Reverse Phase Protein Microarray Research Articles

Abstract P2-04-02: Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using reverse-phase protein microarrays (RPMA)

Abstract Background Inflammatory Breast Cancer (IBC) is a clinicopathologic diagnosis characterized by rapid progression and poor prognosis. Even with the advent of targeted therapies and a multimodal approach, IBC is often treatment refractory and a therapeutic challenge for all subtypes, including ER+ and HER-2 amplified (HER2+) disease (Masuda et al). Therefore identifying mechanisms of resistance to molecularly targeted therapy could provide clues to improve management and outcome. Recent studies comparing the gene expression profiles of IBC tumors with non-IBC demonstrated that HER2+ IBC have increased mTOR signaling compared to their non-IBC counterparts (Iwamoto et al). mTOR activation is a mechanism for Trastuzumab resistance and may contribute to treatment resistance in HER2+ IBC. The availability of molecular diagnostics evaluating phosphoproteins is an appealing approach to predict treatment-sensitivity and select more effective combinations. Methods This is an observational analysis of 12 IBC patients who had tissue biopsy after progression on standard therapies including HER-2 targeted therapies. Tissue analysis for expression of cancer-related phosphoproteins was performed using TheraLink™. The TheraLink™ assay uses reverse-phase protein microarrays (RPMA) to quantify HER1, HER2, and HER3 receptor overexpression; it also evaluates for phosphorylation of the receptor which indicates activation. Phosphorylation of HER downstream signaling pathways such as JAK2, AKT/mTOR and MEK1/2 are also detected. Additionally, next generation sequencing (NGS) using FoundationOne™ was performed if sufficient tissue was available. Results All patients had IBC and most had metastatic disease (83%). According to subtype 25% of patients were ER+/HER2-, 42% ER+/HER2+, 25% ER-/HER2+, 8% ER-/HER2-. 58% of tumors demonstrated HER1 activation, 75% had HER2 activation and 58% had HER3 activation. Interestingly, 83% had mTOR activation, and most of these patients also had accumulation of its downstream proteins, S6 ribosomal protein and 4E-BP-1. 78% of patients with HER2 activation also had mTOR activation. Two of the 4 patients who were HER2- by IHC/FISH had HER2 activation by RPMA. Six patients also had NGS on tissue; 75% had concordance between HER2 activation on TheraLink™ and ERBB2 amplification on NGS, 67% had concordance with mTOR activation on TheraLink™ and mutation in the mTOR pathway (PIK3CA mutation or PTEN loss) on NGS. One patient with triple negative, chemo-refractory IBC who underwent 3 lines of neoadjuvant therapy prior to bilateral mastectomy was found to have HER1, HER2, HER3 and mTOR activation; she was started on lapatinib and capecitabine and remains with no recurrent disease and on treatment. Conclusions Patients with IBC often have activation of members of the HER family and mTOR pathway indicating molecular targets and potential mechanisms of resistance in IBC. The concomitant use of NGS and RPMA is an intriguing approach to molecular diagnostics in this aggressive and treatment refractory disease providing additional information on pathway activation leading to expanded therapeutic options. Future prospective studies should clarify the potential impact in treatment selection and outcome. Citation Format: Laura Austin, Kimberly Limentani, Juan Palazzo, Tiffany Avery, Rebecca Jaslow, Ron Hencin, Emanuel F Petricoin, Massimo Cristofanilli. Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using reverse-phase protein microarrays (RPMA) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-02.

Abstract P3-06-15: Evaluation of HER family protein signaling network as a predictive biomarker for pCR for breast cancer patients treated with neratinib in the I-SPY 2 trial

