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Background: Atypical ductal hyperplasia (ADH) is common finding in the mammographic era, but has wide variation in diagnosis and treatment. ADH is a high risk factor for invasive breast ductal carcinoma (IDC), and is also considered to be a non-obligate precursor to IDC. Although a few studies have revealed some of the common genomic characteristics of ADH, a clear understanding of the molecular changes associated with breast cancer progression has been limited by inadequately powered studies and low resolution methodology (Lopez-Garcia et al., 2010, Histopathology 57: 171-192). Copy number alterations (CNAs), one of the major genetic alterations in cancer, are present in IDC and ductal carcinoma in situ (DCIS) and given a suitable detection method could be used in research and potentially in the clinical setting to predict patient prognosis. Method: We optimised a robust, cost effective low-coverage whole genome sequencing (LCWGS) method for CNA detection, using as little as 5 ng of formalin-fixed paraffin-embedded tissue derived DNA. We applied this novel method to 21 cases of pure ADH (not associated with cancer) as well as 20 ADH with synchronous DCIS and/or IDC. Cases were reviewed independently by two pathologists, followed by micro-dissection and DNA extraction. CNA were analysed using the LCWGS method, using 5-10 ng total DNA input per sample. Results: Highly accurate copy number profiles produced by low input LCWGS are comparable to those obtained from an alternative method, Molecular Inversion Probe arrays, while requiring 10 fold less input DNA (Kader et al., 2016, Genome Medicine 8: 121). Genetic analysis of pure ADH found that 95% had at least one copy number event and the median fraction of the genome altered was 8% (0-19%). Surprisingly, while 29% of pure ADH showed 16q loss and 1q gain (CNAs common in low-grade (LG) breast cancer), 29% of pure ADH showed 8q gain, an event more frequently observed in high grade (HG) cancer. Analysis of ADH with synchronous DCIS and/or IDC showed that 67% of ADH was clonally related with both LG (6/8) and HG carcinoma (4/7). The final analysis of 20 synchronous cases (9 LG, 11 HG) will be presented. Conclusion: Here we report the most detailed molecular taxonomy of this high risk pre-malignant breast lesion with the largest cohort undertaken worldwide. Our observation that 67% of ADH is clonal to breast carcinoma suggests that both LG and HG carcinoma can evolve from a similar ancestor lesion. As ADH has the potential to develop both LG and HG carcinoma, a biomarker signifying breast cancer progression from pre-malignant lesions is highly desirable for the more precise treatment of individual patients diagnosed with ADH. Citation Format: Kader T, Hill P, Opeskin K, Goode DL, Elder K, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Identification of copy number alterations associated with the progression of high risk premalignant breast lesions to breast carcinoma [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-12.

Background: Papillomas are often found co-existing with breast carcinoma yet they are not considered to be a true precursor of the disease. Previous studies have shown that some cases may carry copy number alterations (CNA) or mutations in AKT1/PIK3CA (Troxell et al., 2010, Modern Pathology 23: 27-37) suggesting this lesion may have malignant potential. To date, a detailed study of both pure papillomas (not associated with cancer) and those seen in the same breast as a carcinoma has not been undertaken. Therefore, we set out to investigate the molecular changes associated with this lesion and whether papillomas can be clonally related to synchronous breast carcinoma. Method: Papilloma cases were identified from a hospital database and independently reviewed by consultant pathologists followed by micro-dissection of formalin-fixed paraffin-embedded tumour tissue and DNA extraction. For CNA detection either Affymetrix Molecular inversion Probe (MIP) 330K arrays were used or low-coverage whole genome sequencing using 5-10 ng of DNA (Kader et al., 2016, Genome Medicine 8: 121) where there was insufficient DNA for MIP arrays. We applied either of these 2 methods to 24 cases of pure papilloma as well as 20 papilloma with synchronous ductal carcinoma in situ (DCIS) and/or invasive ductal carcinoma (IDC). Additionally, targeted exon sequencing of breast cancer driver genes was performed for a subset of cases. Results: Among the pure papillomas 31% (5/16) showed CN change with, the most frequent change being 16q loss (2/16). Of the papillomas synchronous with DCIS/IDC analysed to date, 2/5 were shown to be clonal with the co-existing carcinoma. Final CNA analysis will be presented for 24 pure papilloma cases and 20 synchronous cases. Targeted sequencing revealed that all for pure papillomas analysed to date harboured somatic mutations in PIK3CA (3/4 cases) and PIK3R1 (1/4,) suggesting that most papillomas are driven by alterations in the PI3-kinase/AKT pathway. The final sequencing data to be presented will include an additional 5 pure papillomas and 10 synchronous cases. Conclusion: Our observation that 40% of papillomas are clonal to breast carcinoma suggests that DCIS or IDC can arise from a common ancestor as co-existing papillomas, however, most papillomas co-existing with carcinoma are likely to be independent in our cohort. Citation Format: Elder KJ, Kader T, Hill P, Opeskin K, Goode DL, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Genomic analysis of breast papillomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-08.

Abstract Purpose Mammographic density (MD) refers to the extent of radio-opaque breast tissue on a mammogram. Adjusted for a women’s age and body mass index (BMI), it is a strong and independent risk factor for breast cancer (BC). The presentation of mammographically dense breast is not uncommon in the normal female population, and patients’ awareness of its association with BC risk has been growing following mandatory reporting of MD by physicians in several states of America. Women with breasts that have over 75% dense tissue are 4 to 6 times more likely to develop BC than women with breasts that have less than 10% dense tissue. However, the biological basis of how high MD raises BC risk remains elusive. We aimed to examine the histological and molecular differences between high and low dense breast tissues of healthy women, using specimens accrued from prophylactic mastectomy procedures. Method 48 women between 2008 and 2013 underwent prophylactic mastectomy at St Vincent’s Hospital and Peter MacCallum Cancer Centre due to a high BC risk profile. Of these, 41 were eligible for analyses. Tissue slice resected from the mastectomy specimen was X-rayed, and high (HD) and low dense (LD) regions were dissected based on the radiological appearance. The histological composition, immunohistochemistry and proliferation status were assessed on matched high and low MD tissue of the same breast. Signed rank test and paired t test were used for quantitative analyses of potential differences between HD and LD tissue. Result HD tissue demonstrated a significantly greater proportion of stroma (p Conclusion and Significance We found that increased stroma and epithelium proportions contribute to the dense appearance on mammogram. Moreover, dense tissue did not demonstrate differed hormonal receptor expression or proliferation status from non-dense tissue, but showed a preponderance of EMT in the form of co-localisation by both CK-19 and vimentin in some of the epithelial cells. Our study is the first to report EMT phenomenon in benign mammary tissue, and suggests that investigations of the stromal micro-environment, and their interactions with epithelium may be key to improving our understanding on MD-mediated BC risk. Citation Format: Cecilia W Huo, Dexing Huang, Grace L Chew, Prue Hill, Wendy V Ingman, Michael Henderson, Kara L Britt, Rik Thompson. Comparing tissue compositions of within-individual mammographically high and low dense breast tissue [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-11.