Reverse Phase Protein Microarray Technology Research Articles

771 Increased PD-L1 expression in human skin acutely and chronically exposed to UV irradiation

Overexpression of the ligand PD-L1 on tumor cells may represent a hallmark of tumor immune evasion, and such overexpression has been documented in several tumors including cutaneous squamous cell carcinoma (cSCC). While PD-L1/PD-1 activity in the skin has been primarily described in inflammatory models, we focus on PD-L1 expression in human keratinocytes exposed to UV irradiation. We first assessed PD-L1 expression in archived samples from acute human studies implementing different UV exposures in sun protected (SP) skin ranging from 2-3 minimal erythema doses (MED). PD-L1 expression was measured by immunohistochemistry (IHC). From the 20 samples evaluated 14 (70%) demonstrated > 5% epidermal PD-L1 expression at 24 hours. PD-L1 in dermal endothelial cells was also noted to be increased at 24 hrs. Thereafter, we determined that HaCaT cells also respond to acute UV by increasing PD-L1 protein expression in a time- and dose-dependent manner (2-4 fold over control at 24-48 hours), an effect which is markedly reduced in the presence of MEK/ERK or JNK inhibitors. Subsequently, we investigated PD-L1 expression in chronically UV exposed human skin using matched samples representing SP skin, sun damaged skin (SD), actinic keratosis (AK) and cSCC using IHC and reverse phase protein microarray technology (RPPA). The RPPA methodology revealed negligible PD-L1 expression in SP, SD and AK, with significantly measurable expression in cSCC. IHC analysis of PD-L1 in another cohort of matched samples revealed a similar profile, with negligible expression of PD-L1 in SP and SD skin, slight staining in AK (mean 4.2%, +/- 9.5), and significant staining in cSCC (mean 19.5%, +/- 26.6). These findings suggest that upregulation of PD-L1 may be induced by UV through established, pharmacologically targetable stress-signaling pathways in keratinocytes.

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.

Psychiatric patient stratification using biosignatures based on cerebrospinal fluid protein expression clusters

Psychiatric disorders are caused by perturbed molecular pathways that affect brain circuitries. The identification of specific biosignatures that are the result of altered pathway activities in major depression, bipolar disorder and schizophrenia can contribute to a better understanding of disease etiology and aid in the implementation of diagnostic assays.In the present study we identified disease-specific protein biosignatures in cerebrospinal fluid of depressed (n: 36), bipolar (n: 27) and schizophrenic (n: 35) patients using the Reverse Phase Protein Microarray technology. These biosignatures were able to stratify patient groups in an objective manner according to cerebrospinal fluid protein expression patterns. Correct classification rates were over 90%. At the same time several protein sets that play a role in neuronal growth, proliferation and differentiation (NEGR1, NPDC1), neurotransmission (SEZ6) and protection from oxidative damage (GPX3) were able to distinguish diseased from healthy individuals (n: 35) indicating a molecular signature overlap for the different psychiatric phenotypes. Our study is a first step toward implementing a psychiatric patient stratification system based on molecular biosignatures. Protein signatures may eventually be of use as specific and sensitive biomarkers in clinical trials not only for patient diagnostic and subgroup stratification but also to follow treatment response.

Elevated TNFR1 and Serotonin in Bone Metastasis Are Correlated with Poor Survival following Bone Metastasis Diagnosis for Both Carcinoma and Sarcoma Primary Tumors

There is an urgent need for therapies that will reduce the mortality of patients with bone metastasis. In this study, we profiled the protein signal pathway networks of the human bone metastasis microenvironment. The goal was to identify sets of interacting proteins that correlate with survival time following the first diagnosis of bone metastasis. Using Reverse Phase Protein Microarray technology, we measured the expression of 88 end points in the bone microenvironment of 159 bone metastasis tissue samples derived from patients with primary carcinomas and sarcomas. Metastases originating from different primary tumors showed similar levels of cell signaling across tissue types for the majority of proteins analyzed, suggesting that the bone microenvironment strongly influences the metastatic tumor signaling profiles. In a training set (72 samples), TNF receptor 1, alone (P = 0.0013) or combined with serotonin (P = 0.0004), TNFα (P = 0.0214), and RANK (P = 0.0226), was associated with poor survival, regardless of the primary tumor of origin. Results were confirmed by (i) analysis of an independent validation set (71 samples) and (ii) independent bioinformatic analysis using a support vector machine learning model. Spearman rho analysis revealed a highly significant number of interactions intersecting with ERα S118, serotonin, TNFα, RANKL, and matrix metalloproteinase in the bone metastasis signaling network, regardless of the primary tumor. The interaction network pattern was significantly different in the short versus long survivors. TNF receptor 1 and neuroendocrine-regulated protein signal pathways seem to play an important role in bone metastasis and may constitute a novel drug-targetable mechanism of seed-soil cross talk in bone metastasis.

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

Abstract Background: Profiling protein signaling activation in cancer is critical as these proteins represent targets for new molecular therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane- based chemotherapy that longitudinally collected biopsy specimens and molecular and clinical/pathological characterization. Methods: Tumor epithelium was procured by Laser Capture Microdissection from 149 pretreatment frozen biopsy specimens (T1) and 102 frozen biopsy specimens (T2, 1–4 days post-chemotherapy). Reverse Phase Protein Microarray technology was used to quantitatively measure the activation of 39 and 100 key signaling proteins in the T1 and T2 biopsies, respectively, including numerous drug targets for Phase I-III and FDA-cleared therapeutics. Associations between protein activation and response to chemotherapy (RCB 0/1 vs 2/3) and relapse-free survival (RFS) were evaluated for the HR+/HER2− patient population at each time point and for changes between T1 and T2. Significance thresholds for response and RFS were as follows: Wilcoxon rank sum test p < 0.05; Cox proportional hazard model, likelihood ratio p < 0.05. Results: We focused our analysis on protein changes in patients who had poor clinical outcomes (Table 1) We observed systemic activation of HER family signaling and downstream AKT activation in tumors from patients who do not respond (RCB2/3) and/or whom recurred. Increased ERBB2 and ERBB4 levels were observed at T1 and T2, respectively, from patients who did not respond to neoadjuvant therapy. Dynamic changes in ERBB3 and AKT phosphorylation/activation were revealed between the T1 and T2 time points, which were associated with recurrence. Increased phosphorylation of MARCKS, a known PKC substrate, was seen in the T1 biopsy in the non-response cohort. Conclusions: Our analysis revealed activation/increased expression of HER family proteins along with downstream AKT activation in HR+/HER2− patients who have poor clinical response. These events, if validated, point to potential treatment options that could be rationally considered for non-responding HR+/HER2− patients with a number of investigational agents that target ERBB3 and AKT signaling in the clinic today. Increasing HER2 protein expression in a HER2− cohort may appear contradictory, however these findings may point to important subtle increases in HER2 that have important clinical considerations. Given the known clinical benefit from HER2 directed therapy in HER2− patients seen in other studies, our findings may have clinical relevance if validated. Further validation is required to explore the significance of these ongoing findings with the hope that the analysis could lead to molecularly rationalized therapies for patients with HR+/HER2− tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-02.

Reverse phase protein microarray technology in traumatic brain injury

Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology.

Abstract A15: Proteomic profiling of early mammary carcinogenesis: Targeting dysregulated protein pathways with tailored therapies

Abstract Over the last 50 years, the number of cancer related deaths has decreased by only 2%. One of the most promising approaches to reduce breast cancer mortality is to develop tools for early detection and early intervention of breast cancers. Normal mammary gland homeostasis requires the coordinated regulation of signaling networks; whereas, dysregulation of signaling networks occurs during breast cancer initiation. Reverse-phase protein microarray (RPPM) is a high-throughput proteomic tool, developed to test for dysregulation of protein signaling networks in human biopsy specimens. For example, ErbB2/HER2/neu, Erb3, EGFR, Akt, Erk1/2 receptor tyrosine kinases all play a role in cancer cell growth and survival, and developing targeted therapies against these pathways is a promising tailored approach to breast cancer treatment. To test which tyrosine kinases are activated in early mammary carcinogenesis, reverse phase protein microarray analysis (RPPM) was performed on 31 random periareolar fine needle aspirates (RPFNA) obtained from a cohort of asymptomatic high-risk women, testing 59 antibody endpoints related to cell growth and survival pathways, including ER, EGFR, Her2, Erb3, Akt and Erk1/2, to test for patterns of differential protein expression. RPFNA allows for serial sampling of breast cytology and subsequent histology from asymptomatic women at high risk for developing breast cancer and also provides the ability to monitor response to preventative treatments. Epithelial cell clusters from RPFNA samples were isolated using AutoPix automated laser capture microdissection. RPPM uses denatured lysate so antigen retrieval, which is a limitation for tissue arrays, is not a problem. In addition, each sample is printed in serial dilution, providing an internal standard, and RPPM does not require direct labeling of the sample, thus improving reproducibility, sensitivity, and robustness. An interrogation of phosphorylated versus non-phosphorylated state of proteins was performed using a Wilcoxon rank sum test. Spearman rank correlation coefficients were used to estimate the association between RPPM total- and phospho-protein expression as a function of cytology; a 2-sided p value < 0.05 was used to determine if a significant positive or negative association exists. Four distinct clusters of phospho-proteins were identified amongst the 31 RPFNA samples. Varying cell lines, including 15hTert, MDA231, T47D, were then treated with inhibitors based on the protein pathways identified in these clusters. Fluoxetine, a common antidepressant, was shown to affect cancer cell viability as well as the phosphorylation of Erk1/2 pathway in a concentration and time dependent manner. Diagnosing cancers based on proteomic signatures in addition to histopathology will allow for individualized selection of therapeutic combinations that can best target the entire disease-specific errant protein network of a patient. This proteomic signature has great potential for daily clinical practice considering the RPPM technology is commercially available through Theranostics Health and could be used every time a woman undergoes a routine biopsy. In addition, understanding a key protein pathway and how it is wired could have a profound effect on both functional biology and on the understanding of cancer mechanisms as a whole. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A15.

Protein signal pathway mapping of human breast cancer from I-SPY: Correlations with response and genomic subtyping.

10512 Background: Profiling signaling pathway activation in cancer is critical as these pathways represent targets for new molecular guided therapeutics. The I-SPY TRIAL (CALGB 150007/150012, ACRIN 6657) is a trial of neoadjuvant anthracycline- and taxane-based chemotherapy that provides a unique series of biopsy specimens that have been subjected to extensive and detailed molecular characterization, together with detailed clinical and radiographic information. Methods: Purified tumor epithelium from 149 frozen pre-treatment biopsy specimens were procured via laser capture microdissection. Reverse phase protein microarray (RPMA) technology was used to quantitatively measure the activation of 40 key signaling proteins including components of pathways of interest for targeted therapy in breast cancer (HER2, IGFR, EGFR, mTOR, AKT and PLK1). Association between pathway activation and genomic subtype, pCR, residual cancer burden (RCB), and other clinical measures including tumor size, and nodal involvement was examined. Results: Protein pathway activation measured in pretreatment samples divided patients into 5 groups by hierarchical clustering. Activation of HER2 and AKT pathways was associated with pCR and minimal residual disease following neoadjuvant chemotherapy (p < 0.05). Activation of HER2, AKT, PLK1 and EGFR were each associated with HER2 positivity as measured by IHC and/or FISH. Individual pathway activation levels were not observed to be associated with tumor molecular subtype as determined by transcriptome profiling. Patients with global signaling activation had higher rates of cancer recurrence. Conclusions: Using RPMA technology and minimal tissue from core biopsies, it is possible to measure functional pathway activation for which targeted therapies are available. The clinical significance of the five pathway activation subtypes and the activity of individual pathways will be prospectively tested in I-SPY 2, where agents targeted to these pathways will be introduced in the neoadjuvant setting. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Theranostics Health Theranostics Health Theranostics Health

Use of protein pathway mapping of brain metastasis from breast and lung cancer patients to identify new therapeutic targets: A seed/soil study.

10620 Background: Brain metastases are the most common fatal complication of systemic cancer, especially of lung (50%-60%) and breast (15%-20%) cancers. In this era of personalized therapy, there is a critical need to uncover the critical molecular targets for brain metastases, however little is known about what signaling pathways are actually activated in the context of the brain microenvironment. Using a unique study set of brain metastases from patients with breast or lung cancer along with a separate set of primary NSCLC tumors, functional broad-scale pathway activation mapping could be performed. Methods: Epitheliumfrom 89 frozen biopsy samples (10 breast and 32 lung metastasis; 47 primary lung cancer) were collected using Laser Capture Microdissection. The phosphorylation/activation of 128 key signaling proteins involved in important molecular pathways was measured using Reverse Phase Protein Microarray technology. Results: Brain metastasis from breast and lung cancer formed distinct pathway subgroups that consisted of amongst others, c-erbb2, AKT, mTOR, EGFR, SMAD, and ERK-p38 signaling. EGFR phosphorylation did not necessarily correlate with EGFR protein expression levels. Of 128 signaling analytes measured, 30 revealed significant differences in levels of activation/phosphorylation between breast and lung metastases to the brain and 58 were differently activated between lung to brain metastases and primary lung tumors (p < 0.05). Breast cancer metastases showed significantly (p < 0.05) higher activation of many components of the c-erbB2/IGFR-AKT pathway networks compared to lung cancer metastasis whereas lung cancer metastases to the brain exhibited higher relative levels of many members of the EGFR- ERK signaling network. Conclusions: Pathway activation mapping of brain metastasis demonstrates signaling activation portraits with both “seed” and “soil” components. These distinct activation groupings reveal both the heterogeneity of signaling networks in brain metastasis that would require a prior stratification to targeted therapies as well as the requirement of direct analysis of the metastatic lesion. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Theranostics Health Theranostics Health Theranostics Health

Functional proteomic pattern identification under low dose ionizing radiation

High dose radiation has been well known for increasing the risk of carcinogenesis. However, the understanding of biological effects of low dose radiation is limited. Low dose radiation is reported to affect several signaling pathways including deoxyribonucleic acid repair, survival, cell cycle, cell growth, and cell death. The goal of this study is to reveal the proteomic patterns influencing these pathways. To detect the possibly regulatory proteins/kinases, an emerging reverse-phase protein microarray (RPPM) in conjunction with quantum dots nano-crystal technology is used as a quantitative detection system. The dynamic responses are observed under different time points and radiation doses. To quantitatively determine the responsive protein/kinases and to discover the network motifs, we present a discriminative feature pattern identification system (DFPIS). Instead of simply identifying proteins contributing to the pathways, our methodology takes into consideration of protein dependencies which are represented as strong jumping emerging patterns (SJEPs). Furthermore, infrequent patterns, though occurred, will be considered irrelevant. Computational results using DFPIS to analyze ataxia-telangiectasia mutated (ATM) cells treated under six different ionizing radiation doses (0cGy, 4cGy, 10cGy, 50cGy, 1Gy, and 5Gy) are presented. For each dose, the dynamic response was observed at different time points (1, 6, 24, 48, and 72h). The sets of different responsive proteins/kinases at different dose are reported. For each dose, the SJEPs for ATM-proficient and ATM-deficient cells are shown and compared. By using the new RPPM technology and the DFPIS algorithm, we can observe the change of signaling patterns even at a very low radiation dosage where conventional technologies tend to fail.

Periprostatic Adipose Tissue as a Modulator of Prostate Cancer Aggressiveness

Adipose tissue has been suggested to contribute to the pathogenesis of various disease states, including prostate cancer. We investigated the association of cytokines and growth factors secreted by periprostatic adipose tissue with pathological features of aggressive prostate cancer. Periprostatic adipose tissue was harvested from patients undergoing radical prostatectomy and cultured for 24 hours to generate conditioned medium or snap frozen immediately for functional signaling profiling. Multiplex analysis of the periprostatic adipose tissue conditioned medium was used to detect cytokine levels and compared to patient matched serum from 7 patients. Interleukin-6 in serum and periprostatic adipose tissue conditioned medium was further analyzed by enzyme-linked immunosorbent assay and correlated with clinical variables, such as age, body mass index and Gleason score, in 45 patients. Interleukin-6 expression in periprostatic adipose tissue was determined by immunohistochemistry. Reverse phase protein microarray technology was used to analyze cell signaling networks in periprostatic adipose tissue. Interleukin-6 in periprostatic adipose tissue conditioned medium was approximately 375 times greater than that in patient matched serum and levels correlated with pathological grade. This finding was further extended by cell signaling analysis of periprostatic adipose tissue, which showed greater phosphorylation on Stat3 with high grade tumors (any component of Gleason score 4 or 5). Higher Gleason score correlated with high levels of conditioned medium derived interleukin-6. Moreover, cell signaling analysis of periprostatic adipose tissue identified activated signaling molecules, including STAT3, that correlated with Gleason score. Since STAT3 is interleukin-6 regulated, these findings suggest that periprostatic adipose tissue may have a role in modulating prostate cancer aggressiveness by serving as a source of interleukin-6. Also, we found low numbers of inflammatory cells in the fat, suggesting that adipocytes are the major secretors of interleukin-6.

Pathway Biomarker Profiling of Localized and Metastatic Human Prostate Cancer Reveal Metastatic and Prognostic Signatures

Reverse phase protein microarray technology was used to study key signaling pathways thought to be involved in the progression of benign epithelium to the lethal phenotype of prostate cancer. Specimens of androgen-stimulated localized prostate cancer (N = 21) and androgen-deprivation therapy-recurrent local (N = 4) or metastatic (N = 11) prostate cancer were laser capture microdissected prior to analysis. The results showed significant increases in protein expression levels in malignant epithelial cells and patient-matched stromal tissue, which included higher levels of the apoptotic proteins Bax and Smac/Diablo and increased phosphorylation of Bcl2 (S70). The mitochondrial protein Smac/Diablo and the transcription regulatory protein STAT3 (Y705) correlated with Gleason sum and differed statistically in high Gleason grade (8-10) prostate cancers. Distinct metastasis-specific pathways were activated by caspase cleavage activation, ErbB2 phosphorylation, Bax total protein and Bcl-2 phosphorylation while phosphorylation of all three members of the MAPK family, ERK, p38, and SAP/JNK, were reduced significantly in metastatic lesions compared to primary cancers. This study, the most comprehensive pathway analysis ever performed for human prostate cancer, presents evidence of specific pathway biomarkers that may be useful for assessment of prognosis and stratification for therapy if validated in larger clinical study sets.

Molecular network analysis of signaling pathways in metastatic breast cancer in patients treated with erlotinib

10064 Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, is under active clinical investigation. It may have profound effects on the cellular molecular network by affecting downstream signaling and inhibiting tumor cell proliferation. Knowledge about the specificity of drug effects on the signaling pathways might enable therapy tailored specifically for each patient’s defect. We examined the activation of cellular signaling pathways involved in cell survival and proliferation in response to erlotinib treatment using a biopsy study set that comprised patient-matched breast cancer metastases from 8 women obtained prior to treatment with erlotinib and after 1 month of therapy. We procured pure cancer cell lysates from very small amounts of frozen tissue by Laser Capture Microdissection (about 60,000 cells). Matching lysates were printed on nitrocellulose and incubated with antibodies against the endpoints of interest. Using the Reverse Phase Protein Microarray technology we were able to generate a portrait of 18 phosphorylated kinase substrate endpoints as well as total expression levels of ERK, EGFR, Her2/neu, and IGF1R that revealed an upregulation of pro-survival pathway signaling in the post-erlotinib treatment set. Especially the measurement of the pro-survival, apoptosis-inhibiting signal transduction pAkt (S473) protein alone showed a statistically significant stronger staining in post-erlotinib treatment samples (p=0.019) in each of the individual patient-matched specimens as well as the entire post-treatment group, showing concordance with the clinical data of the trial, i.e. progressive disease observed for all participating patients. The results indicate the feasibility of this approach to monitor signaling events and molecular pathways in clinical specimens taken during targeted therapy trials. No significant financial relationships to disclose.

Proteomic profiling identifies prognostic subsets in Rhabdomyosarcoma (RMS)

9502 Background: RMS is the most common soft-tissue sarcoma in childhood and is generally sensitive to currently available chemotherapeutic regimens. At present, there is no way to prospectively identify the 30–40% of patients destined to fail initial therapy. We sought to develop a proteomic signature comprised of signal pathway endpoints to predict outcome in a group of uniformly treated children with Stage III RMS and identify new targets for therapy. Methods: Thirty-four pre-treatment patient samples selected on the basis of tissue availability representing both embryonal and alveolar histologies were analyzed employing laser capture microdissection with multiplexed proteomic analysis using reverse phase protein microarray technology. All patients had been entered onto IRS studies and treated with combined chemotherapy and XRT. A panel of 13 specific antibodies was employed based on ongoing work pointing toward specific cell signaling pathways playing a role in the biological behavior of RMS. Validate...

Use of Reverse Phase Protein Microarrays and Reference Standard Development for Molecular Network Analysis of Metastatic Ovarian Carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.