Abstract Background: We hypothesize that response to the pan-ERBB inhibitor, neratinib (N), may be predicted by pre-treatment HER2-EGFR signaling. In the I-SPY 2 TRIAL, N graduated in the HR-/HER2+ signature. All patients received at least standard chemotherapy. For HER2+ patients, N was administered in place of trastuzumab. We evaluated 18 HER family signaling proteins as biomarkers of N response using reverse phase protein microarray (RPMA) data from pre-treatment LCM purified tumor epithelium. Methods: 168 patients (N: 106, concurrent controls: 62) had RPMA and pCR data. 18 biomarkers relating to HER family signaling were evaluated: AKT S473, AKT T308, EGFR, EGFR Y1068, EGFR Y1148, EGFR Y1173, EGFR Y992, ERBB2, ERBB2 Y1248, ERBB3 total, ERBB3 Y1289, ERK1/2 T202/Y204, Heregulin, mTOR, mTOR S2448, PI3K p85 Y458/p55 Y199, PTEN S380, and SHC Y317. We assessed association between biomarker and response in the N and control arms alone (likelihood ratio test), and relative performance between arms (biomarker x treatment interaction) using a logistic model. Analysis was also performed adjusting for HR/HER2 status. In an exploratory analysis, we selected the marker with the greatest interaction (phosphorylated EGFR (Y1173)) to dichotomize patients optimally based on the data and assessed it in the context of the graduating signature by adding the EGFR Y1173-High patients to the HR-/HER2+ subtype and evaluating the treatment effect in this ‘biomarker-positive’ group. Our study is exploratory with no claims for generalizability of the data and does not account for multiplicities. Statistical calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Results: 7 HER pathway markers (EGFR Y1068, EGFR Y1173, EGFR Y992, ERBB2 total, ERBB2 Y1248, ERBB3 Y1289, SHC Y317) are associated with response in the N but not the control arm. However, the difference in performance between arms did not reach significance by permutation testing. Adjusting for HR/HER2 status, EGFR Y1173 shows a significant biomarker x treatment interaction (p = 0.049). In an exploratory analysis, we dichotomized patients by their EGFR Y1173 levels and evaluated the distribution of pCR rates (Table 1). Neratinib (n=106)Control (n=62)EGFR Y1173 Low (n=31)EGFR Y1173 High (n=75)EGFR Y1173 Low (n=29)EGFR Y1173 High (n=33)HR-/HER2+ (n=28)0 / 412 / 181 / 11 / 5Not HR-/HER2+ (n=140)3 / 2724 / 575 / 285 / 28Table 1 OR between EGFR Y1173 groups in the N relative to control arm is 10.1. When EGFR Y1173 High patients are added to the graduating HR-/HER2+ subset, the OR associated with treatment is 3.2 and is comparable to that in the HR-/HER2+ signature (OR: 2.1), while increasing the prevalence of biomarker-positive patients by ∼50%. Evaluation of EGFR Y1173 under the I-SPY 2 Bayesian model is pending. Conclusion: Our sample size is too small to draw definitive conclusions. Our exploratory analysis reveals that HER family phosphoproteins associate with response to N, but only phosphorylated EGFR Y1173 appears to add value to the graduating signature. Given that this biomarker would expand the patient population that may benefit, it merits evaluation in other ongoing trials of neratinib. Citation Format: Julia D Wulfkuhle, Christina Yau, Denise M Wolf, Rosa I Gallagher, Ashish Sanil, Lamorna Brown-Swigart, Susan Flynn, Gillian Hirst, I-SPY 2 TRIAL Investigators, Meredith Buxton, Angela DeMichele, Nola Hylton, William F Symmans, Laura van't Veer, Douglas Yee, Melissa Paoloni, Laura Esserman, Donald Berry, Minetta C Liu, John W Park, Emanuel F Petricoin III. Evaluation of HER family protein signaling network as a predictive biomarker for pCR for breast cancer patients treated with neratinib in the I-SPY 2 trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-15.

Abstract P2-05-14: New treatment options for metastatic breast cancer revealed by reverse-phase protein microarray and genomic profiling

