Corinne Ramos

Enrichment of PI3K-AKT–mTOR pathway activation in hepatic metastases from breast cancer

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

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. METHODS Following 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. RESULTS 37 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). CONCLUSIONS The 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.

Association of long-term survival in pancreatic cancer (PDAC) with increased proliferative and anti-apoptotic protein activation.

254 Background: 5 year (yr) overall survival (OS) for resected PDAC is typically 20%. To test the potential for molecular prediction of > 5 yr OS in such patients (pts), we analyzed proteomic activation (phosphorylation) patterns in both cancer and stromal cells from initial surgical specimens prior to adjuvant therapy (Rx). Methods: Formalin-fixed, paraffin-embedded tissue from 40 pts (20 pts each with long-term actual disease-free OS [median 65 mo, range 26-137 mo] [LTS] and with early relapse/death 50 signal transduction proteins (STPs) for activation (phosp...

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. METHODS Following 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. RESULTS At 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-). CONCLUSIONS Strong 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.

Abstract C16: Feasibility study: Protein profiling of primary breast cancers from premenopausal women

African American women are more likely to be diagnosed with aggressive breast cancer at a younger age, have a higher risk of death, and have a higher prevalence of obesity than their Caucasian counterparts. Moreover, African American (AA) women diagnosed with triple-negative breast cancer have the lowest survival rates of all racial/ethnic groups. Although these disparities have been attributed to differences in biology, family and reproductive history, mammographic breast density, body mass index, access to healthcare, diet and environment, more etiologic studies are needed to elucidate the complex association between race, biologic factors, and socioeconomic factors. In the present study, we tested the feasibility of using high-throughput reverse phase protein microarray to identify protein(s) that are differentially expressed in the primary invasive breast cancers of premenopausal Caucasian and African American women. We examined the expression of 60 phosphorylated, total, and cleaved proteins in microdissected epithelial cells from 26 pre-treatment primary breast cancers. The impact of obesity on protein expression was also considered by stratifying samples into non-obese and obese based on a body mass index (BMI). Samples that come from women with BMI In this exploratory study, 58% of the primary breast cancers come from Caucasian women, while 42% of the samples come from AA women. The mean age and BMI for the two groups of women did not differ significantly. While the majority of the samples were of luminal subtypes, the triple-negative breast cancers were represented in majority of AA women. The expression of MEK1/2 S217/S221, PKC pan beta S660, and total PTEN were significantly higher in Caucasian women. When the impact of breast cancer subtypes (triple-negative vs. luminal A) on protein expression was examined, only total PTEN was statistically significant. Given the role of obesity in breast cancer, we determined which proteins are differentially expressed in non-obese and obese samples. Few components of the insulin and insulin-like growth factor and inflammatory signaling pathways (e.g. PTEN, acetyl CoA carboxylase, PKC pan beta S660, Stat3) were highly expressed in samples of obese women. This preliminary data demonstrates (a) the feasibility of mapping cell signaling networks in limited number of pre-treatment samples from premenopausal women and (b) the potential of future proteomic and larger cohort studies to identify which women will likely benefit from combination of targeted agents. Citation Format: Catherine Ibarra Drendall, William Barry, Corinne Ramos, Nora Tolbert, Joseph Geradts, Emanuel Petricoin, Victoria Seewaldt. Feasibility study: Protein profiling of primary breast cancers from premenopausal women. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C16. doi:10.1158/1538-7755.DISP13-C16

Abstract P4-04-12: Identification of a common genotype in patients (pts) with HER2-positive, ER-negative, inflammatory breast cancer (IBC) that was primary-refractory to trastuzumab who have had 5+ years of disease control with lapatinib therapy

Background: HER2-amplified inflammatory breast cancer (IBC) that is primary-refractory to trastuzumab has a poor prognosis. The objective of this study was to describe the genotypes of the IBC obtained from 3 pts with high grade, ER-negative, HER2+ classical IBC, primary-refractory to trastuzumab, with chest wall disease, who remain with no evidence of disease (NED) on lapatinib for 5+ years. The 3 postmenopausal pts presented with locally advanced IBC, 2 with disease extending onto the chest wall, and the third with chest wall recurrence while on adjuvant trastuzumab. Two pts were treated with preoperative trastuzumab with multiple chemotherapy agents with no response. They were then treated with preoperative lapatinib and had a clinical partial response. They underwent salvage mastectomy showing extensive residual disease, followed by chest wall/regional radiotherapy (RT), continuing on lapatinib. The third patient received preoperative AC followed by docetaxel and had extensive residual disease at mastectomy. She underwent chest wall/regional RT and then had chest wall recurrence while on adjuvant trastuzumab. She was treated with lapatinib and resection of residual chest wall disease. All 3 pts remain NED on 1250mg lapatinib daily. The 3 pts have a strong family history of breast cancer; two have known wild type germline BRCA1/2. Methods: Following IRB-approved informed consent, targeted next generation DNA sequencing (NGS) was performed using HiSeq-2000 (Illumina) on pts’ FFPE primary IBC at a CLIA-certified laboratory, to characterize all classes of genomic alterations across 4,604 exons of 287 cancer-related genes. Phosphoprotein analysis was performed using a proprietary Reverse Phase Protein Microarray (RPMA) platform on pts9s FFPE IBC to characterize the activity of the targets of anti-HER2 therapy, and their downstream pathways. Results: In 2 pts’ IBCs NGS identified a common genotype with amplified ERBB2 , mutant p53 and PIK3CA (c.3140A>G_p.H1047R), homozygous deletions of both CDKN2A and CDKN2B , and truncated BRCA2 (1 pt) or PALB2 (1 pt). The third pt9s FFPE residual disease from her mastectomy did not yield sufficient DNA from invasive tumor cells for sequencing and we plan to sequence DNA from her original diagnostic core biopsy. Phosphoprotein analysis of one pt9s trastuzumab-refractory, lapatinib-naive IBC showed 3+ expression of HER1 (but not pHER1 Y1068 nor pHER2 Y1248), pAkt S473, pS6 ribosomal S235-236 protein (2+), p4E-BP1 S65 and Notch1. The second pt9s lapatinib-treated IBC showed 3+ overexpression of only p4E-BP1 S65. Conclusions: Pts with a common tumor phenotype who have highly durable responses with a targeted therapy may share a common tumor genotype. Two pts’ trastuzumab-refractory HER2+ IBCs that have been very durably responsive to lapatinib share p53 , PIK3CA , p16 and BRCA2 -related mutations, potentially enabling the prospective identification of trastuzumab-refractory IBC pts who may benefit substantially from lapatinib. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-12.