Reverse Phase Protein Microarray Research Articles

Abstract 3877: A new frontier for molecular profiling of neoplastic bone tissue

Abstract Background: Tissue is alive and reacting to ex vivo stress during “cold ischemia time”. Tumor cells react to the absence of vascular perfusion, hypoxia, acidosis, and accumulation of cellular waste, which can critically mask cancer related signaling network data. Standard formalin fixation of calcified tissue requires decalcification with formic/nitric acid solution, which greatly interferes with downstream molecular analysis. We developed a non-formalin fixative (TheraLin) for preserving biomarker molecules and tissue histomorphology in one step, which greatly facilitates the analysis of phosphoprotein signaling pathways in calcified tumor samples. Methods: Fifty specimens of neoplastic bone were collected at the Istituto Ortopedico Rizzoli (Bologna, Italy). Matched 0.5 cm samples were fixed in TheraLin preservative; or fixed in 4% buffered formalin and decalcified with formic/nitric acid solution. Specimens were processed into paraffin blocks and 5 μm tissue sections cut onto glass slides. Enriched tumor cell populations were procured using Laser Capture Microdissection. Reverse Phase Protein Microarrays were constructed with the microdissected tumor cells and probed with >40 antibodies to unmodified or phosphorylated proteins. Immunohistochemistry (>200 stains in total) was performed on routine clinical protein targets and a subset of phosphorylated proteins. Results: TheraLin fixation greatly facilitated molecular profiling of bony tissues by eliminating the need for decalcification (18.5 hour average reduction in processing time) while maintaining or improving histomorphology (>85% of cases) and immunohistology (>75% of cases). In addition, we were able to demonstrate full compatibility with laser capture microdissection, reverse phase protein microarrays and downstream analysis of cell signal protein activation. Protein extractability from microdissected material was significantly increased (>2.5 fold, p<0.01) compared to formalin fixed, decalcified bone tissue. Conclusion: TheraLin obviates the need for decalcification of bone tissue while simultaneously preserving histomophology and immunohistochemistry. TheraLin enables reliable quantification of phosphorylated signal transduction proteins, the substrates of kinase drug targets, and greatly facilitates tumor cell enrichment compared to standard formalin fixation and decalcification. Citation Format: Claudius Mueller, Lance A. Liotta, Antonella Chiechi, Piero Picci, Marco Alberghini, Lorenzo Memeo, Vincenzo Canzonieri, Virginia Espina. A new frontier for molecular profiling of neoplastic bone tissue. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3877.

Abstract 2859: Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models

Abstract The defining feature of premalignant mammary lesions is proliferation of cells within the intraductal niche. Survival in this stressful duct microenvironment requires sophisticated mechanisms to avoid apoptosis caused by hypoxia, nutrient deprivation, metabolic acidosis, and intracellular calcium overload. Two major survival mechanisms of breast ductal carcinoma cells (DCIS) are 1) the upregulation of autophagy and 2) the enhanced export of calcium via plasma membrane calcium-ATPase isoform 2 (PMCA2) efflux pumps. We hypothesize that calcium efflux, through PMCA2, and activation of autophagic catabolism are critical interdependent mechanisms for the survival of pre-invasive human breast neoplastic cells. To assess the proteomic response to autophagy inhibition and/or elevated intracellular calcium, 41 signal transduction proteins/post-translationally modified proteins related to proliferation, autophagy, and calcium pathways were quantified by reverse phase protein microarrays (RPPA) in time course studies of T-47D metastatic ductal breast cancer cell line, and MCF-10A non-tumorigenic breast cell line, both of which express low basal levels of PMCA2, and in human primary DCIS organoid cultures. Cell lines were treated with chloroquine (50μM), or 1,25 dihydroxyvitamin D3 (Calcitriol) (50nM or 100nM), or chloroquine + Calcitriol, and harvested at periodic intervals over 197 hours. Stoichiometric time course studies (24 hours) were evaluated for Calcitriol as a single agent in both T-47D and MCF-10A cell lines. Cell morphology, adhesion, proliferation and autophagic flux in the DCIS organoids were assessed by Immunohistochemistry and RPPA. Calcitriol alone did not increase proliferation based on PCNA expression within DCIS ducts. Chloroquine markedly increased Cleaved PARP (Chi Sq p = 0.004) and CAMKinase II (p = 0.0068). Chloroquine (50μM) plus Calcitriol (100nM) increased E-Cadherin (p = 0.0379) and phosphoPAK1/2 (p = 0.0163), while PMCA2 levels were not significantly different between treatment groups. Autophagy inhibition and calcium efflux may be exploited therapeutically by co-administration of chloroquine and Calcitrol, to inhibit lysosomal catabolism and increase intracellular calcium levels, respectively, switching them from autophagic survival into apoptotic death. Citation Format: Virginia A. Espina, Solomon Yeon, Joshua N. VanHouten, John Wysolmerski, Lance A. Liotta. Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2859.