Abstract Background: The optimal treatment strategy for patients with metastatic breast cancer (MBC) is currently unknown. Resistance to standard therapies like anthracyclines and taxanes limit the number of treatment options in many patients to a small number of non-cross-resistant regimens. We hypothesized that genomic and proteomic profiling of clinical MBC samples would identify genomic alterations that are linked to targeted therapies, and that this could facilitate a personalized approach to therapy for our patients. Methods: We retrospectively analyzed 31 consecutive metastatic breast cancer patients that had genomic and/or proteomic studies sent over a 4 month period from February to May 2014. All patients were seen in our Genomic Oncology Clinic and subsequently underwent a rebiopsy of a metastatic site. Formalin-fixed, paraffin-embedded (FFPE) tissue was sent to either Foundation Medicine for genomic profiling, Theranostics for reverse-phase protein microarray, or both. Standard immunohistochemistry for ER, PR, and HER2 was also performed on all patients. Results: Genomic or proteomic alterations were identified in all 31 patients. All patients harbored at least one actionable target and a treatment recommendation for a currently available FDA approved drug or drug combination was able to be suggested in all but one patient. The most commonly observed genomic alterations were within PIK3CA (26%), TP53 (23%), CCND1 (19%), and MYC (16%). Over 30 distinct genomic alterations were identified. Proteomic results were available from 16 patients. Activation of the AKT/mTOR pathway was evident in a majority of patients. A change in HER2 status was also found in 26% of patients. 16% of cases underwent a negative to positive conversion in HER2 status while 10% of cases underwent a positive to negative conversion. It is notable that all 5 patients that underwent a negative to positive conversion in HER2 status had biopsies taken from metastatic disease in the liver. Conclusions: All patients in this retrospective study harbored genomic or proteomic alterations that are associated with targeted therapies. Treatment recommendations were suggested in all but one patient and a majority of patients are receiving the suggested therapy. Our data demonstrate that routine genomic and proteomic analysis in a clinical setting makes a significant positive impact for patients. Citation Format: Casey B Williams, Pradip De, Nandini Dey, Kirstin A Williams, Jessica Klein, Michelle King, Misty Small, Brigitte Cyr, Scooter Willis, Yuliang Sun, Jennifer Carlson, Brandon M Young, Amy Krie, Brian Leyland-Jones. New treatment options for metastatic breast cancer revealed by reverse-phase protein microarray and genomic profiling [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-14.

Molecular characterization and targeted therapeutic approaches in breast cancer.

Despite the wide improvements in breast cancer (BC) detection and adjuvant treatment, BC is still responsible for approximately 40,000 deaths annually in the United States. Novel biomarkers are fundamental to assist clinicians in BC detection, risk stratification, disease subtyping, prediction of treatment response, and surveillance, allowing a more tailored approach to therapy in both primary and metastatic settings. In primary BC, the development of molecular profiling techniques has added prognostic and predictive information to conventional biomarkers - estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Moreover, the application of next-generation sequencing and reverse-phase protein microarray methods in the metastatic setting holds the promise to further advance toward a personalized management of cancer. The improvement in our understanding on BC biology associated with the study of the genomic aberrations characterizing the most common molecular subtypes allows us to explore new targets for drug development. Finally, the integration of cancer stem cell-targeted therapies and immune therapies in future combination regimens increases our chances to successfully treat a larger proportion of women with more aggressive and resistant metastatic disease. This article reviews the current state of novel biological markers for BC, the evidence to demonstrate their clinical validity and utility, and the implication for therapeutic targeting.

Impact of upfront cellular enrichment by laser capture microdissection on protein and phosphoprotein drug target signaling activation measurements in human lung cancer: Implications for personalized medicine.

The aim of this study was to evaluate whether upfront cellular enrichment via laser capture microdissection (LCM) is necessary for accurately quantifying predictive biomarkers in nonsmall cell lung cancer tumors. Fifteen snap frozen surgical biopsies were analyzed. Whole tissue lysate and matched highly enriched tumor epithelium via LCM were obtained for each patient. The expression and activation/phosphorylation levels of 26 proteins were measured by reverse phase protein microarray. Differences in signaling architecture of dissected and undissected matched pairs were visualized using unsupervised clustering analysis, bar graphs, and scatter plots. Overall patient matched LCM and undissected material displayed very distinct and differing signaling architectures with 93% of the matched pairs clustering separately. These differences were seen regardless of the amount of starting tumor epithelial content present in the specimen. These results indicate that LCM driven upfront cellular enrichment is necessary to accurately determine the expression/activation levels of predictive protein signaling markers although results should be evaluated in larger clinical settings. Upfront cellular enrichment of the target cell appears to be an important part of the workflow needed for the accurate quantification of predictive protein signaling biomarkers. Larger independent studies are warranted.

Clinical and biological significance of glucocorticoid receptor (GR) expression in breast cancer.

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.

