Abstract PD05-02: Novel mutations in lobular carcinoma in situ (LCIS) as uncovered by targeted parallel sequencing
Authors: De Brot M , Andrade VP , Morrogh M , Berger MF , Won HH , Koslow Mautner S , Qin L-X , Giri DD , Olvera N , Sakr RA , King TA 
Published: August 2014

Abstract
Background: LCIS has traditionally been recognized as a marker of increased risk for the subsequent development of breast cancer, of either the lobular or ductal phenotype, yet due to the incidental nature of LCIS little is known about its underlying biology. Here we describe the first report of novel mutations in LCIS using targeted exome sequencing of fresh frozen tissue samples.
Methods: Fresh frozen tissue samples from patients with a prior history of LCIS undergoing therapeutic or risk-reducing mastectomy from 2003–08 were harvested and systematically reviewed to identify LCIS. Cells from individual LCIS lesions +/− associated cancers were collected by laser capture microdissection. For the purposes of this study, germline DNA (blood) and DNA from 12 unique LCIS lesions were subject to targeted parallel sequencing of exons corresponding to 230 cancer genes using the Illumina HiSeq 2000 platform. DNA from an associated ductal carcinoma in situ (DCIS) and/or an invasive ductal (IDC) or lobular (ILC) lesion was also available for 7 of these cases resulting in 41 samples from 12 pts for mutational analysis. Normalized (RMA) Affymetrix U133A gene expression data were also available.
Results: DNA profiling reliably identified 7 somatic mutations in 5/12 LCIS samples (41.7%). Of these, 4/7 mutations were base substitutions (missense mutations); and the others included: 1 deletion; 1 silent and 1 splice-site mutation. Mutations in LCIS were identified in 5/230 cancer genes analyzed, including: PIK3CA, CDH1, NOTCH4, PREX2 and ARAF. PIK3CA and CDH1 mutations were each identified in two samples, representing 4/7 (57.1%) mutations. Specific mutations found in LCIS and their frequencies are listed (table). Among 3 LCIS-ILC pairs, one shared the G914R mutation in PIK3CA, and 1/3 LCIS-IDC pairs exhibited an identical point mutation (R373W) in the NOTCH4 gene. No shared mutations were observed in 3 LCIS-DCIS pairs. Both CDH1 mutated cases were associated with decreased e-cadherin mRNA levels when compared to non-mutated cases (mean 9.88 vs 11.01), as was the NOTCH4 mutation (mean 6.02 vs 6.47). Mutations in ARAF and PREX2 were associated with increased mRNA levels, mean 7.07 vs 6.52 and 4.82 vs 4.22, respectively. The hotspot PIK3CA mutation (E545K) was also associated with increased gene expression (mean 5.15 vs 4.64) whereas the G914R mutant was associated with decreased expression (mean 4.13 vs 4.64).

Conclusions: This study represents the first targeted exon sequencing analysis of fresh frozen LCIS. Although LCIS has been regarded as a rather genetically stable lesion, somatic mutations were detected in 41.7% of lesions in this small cohort. While CDH1 mutations are expected in lobular neoplasia, this is the first report of mutations in ARAF, NOTCH4, PIK3CA and PREX2. Given the shared relevance of PIK3CA and PREX2 in the PI3K/AKT pathway, these findings suggest novel mechanisms for new chemoprevention strategies among women with LCIS.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD05-02.