Maren K. Levin

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Association of PIK3CA mutation with response (ExRx) to cetuximab (C) in metastatic (met) triple-negative breast cancer (TNBC).

151 Background: 10 to 17% of TNBCs harbor PIK3CA mutations (TCGA Nature 490:61, 2012; Khambata-Ford S. ASCO 2010, abst 1056). Here we report clinical history and molecular findings for a TNBC patient with loss of mutant PIK3CA in a C-refractory metastasis that was present in her primary BC, who has had an ExRx to C.nnnMETHODSnFollowing IRB-approved informed consent, targeted NGS was performed on the pts FFPE primary TNBC and on a C-refractory recurrent lung metastasis at a CLIA-certified laboratory (Foundation Medicine) to characterize all classes of genomic alterations across 287 cancer-related genes. RPPA was performed at a CLIA-certified laboratory (Theranostics Health) where immunostaining with 24 antibodies was directed against HER1/2/3 pathway proteins and AR.nnnRESULTSn37 yo woman presented in 2006 with grade 3 primary TNBC, infraclavicular LNs and lung metastases at 33 weeks gestation. There was no response to preop doxorubicin/cyclophosphamide and following delivery of a healthy baby, she was treated with irinotecan, carboplatin, and C (#NCT00248287) and had near complete response (CR) in her lungs and pathologic CR in breast. In 2008, after 20 mos on C, chest CT showed a new lung met which was resected and she remains disease-free on C alone. NGS: Primary BC: PIK3CA C420R, TP53 mutations, MCL1 amplification (amp), and RAD51D germline mutation; C-refractory lung met: TP53, MLL2 mutations, MCL1, MYC, KDM5A, CCNE1 amp and RAD51D germline mutation (loss of PIK3CAmutation confirmed). RPPA: Primary BC: p-AR S650 (3+); p-ERK (2+), and HER1, p-HER1, p-HER3, p-AKT, p-S6, p-4EBP1 (1+); C-refractory lung met: loss of p-ERK; p-HER1, p-4EBP1 (2+), and p-AR, p-AKT, p-mTOR (1+). PTEN: Primary BC: 60% cells positive by IHC MAb 6H2.1 (Cascade Biosciences).nnnCONCLUSIONSnThe pts ExRx to C was dependent on presence of PIK3CA mutation which was lost in the C-refractory lung met. Loss of RAD51D function may also have contributed (Liping L. Ca Res 68:9141, 2008). High p-AR expression did not preclude response to C. Activating PIK3CA mutations induce an EGFR/ERK paracrine signaling axis in TNBCs (Young CD. Mol Cell Proteomics 2015). A prospective trial of EGFR inhibition in PIK3CA-mutant TNBC is warranted.

Dual inhibition of DNA double strand break (DSB) repair and PI3K pathway with BEZ235 (BEZ) to sensitize refractory metastatic (met) triple negative breast cancer (TNBC) to nab paclitaxel/cisplatin (pac/cis) in a patient with an exceptional response (ExRx).

156 Background: Whether EGFR is a critical target in met TNBC is unknown. Here we report the clinical history & tumor molecular alterations in a patient with refractory metTNBC who had an ExRx to pac/cis.nnnMETHODSnFollowing IRB-approved informed consent, targeted NGS (Foundation Medicine) and WGS (TGEN) was performed on the pts FFPE primary TNBC and 2 recurrent lymph nodes to characterize all classes of genomic alterations in cancer-related genes. RPPA was performed at a CLIA-certified laboratory (Theranostics Health) and George Mason Univ where immunostaining was directed against HER1/2/3 pathway and other proteins.nnnRESULTSnAt age 58 in 2006, pt had T1c 1+ node TNBC treated with FAC/T. Between 2008 and 2011 she had 4 chemotherapy-refractory recurrences in axilla, supraclavicular (SC), internal mammary (IM) LNs treated unsuccessfully with surgery, radiation and multiple cytotoxic agents including carboplatin. In 2011, following SC LN biopsy, she was treated with BEZ, a PI3K/mTOR, ATM, ATR, DNA-PKcs inhibitor, had a 3 mo response, followed by rapidly enlarging progressive disease (PD) in IM LNs pushing sternum anteriorly. She was treated with pac/cis and had an ongoing complete response (CR) of 2.5+ yrs. NGS of 2011 SC LN (pre-BEZ) & 2012 IM LN (post-BEZ): TP53 & BRCA2 (somatic 15% mutant allele freq) mutations, FOXM1 amplification; SMARCA4 (BRG1) deletion RPPA (GMU) 2011 SC LN (Pre-BEZ): 3+ EGFR; 2+ p-EGFR, p-AKT, p-MEK1/2, p-mTOR RPPA (Theranostics) 2012 IM LN (post-BEZ): 3+ p-MEK1/2 (EGFR & p-EGFR 0) (AR-).nnnCONCLUSIONSnStrong EGFR signaling associated with chemo-resistant metTNBC in 2011 SC LN was not present in post-BEZ rapid PD in IM LN which then had durable CR with pac/cis. BEZ inhibits DSB repair, sensitizing cancers to DNA damaging agents (Gil del Alcazar, Clin Ca Res 20:1235, 2014). Progression of p-AKT-activated TNBC following response to inhibitors of PI3K & DNA repair shows DSB repair-deficiency and MAPK activation (Juvekar, Cancer Dis 2:1048, 2012). A prospective trial of BEZ followed at PD by pac/cis in metTNBC is warranted.