Quantitative analysis of proteins of metabolism by reverse phase protein microarrays identifies potential biomarkers of rare neuromuscular diseases

BackgroundMuscle diseases have been associated with changes in the expression of proteins involved in energy metabolism. To this aim we have developed a number of monoclonal antibodies against proteins of energy metabolism.MethodsHerein, we have used Reverse Phase Protein Microarrays (RPMA), a high throughput technique, to investigate quantitative changes in protein expression with the aim of identifying potential biomarkers in rare neuromuscular diseases. A cohort of 73 muscle biopsies that included samples from patients diagnosed of Duchenne (DMD), Becker (BMD), symptomatic forms of DMD and BMD in female carriers (Xp21 Carriers), Limb Girdle Muscular Dystrophy Type 2C (LGMD2C), neuronal ceroid lipofuscinosis (NCL), glycogenosis type V (Mc Ardle disease), isolated mitochondrial complex I deficiency, intensive care unit myopathy and control donors were investigated. The nineteen proteins of energy metabolism studied included members of the mitochondrial oxidation of pyruvate, the tricarboxylic acid cycle, β-oxidation of fatty acids, electron transport and oxidative phosphorylation, glycogen metabolism, glycolysis and oxidative stress using highly specific antibodies.ResultsThe results indicate that the phenotype of energy metabolism offers potential biomarkers that could be implemented to refine the understanding of the biological principles of rare diseases and, eventually, the management of these patients.ConclusionsWe suggest that some biomarkers of energy metabolism could be translated into the clinics to contribute to the improvement of the clinical handling of patients affected by rare diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0424-1) contains supplementary material, which is available to authorized users.

Host response during Yersinia pestis infection of human bronchial epithelial cells involves negative regulation of autophagy and suggests a modulation of survival-related and cellular growth pathways.

Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.

Kinase analysis in alcoholic hepatitis identifies p90RSK as a potential mediator of liver fibrogenesis

ObjectiveAlcoholic hepatitis (AH) is often associated with advanced fibrosis, which negatively impacts survival. We aimed at identifying kinases deregulated in livers from patients with AH and advanced fibrosis in order...

The higher frequency of IgA deficiency among Swedish twins is not explained by HLA haplotypes.

Serum immunoglobulin A (IgA) concentrations were determined in 12 600 adult Swedish twins, applying a high-throughput reverse-phase protein microarray technique. The prevalence of IgA deficiency (IgAD) was found to be 1:241 in monozygotic (MZ) twins and 1:198 in dizygotic (DZ) twins. Hence, the prevalence in twins is markedly elevated as compared with the normal Swedish adult population (1:600). The twins did not show a difference in the frequency of HLA haplotypes in comparison with almost 40 000 healthy Swedish controls. As expected, the risk-conveying HLA alleles A*01, B*08 and DRB1*01 were overrepresented among the IgAD twins and were also associated with significantly lower mean serum IgA concentrations in the twin cohort. In contrast, significantly higher mean IgA concentrations were found among individuals carrying the protective HLA alleles B*07 and DRB1*15. Exome sequencing data from two MZ twin pairs discordant for the deficiency showed no differences between the siblings. Model fitting analyses derived a heritability of 35% and indicate that genetic influences are modestly important for IgAD. The probandwise concordance rates for IgAD were found to be 31% for MZ and 13% for DZ twins.

Fluoxetine induces cytotoxic endoplasmic reticulum stress and autophagy in triple negative breast cancer.

To investigate the mechanism of action of lipophilic antidepressant fluoxetine (FLX) in representative molecular subtypes of breast cancer. The anti-proliferative effects and mechanistic action of FLX in triple-negative (SUM149PT) and luminal (T47D and Au565) cancer cells and non-transformed MCF10A were investigated. Reverse phase protein microarray (RPPM) was performed with and without 10 μmol/L FLX for 24 and 48 h to determine which proteins are significantly changed. Viability and cell cycle analysis were also performed to determine drug effects on cell growth. Western blotting was used to confirm the change in protein expression examined by RPPM or pursue other signaling proteins. The FLX-induced cell growth inhibition in all cell lines was concentration- and time-dependent but less pronounced in early passage MCF10A. In comparison to the other lines, cell growth reduction in SUM149PT coincided with significant induction of endoplasmic reticulum (ER) stress and autophagy after 24 and 48 h of 10 μmol/L FLX, resulting in decreased translation of proteins along the receptor tyrosine kinase/Akt/mammalian target of rapamycin pathways. The increase in autophagy marker, cleaved microtubule-associated protein 1 light chain 3, in SUM149PT after 24 h of FLX was likely due to increased metabolic demands of rapidly dividing cells and ER stress. Consequently, the unfolded protein response mediated by double-stranded RNA-dependent protein kinase-like ER kinase resulted in inhibition of protein synthesis, growth arrest at the G1 phase, autophagy, and caspase-7-mediated cell death. Our study suggests a new role for FLX as an inducer of ER stress and autophagy, resulting in death of aggressive triple negative breast cancer SUM149PT.

