has become increasingly clear that grade (degree of differentiation) is a better predictor of outcome than type [2,12,13]. Therefore, it is not surprising that molecular methods have demonstrated that grade, more than any other clinico-pathological parameter, strongly reflects the extent, complexity, and type of genomic aberrations [2,9,12,13]. The genetic aspects of low- and high-grade breast cancer not only differ in quantitative terms, but are also segregated by the type of aberrations: grade I and tubular breast carcinomas show a low number of genomic alterations with highly recurrent losses of 16q, whereas grade III breast carcinomas show complex genotypes frequently harbouring loss of 11q, 14q, 8p, 13q; gain of 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 and grade III tumours are so different, they suggest that for the most part, progression from low- to high-grade breast carcinoma is rare [2,12,13]. For instance, physical loss of 16q, the most frequent genomic change observed in grade I tumours, is exceedingly infrequent in high-grade breast carcinomas [2,12,13,33]. Although loss of 16q is observed in a subset of grade III carcinomas, it has recently been shown to occur by a distinct mechanism (LOH in combination with mitotic recombination) [34]. CGH and conventional cytogenetic studies have shown that there is some degree of variation in the pattern of genetic alterations between different histological types of invasive breast cancer [2,10,35 46]. Although differences between histological subtypes do exist, this association is not as strong as it happens to be with grade [2,9,12]. Comparative analyses between invasive ductal (IDCs) and lobular breast carcinomas (ILCs) have demonstrated that overall a lower number of genetic changes are found in ILCs relative to IDCs [10,35,36,44 46]. Although some specific chromosomal abnormalities are found at a significantly different frequency in each histological type [10,35,36,44 46], this may only highlight the fact that most ILCs are of lower nuclear grade. Interestingly, several recurrent unbalanced changes, including physical loss of 16q, were common to both types, indicating that ILCs and low-grade IDCs may arise via common pathways of tumourigenesis [35,36,44 46]. This finding has blurred the boundary between ductal and lobular lesions and has raised the question as to whether the designations ductal and lobular are appropriate. In fact, the majority of neoplastic breast diseases arise from the terminal duct-lobular unit (TDLU) and so this terminology is not intended to reflect the micro-anatomical site of origin, but a difference in cell morphology [2,47]. It should also be stressed that although loss of 16q is observed in both grade I IDC and ILC, the culprit genes might differ in these two lesions [47 51]. The likeliest candidate gene involved in loss of 16q in atypical lobular 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 accepted that most of ALH/LCIS and ILC harbour loss of 16q, followed by gene mutation, promoter methylation or further loss of CDH1. Loss of E-cadherin, a critical cell adhesion molecule [2,47,52,53], is reflected at the morphological level by the characteristic discohesive nature of individual cells and overall growth pattern of lobular carcinomas. However, CDH1 is almost certainly not the target gene in grade I IDCs, as loss of E-cadherin expression and CDH1 gene mutations are exceedingly rare in these tumours [48]. The hunt for the gene actually involved in grade I ductal cancers is ongoing and proving exceedingly difficult [48,50,51]. Over the last few years, a pleomorphic variant of lobular carcinoma (PLC) has been described [47,53 57]. Briefly, in pleomorphic LCIS and ILC, neoplastic cells show the typical discohesiveness of lobular neoplasms; however, they are of high grade and show features of apocrine differentiation. Although molecular data on the PLC are scant [47,53 57], these tumours have overlapping genetic changes with both classic ILC and grade III invasive ductal breast carcinomas, harbouring recurrent loss of 16q and lack of E-cadherin expression, but also showing overexpression of Her-2 [47,53 57]. In addition, anecdotal evidence suggests that PLC may have a more aggressive biological behaviour than ILC [53,55 57].