UCN-01 enhances cytotoxicity of irinotecan in colorectal cancer stem-like cells by impairing DNA damage response.

Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide. Despite recent progress, the prognosis of advanced stage CRC remains poor, mainly because of cancer recurrence and metastasis. The high morbidity and mortality of CRC has been recently ascribed to a small population of tumor cells that hold the potential of tumor initiation, i.e. cancer stem cells (CSCs), which play a pivotal role in cancer recurrence and metastasis and are not eradicated by current therapy. We screened CRC-SCs in vitro with a library of protein kinase inhibitors and showed that CRC-SCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. Nonetheless, broad-spectrum inhibition by the staurosporin derivative UCN-01 blocks CRC-SC growth and potentiates the activity of irinotecan in vitro and in vivo CRC-SC-derived models. Reverse-Phase Protein Microarrays (RPPA) revealed that, albeit CRC-SCs display individual phospho-proteomic profiles, sensitivity of CRC-SCs to UCN-01 relies on the interference with the DNA damage response mediated by Chk1. Combination of LY2603618, a specific Chk1/2 inhibitor, with irinotecan resulted in a significant reduction of CRC-SC growth in vivo, confirming that irinotecan treatment coupled to inhibition of Chk1 represents a potentially effective therapeutic approach for CRC treatment.

Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice

Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. SignificanceQuantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating the much lower effects of pMRTP aerosol than cigarette smoke on the mouse lung proteome. The combined analysis of 2D-PAGE and LC-MS/MS data identified an effect of cigarette smoke on the proteasome and actin cytoskeleton in the lung.

Reverse-phase protein microarray (RPPA) analysis of PD-L1 and growth factor receptor signal transduction pathways in hormone receptor (HR)-positive primary breast cancer tissue and correlation with relapse.

558Background: Despite considerabletreatment advances,resistance to hormone therapy remains a major clinical problem. We correlated the protein levels of multiple tyrosine kinase receptors, signal ...

In vivo evidence of mitochondrial dysfunction and altered redox homeostasis in a genetic mouse model of propionic acidemia: Implications for the pathophysiology of this disorder

Accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress in propionic acidemia (PA), an autosomal recessive metabolic disorder caused by the deficiency of mitochondrial propionyl-CoA carboxylase. PA patients exhibit neurological deficits and multiorgan complications including cardiomyopathy. To investigate the role of mitochondrial dysfunction in the development of these alterations we have used a hypomorphic mouse model of PA that mimics the biochemical and clinical hallmarks of the disease. We have studied the tissue-specific bioenergetic signature by Reverse Phase Protein Microarrays and analysed OXPHOS complex activities, mtDNA copy number, oxidative damage, superoxide anion and hydrogen peroxide levels. The results show decreased levels and/or activity of several OXPHOS complexes in different tissues of PA mice. An increase in mitochondrial mass and OXPHOS complexes was observed in brain, possibly reflecting a compensatory mechanism including metabolic reprogramming. mtDNA depletion was present in most tissues analysed. Antioxidant enzymes were also found altered. Lipid peroxidation was present along with an increase in hydrogen peroxide and superoxide anion production. These data support the hypothesis that oxidative damage may contribute to the pathophysiology of PA, opening new avenues in the identification of therapeutic targets and paving the way for in vivo evaluation of compounds targeting mitochondrial biogenesis or reactive oxygen species production.