Abstract B55: Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer

Abstract Background: TRIM28 is a universal transcriptional co-repressor with pleotropic effects in both normal and tumor cells. We have previously shown that varying TRIM28 levels in epithelial cells and stromal fibroblasts have a prognostic value in colorectal cancer patients. The pathophysiological role of TRIM28 in carcinogenesis may therefore be associated with TRIM28 expression levels and cell type of expression within the tumor microenvironment. In this study we aim to dissect the molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment by isolating individual populations of neoplastic epithelial and stromal cells and investigating their protein signaling networks. Methods: TRIM28 expression was analyzed by immunohistochemistry on FFPE tissue from 19 colorectal cancer patients. TRIM28 staining was evaluated in both epithelial and stromal compartments. IHC scoring of at least 2 units of difference in staining intensity between stromal fibroblasts and epithelial cells was defined as a high TRIM28 expression ratio and a low TRIM28 expression ratio was defined as 1 or 0 units of difference in staining intensity. Reverse-phase protein microarrays (RPMA) were constructed from laser capture micro-dissection (LCM) enriched tumor epithelium and stroma isolated from fresh-frozen tissue of the same patient cohort. The protein signaling networks were measured for 32 downstream signaling endpoints. Spearman rank analysis was used to assess the correlations between individual protein pairs. Correlation coefficient ρ ≥ 0.75 with P ≤ 0.01 was considered significant. Results: Immunohistochemical analysis of the FFPE tissue sections identified 10 TRIM28 high ratio cases and 9 TRIM28 low ratio cases. Proteomic networks were assessed in TRIM28 high and low ratio cases in fresh-frozen tissue using RPMA. Spearman ρ rank correlation analyses for the 32 signaling proteins revealed that 184 highly correlated protein pairs exclusive to the TRIM28 high ratio group, whereas 157 protein pairs were found exclusively in the TRIM28 low ratio group. In addition 191 protein pairs were shared across both TRIM28 high and low ratio groups. The caspases 3 and 7, as well as the cell surface receptor RAGE, were prominent in the high TRIM28 ratio group proteomic network, whereas the Metalloprotease MMP9 and the GTPase Ras-GRF1 were exclusive to the low TRIM28 ratio group. Furthermore we found Survivin and JNK to be prominent in the stromal compartment of the high TRIM28 cases. Conclusions: We characterized molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment. Proteomic analysis revealed distinct protein signaling networks associated with varying TRIM28 expression levels in epithelial and stromal compartments. The study presents a novel way of deciphering molecular crosstalk between the epithelial and stromal compartments within the microenvironment. Citation Format: Seán Fitzgerald, Virginia Espina, Katherine M. Sheehan, Robert Cummins, Anthony O'Grady, Dermot Kenny, Richard O'Kennedy, Lance Liotta, Elaine W. Kay, Gregor Kijanka. Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B55. doi:10.1158/1538-7445.CHTME14-B55

Functional characterization of epithelial ovarian cancer histotypes by drug target based protein signaling activation mapping: implications for personalized cancer therapy.

Epithelial ovarian carcinoma (EOC) is a deadly disease, with a 5-year survival of 30%. The aim of the study was to perform broad-scale protein signaling activation mapping to evaluate if EOC can be redefined based on activated protein signaling network architecture rather than histology. Tumor cells were isolated using laser capture microdissection (LCM) from 72 EOCs. Tumors were classified as serous (n = 38), endometrioid (n = 13), mixed (n = 8), clear cell (CCC; n = 7), and others (n = 6). LCM tumor cells were lysed and subjected to reverse-phase protein microarray to measure the expression/activation level of 117 protein drug targets. Unsupervised hierarchical clustering analysis was utilized to explore the overall signaling network. ANOVA was used to detect significant differences among the groups (p < 0.05). Regardless of histology, unsupervised analysis revealed five pathway-driven clusters. When the EOC histotypes were compared by ANOVA, only CCC showed a distinct signaling network, with activation of EGFR, Syk, HER2/ErbB2, and SHP2 (p = 0.0007, p = 0.0021, p < 0.0001, and p = 0.0410, respectively). The histological classification of EOC fails to adequately describe the underpinning protein signaling network. Nevertheless, CCC presents unique signaling characteristics compared to the other histotypes. EOC may need to be characterized by functional signaling activation mapping rather than pure histology.