3 250 PT Simpson et al Studies using expression arrays have also provided new insights into breast cancer taxonomy. Breast carcinomas can be classified into four categories according to their transcriptome: oestrogen receptor (ER)- positive, HER-2-positive, basal, and normal breast-like carcinomas [23]. It is interesting that even this separation is fundamentally into ER-positive and -negative groups, which for the most part fall into low- and highgrade carcinomas, respectively. In following publications, it was shown that these groups have important prognostic implications; namely, patients with HER- 2-positive or basal carcinomas (high-grade) do worse than patients with ER-positive or normal breast-like tumours (low-grade) [24,25]. However, recent data also suggest that each of these groups may be heterogeneous at the genetic and clinical levels [26,58,59]. In addition, it is still unclear whether there is any correlation between the expression profile, molecular genetic features, and histopathological parameters of these tumours. The frequent association and morphological similarities between invasive and some forms of proliferative breast diseases have led pathologists to assume that certain entities would be biologically related (eg LCIS and ILC, DCIS and IDC) [2,5]. The complexity of these relationships has been thoroughly explored using the advancement in molecular pathology. For example, immunohistochemistry, LOH, CGH, and gene expression analysis have recapitulated the genotypic/phenotypic patterns observed in invasive ductal and lobular carcinomas in ADH/DCIS and ALH/LCIS [2,5,9,11,16,44,60 62]. The distinct molecular genetic features found in different grades of invasive carcinomas are also mirrored in pre-invasive lesions of comparable morphology [2,5,11]. In hindsight, it seems clear that there are two major arms in the multi-pathway model of multi-step breast cancer progression (Figure 2): one comprising welldifferentiated DCIS that progress to grade I IDC, and the other encompassing poorly differentiated DCIS that 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, and molecular features. Note the two distinct low- and high-grade pathways of progression. Connectors drawn with continuous lines represent links between morphological entities for which there is supporting morphological and/or molecular data. Connectors drawn with dotted lines represent hypothetical correlations/evolutionary links yet to be proven. Note: boxes show morphological entities. +: presence of immunohistochemical expression or gain of genetic material; : lack of immunohistochemical expression or loss 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 ductal carcinoma; ILC: invasive lobular carcinoma; Interm.-Dif.: intermediately differentiated; LCIS: lobular carcinoma in situ; LOH:lossof heterozygosity; PgR: progesterone receptor; Poor-Dif.: poorly differentiated; Well-Dif.: well differentiated

4 Molecular evolution of breast cancer 251 tumours are of low nuclear grade, usually ER and PgRpositive, negative for Her-2 and basal markers, and harbour low genetic instability and recurrent 16q loss, whereas in the high-grade arm, the lesions show a higher degree of nuclear atypia, are more frequently hormone receptor-negative, frequently positive for either Her-2 or basal markers, and are genetically advanced lesions, showing a combination of recurrent genomic changes including loss of 8p, 11q, 13q, 14q; gain of 1q, 5p, 8q, 17q; and amplifications on 6q22, 8q22, 11q13, 17q12, 17q22 24, and 20q13 [2,5,9,12,13]. Based on their pathological and genetic features, classic LCIS and ILC are remarkably similar to those tumours in the low-grade arm [2,44,47]. However, in contrast to well-differentiated DCIS/grade I IDC, the vast majority of these tumours lack E- cadherin expression owing to genetic and/or epigenetic changes in the CDH1 gene [48,50 52]. On the other hand, the overlapping morphological features of PLC with both classic lobular and grade III carcinomas, and the combination of E-cadherin (16q) loss with occasional Her-2 positivity [53 57] add another level of complexity to these molecular pathways to breast cancer tumourigenesis. Hyperplasias and columnar cell change Apart from ADH and ALH, which bear remarkable morphological and molecular resemblance to welldifferentiated DCIS and LCIS, respectively, the other non-obligate/premalignant lesions have proven more difficult to characterize and establish their actual position along the pathways [2,16,60 67]. Interestingly, ADH and low-grade DCIS show identical immunoprofiles and low numbers of chromosomal abnormalities, comprising recurrent loss of 16q (see ref 2 and refs cited therein). The