GENES, CHROMOSOMES & CANCER 47:1049–1066 (2008) Differential Patterns of Allelic Loss in Estrogen Receptor-Positive Infiltrating Lobular and Ductal Breast Cancer L. W. M. Loo,1 C. Ton,1,2 Y.-W. Wang,2 D. I. Grove,2 H. Bouzek,1 N. Vartanian,1 M.-G. Lin,1 X. Yuan,1 T. L. Lawton,3 J. R. Daling,2 K. E. Malone,2 C. I. Li,2 L. Hsu,2 and P.L. Porter1,2,3* 1Division of Human Biology,Fred Hutchinson Cancer Research Center,Seattle,WA 2Division of Public Health Sciences,Fred Hutchinson Cancer Research Center,Seattle,WA 3Departmentof Pathology,Universityof Washington,Seattle,WA The two main histological types of infiltrating breast cancer, lobular (ILC) and the more common ductal (IDC) carcinoma are morphologically and clinically distinct. To assess the molecular alterations associated with these breast cancer subtypes, we conducted a whole-genome study of 166 archival estrogen receptor (ER)-positive tumors (89 IDC and 77 ILC) using the Affy- metrix GeneChip® Mapping 10K Array to identify sites of loss of heterozygosity (LOH) that either distinguished, or were shared by, the two phenotypes. We found single nucleotide polymorphisms (SNPs) of high-frequency LOH (>50%) common to both ILC and IDC tumors predominately in 11q, 16q, and 17p. Overall, IDC had a slightly higher frequency of LOH events across the genome than ILC (fractional allelic loss 5 0.186 and 0.156). By comparing the average frequency of LOH by chro- mosomal arm, we found IDC tumors with significantly (P < 0.05) higher frequency of LOH on 3p, 5q, 8p, 9p, 20p, and 20q than ILC tumors. We identified additional chromosomal arms differentiating the subtypes when tumors were stratified by tu- mor size, mitotic rate, or DNA content. Of 5,754 informative SNPs (>25% informativity), we identified 78 and 466 individual SNPs with a higher frequency of LOH (P < 0.05) in ILC and IDC tumors, respectively. Hierarchical clustering of these 544 SNPs grouped tumors into four major groups based on their patterns of LOH and retention of heterozygosity. LOH in chro- mosomal arms 8p and 5q was common in higher grade IDC tumors, whereas ILC and low-grade IDC grouped together by vir- tue of LOH in 16q. Published 2008 Wiley-Liss, Inc.† INTRODUCTION recent analyses of Surveillance Epidemiology and Infiltrating lobular carcinoma (ILC) differs both End Results (SEER) data indicate that ILC inci- morphologically and clinically from the more com- dence rates have increased rapidly over the past mon infiltrating ductal carcinoma (IDC) in breast two decades in the United States (Li et al., 2003a), cancer. ILC is more likely to be bilateral, multicen- primarily in women over age 50. This differential tric, and diploid than IDC. Also, ILC tumors have a increase in older women is in keeping with recent pattern of metastasis to bone, gastrointestinal tract, studies that have implicated combined (estrogen/ and ovary that is different from that of IDC (Harris progestin) hormone replacement therapy (CHRT) et al., 1984; Arpino et al., 2004). The widely infiltra- as a risk factor for lobular type breast cancer tive growth of individual and small rows of lobular (Chen et al., 2002; Li et al., 2003b: WHI Steering cancer cells through breast stroma results in a low Committee 2004). detection rate by clinical exam or by mammog- raphy (Porter et al., 1999). Given that ILC typically has favorable prognostic indicators (Arpino et al., Additional Supporting Information may be found in the online 2004), a more favorable relationship with survival version of this article. Supported by: NIH; Grant numbers: RO1 CA095717 and RO1 and recurrence would be expected. A survival CA085913; DOD Breast Cancer Research Program; Grant numbers: advantage has been associated with good prognosis BC045034 and DOD DAMD 17-02-1-0386. in some studies but not in others (Mersin et al., *Correspondence to: Peggy L. Porter, Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 2003; Li et al., 2003b; Arpino et al., 2004; Cristofa- Fairview Ave. N, Seattle, WA 98109, USA. nilli et al., 2005), indicating that other, as yet E-mail: [email protected] unknown, factors might distinguish ILC from IDC. Received 7 March 2008; Accepted 11 July 2008 DOI 10.1002/gcc.20610 Historically, ILC has accounted for between 5 Published online 21 August 2008 in and 14% of breast cancers (Harris, et al., 1992), but Wiley InterScience (www.interscience.wiley.com). Published 2008 Wiley-Liss, Inc. †This article is a US Government work and, as such, is in the public domain in the United States of America. 