Emanuel Petricoin

Adipocyte-derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment

The interactions of transformed cells with the surrounding stromal cells are of importance for tumor progression and metastasis. The relevance of adipocyte-derived factors to breast cancer cell survival and growth is well established. However, it remains unknown which specific adipocyte-derived factors are most critical in this process. Collagen VI is abundantly expressed in adipocytes. Collagen(-/-) mice in the background of the mouse mammary tumor virus/polyoma virus middle T oncogene (MMTV-PyMT) mammary cancer model demonstrate dramatically reduced rates of early hyperplasia and primary tumor growth. Collagen VI promotes its growth-stimulatory and pro-survival effects in part by signaling through the NG2/chondroitin sulfate proteoglycan receptor expressed on the surface of malignant ductal epithelial cells to sequentially activate Akt and beta-catenin and stabilize cyclin D1. Levels of the carboxyterminal domain of collagen VIalpha3, a proteolytic product of the full-length molecule, are dramatically upregulated in murine and human breast cancer lesions. The same fragment exerts potent growth-stimulatory effects on MCF-7 cells in vitro. Therefore, adipocytes play a vital role in defining the ECM environment for normal and tumor-derived ductal epithelial cells and contribute significantly to tumor growth at early stages through secretion and processing of collagen VI.

Breast cancer molecular profiles and tumor response of neoadjuvant doxorubicin and paclitaxel: The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657).

LBA515 Background: I-SPY is a multi-center trial designed to identify predictive markers of pathological complete response (pCR) and survival of women with locally advanced breast cancers (3cm or greater). Women received neoadjuvant doxorubicin and cyclophosphamide then paclitaxel. METHODS 237 women enrolled, 216 completed serial imaging and core biopsies. Pre-treatment assays include: Agilent expression arrays, MIP aCGH, p53 gene chip and sequencing, IHC and reverse phase protein arrays (RPMA). Response to therapy was measured by serial MRI, pCR and residual cancer burden (RCB). Associations among molecular markers, pCR, RCB and survival were evaluated using chi-square test, Kaplan-Meier curves and log-rank test. RESULTS Median tumor size was 6cm, % pCR and RCB 0/1 was 27% and 36% for the entire study; % pCR rate for the 144 Agilent arrays was 25%. Distribution, rates of pCR and RCB 0/1 are shown in the Table for molecular and IHC markers. DFS and OS will be presented. Several molecular subtypes, including NKI 70 gene low, luminal A, 21 gene set low and IHC HR+, define 15-28% of patients with 3-10% pCR, yet excellent early survival. Wound healing, most discriminatory for prognosis, is not predictive of chemotherapy response. By RPMA, patients with pCR had increased phosphorylation of 4EBP1, eNOS, cAbl, STAT5, EGFR, AKT (p<0.05). In ER+ patients with poor MR response, pIRS, pIGFR, p706S were activated (p<0.05). RCB is a more refined way to measure pCR and was more predictive of DFS and OS (p=0.01) than pCR alone with a mean follow up of 3.9 years. MR volume is highly predictive of pCR and RCB. For specific subtypes, e.g. basal, RCB is predictive of DFS (p<0.00001). CONCLUSIONS LABC have aggressive biology. Response to therapy and outcome can be predicted by many biomarkers. The I-SPY data set provides a platform to compare, contrast and combine marker signatures to tailor therapy and demonstrates the power of the neoadjuvant setting. Support: ACRIN U01 CA079778 ; CALGB CA31964, CA33601; NCI SPORE CA58207. [Table: see text] [Table: see text].

Beyond the genome to tissue proteomics.

The ongoing revolution in molecular medicine can be divided into three phases. The first phase is gene discovery, in which the tools of molecular biology are applied to identify and sequence previously unknown genes. Identification of most of the expressed human genes will be accomplished before 2005. The second phase is molecular fingerprinting, which correlates the genomic state, the complementary DNA expression pattern, and the protein repertoire with the functional status of the cells or tissue. The promise of this phase is that expression profiles can uncover clues to functionally important molecules, and will generate information to tailor a treatment to the individual patient. The third phase is the synthesis of proteomic information into functional pathways and circuits in cells and tissues. This must take into account the dynamic state of protein post-translational modifications and protein–protein or protein–DNA interactions. Through an integrated genomic/proteomic analysis, the ultimate outcome will be an actual functional understanding of the molecular events that underlie normal development and disease pathophysiology. This higher level of functional understanding will be the basis for true rational therapeutic design.