similarities between ALH and LCIS are not restricted to the morphological level, but also the immunohistochemical and genetic features [47]. In fact, differentiating between them is arbitrary and subjective, being based on subtle quantitative morphological features. Hence, it is well accepted that both ADH and ALH are non-obligate precursors to the development of well-differentiated DCIS and LCIS, respectively. Alternatively, one could view them just as 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-step model 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 and immunoprofile of HUTs are in stark contrast with those of the accepted precursors, since they are composed of a mixed population of cell types with a variable proportion of ER, PgR-positive luminal cells and myoepithelial/basal marker-positive cells. At the molecular level, no or only rare and fairly random chromosomal changes are observed [2,15,65]. Despite the controversies, there is evidence to suggest that a small proportion of HUTs may be clonal, neoplastic proliferations (adenomas) that may putatively progress to ADH or DCIS, whereas the majority of them fail to show any evidence of a neoplastic/monoclonal nature using existing technology [2,3,16,66]. A more likely precursor to ADH and well-differentiated DCIS are columnar cell lesions (CCLs) [8,68 70], which are characterized by the presence of tightly packed columnar-shaped epithelial cells, with ovoid-to-elongated nuclei and prominent apical cytoplasmic snouts and intraluminal secretions. In fact, they comprise a spectrum of lesions with varying degrees of architectural and mild nuclear atypia. At the lower end of the spectrum are lesions composed of variably dilated acini lined by a single layer of the characteristic cells. At the higher end of the spectrum, lesions resemble ADH, with stratification, bridging, and punched out spaces. Throughout the spectrum, CCLs show an immunoprofile similar to that of ADH/well-differentiated DCIS [8]. However, the degree of proliferation, architectural and cytological atypia are mirrored at the genetic level, with a stepwise increase in the number and complexity of chromosomal copy number changes as defined by CGH [70]. Moreover, the hallmark genetic feature of low-grade lesions, loss of 16q, is the most frequently detected recurrent change and in addition, there is some degree of overlap in the molecular genetic profile of CCL and associated more advanced lesions [69,70]. Interestingly, it is not infrequent to observe ALH/LCIS in the context of multifocal CCLs. Hence CCLs may be the link between normal breast and ADH, as well as between ductal and lobular neoplasia. The precursor of poorly differentiated DCIS has been elusive. Based on morphological, immunohistochemical, and molecular findings, CCL, ADH, and well-differentiated DCIS would be unlikely candidates. Although apocrine change has long been considered a metaplastic process in breast tissues, usually associated with ageing, this concept has come into question with the application of molecular findings [2,17,71 73]. At least a subset of lesions with apocrine morphology show molecular changes, including LOH/allelic imbalance at 1p (MYCL1), 11q (INT2), 13q, 16q and 17q [71,72], and recurrent chromosomal changes 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 in apocrine adenosis and apocrine hyperplasia compared with apocrine cysts. For the large part, these observations have been ignored by the diagnostic pathologist. It is remarkable that a proliferative lesion with the architecture of a micropapillary DCIS is considered benign because it happens to have abundant pink cytoplasm! The lesion regarded as papillary apocrine hyperplasia would be seriously considered DCIS by many if it was not apocrine in nature. Whether this prejudice is justified should be questioned. It may turn out to be wrong but we would suggest that there is

5 252 PT Simpson et al compelling molecular data that at least some of these lesions may be the precursors of high-grade DCIS and invasive cancer. There is a real need for further work in this area. Benign proliferative breast lesions and normal breast Little is known about the presence of molecular genetic changes in benign proliferative breast lesions, such as adenosis, papillomas, and tubular adenomas [2,65,73]. Although gross chromosomal aberrations are not observed in normal breast, most nonproliferative breast lesions, and papillomas, there are some data to suggest that non-recurrent LOH at some loci may be found [2,65]. Although there are changes detected by LOH in some benign proliferative breast lesions, these show little overlap to the most prevalent genetic changes described