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

Abstract 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 &amp;lt; 30 kg/m2 are classified as non-obese, while those with BMI ≥ 30 kg/m2 are considered obese. All statistical analyses were done using SAS Enterprise Guide 5.1 (SAS Institute, Cary, NC). For comparison of 2 groups, t tests were used for continuous variables and Fisher exact tests for categorical variables. ANOVA was used for comparison of more than 2 groups. Two-tailed p-value &amp;lt; 0.05 was considered statistically significant. Unsupervised hierarchical clustering analysis was performed on log 2-transformed values for each protein endpoint, using average linkage of the Pearson correlation coefficient. 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

Whole proteome analysis of mouse lymph nodes in cutaneous anthrax.

This study aimed to characterize a soluble proteome of popliteal lymph nodes during lymphadenitis induced by intradermal injection of Bacillus anthracis Sterne spores in mice using tandem LC-MS/MS and reverse-phase protein microarray with antibodies specific to epitopes of phosphorylated proteins. More than 380 proteins were detected in the normal intra-nodal lymph, while the infectious process resulted in the profound changes in the protein abundances and appearance of 297 unique proteins. These proteins belong to an array of processes reflecting response to wounding, inflammation and perturbations of hemostasis, innate immune response, coagulation and fibrinolysis, regulation of body fluid levels and vascular disturbance among others. Comparison of lymph and serum revealed 83 common proteins. Also, using 71 antibodies specific to total and phosphorylated forms of proteins we carried initial characterization of circulating lymph phosphoproteome which brought additional information regarding signaling pathways operating in the lymphatics. The results demonstrate that the proteome of intra-nodal lymph serves as a sensitive sentinel of the processes occurring within the lymph nodes during infection. The acute innate response of the lymph nodes to anthrax is accompanied by cellular damage and inflammation with a large number of up- and down-regulated proteins many of which are distinct from those detected in serum. MS data are available via ProteomeXchange with identifier PXD001342.

Abstract 1067: Somatic genomic mutations, RNA-Seq, and proteomic signal transduction network analysis of tumor and stroma in metastatic triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous, basal like tumor category for which there is no molecular targeted therapy that has significantly improved overall patient survival. While genomic sequencing has been conducted on primary triple negative breast cancers, these data may not correspond to metastatic TNBC because the metastatic colonies may comprise a subclone of the primary. Moreover, it has been difficult to identify the true driver mutations in the primary tumor. We have hypothesized that evaluation of the protein signal transduction pathways within the tumor-stromal microenvironment of the metastatic tumor may be vastly different than the primary tumor and can provide potential clues to the driver mutations and stromal interactions. Somatic genomic mutations and RNA-Seq profiles were previously cataloged for a set of 14 fresh frozen metastatic TNBC samples. TP53, LRP1B, HERC1, CDH5, RB1, and NF1 were mutated in multiple tumors. FOXM1 gene overexpression was noted in tumor samples compared to non-malignant breast samples. To complement the genetic data, we evaluated more than 70 signal transduction proteins in tumor and stroma for 13 of the metastatic TNBC samples from this cohort. The samples represented lymph node (n=5) and local and distant soft tissue (n=8) metastasis. For proteomic analysis, purified populations of tumor and adjacent stromal cells were collected by Laser Capture Microdissection. Cell signaling pathway proteins were quantified by reverse phase protein microarray. Proteins from diverse pathways were included to: a) correlate our proteomic data with the previous genomic data, b) potentially identify previously unrecognized pathways involved in TNBC metastatic disease, and c) evaluate the tumor-stroma microenvironment in metastatic TNBC. We compared the signaling network of the tumor and its matched stroma to investigate tumor-stroma crosstalk. We also compared the signal pathways to the genomic abnormalities of the matched lesion. We confirmed the down regulation of endocrine (ER, AR, ProgRec) and Her2 pathways. phosphoJAK2 and Her4 (total) were both markedly downregulated. Signal pathway data provided information about the functional importance of the genomic data. Understanding the molecular underpinnings of metastatic TNBC may help elucidate genomic and proteomic alterations which can potentially lead to new strategies for individualizing therapy of metastasis. Citation Format: Virginia A. Espina, Joyce A. O'Shaughnessy, Maren K. Levin, David W. Craig, John D. Carpten, Daniel Von Hoff, Lance A. Liotta. Somatic genomic mutations, RNA-Seq, and proteomic signal transduction network analysis of tumor and stroma in metastatic triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1067. doi:10.1158/1538-7445.AM2014-1067

Abstract 4166: Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies

Abstract Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies Adaptive survival responses to targeted therapy reduce therapeutic benefit, and thus there is a need to identify drug combinations that co-target both oncogenic drivers and critical adaptive responses. We screened a panel of twelve V600E BRAF melanoma cell lines with a library of targeted drugs, and identified the combination of lapatinib (a HER family inhibitor) plus PLX4720 (a RAF inhibitor) as synergistically cytotoxic in the six lines that displayed complete or partial resistance to PLX4720 alone. To understand the mechanism(s) of drug resistance and synergy, and to identify potential biomarkers for utilization of this combination, we have performed an extensive molecular analysis of the cell lines and their responses to treatment with the drugs, singly and in combination. Alamar blue was used to calculate the cytotoxic effects of the drugs, and the cells were profiled by gene expression microarrays and reverse phase protein microarrays, before and after drug treatment. DNA methylation microarrays, and exome sequencing were used to identify the epigenetic and genetic context of the lines. Unsupervised hierarchical clustering analysis of the cytotoxic effects of the single drug and combinatorial treatments revealed four well defined groups: synergistic to combination treatment, PLX4720 sensitive, lapatinib resistant, and insensitive to either drug. The cell lines were analyzed using principal component analysis on the basal gene expression profiles. This analysis revealed four major clusters whose membership was identical to the clusters observed in the cytotoxicity analysis, indicating a strong genotype-drug phenotype relationship (i.e. cell lines that showed similar basal transcriptional programs also demonstrated similar responses to treatment. Differential gene expression analysis was performed between the synergistic group and each of the other groups. The most prominent gene expression differences were between the synergistic and PLX4720 sensitive lines, which correlated with differences in MITF expression and transcriptional output. Changes in gene expression and phosphoproteome levels in response to PLX4720 and lapatinib treatment between the four groups were used to identify genes and pathways associated with drug resistance and synergy. Genes involved in apoptosis and cell cycle regulation were found that could play a role in conferring resistance and synergistic response to PLX4720 and lapatinib treatment. These differing patterns of adaptive responses provide insights into molecular mechanisms of drug resistance and synergy and a pathway to rational construction of drug combinations. Citation Format: Brian J. Capaldo, Devin G. Roller, Mark Axelrod, Alex Koeppel, Michael J. Weber, Aaron Mackey, Dan Gioeli, Stefan Bekiranov. Integrated molecular profiling of melanoma cell lines reveals genotype-drug phenotype associations giving insight on development of combination therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4166. doi:10.1158/1538-7445.AM2014-4166

Abstract 3465: Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine

Abstract Background: The evaluation of intratumor heterogeneity has become a major topic in the oncological field and it has major implications in the delivery of individualized therapy. While a number of genomic studies have reported significant intratumoral heterogeneity the analysis of the activation of protein kinase-driven signaling networks, which constitute the targets of most of the targeted compounds, has been sparse, or did not focus specifically on the tumor cells. This study evaluated the activation/phosphorylation of the protein signaling networks and a number of major precision medicine-oriented drug target to assess whether the intratumoral heterogeneity could impact patient stratification for targeted treatment in the context of metastatic disease. Methods: 3 independent study sets were used for this analysis: 1) metastatic colorectal cancer (mCRC) liver biopsies were collected from 6 patients; for each biopsy, 2 different regions were microdissected, 2) 18 needle biopsies were collected from 2 different regions of the same lesion of 9 mCRC liver metastases, 3) 7 serial liver biopsies were collected from 3 mCRC before and after treatment. Specimens were frozen upon collection, stored at -80°C and subjected to laser capture microdissection to isolate pure tumor epithelium. Protein pathway activation mapping was performed using reverse phase protein microarray. To assess changes in the activation level of the members of the MAPK and the AKT-mTOR pathway, unsupervised hierarchical clustering analysis and fold change between the different areas of the tumors were evaluated. Results: Hierarchical clustering analysis of the activation of 47 key signaling protein drug targets revealed that the signaling activation of any given protein or pathway was largely the same within a patient's tumor, no matter where the biopsy was obtained, but large interpatient variation was observed. When we evaluated activation of the MAPK and AKT-mTOR pathways, we found that the relative levels of pathway activation were found to be similar in different regions of the tumor, while each patient looked distinct. Conclusions: This first of its kind protein drug target activation analysis of microdissected tumor cells from human metastatic tumors revealed that the activated signaling architecture is largely maintained within the same tumor while each patient's tumor has a distinct patient-specific signaling portrait. The data suggest that interpatient protein drug target activation heterogeneity is much larger than intratumoral heterogeneity and point to the need to determine precision medicine based profiling on a patient-by-patient basis. Citation Format: Erika M. Parasido, Alessandra Silvestri, Claudio Belluco, Vincenzo Canzonieri, Maria Grazia Diodoro, Massimo Milione, Lance A. Liotta, Emanuel F. Petricoin, Mariaelena Pierobon. Impact of intratumoral protein signaling network activation heterogeneity: Implication for precision medicine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2014-3465

Abstract 2840: Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology

Abstract With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions. Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification. Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearman's rho &amp;gt;0.85). Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development. Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2014-2840

Abstract 3795: Cabozantinib affects multiple signaling pathways in glioblastoma and is effective in a subset of xenograft tumors

Abstract Background: Glioblastoma (GBM) is the most common and aggressive form of brain tumor characterized by poor prognosis and high recurrence rate. Several new targeted agents are being considered for the adjuvant treatment of GBMs. Cabozantinib, a multi-kinase currently in clinical trials for several malignancies, including glioblastoma, targets c-Met and VEGFR2 receptors, as well as Ret, Kit, Flt-1/3/4, Tie2, and AXL. Our goal is to first evaluate sensitivity and molecular response of patient-derived GBM cancer stem cells (CSCs) to XL184 in vitro. Anti-tumor efficacy of cabozantinib as a single agent and in combination with the DNA-alkylating agent temozolomide (TMZ) in orthotopic xenografts was then evaluated. Methods: Neurospheres enriched in CSCs were cultured from resected GBM tumors. Sensitivity to cabozantinib was determined in vitro. Cells were treated (IC40) in triplicate, and cell lysates were analyzed by reverse phase protein microarrays. GBM CSCs were implanted intracranially into nude mice. Cabozantinib was administered by oral gavage at a dose of 60mg/kg for 4 weeks (5 days/week) as a single agent or in combination with 40mg/kg TMZ. Tumor growth and response to treatment were monitored by non-invasive in vivo biolumiescent imaging (BLI) using the Xenogen IVIS System (Caliper Life Sciences), and overall survival. Results: Sensitivity to cabozantinib treatment varied for the different GBM CSCs. From 70 proteins and phosphoproteins measured, 29 distributed among several signaling pathways were significantly altered after treatment in both resistant and sensitive GBM CSCs. Cabozantinib single agent treatment reduced GBM tumor growth and increased mouse survival in two xenograft lines. Cabozantinib monotherapy reduced tumor size, as measured by BLI, but had no significant effect on overall survival for another xenograft line, however, the combination treatment resulted in sensitization of these xenografts to TMZ treatment. Conclusion: The use of multi-kinase inhibitors is a promising strategy for GBMs, and given the range of responses, identifying predictive biomarkers, though challenging, will be invaluable for patient stratification. Citation Format: Ana C. deCarvalho, Kimberly Arnold, Claudius Mueller, Emanuel F. Petricoin, Laila M. Poisson, Tom Mikkelsen. Cabozantinib affects multiple signaling pathways in glioblastoma and is effective in a subset of xenograft tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2014-3795