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 Neoplasia1

Lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH) of the breast are cytologically similar breast lesions that reportedly carry different relative risks of subsequent development of invasive car cinoma. They are frequently multifocal and bilateral. We have identified the chromosomal copy number changes in 31 LCIS and 14 ALH lesions from 28 cases and also the 7 invasive carcinomas that subsequently developed in 6 of these cases. This was achieved by comparative genomic hybridization analysis of microdissected formalin-fixed, paraffin-embed ded material. There was no significant difference between the aberrations found in the unilateral versus the bilateral cases of LCIS. Loss of material from Iftp,16q, 17p, and 22q and also gain of material from 6q were found at a similar high frequency in LCIS and ALH. Loss of these genomic regions may indicate the locations of genes that predispose to the devel opment of the lesions, and the results are consistent with LCIS and ALH representing the same genetic stage of development. Comparison of the comparative genomic hybridization results from LCIS/ALH with those from ductal carcinoma in situ and invasive cancer showed some similar ities at the chromosomal level, but it also showed significant differences, including gain of Iq and Sq and evidence for genomic amplification, which were not found in LCIS/ALH. A genetic model is postulated for the possible relationships between noninvasive lobular lesions and invasive breast carcinoma, delineating potential roles for specific chromosome copy number changes. INTRODUCTION LCIS3 is a relatively rare and usually asymptomatic lesion that is an incidental finding in breast tissue removed for other clinical reasons (1). Up to two-thirds of LCIS have been shown to be bilateral, and foci of LCIS are multicentric in 60-85% of cases (1). ALH is a cytologically similar lesion to LCIS but is generally considered to be quantitatively less developed than LCIS (2). Existing information on these lesions is not clear regarding a number of issues, including: the relevance of their pathological distinction (3, 4); their relationship to subsequent invasive disease; the genetic basis, if any, for multifocality and bilaterality; and how patients with these lesions should be treated (5-7). The main distinction between LCIS and ALH has arisen from epidemiological data. An estimated 15-25% of women with LCIS Received 5/4/98; accepted 8/18/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by United States Army Grant DAMD 17-94-4066. the Institute of Cancer Research, the Cancer Research Campaign, and the Ludwig Institute for Cancer Research. Y-J. L. and P. O. were supported by BreakThrough Breast Cancer and by a Gilbert Fellowship through the Friends of Hebrew University, respectively. 2 To whom requests for reprints should be addressed, at Molecular Cytogenetics, Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton. Surrey, SM2 5NG United Kingdom. Phone: 44 181 643 8901; Fax: 44 181 6430238; E-mail: [email protected]. 1The abbreviations used are: LCIS, lobular carcinoma in situ; ALH, atypical lobular hyperplasia; LOH, loss of heterozygosity; ILC, invasive lobular carcinoma; CGH, com parative genomic hybridization; DOP-PCR, degenerate oligonucleotide-primed PCR; CI, confidence intervals; DCIS, ductal carcinoma in situ. develop invasive carcinoma over the next 15-20 years (1,6, 8), with almost equal risk for the same and the contralateral breast (6). Ap proximately 50% of the arising carcinomas are of the invasive ductal type (1). Follow-up studies have estimated that the relative risk for subsequent invasive cancer is lower if ALH rather than LCIS is present (1,2, 9). The actual degree of relative risk quoted by different authors varies (2.7-8.9 for ALH; 9-12 for LCIS), and it is generally accepted that the long-term risk for these lesions remains unknown ( 1, 9, 10). It is not known whether the invasive component arises de novo or is derived from the LCIS and ALH (1). Despite repeated attempts to find additional morphological prognostic criteria, it is still not possible to predict which patients with noninvasive lobular disease will develop invasive cancer (2, 4, 6, 10). Molecular analysis has indicated that LOH at Ilql3 was more frequently found in LCIS associated with invasive disease (11) and that loss from chromosomes 16 and 17 is consistent with LCIS having a clonal component and being considered neoplastic (12). Other studies have investigated specific genes, including E-cadherin, which was first found to be mutated in ILC and subsequently shown to be frequently mutated in LCIS, suggesting that this is an early change in lobular neoplasia (13). However, molecular studies on LCIS and ALH are sparse and limited by the small size of the lesions. Because ALH and LCIS are multifocal and bilateral in a high proportion of cases (1), it is possible that there is either a genetic or carcinogenic mechanism underlying their development. An inherited predisposing mutation in a tumor suppressor gene, following Knuudson's classical hypothesis, could be involved. This could correspond to large cytogenetically detectable deletions in one alÃ-ele,as has been demonstrated for the Peutz-Jeghers locus (14), or LOH of a particular locus. Alternatively, a mutation could arise during development and be present in a subpopulation of breast cells, consistent with the type of field changes observed in apparently normal tissue adjacent to breast lesions (15). Various genetic pathways are postulated for the development of ALH and LCIS and subsequent invasive disease, as shown in Fig. \A. As a starting point to investigate the possible relationships between lesions, we have screened over 50 samples from 28 cases for chro mosomal copy number changes. This has been achieved by applying the approach of CGH (16, 17), including a PCR using degenerate primers to generate sufficient representative DNA for analysis from microdissected paraffin-embedded material (18, 19). The CGH results were compared to indicate the genetic relationship between LCIS and ALH in both unilateral and bilateral cases. A comparison with data from invasive lobular and ductal disease was also carried out to determine the chromosome changes that may be specifically associ ated with the development of in situ lobular lesions and invasive breast carcinoma. MATERIALS AND METHODS Tumor and Control Samples and Their Microdissection. Clinical and histological data on the breast lesions studied are summarized in Table 1. 4721 Research. on January 13, 2018. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from CGH ANALYSIS OF LCIS AND ALH OF THE BREAST Epithelium/genetically predisposed epithelium B. Epithelium/genetically predisposed epithelium