in DCIS and invasive carcinomas and are thus insufficient to make them non-obligate precursors of DCIS or invasive breast cancer [2,65,73]. In addition, whilst LOH in neoplastic lesions encompasses all informative markers mapping to a particular chromosome arm, LOH in adjacent normal-appearing epithelium involves only single, random markers across the genome [74,75]. Although the genetic abnormalities found in normal cells show little or no overlap with those of neoplastic lesions, these changes may indicate defective mechanisms for the maintenance of genomic integrity, which may contribute to the carcinogenic process and denote increased risk for developing breast cancer. Conclusion and future prospects Molecular analysis has been extensively utilized in the characterization of breast lesions and has been instrumental in unravelling a direct relationship between genotype and phenotype, identifying new subtypes, illustrating molecular pathways in the progression to invasive carcinoma, defining relationships between different histological variants, and providing invaluable prognostic parameters. Now, the mission of pathologists and scientists alike is to translate our current understanding of the molecular evolution of breast cancer and the complexity of microarray data into practical methods that are suitable for diagnostic pathology and patients management. Acknowledgements J Reis-Filho is the recipient of the Gordon Signy International Fellowship Award and is partially supported by PhD Grant SFRH/BD/5386/2001 from the Fundação para a Ciência e a Tecnologia, Portugal, and Programa Operacional Ciência, Tecnologia e Inovação POCTI/CBO/45157/2002. References 1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: Reis-Filho JS, Lakhani SR. The diagnosis and management of preinvasive breast disease: genetic alterations in pre-invasive lesions. Breast Cancer Res 2003; 5: Farabegoli F, Champeme MH, Bieche I, et al. Genetic pathways in the evolution of breast ductal carcinoma in situ. J Pathol 2002; 196: Vogelstein B, Fearon ER, Hamilton SR, et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988; 319: Shackney SE, Silverman JF. Molecular evolutionary patterns in breast cancer. Adv Anat Pathol 2003; 10: Lakhani SR. The transition from hyperplasia to invasive carcinoma of the breast. J Pathol 1999; 187: Lishman SC, Lakhani SR. Atypical lobular hyperplasia and lobular carcinoma in situ: surgical and molecular pathology. Histopathology 1999; 35: Schnitt SJ, Vincent-Salomon A. Columnar cell lesions of the breast. Adv Anat Pathol 2003; 10: Buerger H, Otterbach F, Simon R, et al. Comparative genomic hybridization of ductal carcinoma in situ of the breast evidence of multiple genetic pathways. J Pathol 1999; 187: Buerger H, Otterbach F, Simon R, et al. Different genetic pathways in the evolution of invasive breast cancer are associated with distinct morphological subtypes. J Pathol 1999; 189: Buerger H, Simon R, Schäfer KL, et al. Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast. Mol Pathol 2000; 53: Buerger H, Mommers EC, Littmann R, et al. Ductal invasive G2 and G3 carcinomas of the breast are the end stages of at least two different lines of genetic evolution. J Pathol 2001; 194: Roylance R, Gorman P, Harris W, et al. Comparative genomic hybridization of breast tumors stratified by histological grade reveals new insights into the biological progression of breast cancer. Cancer Res 1999; 59: Lakhani SR. In-situ lobular neoplasia: time for an awakening. Lancet 2003; 361: Jones C, Merrett S, Thomas VA, Barker TH, Lakhani SR. Comparative genomic hybridization analysis of bilateral hyperplasia of usual type of the breast. J Pathol 2003; 199: O Connell P, Pekkel V, Fuqua SA, Osborne CK, Clark GM, Allred DC. Analysis of loss of heterozygosity in 399 premalignant breast lesions at 15 genetic loci. J Natl Cancer Inst 1998; 90: Jones C, Damiani S, Wells D, Chaggar R, Lakhani SR, Eusebi V. Molecular cytogenetic comparison of apocrine hyperplasia and apocrine carcinoma of the breast. Am J Pathol 2001; 158: Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science 1996; 274: Telenius H, Pelmear AH, Tunnacliffe A, et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 1992; 4: Stoecklein NH, Erbersdobler A, Schmidt-Kittler O, et al. SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples. Am J Pathol 2002; 161: Lage JM, Leamon JH, Pejovic T, et al. Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-cgh. Genome Res 2003; 13: Ma XJ, Salunga R, Tuggle JT, et al. Gene expression profiles of human breast cancer progression. Proc Natl Acad Sci U S A 2003; 100: Perou CM, Sorlie T, Eisen MB, et al. Molecular portraits of human breast tumours. Nature 2000; 406:

6 Molecular evolution of breast cancer Sorlie T, Perou CM, Tibshirani R, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 2001; 98: Sorlie T, Tibshirani R, Parker J, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci U S A 2003; 100: Sotiriou C, Neo SY, McShane LM, et al. Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci U S A 2003; 100: Zudaire I, Odero MD, Caballero C, et al. Genomic imbalances detected by comparative genomic hybridization are prognostic markers in invasive ductal breast carcinomas. Histopathology 2002; 40: Janssen EA, Baak JP, Guervos MA, van Diest PJ, Jiwa M, Hermsen MA. In lymph node-negative invasive breast carcinomas, specific chromosomal aberrations are strongly associated with high mitotic activity and predict outcome more accurately than grade, tumour diameter, and oestrogen receptor. J Pathol 2003; 201: Tanner MM, Tirkkonen M, Kallioniemi A, et al. Amplification of chromosomal region 20q13 in invasive breast cancer: prognostic implications. Clin Cancer Res 1995; 1: Hermsen MA, Baak JP, Meijer GA, et al. Genetic analysis of 53 lymph node-negative breast carcinomas by CGH and relation to clinical, pathological, morphometric, and DNA cytometric prognostic factors. J Pathol 1998; 186: van t Veer LJ, Dai H, van de Vijver MJ, et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002; 415: van de Vijver MJ, He YD, van t Veer LJ, et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 2002; 347: Roylance R, Gorman P, Hanby A, Tomlinson I. Allelic imbalance analysis of chromosome 16q shows that grade I and grade III invasive ductal breast cancers follow different genetic pathways. J Pathol 2002; 196: Cleton-Jansen AM, Buerger H, Haar N, et al. Different mechanisms of chromosome 16 loss of heterozygosity in well- versus poorly differentiated ductal breast cancer. Genes Chromosomes Cancer 2004; 41: Nishizaki T, Chew K, Chu L, et al. Genetic alterations in lobular breast cancer by comparative genomic hybridization. Int J Cancer 1997; 74: Loveday RL, Greenman J, Simcox DL, et al. Genetic changes in breast cancer detected by comparative genomic hybridisation. Int J Cancer 2000; 86: Waldman FM, Hwang ES, Etzell J, et al. Genomic alterations in tubular breast carcinomas. Hum Pathol 2001; 32: Osin P, Lu YJ, Stone J, et al. Distinct genetic and epigenetic changes in medullary breast cancer. Int J Surg Pathol 2003; 11: Thor AD, Eng C, Devries S, et al. Invasive micropapillary carcinoma of the breast is associated with chromosome 8 abnormalities detected by comparative genomic hybridization. Hum Pathol 2002; 33: Cingoz S, Altungoz O, Canda T, Saydam S, Aksakoglu G, Sakizli M. DNA copy number changes detected by comparative genomic hybridization and their association with clinicopathologic parameters in breast tumors. Cancer Genet Cytogenet 2003; 145: Jones C, Foschini MP, Chaggar R, et al. Comparative genomic hybridization analysis of myoepithelial carcinoma of the breast. Lab Invest 2000; 80: Jones C, Nonni AV, Fulford L, et al. CGH analysis of ductal carcinoma of the breast with basaloid/myoepithelial cell differentiation. Br J Cancer 2001; 85: Diallo R, Schaefer KL, Bankfalvi A, et al. Secretory carcinoma of the breast: a distinct variant of invasive ductal carcinoma assessed by comparative genomic hybridization and immunohistochemistry. Hum Pathol 2003; 34: Lu YJ, Osin P, Lakhani SR, Di Palma S, Gusterson BA, Shipley JM. Comparative genomic hybridization analysis of lobular carcinoma in situ and atypical lobular hyperplasia and potential roles for gains and losses of genetic material in breast neoplasia. Cancer Res 1998; 58: Gunther K, Merkelbach-Bruse S, Amo-Takyi BK, Handt S, Schroder W, Tietze L. Differences in genetic alterations between primary lobular and ductal breast cancers detected by comparative genomic hybridization. J Pathol 2001; 193: Richard F, Pacyna-Gengelbach M, Schluns K, et al. Patterns of chromosomal imbalances in invasive breast cancer. Int J Cancer 2000; 89: Simpson PT, Gale T, Fulford LG, Reis-Filho JS, Lakhani SR. The diagnosis and management of pre-invasive breast disease: pathology of atypical lobular hyperplasia and lobular carcinoma in situ. Breast Cancer Res 2003; 5: Roylance R, Droufakou S, Gorman P, et al. The role of E-cadherin in low-grade ductal breast tumourigenesis. J Pathol 2003; 200: Etzell JE, Devries S, Chew K, et al. Loss of chromosome 16q in lobular carcinoma in situ. Hum Pathol 2001; 32: Cleton-Jansen AM. E-cadherin and loss of heterozygosity at chromosome 16 in breast carcinogenesis: different genetic pathways in ductal and lobular breast cancer? Breast Cancer Res 2002; 4: Rakha EA, Pinder SE, Paish CE, Ellis IO. Expression of the transcription factor CTCF in invasive breast cancer: a candidate gene located at 16q22.1. Br J Cancer 2004; 91: Droufakou S, Deshmane V, Roylance R, Hanby A, Tomlinson I, Hart IR. Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast. Int J Cancer 2001; 92: Palacios J, Sarrio D, Garcia-Macias MC, Bryant B, Sobel ME, Merino MJ. Frequent E-cadherin gene inactivation by loss of heterozygosity in pleomorphic lobular carcinoma of the breast. Mod Pathol 2003; 16: Eusebi V, Magalhaes F, Azzopardi JG. Pleomorphic lobular carcinoma of the breast: an aggressive tumor showing apocrine differentiation. Hum Pathol 1992; 23: Sneige N, Wang J, Baker BA, Krishnamurthy S, Middleton LP. Clinical, histopathologic, and biologic features of pleomorphic lobular (ductal-lobular) carcinoma in situ of the breast: a report of 24 cases. Mod Pathol 2002; 15: Bentz JS, Yassa N, Clayton F. Pleomorphic lobular carcinoma of the breast: clinicopathologic features of 12 cases. Mod Pathol 1998; 11: Middleton LP, Palacios DM, Bryant BR, Krebs P, Otis CN, Merino MJ. Pleomorphic lobular carcinoma: morphology, immunohistochemistry, and molecular analysis. Am J Surg Pathol 2000; 24: Korsching E, Packeisen J, Agelopoulos K, et al. Cytogenetic alterations and cytokeratin expression patterns in breast cancer: integrating a new model of breast differentiation into cytogenetic pathways of breast carcinogenesis. Lab Invest 2002; 82: Jones C, Ford E, Gillett C, et al. Molecular cytogenetic identification of subgroups of grade III invasive ductal breast carcinomas with different clinical outcomes. Clin Cancer Res 2004; 10: Lakhani SR, Collins N, Stratton MR, Sloane JP. Atypical ductal hyperplasia of the breast: clonal proliferation with loss of heterozygosity on chromosomes 16q and 17p. J Clin Pathol 1995; 48: Amari M, Suzuki A, Moriya T, et al. LOH analyses of premalignant and malignant lesions of human breast: frequent LOH in 8p, 16q, and 17q in atypical ductal hyperplasia. Oncol Rep 1999; 6: Gong G, DeVries S, Chew KL, Cha I, Ljung BM, Waldman FM. Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization. Clin Cancer Res 2001; 7:

7 254 PT Simpson et al 63. Aubele M, Cummings M, Walsch A, et al. Heterogeneous chromosomal aberrations in intraductal breast lesions adjacent to invasive carcinoma. Anal Cell Pathol 2000; 20: Aubele MM, Cummings MC, Mattis AE, et al. Accumulation of chromosomal imbalances from intraductal proliferative lesions to adjacent in situ and invasive ductal breast cancer. Diagn Mol Pathol 2000; 9: Boecker W, Buerger H, Schmitz K, et al. Ductal epithelial proliferations of the breast: a biological continuum? Comparative genomic hybridization and high-molecular-weight cytokeratin expression patterns. J Pathol 2001; 195: Lakhani SR, Slack DN, Hamoudi RA, Collins N, Stratton MR, Sloane JP. Detection of allelic imbalance indicates that a proportion of mammary hyperplasia of usual type are clonal, neoplastic proliferations. Lab Invest 1996; 74: Werner M, Mattis A, Aubele M, et al. 20q13.2 amplification in intraductal hyperplasia adjacent to in situ and invasive ductal carcinoma of the breast. Virchows Arch 1999; 435: Fraser JL, Raza S, Chorny K, Connolly JL, Schnitt SJ. Columnar alteration with prominent apical snouts and secretions: a spectrum of changes frequently present in breast biopsies performed for microcalcifications. Am J Surg Pathol 1998; 22: Moinfar F, Man YG, Bratthauer GL, Ratschek M, Tavassoli FA. Genetic abnormalities in mammary ductal intraepithelial neoplasia-flat type ( clinging ductal carcinoma in situ ): a simulator of normal mammary epithelium. Cancer 2000; 88: Simpson PT, Gale T, Jones C, et al. Columnar cell lesions of the breast a morphological and molecular analysis. Pathol Int 2004; 54(S2): A5 (abstract). 71. Selim AG, Ryan A, El-Ayat G, Wells CA. Loss of heterozygosity and allelic imbalance in apocrine metaplasia of the breast: microdissection microsatellite analysis. J Pathol 2002; 196: Selim AG, Ryan A, El-Ayat GA, Wells CA. Loss of heterozygosity and allelic imbalance in apocrine adenosis of the breast. Cancer Detect Prev 2001; 25: Washington C, Dalbegue F, Abreo F, Taubenberger JK, Lichy JH. Loss of heterozygosity in fibrocystic change of the breast: genetic relationship between benign proliferative lesions and associated carcinomas. Am J Pathol 2000; 157: Larson PS, de las Morenas A, Bennett SR, Cupples LA, Rosenberg CL. Loss of heterozygosity or allele imbalance in histologically normal breast epithelium is distinct from loss of heterozygosity or allele imbalance in co-existing carcinomas. Am J Pathol 2002; 161: Lakhani SR, Chaggar R, Davies S, et al. Genetic alterations in normal luminal and myoepithelial cells of the breast. J Pathol 1999; 189: