Abstract Introduction: Lobular carcinoma in situ (LCIS) is considered both a risk factor and non-obligate precursor of invasive breast cancer. We sought to determine the genomic landscape of LCIS and the mutational processes involved in the clonal evolution and progression from LCIS to ductal carcinoma in situ (DCIS) and invasive lobular carcinoma (ILC). Methods: Patients with a history of LCIS undergoing therapeutic or prophylactic mastectomy were prospectively enrolled in an IRB approved protocol. Frozen tissue blocks were collected, screened for lesions of interest (LCIS, DCIS, ILC, invasive ductal carcinomas (IDC)) and subjected to microdissection and DNA/RNA extraction. Matched germline DNA was available for all cases. Whole exome sequencing was performed on a HiSeq2000 and data were aligned to the reference human genome and processed using GATK. Single nucleotide variants (SNVs) and small insertions/deletions were identified using MuTect and Varscan, respectively. Purity and ploidy estimates were calculated using ABSOLUTE. Clonal frequencies were estimated using Pyclone and the clonal structure of each sample was reconstructed using SubcloneSeeker. Shannon index and Simpson index metrics were used to calculate heterogeneity levels. Mutational signatures were defined according to their mutational trinucleotide context, and the expression levels of APOBEC gene family members were assessed by quantitative reverse transcription (qRT)-PCR. Results: 30 LCIS, 10 ILCs, 7 DCIS and 5 IDCs from 15 patients qualified for data analysis. CDH1 was the most frequently mutated gene and found to be targeted by mutations in 26 LCIS samples (23 somatic, 3 germline). The repertoire of somatic mutations in LCIS was similar to that of luminal A breast cancers, with the exception of the significantly higher frequency of CDH1 mutations and the lower prevalence of TP53 mutations. ILCs were clonally related to at least one LCIS in 10 patients, and in 3/7 patients, DCIS was clonally related to at least one LCIS. Clonal composition analysis revealed that the presence of a minor clone(s) in LCIS, and the levels of intra-tumor genetic heterogeneity were significantly higher in LCIS clonally related with DCIS/ILC than in LCIS unrelated to DCIS/ILC. In two cases, a minor LCIS subclone constituted the major clone in the associated DCIS/ILC. A comparative analysis of the mutational signatures in the truncal and branch mutations of these cases revealed that whilst the truncal mutations displayed an aging signature, branch mutations were enriched for the APOBEC signature. qRT-PCR analysis demonstrated that cases displaying the APOBEC signature also harbored significantly higher levels of APOBEC3B expression than samples with the aging signature. Conclusions: LCIS displays intra-lesion genetic heterogeneity, and the progression from LCIS to DCIS or ILC may involve the selection of clones resulting from distinct mutational processes during clonal evolution. Our findings also suggest that cytodine deamination driven by the overexpression of APOBEC3B may drive the progression of LCIS to DCIS/ILC in a subset of cases. Citation Format: Reis-Filho JS, Schizas M, Piscuoglio S, Sakr RA, Ng CKY, Lim RS, Carniello JVS, Towers R, Martelotto L, Giri DD, de Andrade VP, Viale A, Solit DB, Weigelt B, King TA. Lobular carcinoma in situ displays intra-lesion genetic heterogeneity and its progression to invasive disease involves clonal selection and variations in mutational processes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-04.