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

Abstract 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<0.05). Markers are analyzed individually; p-values are descriptive and were not corrected for multiple comparisons. 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, van't 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 P2-05-21: The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer

Abstract Background: The identification of organ-specific targetable signatures may help design more effective treatment for patients with metastatic breast cancer (MBC). We took a multi-OMIC approach to assess whether the AKT-mTOR pathway is globally activated during metastatic progression or whether it represents an organ-specific target. Methods: Snap frozen biopsies from 25 MBC patients enrolled in a prospective phase II trial were used. Sites of metastasis were classified as liver (n=8) and others (n=17), the latter including cutaneous, lung, lymph nodes, and intra-abdominal lesions. Signaling analysis of the 25 cases was performed using Reverse Phase Protein Microarray (RPPA) coupled with Laser Capture Microdissection. Activation of the AKT-mTOR pathway was quantified as phosphorylation of AKT (S473) and the mTOR target p70S6 (T389). Matched exome (WES) and RNASeq data were available for 17 of 25 patients, five with liver metastases. Sequencing data was processed using an in-house developed pipeline to identify somatic events including coding mutations, copy number alterations, gene fusions, and differential expression. Activation of the AKT-mTOR pathway and sequencing data were compared between hepatic and non-hepatic lesions using an integrated RPPA and genomic approach. Results: Among liver metastases, AKT was activated in 4 of the 8 (50.0%) patients, while 6 of the 8 cases (75.0%) showed activation of p70S6. Sequencing data revealed mutation of PIK3CA in 4 of the 5 liver metastases (80.0%). Three of the PIK3CA mutated specimens with catalytic domain mutations (codons 1023 and 147) demonstrated co-activation of AKT and p70S6, while the fourth case, containing a helical domain mutation (E542K), had activation of p70S6 only. The PIK3CA wild-type liver metastasis demonstrated low activation of AKT and p70S6. For non-hepatic metastases AKT was activated in 2 of the 17 cases (11.8%) and p70S6 in 5 of the 17 patients (29.4%). Discussion: Although these results need further validation, activation of the AKT-mTOR pathway appears to be a hepatic specific signature in MBC and could be used for the selection of targeted agents for hepatic lesions. Citation Format: Pierobon M, Wong S, Reeder A, Anthony SP, Robert NJ, Northfelt DW, Jahanzeb M, Vocila L, Wulfkuhle J, Dunetz B, Aldrich J, Byron S, Craig D, Liotta L, Petricoin EF, Carpten J. The AKT-mTOR pathway as a potential organ-specific drug target signature of hepatic metastases from breast cancer. [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 P2-05-21.

Normalization of Reverse Phase Protein Microarray Data: Choosing the Best Normalization Analyte.

Reverse phase protein microarray (RPMA) are a relatively recent but widely used approach to measure a large number of proteins, in their original and posttranslational modified forms, in a small clinical sample. Data normalization is fundamental for this technology, to correct for the sample-to-sample variability in the many possible confounding factors: extracellular proteins, red blood cells, different number of cells in the sample. To address this need, we adopted gene microarray algorithms to tailor the RPMA processing and analysis to the specific study set. Using geNorm and NormFinder algorithms, we screened seven normalization analytes (ssDNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), α/β-tubulin, mitochondrial ribosomal protein L11 (MRPL11), ribosomal protein L13a (RPL13a), β-actin, and total protein) across different sample sets, including cell lines, blood contaminated tissues, and tissues subjected to laser capture microdissection (LCM), to identify the analyte with the lowest variability. Specific normalization analytes were found to be advantageous for different classes of samples, with ssDNA being the optimal analyte to normalize blood contaminated samples.

Abstract B1-14: A machine learning approach applied to Reverse Phase Protein Microarray data for pathways activation mapping of KRAS wild type and mutated adenocarcinomas of the lung

