Abstract PD4-13: Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis
Authors: Geyer FC , Li A , Papanastasiou AD , Smith A , Selenica P , Burke KA , Edelweiss M , Wen H-C , Piscuoglio S , Schultheis AM , Martelotto LG , Pareja F , Kumar R , Brandes A , Lozada J , Macedo GS , Muenst S , Terracciano LM , Jungbluth A , Foschini MP , Wen HY , Brogi E , Palazzo J , Rubin BP , Ng CKY , Norton L , Varga Z , Ellis IO , Rakha E , Chandarlapatty S , Weigelt B , Reis-Filho JS 
Published: February 2018

Abstract
Introduction:Adenomyoepithelioma (AME) of the breast is a rare biphasic tumor, characterized by epithelial and myoepithelial differentiation. Although AMEs have an indolent clinical course, a subset may progress to carcinoma and metastasize. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent pathogenic mutations identified in these tumors.
Methods: Thirty-one AMEs were subjected to whole-exome sequencing (WES, n=8) or massively parallel sequencing targeting all coding regions of 410 key cancer genes and intronic and regulatory regions of selected genes (n=23). Somatic genetic alterations were defined using state-of-the-art bioinformatics algorithms. In an additional set of 12 AMEs, Sanger sequencing analysis of HRAS, PIK3CA and AKT1 was performed. Non-tumorigenic estrogen receptor (ER)-negative mammary epithelial cells (i.e. MCF10A, MCF10A with a PIK3CA H1047R mutation knock-in and MCF12A) were utilized for functional studies using both conventional monolayer and three-dimensional (3D) culture assays.
Results: 27 (63%) and 16 (37%) AMEs were ER-positive and ER-negative, respectively. ER-negativity was significantly associated with histologic features predictive of a more aggressive behavior, with a higher number of mutations and copy number alterations, and with a distinct mutational profile as compared to ER-positive AMEs. Of the 27 ER-positive AMEs, 12 cases (44%) harbored PIK3CA hotspot mutations, and 5 PIK3CA wild-type cases displayed E17K AKT1 hotspot mutations. By contrast, of the 16 ER-negative AMEs, 9 (56%), 9 (56%) and 3 (19%) harbored HRAS, PIK3CA (mostly E545K and H1047R hotspots) and PIK3R1 mutations, respectively. Strikingly, all HRAS mutations were restricted to ER-negative AMEs, affected the hotspot codon Q61 (Q61R/K), and all but one co-occurred with PIK3CA or PIK3R1 mutations. In addition, HRAS Q61 hotspot mutations were significantly associated with necrosis (p=0.01) and high mitotic rates (p=0.03). CDKN2A homozygous deletions were also detected only in ER-negative AMEs (19%) and found to be significantly associated with progression to carcinoma (p=0.001). Forced expression of HRAS Q61R in MCF10A and MCF12A cells resulted in i) increased proliferation and transformation, ii) an irregular growth pattern in 3D organotypic cell cultures, iii) partial loss of the epithelial phenotype, and iv) acquisition of myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. HRAS Q61Rinduced hyperactivation of the PI3K pathway, but both PI3K and MAPK pathways likely contributed to the RAS-mediated proliferation, which was completely arrested by combined AKT and MEK inhibition.
Conclusion: AMEs are phenotypically and genetically heterogeneous. Whilst pathogenic mutations in PI3K pathway-related genes occur across the spectrum of lesions, HRAS Q61 hotspot mutations are restricted to ER-negative AMEs. Our genomic and functional analyses indicate that HRAS Q61 mutations are driver events in the pathogenesis of ER-negative AMEs and, in conjunction with mutant PIK3CA, may lead to the acquisition of myoepithelial differentiation in breast epithelial cells.
Citation Format: Geyer FC, Li A, Papanastasiou AD, Smith A, Selenica P, Burke KA, Edelweiss M, Wen H-C, Piscuoglio S, Schultheis AM, Martelotto LG, Pareja F, Kumar R, Brandes A, Lozada J, Macedo GS, Muenst S, Terracciano LM, Jungbluth A, Foschini MP, Wen HY, Brogi E, Palazzo J, Rubin BP, Ng CKY, Norton L, Varga Z, Ellis IO, Rakha E, Chandarlapatty S, Weigelt B, Reis-Filho JS. Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis [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 PD4-13.