Genomic and Proteomic Pathway Mapping Reveals Signatures of Mesenchymal-Epithelial Plasticity in Inflammatory Breast Cancer

1.1 Inflammatory breast cancer as a distinct clinicopathologic entity There are several clinically distinct types of breast cancer, which include early stage breast cancer, locally advanced breast cancer (LABC) and metastatic breast cancer. The most rare but lethal form of LABC is inflammatory breast cancer (IBC) (reviewed in 1). This type of breast cancer accounts for an estimated 25% of all breast cancers in the United States and up to 20% of all breast cancers globally (2-4). Although primary IBC is less commonly diagnosed than other types of breast cancer, IBC is responsible for a disproportionate number of breast cancer-related deaths that occur each year world-wide due to its propensity to rapidly metastasize. (2-4). Women diagnosed with IBC have a significantly shorter median survival time (~ 2.9 years) than women with either LABC (~ 6.4 years) or non-LABC breast cancer (>10 years). The clinical diagnosis of IBC is based on the combination of the physical appearance of the affected breast, a careful medical history, physical examination, and pathological findings from a skin biopsy and/or needle or core

Evaluation of the HER/PI3K/AKT Family Signaling Network as a Predictive Biomarker of Pathologic Complete Response for Patients With Breast Cancer Treated With Neratinib in the I-SPY 2 TRIAL

Purpose In the I-SPY 2 TRIAL (Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2), the pan-erythroblastic oncogene B inhibitor neratinib was available to all hormone receptor (HR)/human epidermal growth factor receptor 2 (HER2) subtypes and graduated in the HR-negative/HER2-positive signature. We hypothesized that neratinib response may be predicted by baseline HER2 epidermal growth factor receptor (EGFR) signaling activation/phosphorylation levels independent of total levels of HER2 or EGFR proteins. Materials and Methods Complete experimental and response data were available for between 130 and 193 patients. In qualifying analyses, which used logistic regression and treatment interaction analysis, 18 protein/phosphoprotein, 10 mRNA, and 12 DNA biomarkers that related to HER family signaling were evaluated. Exploratory analyses used Wilcoxon rank sum and t tests without multiple comparison correction. Results HER pathway DNA biomarkers were either low prevalence or nonpredictive. In expression biomarker analysis, only one gene (STMN1) was specifically associated with response to neratinib in the HER2-negative subset. In qualifying protein/phosphoprotein analyses that used reverse phase protein microarrays, six HER family markers were associated with neratinib response. After analysis was adjusted for HR/HER2 status, EGFR Y1173 (pEGFR) showed a significant biomarker-by-treatment interaction (P = .049). Exploratory analysis of HER family signaling in patients with triple-negative (TN) disease found that activation of EGFR Y1173 (P = .005) and HER2 Y1248 (pHER2) (P = .019) were positively associated with pathologic complete response. Exploratory analysis in this pEGFR/pHER2-activated TN subgroup identified elevated levels of estrogen receptor α (P < .006) in these patients. Conclusion Activation of HER family phosphoproteins associates with response to neratinib, but only EGFR Y1173 and STMN1 appear to add value to the graduating signature. Activation of HER2 and EGFR in TN tumors may identify patients whose diseases respond to neratinib and implies that there is a subset of patients with TN disease who paradoxically exhibit HER family signaling activation and may achieve clinical benefit with neratinib; this concept must be validated in future studies.

Abstract P3-07-48: Prediction of complete pathologic response to veliparib/carboplatin plus standard neoadjuvant therapy in HER2 negative breast cancer: Exploratory protein pathway marker results from the I-SPY 2 trial

Background: In the I-SPY 2 TRIAL, HER2- patients were randomized to receive standard chemotherapy or chemotherapy plus the oral PARP inhibitor veliparib in combination with carboplatin (V+C), which graduated in the HR-/HER2- arm. Exploratory analysis of protein signaling was performed to identify biomarker candidates that correlated with pCR in the HER2- population. We evaluated 110 key signaling proteins using reverse phase protein microarray (RPPA) data from pre-treatment LCM purified tumor epithelium. Methods: Of 115 patients, 97 (V+C: 61 controls: 36) had RPPA and pCR data. RPPA data was correlated to pCR in both the treated and control patients using parametric (t-test) or non-parametric (Wilcoxon) statistical analysis, depending on data distribution. Only analytes whose pre-treatment levels were associated with response in the V+C but not the control arm were identified (P Results: 11 protein/phosphoprotein markers were significantly associated with pCR in the V+C arm but not in controls. Two were positive predictors of response: YAP S127 p= 0.03 and LC3B p=0.04. Negative predictors of response included Cyclin D1 p=0.001, and a number of phosphorylated RTKs: ROS Y2274 p=0.03, IGF1R Y1135/Y1136-IR Y1150/Y1151 p=0.03, ERBB4 Y1284 p=0.002, total HER2 p=0.04, and total IGF1R p=0.01. Moreover, a number of AKT-mTOR pathway proteins were found to be negative predictors of V+C response: ACC S79 p=0.005, p70S6K S371 p=0.01, and B-RAF S445 p=0.01. Conclusion: Our sample size is too small to draw definitive conclusions and the results are exploratory. Coordinated RTK-mTOR pathway activation appears to be a hallmark signature of lack of response to veliparib in HER2- tumors. We also found that HER2 levels were correlated paradoxically with lack of response in this HER2- population, suggesting potential added clinical value of quantitative HER2 measurement techniques. Such exploratory results merit evaluation in larger trials with HER2- breast cancer patients. Citation Format: Wulfkuhle JD, Yau C, Wolf DM, Gallagher RI, Deng J, Brown-Swigart L, Hirst G, I-SPY 2 Trial Investigators, Rugo H, Olopade OI, Esserman L, Berry D, van9t Veer L, Petricoin EF. Prediction of complete pathologic response to veliparib/carboplatin plus standard neoadjuvant therapy in HER2 negative breast cancer: Exploratory protein pathway marker results from the I-SPY 2 trial. [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 P3-07-48.

Abstract P6-04-01: Protein pathway activation mapping of LCM tumor epithelium from I-SPY 1 TRIAL surgical specimens reveals activation of receptor tyrosine kinase drug target signaling networks in the non-responding patient cohort

Background: Identification of alternative therapeutic strategies is critically needed for patients who do not achieve complete clinical response in the neoadjuvant setting, and in particular, from that cohort with the most aggressive disease (shortest relapse-free survival (RFS)). The I-SPY 1 TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and extensive molecular and clinical/pathological characterization of non-responding patients. Methods: Tumor epithelium was procured by Laser Capture Microdissection (LCM) from frozen surgical specimens of 27 patients who did not achieve complete pathological response in the I-SPY1 TRIAL. Reverse Phase Protein Microarray (RPPA) technology was used to measure the levels of 120 key signaling proteins including drug targets for Phase I-III and FDA-approved therapeutics. Cox proportional hazard univariate and multivariate analyses were used to evaluate RFS correlates. Significance threshold was p = <0.05. Results: Univariate analysis of pathway activation revealed a number of actionable/druggable targets, particularly receptor tyrosine kinase (RTK) activation, where increased phosphorylation of ALK (Y1586), EGFR (Y1173), ERBB3 (Y1289), cABL (T735), and FAK (Y576/Y577) was significantly associated with aggressive disease (shorter RFS). Higher levels of SRC (Y527) inhibitory phosphorylation were also significantly associated with early recurrence. Coordinate activation of downstream targets of these RTKs, such as RAS-GRF S916, SHC (Y317), MEK (S217/221) and the Aurora kinases were also associated with early relapse in this study set. In multivariate analyses controlling for receptor subtype (HR+/HER2-, triple-negative (TN), HER2+), the associations between shorter RFS and RTK pathway activation including EGFR, ERBB3, cABL and FAK activation and SRC inhibition, as well as RAS-GRF and SHC phosphorylation remained significant. Conclusions: This unique functional protein pathway/proteomic analysis of LCM-isolated tumor epithelium by RPPA in surgical specimens taken from patients who did not achieve complete pathological response provides evidence for a significant number of druggable RTK pathway targets in patients with residual disease who are most at risk to die from breast cancer. Activation of ALK, EGFR, ERBB3, ABL, FAK, along with downstream RAS-ERK and Aurora kinase signaling activation in this cohort provide new insights into tumor progression along with distinct, actionable information on new therapeutic modalities to test in patients with progressing disease. Identification of new therapeutic strategies for patients who progress or recur early after neoadjuvant therapy is critical and, if validated in larger independent cohorts, could lead to rationalized personalized therapy-based trials. This strategy is being utilized in the I SPY 2 TRIAL as we introduce pathway-directed therapeutics in the neoadjuvant setting and are employing the RPPA platform to determine whether these agents are impacting their intended pathway. This work was partially supported by NIH grants CA31946 and CA33601. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-01.

Abstract P3-04-02: Protein pathway activation mapping of I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with HR+/HER2− tumors

Background: Profiling protein signaling activation in cancer is critical as these proteins represent targets for new molecular therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane- based chemotherapy that longitudinally collected biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens (T2, 1–4 days post-chemotherapy). Reverse Phase Protein Microarray technology was used to quantitatively measure the activation of 39 and 100 key signaling proteins in the T1 and T2 biopsies, respectively, including numerous drug targets for Phase I-III and FDA-cleared therapeutics. Associations between protein activation and response to chemotherapy (RCB 0/1 vs 2/3) and relapse-free survival (RFS) were evaluated for the HR+/HER2− patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of HER family signaling and downstream AKT activation in tumors from patients who do not respond (RCB2/3) and/or whom recurred. Increased ERBB2 and ERBB4 levels were observed at T1 and T2, respectively, from patients who did not respond to neoadjuvant therapy. Dynamic changes in ERBB3 and AKT phosphorylation/activation were revealed between the T1 and T2 time points, which were associated with recurrence. Increased phosphorylation of MARCKS, a known PKC substrate, was seen in the T1 biopsy in the non-response cohort. Conclusions: Our analysis revealed activation/increased expression of HER family proteins along with downstream AKT activation in HR+/HER2− patients who have poor clinical response. These events, if validated, point to potential treatment options that could be rationally considered for non-responding HR+/HER2− patients with a number of investigational agents that target ERBB3 and AKT signaling in the clinic today. Increasing HER2 protein expression in a HER2− cohort may appear contradictory, however these findings may point to important subtle increases in HER2 that have important clinical considerations. Given the known clinical benefit from HER2 directed therapy in HER2− patients seen in other studies, our findings may have clinical relevance if validated. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with HR+/HER2− tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-02.

Abstract P3-04-01: Protein pathway activation mapping of the I-SPY 1 biopsy specimens identifies new network focused drug targets for patients with triple negative tumors

Background: Identification of new therapies and predictive biomarkers is needed for patients with triple negative (TN) breast cancer. Elucidating pathway activation in cancer is critical as these proteins represent targets for many of the new molecular therapeutics and companion diagnostic markers. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides longitudinal biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens collected 1–4 days after first chemotherapy treatment (T2). Reverse Phase Protein Microarray was used to measure the activation of 39 and 100 signaling proteins in the T1 and T2 biopsies, respectively, including drug targets for Phase I-III and FDA cleared therapeutics. Associations between pathway activation and response to chemotherapy (RCB 0/1 vs. 2/3) and relapse-free survival (RFS) were evaluated for the triple-negative (TN) patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of receptor tyrosine kinases such as MET and ALK in the T2 biopsies from patients that recurred, and VEGFR activation was seen in the T1 relapsed tumors. Activation in the AURORA-PLK1 cell cycle signaling network was seen with increased activation in the T2 biopsy from patients who did not respond to therapy and had poor RFS. Dynamic changes in ERK activation were revealed with decreased ERK activation between T1 and T2 time points associated with recurrence. Conclusions: Our analysis has revealed activation and increased expression of proteins involved in proliferation (Ki67), DNA repair (phosphorylated p53) and protein kinases in patients with TN disease who had poor clinical response. These events, if validated, point to potential treatment options that could be considered for non-responding TN breast cancer patients. VEGFR, ALK and MET, have FDA cleared therapeutics that could be rationally proposed for rapid clinical investigation, and there are numerous investigational agents that target AURORA and PLK1 in the clinic. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with TN tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-01.

Development and Implementation of Array Technologies for Proteomics: Clinical Implications and Applications

Array-based technologies, providing “-omic” level understanding of tumors at the DNA, RNA, and protein levels, have led to the uncovering of new disease susceptibility genes, therapeutic targets, expression profiles of genes or proteins related to disease outcomes as well as markers of therapeutic sensitivity and resistance. Analysis of signaling network activation at the protein level is of critical importance because nearly all current molecular-targeted therapeutics directed at modulating protein kinase activity, hence, the proteins themselves are the drug targets. Newer array-based and multiplexed approaches that can measure signaling network activation in very small tissue samples of the patient, and can perform broad-scale pathway mapping, will be the best to deliver effectively the needed predictive, prognostic, and therapy-guiding information to the bedside. The power of protein microarrays lies in their ability to provide a “map” of known cellular signaling proteins that generally reflect the state of information flow through protein networks in individual specimens. Combined with continued efforts to identify and monitor protein markers indicative of therapeutic response or resistance, protein array-based technologies are uniquely poised to provide direct functional information for individual patient tumors in time frames which was never before possible and could have a tremendous positive impact on therapeutic decision-making and ultimately on disease outcome.