Abstract Background: KRAS proto-oncogene is one of the most commonly mutated genes in Non-Small Cell Lung Cancer (NSCLC) with greater frequency in the adenocarcinoma (AD) histotype. Patients with mutant KRAS often do not benefit from standard therapy and effective targeted therapy are still not available for these patients. Little is known about which pathways are activated in KRAS mutant ADs. Understanding which interactions drive KRAS mutant lesions can lead to the identification of novel targets for treating more effectively patients harboring a KRAS mutation. The aim of this study was to apply machine learning techniques to Reverse Phase Protein Microarray (RPPA) data to select proteins whose activity can better describe the KRAS status (wild type or mutated). Materials and methods: A total of 58 samples (24 KRAS wild type and 34 KRAS mutated) were collected from surgically treated AD patients of the lung at the H. Lee Moffitt Cancer Center & Research Institute (Tampa, FL) and at the S. Maria della Misericordia Hospital (Perugia, Italy). Tumor cells were isolated with laser capture microdissection and RPPA was performed to quantitatively measure the expression/activation levels of 155 proteins. Recursive Feature Elimination with Support Vector Machine (RFE-SVM) was used to rank proteins according to the absolute value of their weight in the hyperplane defined by the SVM to separate the 2 groups (KRAS wild type or mutated). LSimpute algorithm was used to impute missing data due to depletion of biological sample. Stability and robustness of the results were achieved using the RFE-SVM algorithm within an ensemble feature selection framework. Results: The LSimpute algorithm was applied to impute missing data in 11 patients that presented a number of missing proteins between 1% and 48% in the single record and of 5% of the overall dataset. The tested algorithm accuracy was 0.90. The RFE-SVM algorithm was then applied to the entire dataset (58 samples). The analysis of the RPPA data revealed that the activation of many signaling proteins involved in the ERK pathway is also discriminative relatively to KRAS WT/MUT. Among the proteins with higher rank were found p70S6K, ERK1/2T202/Y204, EGFR, PP2A and Akt S473. Stability and robustness of the output of the algorithm was confirmed in the completed dataset RFE-SVM algorithm. Conclusion: The proposed methodology is the first example of computational approach based on machine learning algorithms applied to the analysis of proteomic data in cancer translational research. The output of the procedure is a ranking of proteins that could play potential key roles in the signal pathways of patients harboring KRAS mutations when compared to KRAS wild type patients. Results obtained from this study could make important contributions to the identification of proteins that can be targeted to develop more effective treatments for AD patients with KRAS mutations. Furthermore this methodology can overcome the issue of missing values in RPPA datasets generating a stable and robust complete output. Citation Format: Fortunato Bianconi, Elisa Baldelli, Federico Patiti, Paolo Valigi, Eric B. Haura, Lucio Crinò, Vienna Ludovini, Emanuel Petricoin, Mariaelena Pierobon. A machine learning approach applied to Reverse Phase Protein Microarray data for pathways activation mapping of KRAS wild type and mutated adenocarcinomas of the lung. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-14.

Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors.

Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets.Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to Laser Capture Microdissection and Reverse Phase Protein Microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC).Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02).This finding was verified in an independent population by IHC (p=0.03).KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications.

Abstract B52: Pilot study: Random periareolar fine needle aspiration (RPFNA) accurately depicts biochemical phenotype displayed in breast tissues of high risk African American women

Abstract Background: There are more than 2.9 million breast cancer survivors in America today. However African American women have a 79% five-year survival while survival rates for European American women are at 92%. While early detection increases the survivability rates of most breast cancer, the standard mammogram fails to identify the earlier stages of some of the more aggressive types of breast cancer. Short-term breast cancer risks in women are increasingly being identified by Random Periareolar Fine Needle Aspiration (RPFNA). While the reproducibility of this technique is no longer the primary concern for its usage; RPFNA's small sample-sets and the inability to accurately correlate pathological and biochemical findings within the entire breast inhibits full acceptance of this technique. The purpose of this study was to determine if the utilization RPFNA accurately sampled the entire breast tissue. Methods: In this pilot study 10 high risk African American women who were high risk for breast cancer (known or suspected BRCA1 mutation carriers) had breast tissues collect by RPFNA. These samples were laser captured and the RPFNA protein expression for wide array of biomarkers (stem cell, epithelial- mesenchymal transition (EMT), proliferation, apoptosis and senescence) were assessed utilizing reverse-phase protein microarray in mammary epithelial cells. The same 10 high risk African American women either underwent prophylactic mastectomies or had a mastectomy after cancer diagnosis. Mastectomy specimens were serial sliced into 2.5 centimeter slices and 2 to 3 cubic centimeter samples were excised from a specified section of each slice of breast tissue. On average 103 samples were collected from each breast and the same (RPFNA) biomarkers were analyzed. Results: In this exploratory study, we have determined there are no significant differences between the expression of the biomarkers we have tested in RPFNA samples and the whole breast tissue sampling. Specifically we have tested for EMT markers (Vimentin and Snail), p53 and additional markers involved in proliferation and apoptosis. Conclusions: This study shows that RPFNA is an accurate measure of mammary breast epithelial and this technique directly correlates to the measurements found throughout the entire breast tissues. RPFNA did not show any significant differences in the expression of specific biomarkers tested, suggesting that RPFNA samples could be utilized to develop more robust breast cancer risk profiles. Citation Format: Christopher Sistrunk, Adrian Ambrose, Amira Carter, Victoria Seewaldt. Pilot study: Random periareolar fine needle aspiration (RPFNA) accurately depicts biochemical phenotype displayed in breast tissues of high risk African American women. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B52.

The intracellular signalling pathway signature (the signalome) in PBMCs in the presence of a common TRAPS-associated genetic variant, TNFRSF1A p.(Arg121Gln) (legacy p.R92Q) is distinct from normal PBMCs and from other pathogenic variants

Question Like other disorders with a strong genetic association, a growing sequence dataset from various autoinflammatory syndrome patients continues to identify many variants of uncertain significance (VUS), i.e. missense and intronic variants or small insertions/deletions, for which the impact on protein function and pathways, and therefore the clinical significance, is unknown. Since it is unclear how these variants are associated with an increased risk of autoinflammatory disease, the clinical management of carriers of VUS is complicated. Therefore, there is a strong demand for reliable tests to rapidly assess the clinical significance of VUS, providing carriers of these variants with the necessary information to make an informed clinical decision and refining treatment by stratified therapy strategies. In many cases, a novel VUS is not common enough to evaluate its significance. Common variants with apparently variable penetrance can be more accessible as a model to test functional aspects. A missense variant in TNFRSF1A rs41495584 (“p.R92Q”) is the most commonly identified variant associated with TRAPS within our mainly Caucasian UK patient population. The aim of this study was to comprehensively examine the intracellular signalling pathways that are affected by the presence of p.R92Q, in comparison to normal cells and to wellknown symptomatic variants such as p.C33Y. Methods PBMCs were collected from patients and healthy volunteers with informed consent. Reverse-phase protein microarray was applied to examine a large number of signalling molecules and inflammatory cytokines, using a feature subset selection process to identify distinctive subsets.

Abstract 4847: Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using next-generation sequencing (NGS) and reverse-phase protein microarrays (RPMA)

Abstract Background Inflammatory Breast Cancer (IBC) is a clinicopathologic entity characterized by rapid progression and poor prognosis, even with the advent of targeted therapies and a multimodal approach. Gene expression profiles of IBC tumors vs. non-IBC demonstrated that HER2+ IBC have increased mTOR signaling compared to non-IBC (Iwamoto et al). mTOR activation is a mechanism for both HER-2 targeted and endocrine resistance. Combining the molecular diagnostics of next-generation sequencing (NGS) and reverse-phase protein microarray (RPMA) may identify additional therapeutic targets as well as mechanisms of resistance to targeted therapy. Methods This is an observational analysis of 12 IBC patients who had tissue biopsy after progression on standard therapies. All patients had tissue analysis by NGS and RPMA. NGS was performed by either FoundationOne™ or our in-house hot spot panel of 45 genes. RPMA was performed using TheraLink™ to identify expression of cancer-related phosphoproteins; it quantifies HER1, HER2, and HER3 receptor overexpression, and 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. Subset of 7 patients had AR, MET, ALK and PDL1 expression evaluation. Results All patients had IBC and 75% had metastatic disease. 17% of patients were ER+/HER2-, 42% ER+/HER2+, 25% ER-/HER2+, and 17% TNBC. All HER2+ patients by IHC had activation of HER2 on RPMA, and 75% of those patients had confirmed ERBB2 amplification on NGS. 75% of patients had mTOR activation on RPMA, 78% of whom had a concomitant alteration in the PIK3CA pathway on NGS. 71% of the patients evaluated had ALK expression and 80% of those patients had concomitant mTOR activation. Also, both TNBCs had HER2 activation by RPMA. HER2 targeted therapy was continued on 2 patients after RPMA confirmed activation of HER2. 4 patients were initiated on combinations with an mTOR inhibitor, Everolimus (Afinitor®), and one of whom initially obtained a clinical response, upon progression had repeat RPMA demonstrating only persistent mild activation of mTOR with a decrease in downstream proteins. Another patient with TNBC IBC who underwent 3 lines of neoadjuvant therapy prior to surgery was found to have HER1, HER2, HER3 and mTOR activation; she was started on adjuvant lapatinib and capecitabine and remains with no recurrent disease. 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 as they provide complimentary data on molecular pathways activation which expands therapeutic options, predicts treatment-sensitivity and selects more effective combinations. Citation Format: Laura K. Austin, Keithe Shensky, Juan Palazzo, Tiffany Avery, Rebecca Jaslow, Corrine Ramos, Nicholas Hoke, Emanuel Petricoin, Massimo Cristofanilli. Identifying molecular targets and mechanisms of treatment resistance in inflammatory breast cancer (IBC) using next-generation sequencing (NGS) and reverse-phase protein microarrays (RPMA). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4847. doi:10.1158/1538-7445.AM2015-4847

Abstract LB-112: A retrospective study to assess the potential for the TheraLink® HER family assay, a reverse-phase protein microarray assay, to predict treatment benefit in breast cancer

Abstract Background A significant percentage of patients with HER2+ breast cancer exhibit de novo resistance or develop an acquired resistance to anti-HER therapy. HER2 overexpression is currently used to guide anti-HER2 therapy by either IHC to measure the total amount of HER2 protein present, or FISH to measure the number of copies of the HER2 gene. Several potential mechanisms for this resistance have been proposed, ranging from mechanisms intrinsic to the target itself to the involvement of compensatory signaling pathways. As current therapy-guiding assays provide a measure of the level of HER2 protein, a better predictor of response may be possible through an assessment of not only HER2 activation (phosphorylation status), but also the protein levels of the other HER family members, their activation, and the activation of downstream resistance signaling pathways. To provide evidence that HER receptors are actively signaling, and to identify alternative therapies, a measure of the activation status of key signal transduction pathways downstream of these receptors was performed. The availability of molecular diagnostic assays that can evaluate phosphoproteins is an appealing approach to predicting treatment-sensitivity and to select more effective therapies. Methods De-identified breast tissue samples, including a subset of samples having their HER2 status identified by IHC and FISH were received for phosphoproteomic analysis using the TheraLink® HER Family Assay. The assay utilizes reverse-phase protein microarray (RPMA) to measure the total protein level and activation status of multiple proteins directly from a FFPE-biopsy lysate. The TheraLink® Assay measures the protein levels of EGFR, HER2, and HER3, their phosphorylation status, and the activation status of proteins in three downstream signaling pathways: AKT/mTOR; Mek/Erk; and Jak/STAT. Results The TheraLink® Assay's panel identified the cohort of tumors with known HER2 status (100% concordance between HER2 level measured by RPMA platform and IHC/FISH). Moreover, unsupervised clustering of this subset of samples identified three patterns of unique signaling. The first group showed high phosphorylation levels of HER2 and EGFR protein, suggesting the potential use of a dual kinase inhibitor therapy. The PI3 kinase and MAPK pathways were also elevated in this cohort. A second group that might not benefit from mono-therapy was observed. This group showed high levels of HER3 protein, a well-known dimerization partner for HER2. Therefore, this group might benefit from an anti-dimerization therapy, like pertuzumab. The PI3 Kinase and Jak/Stat pathways were also highly activated in this cohort. A third unique group exhibiting only activation of HER2 and HER3 was observed, with no downstream activity. Although a pan-HER kinase inhibitor has therapeutic potential, further downstream pathways, as well as other tyrosine kinase receptors, should be further examined. Conclusion The TheraLink® Assay has the potential to identify therapeutic strategies that might provide benefit to patients that are HER2 positive but failing on anti-HER2 therapy. Alternative therapeutics can be more precisely identified using the TheraLinkTM HER Family Assay's panel, based upon the molecular uniqueness of the groups described. Citation Format: Corinne Ramos, Nicholas Hoke, George J. Snipes, Pinar Yurt, Cody Thomas, Tuan Tran. A retrospective study to assess the potential for the TheraLink® HER family assay, a reverse-phase protein microarray assay, to predict treatment benefit in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-112. doi:10.1158/1538-7445.AM2015-LB-112

Abstract 3817: Suicidal autophagy induced by immunotherapy targeting folate receptor in ovarian cancer

Abstract Objective Cancer cells depend on folate metabolism for DNA replication, making folate receptor alpha (FRα) an important therapeutic target. Blocking FRα with a humanized anti-FRα monoclonal antibody, MORAB-003 offers a potent clinical opportunity; however, the mechanisms underlying its efficacy were not well understood. We investigated the functional mechanisms underlying MORAB-003 induced tumor inhibition in ovarian cancer. Methods Using FACS analysis and real-time PCR, FRα expression was determined in an array of human ovarian cancer cells. Next, we examined the effects of MORAB-003 monotherapy and combination with docetaxel in tumor growth using the xenograft mouse models. In vitro, we analyzed ovarian cancer cells with 3-dimensional (3-D) spheroid-like architecture. We next performed systems-based analysis including reverse-phase protein and genomic microarrays, and discovered an array of factors/genes involved in autophagy was upregulated by MORAB-003. Functional assays including FACS with AO staining, tandem mRFP/mGFP-LC3 fluorescence microscopy, transmission electron microscopy (TEM) and siRNAs knockdown were performed to further investigate MORAB-003-induced autophagy in tumor inhibition. Results IGROV1 and SKOV3ip1 cells express high-level FRα and A2780 cells express low-level FRα. In vivo, MORAB-003 monotherapy or a combination of MORAB-003 and docetaxel led to significantly decreased tumor growth in IGROV1 and SKOV3ip1 models, but not in A2780 model. Immunohistochemistry analysis on tumor sections indicated that MORAB-003 significantly reduced proliferative cancer cells, but had minimum effects on tumor cell apoptosis or angiogenesis. Network analysis on protein and gene expression profiles in IGROV1 and SKOV3ip1 tumors revealed that MORAB-003 significantly upregulated a number of factors/genes involved in autophagy, including LC3II, ATG3, ATG4B, S116-PEA-15 and BECN1. Furthermore, S116-PEA-15 and Beclin-1 were required for caspase-3-activated cell death in 3-D IGROV1 and SKOV3ip1 spheroids treated with MORAB-003. Results from autophagic flux assays indicated that MORAB-003 treatment led to an abundant accumulation of late-stage autophagic vacuoles. Blocking the autophagic vacuolization or fusion using bafilomycin A1 or hydroxychloroquine reversed the MORAB-003 induced reduction of cell viability in 3-D IGROV1 and SKOV3ip1 spheroids. Furthermore, queries from the Cancer Genome Atlas data (TCGA) revealed that copy-number gains of the FOLR1 gene significantly correlated with shorter disease-free survival in patients with the immunoreactive subtype of ovarian serous carcinoma and uterine carcinoma. Conclusion MORAB-003 inhibits tumor growth via a non-canonical autophagy associated with programmed cell death. This finding has substantial implications for clinical development of anti-FRα therapies, especially with regard to novel combinations and predictive biomarkers. Citation Format: Yun-Fei Wen, Anil K. Sood. Suicidal autophagy induced by immunotherapy targeting folate receptor in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3817. doi:10.1158/1538-7445.AM2015-3817

Abstract 1994: Impact of laser capture microdissection on cancer signaling proteins and phosphoproteins