1050 LOO ET AL. Although a few studies have compared molecu- ticipants consented to tumor- and blood-sample lar alterations between the two subtypes and have testing. generated some information about differences in the frequency and locations of genomic alterations, Pathology Review for Histological the small number of ILC tumors and the limited Diagnosis/Tumor Characteristics number of loci interrogated in most studies have To verify histological diagnoses for all tumors, limited the comprehensive evaluation of genomic pathology reports and histology slides underwent differences between ILC and IDC (Gunther et al., centralized pathology review by the four patholo- 2001; Loo et al., 2004; Roylance et al., 2006). gists (PLP, MGL, XY, TL), using published criteria One of the most common alterations in breast for ILC and IDC tumors (Page and Anderson, cancer is allelic imbalance or loss of heterozygosity 1987). Histological grade was assessed for IDC (LOH). An LOH event can be associated with tumors using the Nottingham grading scheme either a gene copy number loss or amplification. (Elston and Ellis, 1991). After agreement by all LOH has been shown to occur in precancer lesions study pathologists on diagnostic criteria, the histol- of the breast and in normal epithelium of women ogy of each case was assessed by review of all sub- with breast cancer (Deng et al., 1996; O’Connell et mitted slides by one of two staff pathologists al., 1998). The profile of the most common LOH (MGL, XY). A second review of the diagnosis and events in breast cancer includes losses in chromo- histological grade of each case was done by the some arms, 1q, 3p, 6q, 11p, 13q, 16q, 17p, 17q, and study pathologist (PLP). Discrepancies between 18q (Devilee and Cornelisse, 1994), but the contri- the initial and second assessments were resolved bution that ILC makes to that overall profile is by consensus review or submitted for additional small, given the low number of lobular cancers review to the study’s consulting pathologist (TL). included in virtually all studies assessing allelic All reviews were conducted independently. imbalance. The components of clinical stage: tumor size, To gain a better understanding of allelic imbal- lymph node metastasis, and distant metastasis ance events inherent in the lobular subtype of were obtained from SEER and/or original pathol- breast cancer, we compared LOH profiles of flow ogy reports. cytometrically sorted ER-positive ILC and IDC tumors derived from a population-based sample. Immunohistochemistry Restricting the analysis to ER-positive tumors To verify the steroid-receptor status of all allowed us to assess similarities and differences in tumors, immunohistochemistry (IHC) for the LOH events between the subtypes not con- expression of estrogen receptor (ER) (ER1D5, founded by differences due to the ER status of the AMAC, Inc) (Andersen and Poulson, 1989) and tumors. progesterone receptor (PR) (1A6 NovoCastra Lab) (Giri et al., 1988) was done on a representative block for each tumor using standard IHC techni- MATERIALS AND METHODS ques including antigen retrieval (Hsu et al., 1981; Taylor et al., 1994). Positive and negative controls Patient Population were included for each antibody. Participants in this study were drawn from a population-based case–control study designed to Flow Cytometric Cell Sorting and DNA Extraction examine the relationships of risk factors and histo- Tumor cells were collected by flow sorting based logical type of breast cancer. The methods used to on cytokeratin and DNA content, which enriches select, enroll, and interview participants have been the sample for tumor cells while excluding contam- described in detail elsewhere (Li, et al., 2008). inating stromal and lymphocytic cells as described The study included ILC (ICD-O histology code previously (Glogovac et al., 1996). Cell populations 8520/3) and IDC cases (ICD-O histology code were classified as diploid/near diploid (1.0 DNA 8500/3) 55–74 years of age, ascertained through our Index (DI) 1.39) and aneuploid (DI > 1.39 or DI population-based cancer registry for 13 western < 1.0). If a tumor contained multiple populations Washington counties and diagnosed between Janu- with different ploidy, the most prevalent aneuploid ary 2000 and March 2004. This analysis was re- population was assayed. DNA was extracted from stricted to the 77 ILC and 89 IDC cases for whom the sorted tumor cells (a minimum of 100,000 cells we had sufficient tumor tissue to conduct this work per tumor) using the QIAamp DNA Micro kit and who had an available blood specimen. All par- (Qiagen, Valencia, CA) with modifications. Briefly, Genes, Chromosomes & Cancer DOI 10.1002/gcc ALLELIC LOSS IN ER-POSITIVE BREAST CANCER 1051 tumor cells were extracted in Qiagen ATL buffer samples and excellent reproducibility in four sam- with the addition of proteinase K (20 lg/ll), and ples done in duplicate on different days (data not two additional proteinase K applications at 6 and shown). 24 hr, while being mixed at 350 rpm (Eppendorf Thermomixer R, Westbury, NY) at 568C for a total Short Tandem Repeat (STR) Validation Assays of 45 hr. Tumor DNA was then extracted based To validate the array findings, we assayed for on the manufacturers’ recommendation.