Abstract Background: Tissues are heterogeneous in terms of cell composition and the percentage of tumor epithelium can vary tremendously in any clinical biopsy. Given the importance and focus on the cancer phosphoproteome due to the mechanism of action of nearly all targeted therapies, we and others have shown the importance of upfront cellular enrichment strategies such as laser capture microdissection (LCM). However, despite the improved data quality, retention of dynamic range, and correlations with IHC and genomic data shown with LCM based analysis; questions remained about the overall impact of LCM on protein and phosphoprotein measurements. The aim of the study was to investigate whether a real-world tissue processing workflow using infrared (IR) based LCM for the isolation of pure cell populations affect the overall expression and activation/deactivation levels of protein signaling molecules that are important in druggable target analysis and companion diagnosis. Materials and methods: Five Non Small Cell Lung Cancer samples were used for this study. Eight micron sections were cut and mounted on glass slides. Samples were microdissected or lysed within 30 minutes once stained. For each sample, using alternating adjacent slides, three different protein lysates were collected: the first lysate was generated after tumor epithelium enrichment was performed via LCM, the second one was obtained by lysing of the undissected whole tissue directly from the glass slide (TL) and the last was obtained by microdissecting the entire tissue slide (LCM-TL) without regard to the underpinning cell population. Lysates were then analyzed by Reverse Phase Protein Microarray to evaluate the expression/activation levels of 87 proteins. Unsupervised hierarchical clustering analysis was used to compare the expression/activation of the 87 analytes across the different collection methods. Results: Tumor percentage was estimated as 50% (n = 1), 60% (n = 1) and 70% (n = 3). Unsupervised hierarchical clustering analysis showed that for all five pairs (100%), the TL and matched LCM-TL samples clustered within the same arm of the dendrogram while only one of the LCM samples (with tumor percentage of 70%) clustered in the same arm of the dendrogram as the matched LCM-TL. On the other hand, two of the five LCM samples were contained in the same cluster. Conclusion: These results confirm the importance of using LCM for upfront cellular enrichment for the accurate measurement of the expression/activation levels of proteins for biomarker discovery, even when the tumor cells percentage in the specimen is high. Importantly, based on this pilot study we conclude, that the LCM process, using an IR based direct capture laser system, itself does not appear to have a significant impact on the integrity of proteome and phosphoproteome. Citation Format: Elisa Baldelli, Vienna Ludovini, Lucio Crinò, Lance Liotta, Emanuel Petricoin, Mariaelena Pierobon. Impact of laser capture microdissection on cancer signaling proteins and phosphoproteins. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1994. doi:10.1158/1538-7445.AM2015-1994

A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection.

Burkholderia is a diverse genus of gram-negative bacteria that causes high mortality rate in humans, equines and cattle. The lack of effective therapeutic treatments poses serious public health threats. Developing insights toward host-Burkholderia spp. interaction is critical for understanding the pathogenesis of infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray technology was previously proven to identify and characterize novel biomarkers and molecular signatures associated with infectious disease and cancer. In the present study, this technology was utilized to interrogate changes in host protein expression and phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections of which eight proteins were selected for further characterization by immunoblotting. Increased phosphorylation of AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune response. Modulating the inflammatory response by perturbing their activities may provide therapeutic routes for future treatments.

Potent organo-osmium compound shifts metabolism in epithelial ovarian cancer cells

The organometallic "half-sandwich" compound [Os(η(6)-p-cymene)(4-(2-pyridylazo)-N,N-dimethylaniline)I]PF6 is 49× more potent than the clinical drug cisplatin in the 809 cancer cell lines that we screened and is a candidate drug for cancer therapy. We investigate the mechanism of action of compound 1 in A2780 epithelial ovarian cancer cells. Whole-transcriptome sequencing identified three missense mutations in the mitochondrial genome of this cell line, coding for ND5, a subunit of complex I (NADH dehydrogenase) in the electron transport chain. ND5 is a proton pump, helping to maintain the coupling gradient in mitochondria. The identified mutations correspond to known protein variants (p.I257V, p.N447S, and p.L517P), not reported previously in epithelial ovarian cancer. Time-series RNA sequencing suggested that osmium-exposed A2780 cells undergo a metabolic shunt from glycolysis to oxidative phosphorylation, where defective machinery, associated with mutations in complex I, could enhance activity. Downstream events, measured by time-series reverse-phase protein microarrays, high-content imaging, and flow cytometry, showed a dramatic increase in mitochondrially produced reactive oxygen species (ROS) and subsequent DNA damage with up-regulation of ATM, p53, and p21 proteins. In contrast to platinum drugs, exposure to this organo-osmium compound does not cause significant apoptosis within a 72-h period, highlighting a different mechanism of action. Superoxide production in ovarian, lung, colon, breast, and prostate cancer cells exposed to three other structurally related organo-Os(II) compounds correlated with their antiproliferative activity. DNA damage caused indirectly, through selective ROS generation, may provide a more targeted approach to cancer therapy and a concept for next-generation metal-based anticancer drugs that combat platinum resistance.

HER3, PI3K, and JAK2 pathway activation on reverse phase protein microarray (RPMA) in HER2-amplified residual disease (RD) refractory to preoperative chemotherapy plus trastuzumab (H), lapatinib (L), or both (HL).

621 Background: Pretreatment (preRx) biopsies of HER2-amplified breast cancers (BCs) that did not achieve pCR with preop chemotherapy plus H vs L vs HL had PI3K pathway activation with high p-FOXO ...